The Effects of Metallic Ions on Citrate Oxidation

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m n EFFECTS OF METALLIC IONS ON CITRATE OXIDATION

by W illia m Jo se p h H artm an

A t h e s i s s u b m itte d i n p a r t i a l f u l f i l l m e n t o f th e r e q u ir e m e n ts f o r t h e d e g re e o f D o c to r o f P h ilo s o p h y i n t h e D ep artm en t o f B io c h e m is try i n th e G ra d u ate C o lle g e o f t h e S t a t e U n iv e r s it y o f Iowa# A u g u s t, 1950

ProQuest Number: 10666170

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T m o

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The a u th o r w ish e s t o e x p r e s s h i s s i n c e r e a p p r e c i a t i o n f o r t h e e x c e l l e n t s u p e r v is io n an d tim e ly en co u rag em en t o f H r. 0 e o rg e K a ln lts k y a t lahose s u g g e s tio n t h i s s tu d y was made.

u ii

$m m

op

c g m c tts

C h a p te r I II III

p ag e

In tro d u c tio n * » • • * , * » « , • » • « • * •

I

H is to ric a l*

. . • * • * * . * * * • * * . . .

2

M ethods . . . . . . . . . . . . . . . . . . .

IP

p r e p a r a t i o n o f Mnayme. System s P r e p a r a t i o n o f S o lu tio n s # . . A n a l y t i c a l M ethods# • * * # * M anom etric M ethods* • • * * ;# 17

R e s u lts and D is c u s s io n *

. . . . . . . . . . . . * * * * . # *. * * * • #

* * * # # # * * # . *

In tro d u c tio n . . • M anganese and Magnesium, C o m p e titio n .* • M anganese an d M ercu ry I n h i b i t i o n . * .• . I n h i b i t i o n b y V a rio u s M etals* * * * * * ♦ A n a ly s is o f t h e ' Kg** I n h i b i t i o n * * ** • A n a ly s is o f c o p p e r I n h i b i t i o n # • * . * * M anganese Sodium and M anganese P o ta s s iu m In h ib itio n . * * * * * * * ' * • * • • • ' * * lo c a l i s a t i o n o f In h ib itio n * « • • • • • • N on-E nzym atie E f f e c t o f M anganese * * * *

IP 22 25 26 SB 28 33 35 38 38 43 43 53 60

?

Summary . * » » * * • » • * . * * « * # • # •

73

VI

B ib lio g ra p h y * . . . . . . . . . . . . . . . .

75

111

f ABU OF f ABUS

fa b le

P&g*

X C o m p e titiv e E f f e c t s o f Mg** an d Mh** i n C i t r a t e O x id a tio n I * * * * * * * * * . • * « II III

IV V VI V II V III IX X XI X II

32

C o m p e titiv e E f f e c t s o f Mn** and Mg** i n S i t u a t e O x i d a t i o n 'l l # • # * « * • * * * • • * *

34

C o m p e titiv e E f f e c t s o f Ig * * an d Mh** i n C i t r a t e O x id a tio n w ith V a r i a t i o n i n f i n e o f A d d itio n o f C i t r a t e an d Go^f a c t o r s * » • « * « • , * • • • •

36

C o m p e titiv e E f f e c t s o f M ercury and M anganese i n C i t r a t e O x id a tio n * . « « • * • * * * * • * * *

37

C o m p e titiv e E f f e c t s o f Mn** and En**# C a**, 0u**# and Ha* I n C i t r a t e O x id a tio n * » • . * » . * * • 39 The E f f e c t s o f V a ry in g C o n c e n tr a tio n s o f Mn** i n O vercom ing th e I n h i b i t i o n C aused b y Mg** * •

41

C o m p e titiv e E f f e c t s o f On** an d Mn** i n C i t r a t e O x id a tio n I . * « * * * • « « . • * * • • * * *

44

C o m p e titiv e I f f a c t s o f Cu** and Mn*+ i n C i t r a t e O x id a tio n X I * * • * * • * • « • * . • • • * • .

46

C o m p e titiv e E f f e c t s b e tw e e n Mh** an d V a rio u s C o n c e n tr a tio n s o f Ha* i n C i t r a t e O x id a tio n * * •

49

Rf f o o t s o f V a ry in g C o n c e n tr a tio n o f Mn** i n O vercom ing I n h i b i t i o n P ro d u ced b y Ha* • • • • •

30

C o m p e titiv e E f f e c t s o f K* and Mn*+ i n C i t r a t e O x id a tio n * * * * • • « • • • « • • • • * . • «

32

R e la t io n s h ip B etw een I n h i b i t i o n and P o s i t i o n o f M e ta l i n P e r i o d i c f a b l e * » « « • • • * • * * •

34

TABLE OF TABLES (O o at* d .) fa b le X III XIV

p ag e X f f e e tg o f % * * an d Mn** I n O x& losucoim t© D e c a r b o x y la tio n . * « * * « * * * « • • * * * * • E f f e c t s o f V a rio u s C o - f a c t o r s on O x id a tio n o f a - K e t o g l u t a r i c A cid I « • • « • • • » • • • • •

SB .

SB

I f f a c t s o f V a rio u s C o - f a c to r s on O x id a tio n o f a - K e t o g l n t a r i c A cid I I . .................................... * * *

@9

A c t i v i t y o f o - K e t o g l u t a r a t e D eh y d ro g en ase i n th e P re s e n c e o f V a ry in g C o n c e n tr a tio n s o f Mn** an d Mg** * * , * * * * * * * , * * * * * * * * * * *

61

C o m p e titiv e I f f a c t s o f B o th ' Sfg** and in * * i n a X et o g lu t a r a t e O x id a tio n * • . » * * . » * • * « «

6$

The I f f a c t s o f M e ta ls On c-K e t o g l u t a r a t e O x id a tio n I n P ig e o n B r e a s t M uscle* * * * * * * *

64

The E f f e c t o f Mg** and in * * on th e O x id a tio n o f S u c c in a te i n B a b b it K idney C o rte x * « » * • • « •

68

XX f h e E f f e c t o f % * * an d Mn** on D e c a r b o x y la tio n o f Q x a la c e ta te i n B a b b it K idney C o rte x * * * * *

VO

XV XVI

XVII X V III XIX

XXI

E f f e c t s o f 1%** and Mn** on t h e O x id a tio n o f P y ru v a te I n E s c h e r i c h ia C o ll * * * * * * * * * *

V

71

»|1a

All1 T3T

F ig u r e I

F r a c t i o n a l C e n t r i f u g a t i o n o f C e l l u l a r C om ponents»

8

f h e I f f e c t a o f V a ry in g G o n e e n tra t 1 ons o f Mn*+l a O vercom ing th e I n h i b i t i o n C aused b y £%#*« * * * •

$

fit© Wtf©etjr of Varying Ooneanbrationa of In 0v©robmlng ih© in h ib it Ion Cant ed by Cu**# » * * *

1

ISTBOBUCTIOH A lth o u ^ i m in e r a ls make u p o n ly a s m a ll p o r t i o n o f t h e c o n s t i t u e n t s o f t h e b o d y , l i k e t h e v i t a m i n s , th e y e n t e r i n t o many v i t a l p h y s i o l o g i c a l f u n c tio n s *

P ro g re ss i n u n d er­

s t a n d i n g t h e r o l e o f i n o r g a n ic io n s i n l i v i n g t i s s u e h a s b e e n s lo w , d u e I n p a r t t o t h e d i f f i c u l t y i n d e v i s i n g e x p e r im e n ta l m ethods o f a p p r o a c h , t h e c o m p lic a tio n s In v o lv e d i n d e te r m in ­ in g a n e x a c t p o i n t o f a c t i o n an d i n i s o l a t i n g th e m e t a l l i c com pounds i n t h e i r n a t u r a l form * I t i s known t h a t some m e t a l l i c I o n s c a n f u n c t i o n a s c a t a l y s t s f o r num erous e n z y m a tic r e a c t i o n s *

S in c e i t

h a s b e e n d e m o n s tra te d t h a t compounds s t r u c t u r a l l y r e l a t e d t o m e ta b o lic in t e r m e d i a t e s c o m p e t i t i v e l y i n t e r f e r e w ith n o rm al m e ta b o lic p r o c e s s e s , t h i s s tu d y was u n d e r ta k e n t o d e te r m in e w h e th e r c o m p e titiv e e f f e c t s c o u ld b e d e m o n s tra te d w ith m e t a l l i c io n s *

T he r e s u l t s o f t h i s p a p e r show t h a t

Mg*+, 0u*4> O a**, Zn++ an d Hg++ com pete w ith MrvH- f o r th e enzyme s y s te m s w h ich o x id iz e c i t r a t e * The enzym e, a - k e t o g l u t a r i c d e h y d ro g e n a s e , I s p r i m a r i l y I n v o lv e d i n t h e s e c o m p e titiv e e f f e c t s *

2 HISTORICAL A u m b e r o f s t u d i e s c o n c e r n in g t h e r o l e o f m in­ e r a l s I n n u t r i t i o n h a s d e m o n s tra te d t h a t t h e a n im a l body r e q u i r e s s e v e n p r i n c i p a l m in e r a l e le m e n ts ! c a lc iu m , mag­ n e s iu m , so d iu m , p o ta s s iu m , p h o s p h o ru s , s u l f u r and c h l o r in e * I n a d d itio n , a t l e a s t s ix o th e r m in e ra ls , th e s o - c a lle d "trace** e le m e n ts , a r e n e e d e d i n s m a lle r a m o u n ts | n am ely , i r o n , c o p p e r , i o d i n e , m an g an ese, c o b a l t and z in c #

I t is

n o t known w h e th e r f l u o r i n e , alum inum an d b a r o n a r e e s s e n ­ t i a l f o r a n im a ls o r man {1 } ( 2 )# T he n e c e s s i t y f o r t h e s e v a r i o u s m in e r a ls h a s a l s o b e e n shown b y t h e p h y s i o l o g i s t I n s t u d i e s on i s o l a t e d t i s s u e s su c h a s n e r v e an d m u scle*

I f th e s e ti s s u e s a r e

p u t I n t o a n o n - io n ic m edia and th e n s t i m u l a t e d , th e y f a l l t o r e s p o n d , b u t i f a s m a ll am ount o f a n y a l k a l i Io n i s ad d ed t o t h e m e d ia , a r e s p o n s e c a n b e e l i c i t e d #

T h is r e s ­

p o n se v a r i e s w ith t h e k in d , t h e d u r a t i o n , and t h e e o n c e n tm t i o n o f th e io n added#

