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EXPERIENTIA 32/11

Table II. Effect of temperature on the kinetic constants of the reaction catalyzed by the citrate synthase from Bacillus stearothermophilus. Temperature (~ 30 37 43.5 50 55.4 62

Apparent K,~ for aeetyl-CoA ( ~ z M ) 3.7 4.4 6.1 6.9 9.1 13.5

Apparent K,,~ for oxaloacetate ( [ z M )

Turnover number (min-1)

1.1 1.3 2.1 2.9 3.3 3.3

4000 7000 11100 21200 28200 38800

The reaction mixtures were as previously described 1~, except that 20 mM potassium phosphate buffer (pH 7.6) was used instead of Tris-HC1, 0.1 M KC1 was present, and the concentrations of aeetyl-CoA or oxaloacetate were varied. When used as the fixed substrate, the concentrations of acetyl-CoA and oxaloaeetate were 0.05 and 0.25 mM, respectively. The turnover number values were calculated fronl the V,,~ values obtained from the double-reciprocal plots for acetyl-CoA at a saturating concentration of oxaloacetate, employing the molecular weight of 100,000 estimated from gel filtration experiments. several c o n c e n t r a t i o n s of t h e c o - s u b s t r a t e i n t e r s e c t e d a t t h e abcissa, s h o w i n g t h a t t h e Km values were i n d e p e n d e n t of t h e c o n c e n t r a t i o n of t h e c o - s u b s t r a t e . As in t h e case of t h e c i t r a t e s y n t h a s e s isolated f r o m o t h e r G r a m p o s i t i v e bacteriaS0, 21, t h e t h e r m o p h i l i c e n z y m e was n o t i n h i b i t e d b y N A D H or e - o x o g l u t a r a t e c o n c e n t r a t i o n s u p to 0.5 m M or 5 mM, respectively, b u t was i n h i b i t e d b y A T P a n d less effectively b y A D P ; A M P was ineffective a t c o n c e n t r a t i o n s up to 5 raM. T h e i n h i b i t i o n b y 5 m M A T P or A D P (62% a n d 28%, respectively) was t h e s a m e e i t h e r in t h e p r e s e n c e or in t h e a b s e n c e of KCI (0.1 M), a t 30~ or a t 62 ~ As in t h e case of o t h e r b a c t e r i a l c i t r a t e s y n t h a s e s , w i t h t h e e x c e p t i o n of t h e e n z y m e f r o m B. subtilis H S 2A2 ~, t h e i n h i b i t i o n b y b o t h nucleotides a t 30~ was s t r i c t l y c o m p e t i t i v e t o w a r d s acetyl-CoA, a n d n o n - c o m p e t i t i v e t o w a r d s o x a l o a c e t a t e . The a p p a r e n t Km for acetyl-CoA (27 ~zM oxaloacetate) was increased f r o m 3.7 a M in t h e absence of i n h i b i t o r s to 10.0 ~zM a n d 31.2 ~zM in t h e p r e s e n c e of 3 m M A D P or A T P , respectively, w i t h o u t c h a n g e in t h e a p p a r e n t Vm,~. The a p p a r e n t Km for o x a l o a c e t a t e (50 a M acetyl-CoA), on t h e o t h e r h a n d , was n o t c h a n g e d in t h e p r e s e n c e of 3 m M A D P or A T P , whereas t h e a p p a r e n t V ~ d e c r e a s e d b y 19~o a n d 45%, respectively. The c i t r a t e s y n t h a s e f r o m B. stearothermophilus s h o w e d considerably greater thermal stability than the enzyme f r o m pig h e a r t , w h i c h has similar kinetic a n d r e g u I a t o r y p r o p e r t i e s . T h e latter, as a 20 ~g/ml solution in 20 m M p o t a s s i u m p h o s p h a t e buffer (pH 7.6) a n d 1 m M E D T A , was c o m p l e t e l y i n a c t i v a t e d a f t e r h e a t i n g for 2 m i n a t 50~ A 10 Izg/ml solution of t h e t h e r m o p h i l i c e n z y m e in t h e s a m e buffer, on t h e o t h e r h a n d , d e c a y e d only b y 32% a f t e r h e a t i n g for 20 m i n a t 74~ Half-lives were n o t calculated, since t h e d e c a y of t h e t h e r m o p h i l i c e n z y m e did n o t follow f i r s t - o r d e r kinetics, being considerable