The a l k a l i n e e a r t h I o n s c a n n o t

m a in ta in a r e s p o n s e when ad d ed t o th e n o n - io n ic m e d ia , b u t th e y c a n overcom e i n h i b i t i o n s c a u se d b y a n e x c e s s o f th e a l k a l i Io n s*

T h is h a s b e e n shown i n t h e c l a s s i c a l e x p e r i ­

m ents on t h e b e a t i n g h e a r t , w here t h e p a r a l y z i n g e f f e c t o f K+ h a s b e e n r e v e r s e d b y v e r y s m a ll am ounts o f Ca-M», Sr-f-f,

3 Mg++* o r Ba-fr**

t h i s r e v e r s a l h a s a l s o b e e n o b ta in e d b y u s ­

i n g su c h I o n s a s 0 o++# "Hn*** am i Hi-M-*

th e a n ta g o n is tic

e f f e c t b e tw e e n t h e io n s ha® b e e n a t t r i b u t e d t o t h e i r i n f l u ­ e n c e on t h e d e g r e e o f h y d r a t i o n o f t h e p r o t e i n { $ )• M e t a l l i c io n s h a v e a l s o b e e n shown t o b e n e e d e d b y l i v i n g t i s s u e s f o r t h e f o r m a tio n o f su c h s p e c i f i c p r o ­ t e i n co m p lex es a s h e m o g lo b in , m y o g lo b in , c y to c h ro m e , e r y t h r o c r u o r i n an d c h l o r o c r u o r in *

T h e se compounds a r e a l l

ir o n - p o r p h y r I n - p r o t e i n co m p lex e s and a r e c o n c e rn e d w ith t h e r e s p i r a t o r y f u n c t i o n o f t h e v e r t e b r a t e s an d I n v e r t e b r a t e s * The s t r u c t u r e s o f t h e s e com plex s u b s ta n c e s h a v e b e e n s t u d i e d i n g r e a t d e t a i l a® t o i r o n c o n t e n t (4 ) ( 5 ) , m o le c u la r w e ig h t ( 6 ) ( 7 ) , k in d an d au s& er o f p o r p h y r in s ( 8 ) (9 ) ( 7 ) , an d t h e am ino a c id c o n t e n t o f t h e p r o t e i n (1 0 ) (1 1 ).

T he l i n k a g e b e tw e e n t h e i r o n an d p r o t e i n h a s b e e n

s t u d i e d b y m a g n e to m e trie ( 1 2 ) # p o t© n tio m e tr ie and s p e c t r o p h o to m e tr lc means (1 3 ) (1 4 )*

T h e se v a r io u s m eth o d s h av e

shown t h a t t h e i r o n i s i n c o m b in a tio n w ith tw o im id a z o le g ro u p s o f t h e p r o t e i n l,g l o b i n ,># A n o th e r ty p e o f r e s p i r a t o r y p ig m en t fo u n d i n c e r t a i n m a rin e i n v e r t e b r a t e s i s h em o cy an in (1 5 )*

T h is p ig ­

m ent c o n t a i n s c o p p e r I n c h e m ic a l c o m b in a tio n w ith a p o ly ­ p e p t i d e p r o s t h e t i c g ro u p o f a l a r g e r p r o t e i n m o le c u le (1 6 )* O th e r p r o t e i n s fo u n d I n t h e a n im a l body w hich

4

c o n t a i n F e* + , JSn-M*, Ca** and

a re re s p e c tiv e ly ? f e r r i t i n

( 1 7 ) , i n s u l i n ( 1 8 ) , m y o sin and a c t i n (1 9 )* A lth o u g h t h e s t r u c t u r e s o f t h e p r e c e d in g m e ta llo * p r o t e i n s a r e i n t e r e s t i n g and c a n s e r v e a s a g u id e t o f u r t h e r s tu d y o f o t h e r p u r e m e ta llo ~ p r o t© in s # t h i s p a r t i c u l a r s tu d y I s c o n c e rn e d w ith th e m e t a l l i c io n s w hich a r e in v o lv e d i n e n s y m a tic r e a c t i o n s * A l a r g e number o f ensym es n ee d s m e t a l l i c io n s f o r a c tiv ity *

A few su c h en&ymes a r e l i s t e d a c c o r d in g t o th e

m e ta l r e q u ir e d * Ensvmes C o n ta in in g I r o n C a ta l a s e

8 heme— 1 i r o n

(8 0 )

H o r s e r a d is h p e r o x id a s e

hem e-c& rboxyl

(8 1 )

C ytochrom e A^, Ag, Ag

heme

( 88)

C ytochrom e b

n o t heme

(88)

O ytochrom e c

*43$ Fe a s heme

(8 2 )

C ytochrom e o x id a s e

heme

(2 5 )

C ytochrom e 0 p e r o x id a s e

3$ Fe a s heme

(2 4 )

Myelo p e r o x id a s e

1$ Fe a s heme

(2 5 )

Ziacto p e r o x id a s e

1 heme

(2 6 )

*2$ Cu++

(2 7 )

Snzymes C o n ta in in g Copper T y r o s in a s e

Sagas?.?. fflWtatoibMt Copper (C o n 'td .) taa c c a s e

*24^ Cu++

(2 7 )

A s c o rb ic A cid O x id ase

*24# Gu++

(2 7 )

P o ly p h e n o l O x id ase

011+4*

(2 8 )

C a rb o n ic Arihydras©

*54# 2n++

(2 9 )

K id n ey P h o s p h a ta s e

&n++

(SO)

I n t e s t i n a l P h o s p h a ta s e

Zn++

(3 0 )

e ric a s o

•13$ Zn++

(» 1 )

Enzymes C o n ta in in g Z t m

Bngymes in C arbohydrate M etaboll am C ontaining Mn++ o r Mg»» Mali© a c i d d e c a r b o x y la s e

Mn++

(3 2 )

O x a lo s u c c in ic c a r b o x y la s e

Mn++

(3 3 )

O xaX aootate c a r b o x y la s e

Mn++# Mg++

(3 4 )

P y ru v ic d e h y d ro g e n a se

Mn++* Mg++

(3 5 )

C a rb o x y la s e

Mg++, Mn++, JPe++1 CU++# (3 6 ) Gd++# 2n++# Co++

Phosphogliacom utas©

Mg++* $tn++# 00++ # Mgf+ (3 7 )

B e x o k in a se

Mg++

(3 8 )

Frueto**kinas©

Mg++

(3 9 )

P h o s p h o ry la s e

Mg++

(4 0 )

P h o s p h a ta s e

Mg++# Mn++

(4 1 )

B n o la se

Mg++, Mn++# 2n++

(4 2 )

6

Bnzvmes I n C a rb o h y d ra te M etab o lism C o n ta in in g fe * * o r Ma** A d e n o sin e p o ly p h o s p h a te s e

Mg**, Mn**

(4 3 )

f h o s p h o * e n o l* tr a n s p h o s p h o r y la s e

Mn**,

a k o to g l u t a r i c d e h y d ro g e n a se

Mg**

(4 5 )

G lu c o se p h o s p h a te d e h y d ro g e n a se

Mn**

(4 6 )

K*

(4 4 )

Hnsymes i n P r o t e i n M etah o lism C o n ta in in g Mn** o r fe * * P a ra am ino h i p p u r i c a c i d h y d ro ly s is

K*, Mg**

(4 7 )

Zn**, Mg**, Mn**, Co**

(4 8 )

M ic ro b ia l p o ly p e p tid a s e s

Z n**, H i*-

(4 9 )

P e p tid a s e o f L» m e s e n te rio d e s

Zn**, Pb**, Cu**, f e * * , Sn**

(so )

P ro lid a s e

fe * *

(S I)

A rginas©

Fe** , Co**, Mn**, Ml**

(5 2 )

Co**, Mb**

(S 3 )

G lutam ines©

Mg-M-, K*

(5 4 )

Oys t a t h i o n l n e - a s e

Mgt+

(5 5 )

C h o lin e s c e t y l a s e

K*, Mg**, Mn**

(5 6 ) (5 7 )

C h o l ln e s t e r a s e

d i v a l e n t io n s

(5 7 )

Bone p h o s p h a ta s e

Mn**

(5 8 )

D ip e p tid a s e s

J a c k b e a n a rg in a s © fezvm es C o n ta in in g P a r lo u s M etals

7

t e m » s C o n ta in in g V a r io u s M e ta ls (C o n ’ t d . ) P re th ro m b i a

Ca-n*

(5 9 )

Some -very i n t e r e s t i n g w ork h a s b e e n d o n e w i t h i n t h e l a s t few y e a r s to w a rd e x p l a i n i n g t h e f u n c t i o n o f m e t a l l i c io n s i n e n z y m a tic r e a c t i o n s . The w ork o f E m il S m ith on p e p t i d a s e s o f a n im a l t i s s u e s i s th e m ost s y s t e m a tic and c o m p le te (5 1 ) {©G-6 8 } . He h a s u s e d p u r i f i e d p r e p a r a t i o n s o f enzym es an d h a s fo u n d d e f i n i t e r a t i o s b e tw e e n t h e am ount o f m e ta l r e q u i r e d an d t h e am ount o f p r o t e i n ( 6 5 ) .

He h a s shown t h a t c e r t a i n

p e p tid a s e ® may f u n c t i o n i n t h e p r e s e n c e o f v a r io u s m e t a l l i c io n s ( 6 5 ) # and i h a t enzymes fro m d i f f e r e n t t l s ~ s u e s w h ich h y d r o ly s e t h e same d i p e p t i d e n e e d d i f f e r e n t m e ta ls i n o r d e r t o f u n c t i o n ( 6 2 ) .

I t h a s b e e n o b s e rv e d

t h a t m e t a l l i c io n s may r e q u i r e a s lo n g a s f o u r h o u rs t o fo rm a com plex w ith a n enzym e, th u s i n d i c a t i n g a d e f i n i t e c h e m ic a l com plex f o r m a tio n ( 6 1 ) .

A ls o , t h e d i s s o c i a t i o n

c o n s t a n t o f th e g ly e y l^ J ^ le u e in® d ip e p tld a s e - m e t& l com plex o f h o g m ucosa h a s b e e n d e te rm in e d ( 6 4 ) .

By s tu d y in g th e

e f f e c t s o f g l y c y l g l y c l n e d ip e p tld a s © and c o b a l t on m e th y la te d g l y c y l g l y c l n e , S m ith h a s shown t h a t a t l e a s t one h y d ro g e n o f th e am ino g ro u p i s n e c e s s a r y f o r com plex f o r m a tio n an d h y d r o l y s i s , a l s o t h e h y d ro g e n on th e p e p tid e

8

n i t r o g e n I s in v o lv e d I n t h e com plex*

T he n e c e s s i t y o f t h e

c a r b o x y l g ro u p i s s t i l l I n q u e s tio n *

E x p e rim e n ts w ith

l e u c i n e am ino e x o p e p tid a s e show t h a t t h e h y d r o l y t i c pow er o f t h e enzyme i s p r o p o r t i o n a l t o t h e m ass o f th e s i d e c h a in s o f t h e am in o a c i d s ( 6 8 ) .

T he S te r e o c h e m ic a l c o n f i g u r a t i o n

o f t h e p e p t i d e i s a l s o a f a c t o r i n s p e c i f i c i t y s in c e B -le u cin & m id e i s h y d r o ly s e d 10*000 tim e s s lo w e r th a n L~ le u c in a m ld e *

A n o th e r i n t e r e s t i n g e x p e rim e n t w ith J> leu ey X -L

a l a n i n e a n d 3> i© u e y i~ D ~ a la n in o i n d i c a t e s t h a t t h e m e th y l g ro u p o f a l a n i n e i n th e I*~B compound s t e r l c a l l y h i n d e r s t h e f o r m a tio n o f t h e m e t a l l i c com plex*

Thus* i n t h e m e ta l*

a c t i v a t e d p e p t id a s e s * t h e r e a r e a t l e a s t f i v e f a c t o r s w hich I n f l u e n c e s p e c i f i c i t y o f t h e enzyme (6 9 )*

( 1 ) The enzyme

m ust h a v e tw o p o i n t s o f a tta c h m e n t t o th e s u b s t r a t e ; ( 2 ) t h e s i d e c h a i n o f t h e s u b s t r a t e m ust b© i n c o r r e c t s t e r e o ­ c h e m ic a l r e l a t i o n s h i p t o t h e enzyme a t t h e tim e o f a t t a c h ­ m ent* and ( 3 ) m ust b e o f a c e r t a i n m a ss; (4 ) t h e s i d e c h a i n o f t h e u n s p e c i f i c p o r t i o n o f t h e s u b s t r a t e m ust n o t i n t e r f e r e w ith t h e com plex f o r m a tio n an d f i n a l l y ( 6 ) th e f r e e e n e rg y o f t h e b o n d t o b e h y d r o ly s e d m ust b e s u f f i ­ c i e n t l y low* T h e re h a s b e e n v e ry l i t t l e

o f t h i s ty p e o f

i n v e s t i g a t i o n c a r r i e d o u t i n t h e f i e l d o f c a rb o h y d r a te m e ta b o lism *

B e fo re d i s c u s s i n g t h e w ork w hich h a s b e e n

9

d o n e a lo n g t h i s l i n e on th e © nsym atic r e a c t i o n s I n t h e K reb s t r i c a r b o x y l i c a c i d c y c l e , th e r e a c t i o n s o f th e c y c le w i l l b e c o r r e l a t e d I n a s im p le d ia g ra m s mmwm f a t t y a c id s c a r b o h y d r a te p ro te in \ \ \ _ \ a s p a r ti c a c id a c e to a c e tic a c id p y r u v ic a c l& ± = £ a la n in e an d a c e t i c a c i d / O n * * / •*CGg o r/ Mg*Ho x a l a c e t i c a c i d + 2 c a rb o n compound 1 A 02 ~HgO

c i t r i c a c id 4-HgO

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Is© c i t r i c a c id fu ra a r ic a c i d A |A l A Og 1 /2 Og -RgO *HgO t 1 /2 Og H302 ^ ©xalosuccinic succinic acld e n o l e n o l 4* c a t i o n ~ ~ 7> com plex com plex ■< £ a k e to ae id ^ C O g ^ c atIo n The mode o f c o m b in a tio n b etw e en t h e t h r e e e le m e n ts o f th e d e c a r b o x y la t io n was n o t p o s tu la te d * The a k e t o g l u t a r i c a c i d form ed b y th e d e c a r b o x y l­ a t i o n o f o x a lo s u c c in ic a c i d i s a t t a c k e d b y a k e t o g l u t a r i c d e h y d ro g e n a se *

A c e l l - f r e e s u s p e n s io n o f w ashed h e a r t

m u scle c o n t a i n s a v e ry a c t i v e a k e t o g l u t a r i c d e h y d ro g e n a se w hich w i l l o x id is e a k e t o g l u t a r i c a c i d t o s u c c i n i c a c id a n d c a rb o n d io x id e (*77)* 0*

0,

/>

NC-C-GH

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\

HCH H

a k e t o g l u t a r i c a c id

*C-GB MTH-+ o r Mg»»

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a k e to g lu ta ric d e h y d ro g e n a se P04= + ATP

HC-C-OH

+C0g

s u c c in ic a c id

13

C o e a r b o x y la s e , I n o r g a n ic p h o s p h a te s , m agnesium io n s an d a d e n y lic a d d o r A ,T ,P # a r e n eed ed f o r i t s a c t i v i t y , arid Oytoohrom© o c a n a c t a s a n e l e c t r o n c a r r i e r i n t h i s r©«* a c tio n *

M anganese io n s a l s o a c t i v a t e t h e d e h y d ro g e n a se ,

h u t n o t t o a s g r e a t a n e x t e n t a s magnesium#

The s u c c in ic

a c i d fo rm ed i n t h i s r e a c t i o n i s th e n o x id iz e d b y s u c c i n i c d e h y d ro g e n a s e , a n enzyme d is c o v e r e d b y T hunberg (7 8 ) i n 1909*

Th© p ro d u c t o f t h i s o x id a tio n i s fu m a ric a c id *

H 4$

«®)

HC«*C**0i! I ^0 HC~C~0H H

________________ ^ «-----aucc i n i c d e h y d ro g e n a se

s u c c i n i c a c id

%

HOC-QH U ^0 EG~G~GH fu m a ric a c id

S u c c in ic d e h y d ro g e n a se o c c u rs i n m ost p l a n t s , a n im a ls , an d m ic ro -o rg a n is m s *

The g r e a t e s t amount i s fo u n d i n

k id n e y , l i v e r a n d h e a r t .

I t i s i n a c t i v a t e d by s e l e n i t e ,

a r s e n i t © , p y ro p h o s p h a te , m a lo n lc a c i d , o x a l o a c e tic a c i d , and c y s t i n e ( 7 9 ) ,

The n e x t enzyme f u n c tio n i n g i n t h e

o x i d a t i v e c y c le i s fu m arase* a c i d fo rm in g m a lic a c i d .

T h is enzyme h y d r a te s fu m a ric

The enzyme h a s n o t b e e n shown t o

r e q u i r e a m e ta l f o r a c t i v a t i o n * M alle d e h y d ro g e n a se w hich c o n v e r ts m a lic a c i d t o © x a la c e tic a d d d o es n o t r e q u i r e a m e ta l f o r a c t i v i t y e ith e r*

H ow ever, Ochoa e t a l» h a s d is c o v e r e d a n enzyme

i n p ig e o n l i v e r w h ich a p p e a rs t o d e c a r b o x y la te o x i d a t i v e l y

14

8.6I d t o p y r u v ic a c id and OOg i n th© p r e s e n c e o f T .P .H * an d Mn** (5 0 )* hoo^ c ^ qb

(s)

,0 4 T .f M t. I B e-o-oB OK ox*

.

Mn**

HOE

M alic d e c a rb oxy3 a se m a lic a c i d

re d .

H

* OOg

p y ru v ic a c id

O x a la c e tic a c i d d e c a r b o x y la tio n was f i r s t i n v e s t ! g a te d b y I r a m p l t s and Workman i n 1941 ( 3 4 ) .

They d i s -

c o v e re d a n enzyme i n M icro co ccu s L y c o d e ik tic u a w hich decom posed o x a l a c e t i c a c id i n t o p y ru v ic a c id and c a rb o n d io x id e * 0*O»C00H

O»0H300H {? >

H-C-dOOE E

Mh+* o r Mg*+>

o x a la c e tic a c id

H-C-H H

*00$

p y ru v ic a c id

T hey a l s o fo u n d t h a t f o r t h i s p r o c e s s m agnesium o r m anganese io n s w ere n e c e s s a r y , an d t h a t th ia m in o r co» c a r b o x y la s e was n o t n e c e s s a r y * Th*$ en z y m atic and ncjr*© nzy® atic d e c a r b o x y la tio n o f o x a l a c e t i c a c i d h a s b e e n e x t e n s i v e l y s tu d i e d b y J . F . S peck ( 8 0 ) .

I© fo u n d t h a t m e ta ls a c t i v a t e d t h e non**

e n z y m a tic d e c a r b o x y la tio n o f o x a la e e ta t© i n a d i r e c t r e l a t i o n t o t h e i r a to m ic w e ig h t*

The atom s w ith h i g h e s t

a to m ic w eight© c a u se d a f a s t e r s p o n ta n e o u s d e c a r b o x y la tio n

15

o f o x a l a c e t i c a c i d th a n atom s o f a lo w e r w e ig h t *

On t h e

o t h e r h a n d , u s in g th e o x a l a c e t i c d e c a r b o x y la s e fro m p a r s l e y r o o t , he fo u n d t h a t t h e r a t e s o f e n g y m a tic a c t i v i t y w ith v a r io u s m e ta ls h ad no d l s c e r n a b l e r e l a t i o n * s h i p t o th e a to m ic ^ e i g h ts *

The m ost a c t i v e m e ta l was

Mm**** an d th e optimum c o n c e n t r a t i o n f o r t h i s m e ta l was 1 x K T S M*

O th e r Io n s su c h a s 0 a * * , H I* * , and Fe**

showed optim um c o n c e n t r a t i o n a t a p p r o x im a te ly 1 x 10~® b u t t h e i r r a t e s o f a c t i v i t y w ere much lo w e r th a n t h a t o f In* * #

The optim um c o n c e n t r a t i o n o f Mg** and Ba** w ere

h ig h e r t h a n 5 x 10-3* Jl w h ich was t h e h i g h e s t c o n c e n t r a t i o n o f th e s e m e ta ls t h a t w as t r i e d i n t h e s e e x p e r im e n ts .

The

m o st i n a c t i v e m e ta ls G a**, 0d * * # Zn** an d ?b++ showed optim um r a t e s o f d e c a r b o x y la t io n a ro u n d 1 x 1Q~^ M*

B o th

e n s y m a tic and non^ensym & tic r e a c t i o n s f o llo w e d f i r s t o r d e r r a t e s f o r a l l d i v a l e n t m e ta ls * The n a t u r e o f th e com plex b e tw e e n m e t a l l i c io n s and o x a l a c e t i c a c id h a s r e c e n t l y b e e n s t u d i e d b y Stein** b e r g e r and W esthelm er (8 1 )*

T h ese i n v e s t i g a t o r s p r e p a r e d

d e r i v a t i v e s o f o x a l a c e t i c a c i d su c h a s ( 1 ) a a d im e th y l o x a l a c e t i c a c i d and (B) i t s mono e t h y l e s t e r i n o r d e r t o show w h ich c a rb o x y l g ro u p o f th e a c i d was in v o lv e d an d d e te r m in e w h e th e r th e e n o l fo rm o f th e k e to g ro u p was in v o lv e d i n th e com plex*

16

(1 )

'b-cf-Q H

pH g - 7

O H g«C -C -O H

gg® q .

o

®S

J* I

P

C H s -O C -O H I

C H g -C -H

♦ OOg

SHg

0

C -O -O -E t (8 )

° 0 -b -0 H

pH 8 - V

■ ■ ■■ > 28® 0 .

0

'b - ( f - 0 - l l t

I OHg-OH I CHs



B o th ao lcls d e o a r b o x y la te d sm o o th ly a t 2 8 ° C. a n d a pH o f 2*7*

When m e t a l l i c I o n s w ere a d d e d , t h e

d e c a r b o x y la t i o n o f th e d i a c i d was s t r o n g l y c a t a l y z e d , b u t t h e r a t© o f th e m o n o -e th y l e s t e r was n o t a f f e c t e d *

From

t h i s th e y c o n c lu d e d t h a t t h e e n o l fo rm was n o t n e c e s s a r y f o r d e c a r b o x y la t io n an d t h a t th e a e id g ro u p a t o t h e k e to g ro u p was t h e one in v o lv e d I n th e complex* T he p y r u v ic a c id w hich i s fo rm ed b y th e d e c a r b o x y la t io n o f o x a l a c e t i c a c id c a n b e o x id is e d i n s e v e r a l ways* P y ru v ic d e h y d ro g e n a se o x i d i s e s p y r u v ic a c id t o a c e t i c a c i d an d COg (5 5 )*

17

G-CB

ton**, %*•+, or Co++ (8 )

BOB p y r u v ic dehy& rogenaae

H

HCH 1

P04 *

p y r u v ic a c i d

a c e tic a c id

T h is o x i d a t i o n r e q u i r e s c o -o a rb o x y l a s e , m agnesium , m an g an ese, o r c o b a l t I o n s , an d in o r g a n ic p h o sp h a te * The o x i d a t i o n b y t h i s sy ste m i s c o u p le d w ith t h e p h o s* p h o r y l a t l o n o f a d e n y lic a c id * C a rb o x y la s e c a n d e c a rb o x y la t© p y r u v ic a c id t o a c e ta ld e h y d e ( 8 8 )* %

P

C~C~0H Mb** o r Mg**

(9 )

HOH H

c a r b o x y la s e

p y r u v ic a c id

CH I +COg BOH H

a c e ta ld e h y d e

D rie d y e a s t w hich ha® b e e n w ashed w ith a l k a l i n e p h o s p h a te l o s e s i t s power t o d e c a rb o x y la t© p y ru v ic a c i d (8 3 )*

The

s u b s ta n c e w hich w as w ashed o u t was th e n c a l l e d !fc o c a rb o x y la a © ,, (3 6 )*

A t th e same tim e a s t h e d is c o v e r y o f

c o - c a r b o x y la s e , i t was fo u n d t h a t m agnesium io n s w ere n eed ed t o a c t i v a t e t h e c a rb o x y la s e *

M anganese io n s w ere

fo u n d t o b e e v e n m ore a c t i v e th a n m agnesium io n s* C a rb o x y la s e i s i n a c t i v a t e d b y s i l v e r , c o p p e r, m e rc u ric and a n tim o n y io n s ( 8 4 ) ; sodium n i t r i t e , io d in e an d b ro m in e a l s o i n h i b i t ( 8S)*

The enzyme i s found i n y e a s t , b a c t e r i a ,

18

f u n g i an d p l a n t s and i s n o t a b s o l u t e l y s p e c i f i c , f o r i t c a n a l s o s p l i t a k e t o b u t y r i c and a k e t o v a l e r i o a c id s *

fh e

p u r e s t c a r b o x y la s e t h a t h a s b e e n made c o n t a i n s one gram ato m o f m agnesium , on© mol© o f th ia m in d ip h o s p h a te an d 7 i , 000 gram s o f p r o t e i n (8 0 )* S in c e t h e r e a c t i o n s i n th e K rebs t r i c a r b o x y l i c a c id c y c le w h ich n eed In * * a l s o may u s e Mg** and t h e eon-* e e n t r a t i o n s o f optimum a c t i v i t y f o r t h e s e two io n s a r e q u it© d i f f e r e n t , i t seemed p l a u s i b l e t h a t on© m e ta l m ig h t a c t i v e l y com pete w ith t h e o th e r f o r th e enzyme sy ste m s* f h e e x p e rim e n ts i n t h i s t h e s i s show t h a t Mg** and s e v e r a l o t h e r m e t a l l i c io n s c a n com pete w ith Mn** f o r th e a c t i v e g ro u p s on t h e enzymes in v o lv e d i n th e o x i d a t i o n o f c i t r i c a c id *

29

W IO D 0 P r e p a r a t i o n o f Bnzyta© S y s te m The enzyme sy ste m was fo u n d i n m ito c h o n d ria p r e p a r e d e s s e n t i a l l y a s d e s c r ib e d b y C lau d e ( 8 8 ) ( 8 9 ) , H ogeboon (9 0 ) an d S c h n e id e r (9 1 )#

A r a b b i t was s tu n n e d

b y a blow on t h e h ea d an d th e n d e c a p it a te d * w ere c h i l l e d on ic e *

The k id n e y s

t h e c o r t e x was removed* w eig h ed and

gro u n d i n a m o r ta r and p e s t l e w ith two p a r t s 0*154 M flaO l p lu s one p a r t 0*154 M KOI a t 0 ° 0*

The r e s u l t i n g

past© was hom o g en ised i n a P o t t e r E lv eh jem gro u n d g l a s s h o m o g e n ise r (9 2 ) f o r t h r e e m in u tes*

T h is hom ogonate

was f r a c t i o n a l l y c e n t r i f u g e d a c c o r d in g t o t h e p ro c e d u re o u t l i n e d ( F ig u r e 1 ) i n o r d e r t o i s o l a t e t h e m ito c h o n d r ia * The m ltr o c h o n d r ia w ere d i s p e r s e d i n 20 ml o f HaCl-KCl s o l u t i o n m aking a s u s p e n s io n c o n t a in i n g a p p ro x im a te ly 10 mg* d r y w e ig h t p e r ml*

T h is p r e p a r a t i o n w as th e n

d i a l y s e d i n a c e llo p h a n e s a c k f o r n i n e t y m in u te s a g a i n s t tw e n ty l i t e r s o f r e d i s t i l l e d w a te r*

The d i a l y s i s b ag

was t i e d o v e r a s lo w ly r o t a t i n g b e n t g l a s s ro d i n su ch a way t h a t t h e d i a l y s i s b a g tu r n e d end o v e r end* th u s a g i t a t i n g t h e c o n t e n ts o f th e b a g a s w e ll a s t h e d i a l y s i s w a te r*

A l l t h e abo v e o p e r a t i o n s w ere c a r r i e d o u t i n a

c o l d room a t 0° 0 *

20

F ig u r e 1 R a b b it K id n ey C o rte x H om ogenate 5 m in u te s 1500 x g r a v i t y R e s id u e

S u p e r n a ta n t

N u e le i, w hole c e l l s , d e b r i s

M ito c h o n d ria

KCl-UaOl 5 m in u te s 1500 x g r a v i t y R e sid u e

S u p e r n a ta n t M ito c h o n d ria

J f u c le i, w hole c e l l s and d e b r i s

25 m in u te s 2400 x g r a v i t y

r

S u p e r n a ta n t

R e sid u e

1

M ito c h o n d ria

d is c a r d e d

KGl-RaCl 5 m in u te s 1 9 ,0 0 0 x g r a v i t y R e s id u e

S u p e r n a ta n t 1

w ashed M ito c h o n d ria

d is c a r d e d

waahc i 5 tim e s 1 9 ,0 0 0 x g r a v i t y F r a c t i o n a l O s n tr tf u & a tio n o f C e l l u l a r Components

21

T he r a b b i t h e a r t was p r e p a r e d a c c o r d in g t o th e m ethod o f Ochoa (7 7 ) *

The h e a r t was rem oved fro m t h e

r a b b i t an d c h i l l e d * f i n e l y m in c e d , th e n w ashed w ith *9 p e r c e n t KOI an d c e n tr if u g e d *

The s u p e r n a ta n t was d is c a r d e d

a n d t h e m in ce w ashed two m ore tim e s w ith KOI o r u n t i l s u p e r n a ta n t s o l u t i o n h ad no r e d c o l o r a t i o n #

The m ince

w as th e n ground i n a m o r ta r w ith gro u n d g l a s s u n t i l a t h i n p a s t e was o b ta in e d *

T h is p a s t e was c e n t r i f u g e d a t

low sp e e d i n t h e c e n t r i f u g e and t h e c lo u d y s u p e r n a ta n t f l u i d was d la ly & e d a s d e s c r ib e d f o r tw o h o u r s i n tw e n ty l i t e r s o f r e d i s t i l l e d w a te r a t 0° 0 * The b a c t e r i a l p r e p a r a t i o n was p r e p a r e d fro m E s c h e r i c h ia O o li*

The E s c h e r ic h ia C o l l w ere in n o c u la te d

i n t o tw e n ty l i t e r s

o f s t e r i l e b r o th w hich was made o f th e

f o llo w in g c o n s t i t u e n t s i 10 20 15 12 5

gm p e p to n e gm y e a s t em KOI gm K^ETF04 gm b e e f e x t r a c t

5 gm Hag|!F04 *1 gm n o c ta & e c y l a l c o h o l ,5 1 t a p w a te r 9*5 1 d i s t i l l e d w a te r

T he b a c t e r i a w ere a e r a t e d and in c u b a te d a t 3 0 ° 0* f o r tw e n ty -tw o h o u rs #

T hey w ere th e n s e p a r a te d fro m th e

b r o t h i n a S h a r p ie s s u p e r - c e n t r i f u g e * The b a c t e r i a l p a s t e was th e n w eig h ed an d m ixed w ith a p p ro x im a te ly two p a r t s o f gro u n d g la s s *

T he r e s u l t i n g t h i c k p a s te was p u t i n t o a

c o n e ty p e ground g l a s s h o m o g en izer and gro u n d i n t o

03

t w e n t y - e i g h t m l o f w a te r*

t h e g ro u n d b a c t e r i a w ere m ixed

w e l l w ith t h e w a te r an d a llo w e d t o e x t r a c t , w ith o c c a s i o n a l s t i r r i n g , f o r f i f t e e n m in u te s *

t h e r e s u l t i n g f l u i d was

c e n t r i f u g e d a t 1 9 ,0 0 0 tim e s g r a v i t y f o r f i f t e e n m in u te s and t h e n th e s u p e r n a ta n t was d i a l y s e d a s d e s c r i b e d f o r n i n e t y m in u te s i n tw e n ty l i t e r s o f r e d i s t i l l e d w a te r* f f r e n a r a tio n o f S o lu tio n s T he v e r o n a l b u f f e r was p r e p a r e d a c c o r d in g t o th e m ethod o f M ic h a e lls ( 9 3 ) .

The s o l u t i o n was 0 . 1M sodium

v e r o n a l i n 0 . 1M HOI* When C. P . sodium c i t r a t e was u s e d a s s u b s t r a t e , t h e s t o c k s o l u t i o n was made t o 0 *1M* Th© s o l u t i o n was made o n ly i n 30 c * c . am ounts and k e p t i n th e c o ld *

On th e

a p p e a ra n c e o f m o ld , a new s o l u t i o n was p r e p a r e d . 0 * P . sodium s u c c i n a t e was p re p a re d i n th e same m anner a s t h e c i t r a t e . Sodium o x a la c e ta t© was p r e p a r e d b y h y d r o ly s in g th e © th y l e s t e r t o t h e a c i d a c c o r d in g t o th© m ethod o f Kramp i t s and Workman (3 4 )*

The o x a l a c e t i c a c i d was

n e u t r a l i s e d w ith p o ta s s iu m h y d ro x id e t o a n o ra n g e c o l o r w ith p h e n o l r e d (pH 7 ) Im m e d ia te ly b e f o r e a d d i t i o n t o th e s i d e arm o f th e W arburg v e s s e l .

Only 5 o .c * o f a *1|§

25

s o l u t i o n was p r e p a r e d b e c a u s e o f t h e i n s t a b i l i t y o f th© compound* A lpha k e t o g l u t a r i c a c i d was o b ta in e d fro m th e n u t r i t i o n a l B io ch em ical® C o r p o r a tio n and t h i s was n e u t r a l i s e d t o p h e n o l r e d a s d e s c r ib e d *

A *1M s o l u t i o n was p r e ­

p a re d i n 5 e*c* am o u n ts im m e d ia te ly b e f o r e u se * O x a lo s u e o in ic a c i d was p r e p a r e d b y c o n d e n sin g d i e t h y l o x a l i c e a t e r w ith d i e t h y l s u c c in ic e s t e r (9 4 )* Th© r e s u l t i n g t r i e t a y l ox& logm ccInlc e s t e r was h y d ro ly z e d i n c o n c e n tr a t e d h y d r o c h lo r ic a c id a c c o r d in g t o th e m ethod d e s c r i b e d b y Ochoa (7 5 ) •

The o x a lo s u c c in ic a c id was

p r e c i p i t a t e d a s t h e b a riu m © a lt an d s t o r e d i n th e c o ld u n d e r vacuum*

Im m e d ia te ly b e f o r e u&©# th e b a riu m was

p r e c i p i t a t e d b y s u l f u r i c a c id and th© s o l u t i o n n e u t r a l i s e d t o p h e n o l r e d w ith p o ta s s iu m h y d ro x id e * 0 . P , L ith iu m p y r u v a te (9 5 ) was u s e d a s a sub­ s t r a t e i n th e p y r u v ic d e h y d ro gem s© e x p e rim e n ts * A d en o sin e t r i p h o s p h a t e was p r e p a r e d fro m r a b b i t m u sc le a c c o r d in g t o t h e m ethod d e s c r i b e d i n b i o c h e m i c a l P r e p a r a t i o n s 1* (9 6 )*

The A*T.P* was s t o r e d i n a d i s i c c a t o r

i n t h e c o ld a s t h e d i- b a r iu m s a l t .

The b a riu m was p r e ­

c i p i t a t e d w ith s u l f u r i c a c i d and th e s o l u t i o n n e u t r a l i s e d t o p h e n o l r e d w ith p o ta s s iu m h y d ro x id e b e f o r e u se * Th© m e t a l l i c io n s o l u t i o n s w ere p re p a re d by

84

d i s s o l v i n g r e a g e n t g ra d e s a l t s i n t r i p l e d i s t i l l e d w ate r* The c o n c e n t r a t i o n r a n g e s w ere c o v e re d i n u n i t s o f t e n , a s c a n h e s e e n I n th© f o llo w in g ta b l e s *

s to c k s o l u t i o n s w ere

made t o a c o n c e n t r a t i o n o f *5 M an d w ere rem ade a b o u t tw ic e a m o n th , o r w h en ev er a p r e c i p i t a t e o r d i s c o l o r a t i o n i n d i c a t e d t h e s o l u t i o n was d e t e r i o r a t i n g * w e re k e p t a t 1 ° o*

A ll s o l u t i o n s

The f o llo w in g r e a g e n t grad© s a l t s w ere

u s e d i n t h e s e e x p e r im e n ts 8

Mn$04 , MnGlg* MgS04 , GUSO4 ,

NaCl* KOI* OaOlg# F®$04 , C rC l3 , OoGlg , B aC lg , ZnSQ4 and

The p h o s p h a te s o l u t i o n w as p r e p a r e d a s d i r e c t e d f o r K[email protected] bs«*Klnger~Fhosphate (9 4 )*

Th© s o l u t i o n was 0 *1M

w ith r e s p e c t t o HaSHP04 I n a Q«08M HOI s o l u t i o n , pH 7*4* H sin g 0*1 t o 1*0 o*e* o f t h i s s o lu t io n * a p h o sp h a te ra n g e fro m 0*005 M t o 0*05 Jt c o u ld b e o b ta in e d * The s a l i n e s o l u t i o n was p re p a re d a s d e s c r ib e d b y K a ln i ts k y ( 9 7 ) , c o n t a i n i n g two p a r t s H a d (* 9$ o r 0*154 M) an d one p a r t M l (1* 1 5 $ o r 0 .1 5 4 M ),

OaGlg*

4,

M^S04 * an d HaH00$ w ere n o t in c lu d e d i n t h i s s a l i n e s o l u t i o n b e c a u s e o f t h e i r d i s t u r b i n g in f l u e n c e on th e f o llo w in g e x p e rim e n ts * A sodium m alonat© s o l u t i o n was p r e p a r e d t o g iv e a c o n c e n t r a t i o n o f 0 ,3

0 *5 ©#g* t o 0*1 c*c« o f th e

25

s o l u t i o n gave a c o n c e n t r a t i o n r a n ® fro m 0 .0 5 t o 0*01 |J . A 115 d i l u t i o n gav e a rang® fro m 0 .0 1 M t o 0*005 M* A n a l y t i c a l M ethods 01 t r i o A cid was d e te r m in e d b y th© c o l o r i m e t r i c m ethod o f S a f f r a n an d D e n e ta d t ( 9 8 ) .

The ro d c o l o r was

d e v e lo p e d i n a c e t i c a n h y d r id e b y th© a c t i o n o f p y rid in © on c i t r i c a c id * an d r e a d a t 420 mu* i n th e Coleman J r * s p e c tr o p h o to m e te r . A lp h a k e t o g l u t a r i c a c i d w as d e te rm in e d by th e c o l o r i m e t r i c m ethod o f F ried m an an d E augen (9 9 )*

The

a k e t o g l u t a r i c a c id was p r e c i p i t a t e d w ith 2 ,4 d i n i t r o p h e n y l h y d r a z i n e , e x t r a c t e d , and th e r e d c o l o r d e v e lo p e d in a s tro n ly a lk a lin e s o lu tio n .

Th® o p t i c a l d e n s i t y was

r e a d a t 420 mu. G x a lo s u c c in ic a c id o r o x a l a c e t i c a c id was d e t e r ­ m ined b y th e a n i l i n e c i t r a t e m ethod d e s c r ib e d by M so n (1 0 0 )«

A f t e r th e e n z y m a tic r e a c t i o n i n th e W arburg v e s s e l

h ad c o n tin u e d f o r 10»*20 m in u te s , 50 p e r c e n t c i t r i c a c id was t i p p e d i n t o th e m ain com partm ent fro m th e s i d e arm , th u s s to p p i n g th® e n z y m a tic r e a c t i o n and l i b e r a t i n g th e c a rb o n d io x id e i n s o l u t i o n *

When th e m anom eter r e a d in g s

c e a s e d t o ch an g e on c o n tin u e d s h a k in g , th© m anom eter and

f l a s k s w ere rem oved fro m th© w a te r b a t h , th e s to p p e r o f one ©id® arm o f th© f l a s k w as rem oved and .4 c .c * o f a s o l u t i o n c o n s i s t i n g o f 50 p e r c e n t a n i l i n e and 50 p e r c e n t c i t r i c a c i d w as added*

The s to p p e r was s e a le d i n p la c e and

t h e m anom eter and v e s s e l r e t u r n e d t o t h e w a te r b a th *

A f te r

te m p e r a tu r e e q u i l i b r a t i o n , th e m anom eters w ere r e s e t and th© a n i l i n e c i t r a t e tip p e d i n fro m th e s id e arm*

The OOg

e v o lv e d was fro m th© d e c o m p o s itio n o f th© re m a in in g sub­ s t r a t e ( o x a l o s u c c in a te o r o x a la c e ta t© ) i n t h e v e s s e l . H an o m etrio M ethods T he oxygen and c a r b o n d io x id e a n a ly s e s f o r t h e s u b s t r a t e u t i l i z e d d u r in g th© e n z y m a tic p r o c e s s was m e asu red b y r e c o r d i n g m anom eter r e a d i n g s on th e con­ v e n t i o n a l W arburg a p p a r a tu s *

T e m p e ra tu re was 30*1° C .,

e x c e p t when o th e r te m p e r a tu r e s a r e I n d i c a t e d .

F la s k s w ith

two s i d e arm s w ere n eeded I n o r d e r t o v a ry tim e o f a d d i­ t i o n o f v a r io u s c o n s t i t u e n t s *

The s t r o k e r a t e was 120

s t r o k e s p e r m in u te* V e ro n a l and p h o s p h a te b u f f e r s w ere u s e d a s i n ­ d i c a t e d i n th© t a b l e s ; 0*3 c . c . o f e i t h e r gav e a f i n a l c o n c e n t r a t i o n o f 0 .0 1 M when made u p t o f i n a l volume o f 3 c .o * i n th e W arburg f l a s k .

B u f f e r s and c o - f a c t o r s w ere

37

a d d e d t o t h e m ain com partm ent o f t h e W arburg f l a s k , a s w ere r e d i s t i l l e d w a te r and enzyme*

S u b s tra te , u s u a lly

0*3 c # c . o f 0*1 M w as tip p e d i n fro m th e s i d e arm a f t e r e q u i l i b r a t i o n on t h e b a t h f o r t e n m in u te s*

M e ta llic io n s

w ere t i p p e d i n fro m th e a id e arm a s in d ic a te d *

Oxygen

u p ta k e w as r e c o r d e d e v e ry f i f t e e n m in u te s f o r on© h o u r , u n l e s s o th e r w is e i n d ic a t e d *

C arbon d io x id e e v o l u t i o n was

u s u a l l y m easu red f o r t e n m in u te s*

I n h i b i t o r s w ere added

t o th e m ain com partm ent i n th o s e e x p e rim e n ts w here Ih h ib » i t o r s w ere r e q u ir e d *

A l l m it r o c h o n d r i a l p r e p a r a t i o n s and

r e a g e n t s w ere m easu red I n t o t h e W arburg v e s s e l s b y means o f c a l i b r a t e d m ic ro p i p e t t e s , w h ich a r e a m o d if ic a tio n of t h o s e u s e d b y M n d b e rg and Hummel (1 0 1 )*

28

H B arars abb D is c u s s io n I n tro d u c tio n

H om ogem tea o f r a b b it- h e a r t- m u s c l© o x id iz e d c i t r a t e a t a p p r o x im a te ly th e same ra t© a s d id k id n e y c o r t e x h o m o g e n a te sj l i v e r horaogenate® w ere a p p ro x im a te ly o n e - h a l f a s a c t i v e a s h e a r t m u scle an d k id n e y c o r t e x , w h ile h o m o g en ates o f b r a i n and s k e l e t a l m uscle showed l i t t l e o r no a c t i v i t y u n d e r th e same c o n d itio n s #

S in c e k id n e y

c o r t e x w as m ore p l e n t i f u l th a n h e a r t m u scle an d was e a s i e r t o w ork w i t h , k id n e y c o r t e x w as em ployed I n th e e x p e rim e n ts* The e f f e c t s o f v a r y in g c o n c e n t r a t l n s o f k id n e y c o r t e x w ere exam ined#

One h u n d re d tw e n t y - f iv e mg# w et

w e ig h t t i s s u e p e r f l a s k a p p e a re d t o b e th© optimum con­ c e n t r a t i o n u n d e r th e c o n d i tio n s o f t h e e x p e rim e n ts # T h e r e f o re * t h i s am ount o f t i s s u e was em ployed i n th e e x p e rim e n ts *

W ith i n c r e a s i n g c o n c e n tr a t io n s o f c i t r a t e ,

up t o 0*01 M, oxygen u p ta k e in c re a s e d * t i o n s h a d no f u r t h e r e f f e c t *

H ig h er c o n c e n tr a ­

V e ro n a l a t 0*01 if c o n -

c e n t r a t i o n was an e f f e c t i v e b u f f e r and was n o t to x ic t o th© system * A f t e r d i a l y s i s o f t © ground tis s u © p r e p a r a t io n f o r 50 m in u te s , t h e n e c e s s i t y o f Mh++ o r Mg++ f o r c i t r a t e o x i d a t i o n c o u ld b e d e m o n s tra te d i n a p p ro x im a te ly th© same

m

optim um c o n c e n t r a t i o n s a s o iig i n a ll y r e p o r t e d b y A d le r ©t

a l » T he optim um f o r Mr*** was a p p r o x im a te ly 5 x 10*“® M i n t h i s s y s te m , and t h a t f o r Mg**, 2*5 x 10*® m % ** was c o m p le te ly i n a c t i v e a t a c o n c e n t r a t i o n o f 5 x 10*® m# I n o r g a n ic p h o s p h a te w hich was n e c e s s a r y f o r c i t r a t e o x id ­ a t i o n t o p ro c e e d was a l s o rem oved on d i a l y s i s ; a c t i v i t y c o u ld b e r e s t o r e d o n a d d i t i o n o f v a r y in g c o n c e n tr a t io n s o f p h o s p h a te « S in c e Mg** was a c t i v e a t h ig h e r c o n c e n tr a t io n s b u t was i n a c t i v e a t lo w er c o n c e n tr a t io n s ( 7 6 ) , Mg** c o u ld c o n c e iv a b ly com pete w ith Mn** f o r th© enzyme sy stem in v o lv e d *

T h is c o m p e titiv e e f f e c t b etw e en Mg** and Mn**

w as d e m o n s tr a te d (T a b le 1 )*

The d i a ly z e d t i s s u e p re p ­

a r a t i o n h a d a v e ry low endogenous oxygen u p ta k e .

On th e

a d d i t i o n o f c i t r a t e , t h e oxygen u p ta k e was in c r e a s e d and m s f u r t h e r I n c r e a s e d on th e a d d i t i o n o f Mn** b u t n o t on th e a d d i t i o n o f Mg**•

When Mn** (5 x 10*® M) was

I n c u b a te d w ith th® d i a l y z e d t i s s u e f o r 15 m in u te s and th e n Mg** {5 x 1 0 M) and f i n a l l y c i t r a t e war© a d d e d , th e I n c r e a s e d ©xy @n u p ta k e d u e t o t h e p re s e n c e o f th e m e ta ls was 101 an d 109 u l . I n d u p l i c a t e v e s s e l s (T a b le I ) , Mg** w ith o u t Mh+* h ad no e f f e c t #

I f Mn** and Mg** w ere

ad d e d s im u lta n e o u s ly t o th© d ia ly z e d t i s s u e p r e p a r a t io n fo llo w e d b y th e a d d i t i o n o f c i t r a t e , t h e r e was no

d im i n u tio n i n o x y g en u p t a k e .

H ow ever, when Mg** was

I n c u b a te d w i t h th e d i a l y z e d t i s s u e p r e p a r a t i o n f o r 15 m in u te s , an d th e n Bin** and f i n a l l y c i t r a t e w ere a d d e d , t h e oxygen u p ta k e d u e t o th© p r e s e n c e o f th e m e ta ls was i n h i b i t e d 6 0 -6 5 % (T a b le I ) ,

T h e r e f o r e , Mg** a t low

c o n c e n tr a tio n ® c a n n o t r e a c t i v a t e t h e d i a l y z e d enzyme s y s ­ te m , b u t i t a l s o p r e v e n ts Mn** fro m r e a c t i v a t i n g I t ,

It

seem s p ro b a b le t h a t Mg** fo rm s a n i n a c t i v e , s t a b l e com plex w ith th e d i a l y z e d snsyme s y s te m , p r e v e n tin g t h e e n tr a n c e o f Mn** i n t o t h e s y s te m and th u s i n h i b i t i n g t h e a c t i v i t y o f th e enzym e*

S in c e Mn** i s known t o f u n c t i o n i n th e

d e c a r b o x y la t io n o f o x a lo s u o c in io ( 3 3 ) , a k e t o g l u t a r i c ( 7 7 ) , and o x a l a c e t i c a c i d s ( 3 4 ) , a l l o f w hich a r e i n t e r ­ m e d ia te s i n t h e o x id a tio n o f c i t r a t e , t h i s c o m p e titiv e e f f e c t c o u ld p re su m a b ly ta k e p la c e a t an y one o f th e s e s te p s # T h ese c o m p e titiv e e f f e c t s w ere o b ta in e d In e v e ry one o f a t l e a s t s i x e x p e r im e n ts , w ith a n o c c a s io n a l v a ria tio n *

I n one o r tw o e x p e r im e n ts , t h e in c r e a s e d

oxygen u p ta k e on th e a d d i t i o n o f th© m e ta ls was s l i g h t l y l e s s When b o th Mn** an d Mg** w ere ad d ed s im u lta n e o u s ly t h a n when Mn** was I n c u b a te d w ith th e t i s s u e and Mg** ad d ed a f te r w a r d # T he p o s s i b i l i t y t h a t Mg** m ig h t e x e r t i t s e f f e c t

31

b y p r e c i p i t a t i n g I n o r g a n ic p h o s p h a te a s a n i n s o l u b l e magnesium p h o s p h a te was t e s t e d and eliminated*

The stan­

dard t e x t s on in o r g a n ic a n a l y s i s l a c k a d e q u a te in f o r m a tio n on s o l u b i l i t i e s o f m agnesium p h o sp h a te *

T h e re fo re , a

t u r b l d l m e t r l e s tu d y w as u n d e r ta k e n o f th e s o l u b i l i t i e s o f to * * and Mg** p h o s p h a te s *

Th© s o l u t i o n s d u p l i c a t e d th o s e

u s e d i n t h e p r e v i o u s l y d e s c r ib e d n a n o n io tric e x p e rim e n ts e x c e p t t h a t no t i s s u e homo s e n a te was p r e s e n t*

?%** a t

c o n c e n t r a t i o n s up t o 0*01 M, i n t h e p r e s e n c e o f 0*003 M p h o s p h a te , showed no t u r b i d i t y and t r a n s m i t t e d 100$ o f th e lig h t*

Mn** (5 x 10**® SI) p lu s p h o s p h a te (0*003 M) a l s o

t r a n s m i t t e d 100$ o f t h e l i g h t a s d i d a s o l u t i o n c o n ta in in g to * * (5 x 10~® M) p lu s Mg** (5 x 10**® M) p lu s p h o s p h a te (0*003 M) * T h e r e f o r e , p r e c i p i t a t i o n o f p h o s p h a te c o u ld n o t b e a f a c t o r i n th e c o m p e titiv e e f f e c t s d e s c r ib e d # H ow ever, on i n c r e a s i n g t h e c o n c e n tr a t io n o f to * * fro m 5 x 30*® M to 5 x 10"® M, in th e p r e s e n c e of 0.003 M p h o s p h a te , th e p e r c e n t t r a n s m i s s i o n d e c r e a s e d fro m 100$ t o 55$*

The p h o s p h a te s of Mg** a r e t h e r e f o r e more s o lu b le

th a n t o se o f to * * *

I n th© same e x p e rim e n t, th e a d d i t i o n

o f c i t r a t e t o a tub© c o n t a in i n g 5 x 10*"® M

I v in * * p lu s

0*005 M p h o s p h a te in c r e a s e d th e t r a n s m is s io n r e a d in g t o 8 3 $ , com pared t o 55$ w ith o u t c i t r a t e , t h e r e f o r e demons­ t r a t i n g th e b in d in g o f Mn** b y c i t r a t e #

H ow ever, t h i s

32

ta b a C o m p e titiv e g f f a e t a o f

t and MfrH- I n C i t r a t e O x id a tio n

1

C o n d itio n s O la ly s e d t i s s u e a d d i t i o n ; c i t r a t e I n th© s i d e arm Ho c i t r a t e Ho m e ta l Mg** Mn++ and Mge* Mb* * j 15 min* 15 min* Mg**s $S f i n * Mg**l I S min*

ad d ed s im u lta n e o u s ly l a t e r , Mg+* added l a t e r * MftM» added S a te r, add e l l a t e r , Mof* added

M io ro lite rs Og u p ta k e

' "

@1 151 131 234 2&S 240 ife 173

Change due t o m e ta l

0 103 lo i 10® 37 42

T is s u e { d laly & ed 45 m in*) 3L35 mg* w et w e ig h t p e r f l a s k ; v e r o n a l b u f f e r , pH 7 * 4 , 0*01 M; c i t r a t e 0 *ol M; p h o s p h a te , pfc 7 * 4 , 0*003 SSj p l u s s a l i n e s o l u t i o n t o t o t a l volume o f 3*0 s i * ; KOH i n a l k a l i w e ll* MnS04 and M g804 5 x I0 m® M, a d d ed a s i n d i c a t e d * T im e, 00 min* ~

S3

b i n d i n g e f f e c t c a n n o t b e a f a c t o r I n t h e s e e x p e rim e n ts , s i n c e t h e same c o m p e titiv e e f f e c t s w ere o b ta in e d ( a f t e r I n c u b a t in g MgH- w ith d i a l y z e d t i s s u e ) w h e th e r Mn*+ a d d i t i o n w as f o llo w e d b y c i t r a t e , o r w h e th e r Mn++ and c i t r a t e w ere ad d e d s im u lta n e o u s ly * M anganese and Magnesium C o m p e titio n

I n o r d e r t o e l i m i n a t e e f f e c t s d u e t o e x tra n e o u s p r o t e i n i n t h e c r u d e hom ogenate* t h e f o llo w in g e x p e rim e n ts w ere c a r r i e d o u t w ith a m ito c h o n d r ia l p r e p a r a t i o n w hich w as w ashed f o u r tim e s an d di& ly& ed*

A h ig h e r c o n c e n tr a t io n

o f Mn++ (3 x 10~3 j |) was n ee d ed f o r optimum oxygen u p ta k e w ith t h i s p r e p a r a t i o n ( f a b l e I I ) i n d i c a t i n g t h a t th e s e p r o c e d u r e s w ere more e f f e c t i v e I n rem o v in g Mn+* fro m th® m ito c h o n d r ia t h a n fro m th e c ru d e hom ogancie*

T h is con­

c e n t r a t i o n i s v e r y s i m i l a r t o t h e optimum c o n c e n tr a t io n o f Mn*+ (1*3 x 10"*® j$) n e c e s s a r y f o r o x a lo s u c c in a te d e c a r b o x y la t io n (7 4 )*

A d en o sin e t r i p h o s p h a t e m s a l s o

rem oved b y t h e w a sh in g an d d i a l y s i s p ro c e d u re s and had to b e ad d ed t o a f i n a l c o n c e n t r a t i o n o f 1 x 10"*® mM m* As w ith t h e d i a l y z e d w hole h o m o g en ate, Mg++ i n a con­ c e n t r a t i o n o f 3 x 10"*® M o r 3 x 10~4 M h ad no e f f e c t on oxygen u p ta k e when Mn++ was ad d ed f i r s t .

However, when

. •«! #«i O s i^ a ig

I« 5I :

!

«I *H

!t If to *8 t9 to tO

II

*8 !r! r! QI 8

r !o * * 3

+

»

«

« § i

!I t1 Ii 1111

to to o o0o ogeo to 0 ^to e* 5 ® so rf

!i !iI

H U h m



■*o

i

I

If #O**4I S « !

©I

o

I t a *U

33 -®| 18 m in . l a t e r , Hg++ 3x10-16238 B e** SxlO “ 1 6 j 15 m in . l a t e r . , m * * 3xl0-® 166

Change due t o m e ta l

i i i t i t

C o n d itio n s D la ly s e d t i s s u e a d d i t i o n s ; c i t r a t e i n s i d e arm

H&ah v e s s e l c o n t a i n s 16 mg* o f m ito c h o n d r ia l p r e p a r a t i o n ; v e r o n a l b u f f e r , p i 7 , 4 , *01 M; p h o s p h a te , pH 7 * 4 , *005 My c i t r a t e .0 1 M) A«T »?• *001 M; r e d i s t i l l e d w a te r to volume o f 5 m l . j 1 0 ? KOH I n a l k a l i ~ w e l l $ Og u p ta k e r e c o r d e d f o r 60 m in u te s*

11 Owe— n»WW

43 aeeewi

42

Tvai o n t h e same m ito c h o n d r ia l p r e p a r a t i o n , and th e m ercu ry s o l u t i o n s w ere a l s o mad© fro m t h i s t r i p l e - d i s t i l l e d w a te r , I n o r d e r t o d e m o n s tr a te t h a t t h e I n h i b i t i o n was n o t due t o a n im p u r ity i n th e w a t e r . I n h i b i t i o n By V a rio u s M etals The c o m p e titiv e i n h i b i t o r y e f f e c t s o f Na*,

Ca**,

Z n**, Qu** and Hg** w ere a l s o exam ined and found t o p ro d u ce a t l e a s t SO p e r c e n t i n h i b i t i o n o f oxygen u p ta k e (T a b le V) when ad d ed t o th e W arburg v e s s e l , 15 m in u te s b e f o r e th© n e c e s s a r y 5 * 10**® M Mn***

When added a l o n e , t h e s e con­

c e n t r a t i o n s o f i n h i b i t o r y m e ta ls h ad no e f f e c t on th e oxygen u p ta k e I n r e l a t i o n t o a b la n k w ith no m e ta l (T a b le V)* Th© c o n c e n t r a t i o n s u s e d w ere a s fo llo w s *

Zn**, 5 x 10**® mmr Ms

Ca-M*, 3 x 10"*7 M| Gu.**, 5 x 10*6 Mj N a t, 3 x 10*® J |,

Th©

1Q0 p e r c e n t I in h ib itio n w ith added Na* a t a c o n c e n tr a t io n of 3 x XO**5 '•air M was u n e x p e c te d , b u t t h i s may n o t b e so o d d , when we c o n s i d e r t h a t th© Na* o f th© c i t r a t e and v e r o n a l b r i n g th e c o n c e n t r a t i o n t o n e a r l y ,0 5 M* A n a ly s is o f th e Mg** I n h i b i t i o n The i n h i b i t i o n produced b y Mg++ can b e r e v e r s e d b y a s u f f i c i e n t l y h ig h c o n c e n tr a tio n o f Mh*+, when b o th m e ta ls

39 TABLE V

M i&tfcn

w m ± is&

s a t t # &»** n a i »&♦

I n C i t r a t e O x id a tio n

Ooixiifc Io n a B la ly a e d t i a s u e a d d i t i o n a l c i t r a t e tm a i d # aim

u p ta k e

Change In h ifc i d u e to tle n m e ta l %

t© m e ta l W a a 8xA0**§ Zn++ 3x10*®

A ll 3 24 117

w»«eee 313 6

•*#»#» akWM* «****#

»*+* 5*10“ ®} 15 ra in , l a t e r , Zn++ 8*30"® 8 bw+ 8*10*®} I® a i n . l a t e r , #a»* 5*10*5

37© 170

107 39

#n# m

fl»*e jjjg u r #

104

0

Mn++ 3*10“ 5 j j # l a t e r , Ca++ sx lO * 7 Oa+e 5 * 1 0 " ? , 18 ra in , l a t e r , Mn++ 5*10"®

374 10®

103 44

40 41

A

am4©a©

70 •1 4 •ewera*

100 #»**#»

So n a t a l _ S e e 5*30"® *»*+ 3 x 1 0 * 8 , 18 a i n . l a t e r , So* 3x10*5 S ee 3*1©*®} IS ra in , l a t e r , 8*10*® {ftftN* 0X10*® mS w > S

110 36 39

es

73

95

-1 7 awesera 0

eramraa 100 eran**# wee#ee

200 144

110 04

wrara 00

x J 0 ^ | 10 mtn# l a t e r # One* 3x10*® CU++ 3x10*® f U min# l a t e r # lit*** 5x10"®

10® 33

! • m e ta l ^ One# 3 x 1 0 -?

1M

!&*►* S a tlO ^ i 1® min# l a t e r # On** 3x10*? fih»M» 3 x 1 0 * ? ; 1® min# l a t e r # Mu m * 3 x 1 0 -3

g aeh v a s s a l eont& lns 15 rag. o f m ito o b o n d ria l p re p a ra tio n } v e r o n a l b u f f e r , pH 7 . 4 , .0 1 S j phosphate pH 7 . 4 , .0 0 5 Mj O itr a te ,0 1 M) & .7 .P . .0 0 1 Mj r e d i s t i l l e d w ater t o volurae o f S m l . ; 10# IKB I n a l k a l i w e ll] Og uptake re co rd ed f o r 60 rain.

40

a r e ad d e d a t t h e same tim e ( f a b l e V I) • t h a t t h e i n h i b i t i o n m e c o m p e titiv e *

T h ia w ould I n d i c a t e L In ew eav er and Burke

( 1 0 0 ) h a v e eh own t h a t when th e r e c i p r o c a l o f th® v e l o c i t y ( i / v ) 1® p l o t t e d a g a i n s t th e r e c i p r o c a l o f th e s u b s t r a t e c o n c e n t r a t i o n (X /(« ) ) 9 c o m p e titiv e I n h i b i t i o n I s I n d ic a te d b y a n in c r e a s e i n th e slop® o f t h e lin e * accom panied by no s i g n i f i c a n t ch a n g e I n th e i n t e r c e p t #

U sin g t h i s method*

t h e i n h i b i t i o n was shown t o b e c o m p e titiv e * w ith in th e ©one e n t r a t l o n s u s e d i n a l l e x p e rim e n ts i n t h i s r e p o r t ( F ig u r e B)* H ig h e r c o n c e n t r a t i o n s o f MU** w ere i n h i b i t o r y ( F ig u r e 2) • A c c o rd in g t o M lc h a e lls and M enten (1 0 3 ) and I»inew eav er an d B u rk e (iOB)* t h e v e l o c i t y o f a n ©nsym&tie r e a c t ion i n c r e a s e s a s t h e c o n c e n t r a t i o n o f t h e s u b s t r a t e in c r e a s e s u n t i l a maximum v e l o c i t y I s a t t a i n e d when t h e ensyme i s ^ s a tu ra te d ® b y s u b s t r a t e *

f h l s v e lo c ity of re a c tio n I s

c o m p e titiv e ly d e c re a se d b y th e a d d itio n o f an I n h ib ito r Which i s s i m i l a r I n c o n f i g u r a t i o n t o th® s u b s t r a t e , e q u a tio n s u s e d i n t h e i r a n a l y s i s w ere a s f o llo w s ? 1 ♦

e f

and 38

I*=#E I

Our e q u a tio n s w ould n e c e s s a r i l y b e j 1 4- M±=*E~M + B * M%±=r B-Sffi * S 2^ S -M i-s

♦ F and

fh e

41

TABLE VI gfeft SgggQM ttL TMaftnx C o n c e n tra tio n s o f Mn++

la sxsssasfM Tte .MMftiUm S&nsssi Sx m*±

Hn** done# (M -)

Mg** Gone* (M .)

3 3 3 1 8 1

x x X x x x

1 0 -6 1 0 -* 1 0 -* IB *5 10*6 1 0 -2

#*«k«Ni-

3 3 6 1 3 1

x x x x x x

1 0 -6 10“ * 10- * 1 0 -6 10 -6 10-®

5 X 10 ^

«*«*«»

u l 0^ u p ta k e

til % «*blank

In h ib itio n %

17$ 586 400 450 360 174

12 22$ 237 267 197 11

» n

161 304 360 410 $74 146

0 141 197 247 211 0

100 33 17 7

•ee-MCse

16$

0

mrnm*

*#*» 4NNNM ND

a

•« «

M ito c h o n d ria d i a l y s e d 90 wXn*$ 10 isg* la lb o e h a n d ria l p r o t e i n d r y wi*$ #01 M v e r o n a l b u ffe r® pH 7*4$ .0 1 M c i t r a t e ; #003 lj| K gW O j pH 7 # 4 ; Mn** and 2£g** con©* a s i n d i c a t e d 001 1 JU f *F#| HgO t o volum e $ ©•©*; K0H i n c e n t e r w e l l ; t i k e 1 h r # ; g a s , a i r , Mg**- and ?&*** added s lm u lta n e o u s ly #

42

PISURE 2

.008

1

.0 0 5

V .0 0 4

.002

I XI O3

2XI03

3 X I0 3

( M n++)

Th® E f f e c ta o f V arying C o n cen tratio n s o f Mn++ in Overcoming The I n h i b i tio n Caused By Mg++

43

w here*

s ** ensyme S * s u b s tra te I * I n h ib itin g s u b s tra te f m p ro te s ts M m a c t i o s m e ta l {$&*** J Ml m i n h i b i t i n g m e ta l {Mg++)

T he v e l o c i t y o f t h e e n a y m a tie r e a c t i o n s i n o u r sy stem i n c r e a s e s w ith t h e c o n c e n t r a t i o n o f th e m e ta l (Mh+*) u n t i l

a maximum v e l o c i t y i s a t t a i n e d when t h e ensyme i s " s a t u r a t e d ” w ith t h e m e ta l*

T h is v e l o c i t y i s c o m p e t i t i v e l y d e c r e a s e d

b y a n i n a c t i v e m e ta l su c h a s Mg-M* ♦ The o b v io u s d i f f e r e n c e b e tw e e n o u r sy stem an d t h a t o f L in e w e a v e r a n d B urke i s t h a t i n t h i s c a s e , we a r e deal-* I n g w ith a m e t a l l i c c o - f a c t o r w h ich may b e r e g e n e r a te d and w h ich i s t o x i c a t h ig h e r e o n e e n t r a t l a n s * A more co m p lete m a th e m a tic a l r e p r e s e n t a t i o n o f th e sy ste m i s n e e d e d , s in c e t h e c o n v e n tio n a l fo rm d o e s n o t t a k e t h e s e f a c t o r s i n t o a c c o u n t* A n a ly s is b f P o p p er I n h i b i t i o n S e v e r a l e x p e rim e n ts w ere c a r r i e d o u t t o show t h e same ty p e o f c o m p e titiv e i n h i b i t i o n b e tw e e n Cu*h* and I n o r d e r t o d e te r m in e t h e a p p r o p r i a t e c o n c e n tr a t io n o f Gu*Mf o r t h e c o m p e titio n s t u d y , p r e lim in a r y e x p e rim e n ts su ch a s a r e shown i n T a b le V II w ere u n d e rta k e n *

I n th e s e e x p e r i ­

m e n ts , Cue* a t a c o n c e n t r a t i o n o f 3 x 1 0 M p ro d u ced a n

44

TABUS VII S £ 2 2 M fit S2m

SSt Stt£ IS m s & te . Oxidation I

Cue* Cons. ( 1 *)

Cu-H-Tissue Incubation

Mn++ Cono. (It.)

mm**

****«#

**#»*».

$ x2 0 m®

15 lain# .*#tO £ G O%

A S J ^

10 w

S

H 48

H

60

60 rl

§

© o

.§*1! O © ri HP £ 1

t> ^ < 9W $ H

to to d S

ri © t o o 0- t> *2 00 «J 0 OJ 01 ,- n n

*>€» HtQ 03 o5

to 0 to 10 H 01

*»G O

, , ® ■** M l p

©*®*D

O © © ©iJt C? © P ri Pi

0 0

Hi

H

frt

§ A § 5 © © $ P « p © *rt 0 m © s H

« «rt ; 1 * «q t HI

to 60 ^

* a vf

©* €0 to to ©•

*

n *

t 10 t H

! i H

0 60 O E60 60

0 03

t £ «H S t 1 to 1 ri

• £ a 1

■§ j

to ! ri

to I H

8 $ tO©1

1O1§ A3 3 < S®1

Gug *< © © © O P H » P ©HO © %p HI ©H

H

*

*

fit © ri

m

8 x 1 0 -3 3 x 1 0 -3

* m

* *

Mm*

U l Op u p ta k e

* -+ * *

185 264 184 250

m m

440 288

C o n d itio n s ; M ito c h o n d ria w ashed 4 tim e s , d i a l l e d 90 m in*; X0 mg m ito c h o n d r ia l p r o t e i n d r y w e ig h t; *01 M v e r o n a l b u f f e r pH 7 . 4 ; *01 M a k e t o g lu ta r a t © ; * \0 0 1 M A*T*P#; #01 M m a lo n a te ; KgH POg b u f f e r pH 7 .4 a s i n d i c a t e d ; MgM* con* c e n t r a t i o n 5x10 ® M; Mn++ c o n c e n t r a t i o n 5x10*® M; KOH i n © e n te r w e l l ; tim e 1 h r * ; g a s , a ir# ~

59

SABIS XV Of Various C o-factors on Oxidation o f Alpha K eto^Iutaric Aoid^ I I L

IS elo n ate

m m

*

M g - tls s u e I n c u b a tio n

M h -tis s u e I n c u b a tio n

u l 00 u p ta k e

15 mln#

15 min* IS ml n *

465 415 530

564 314 229

27*3

217 172 101

116 71

38*7

******

15 mln# « * « •« *

*******

IS mln*

15 mln* *

«*«*«*

«*«»*»

Conditions ? the same a® Table XIV*

Change due t o m e ta l

In h ib it tio n t ****** ******

60

o f e a c h o f t h e s e io n s f o r a k e t o g l u t a r a t e o x i d a t i o n , showed t h a t Mg** was th e m ore a c t i v e m e ta l ( f a b l e XVX)*

I n c u b a tin g

t h e m ito c h o n d r ia w ith a n e f f e c t i v e Mg** c o n c e n tr a t io n (3 x 1 0 -3 m) and th e n a d d in g a n i n a c t i v e c o n c e n t r a t i o n o f t o (3 x KT® M) r e s u l t e d i n good a c t i v i t y *

H ow ever, in e u b a t*

in g t h e m ito c h o n d r ia w ith th e i n a c t i v e t o o o n c e n tr a tlo n f i r s t * and th e n a d d in s t h e optimum Mg c o n c e n tr a tio n * g av e lo w e re d a c tiv ity , or an in h ib itio n .

I n o th e r w o rd s, u n d e r t h e s e

c o n d iM m a » to * * was th e i n h i b i t i n g m etal* and n o t Mg** • By s im p ly r e v e r s i n g t h e c o n c e n tr a t io n s o f t h e m e t a l l i c i o n s , Mg** c o u ld a g a in b e shown t o b e i n h i b i t o r y ( f a b l e X V II), w ith t h e same p r e p a r a t io n * t o a k e t o g l u t a r a t e d e h y d ro g e n a se was p re p a re d fro m r a b b i t h e a r t i n t h e same m anner a s d e s c r ib e d b y Ochoa (7 7 )* f i l l e r © o n een trafcio n s o f m a lo n a te w ith t h i s tis s u e * a l s o i n h i b i t e d a k e t o g l u t a r a t e o x id a tio n *

f h e c o m p e titiv e

e f f e c t o f Mg+* was shown w ith t h i s t i s s u e I n th e a b s e n c e o f m a lo n a te * Hon B naym atic 25ESSSSS5-SSS B f f e e t 2SS5? of M anganese J -!■ -i '-W -' -■ir.qi*r.:r*» t o i n v e s t i g a t i o n o f th e a k o to g l u t a r l c d eh y d ro * g e n a se o f p ig e o n b r e a s t m uscle was a l s o u n d e r ta k e n b e c a u s e i t h a d b e e n r e p o r te d b y 0 re e n s t * a l » (104) t h a t t h e

61

TABLE XVI

A c t i v i t y o f g , J f o t o ^ u t a y a t o D eh y d ro g en ase I n t h e F re s e n e e off V a ry in g G o n c e n tra tio n s o f ih » * and fe»+

MfrH* Oone» (M .)

Ig*+ Oono *

mm-m

S x lO -3 3 x l0 ~ 5 nwr■«'■» *»**#* «•«*««



5x10*$ 3 x l0 ~ 4 3x10^

C o n d itio n s ?

t a 02 u p ta k e

Change d u e t o m e ta l

01 140 128

«*«*«*

417 172 108

526 81 14

49 57

t h e same a s T a b le XV$ m e ta ls added a s in d ic a te d *

62

13 O«H Jaw .

M h 9 © 49 I I I

sW i4

si

4> t 1- COto *0 t* H VJ fH «»$(30 60 !"60 ■* n H

tOH H

h

tJ © q 43 H i© jd

S

t>5

%

a

A

© o(3

f .j

0 •» A 0 _ 0 q *h * *d »t xi © ■ 6t ® t» N fj .

r*f 0

*

S 3