w i t h i n t h e first 3 m i n of i n c u b a t i o n , a n d b e c o m i n g m u c h slower a f t e r w a r d s . A similar finding h a s b e e n r e p o r t e d for t h e 5 , 1 0 - m e t h y l e n e t e t r a h y d r o f o l a t e d e h y d r o g e n a s e f r o m Clostridium tetanomorphum 22, a n d has b e e n interp r e t e d as a possible c o n v e r s i o n of t h e e n z y m e into a m o r e stable, b u t less active, f o r m d u r i n g h e a t i n g . The a p p a r e n t Km values for b o t h s u b s t r a t e s increased a b o u t 3-fold w h e n t h e t e m p e r a t u r e was raised f r o m 30 ~ to 62 ~ (Table II). Tile t u r n o v e r n u m b e r of t h e t h e r m o philic e n z y m e a t 62~ 38,800 rain -1 (Table II) was 3 t i m e s h i g h e r t h a n t h e v a l u e r e p o r t e d for t h e r a t h e a r t e n z y m e a t 28~ 13,000 m i n -I (ref. s). The A r r h e n i n s p l o t of t h e n a t u r a l l o g a r i t h m of t h e t u r n o v e r n u m b e r values given in T a b l e I I as a f u n c t i o n of t h e reciprocal a b s o l u t e t e m p e r a t u r e values was linear f r o m 30~ to 62~ a t h i g h e r t e m p e r a t u r e s (not shown), a decrease was observed. The a c t i v a t i o n e n e r g y calculated f r o m t h e A r r h e n i u s p l o t was 15 kcal/mol. The c o r r e s p o n d ing value for t h e pig h e a r t e n z y m e was 9.7 kcaI/mol a t t e m p e r a t u r e s h i g h e r t h a n 20~ a n d 13.7 k c a l / m o l a t t e m p e r a t u r e s lower t h a n 20 ~ (ref. 2a). The results d e s c r i b e d in t h i s p a p e r show t h a t t h e c i t r a t e s y n t h a s e f r o m B. stearothermophilus shares t h e kinetic and r e g u l a t o r y p r o p e r t i e s a n d t h e molecular w e i g h t of t h e e n z y m e s isolated f r o m suitable mesophilic c o u n t e r p a r t s ; o n l y its t h e r m a l p r o p e r t i e s , p a r t i c u l a r l y its considerable stability, seem to differ f r o m t h o s e of t h e c i t r a t e s y n t h a s e s f r o m mesophilic G r a m p o s i t i v e b a c t e r i a a n d eucaryotes. 2o p. D. J. WEITZ.~IANand D. JoNEs, Nature, Lond. 219, 270 (1968). 21 p. D. J. WEITZMAN and P. DE,MORE, FEBS Lett. ,3, 265 (1969). 22 W. E. O'BRIEN, J. M. BREWER and L. G. LJUNGDAHL, J. biol. Chem. 2~t8, 403 (1973). 23 G. W. KoSleKi arid P. A. SRERE, J. biol. Chem. 236, 2560 (1961).

Regional and Subcellular Distribution of Superoxide Dismutase in Brain T. C. LOOMIS, G. YEE a n d W. L. STAHL 1

Neurochemistry Laboratory, Veterans Administration Hospital and Departments o/ Physiology and Biophysics and Medicine (Neurology), University o/ Washington School o/ Medicine, Seattle Washington 98108, USA), 17 May 1976. Su~zmary. S u p e r o x i d e d i s m u t a s e has been f o u n d t o be widely d i s t r i b u t e d a n d of a p p r o x i m a t e l y t h e s a m e specific a c t i v i t y ill all regions of h u m a n b r a i n e x a m i n e d . I t is n o t r e d u c e d d u r i n g d e g e n e r a t i o n of t h e basal ganglia ill H u n t i n g t o n ' s Chorea. A f t e r snbcellular f r a e t i o n a t i o n of h u m a n a n d guinea-pig cerebral cortex, t h e h i g h e s t specific a c t i v i t y of t h e e n z y m e was f o u n d in t h e soluble fraction. S u p e r o x i d e d i s m u t a s e (SOD) has b e e n d e m o n s t r a t e d in a v a r i e t y of tissues a n d cell t y p e s a n d a p p e a r s t o prot e c t a g a i n s t t h e t o x i c effects of t h e O~-radical z. R e c e n t s t u d i e s ~ 6 i n d i c a t e t h a t v e r y h i g h levels of a c t i v i t y are p r e s e n t ill liver, while t h e adrenals, k i d n e y a n d red blood

cells h a v e i n t e r m e d i a t e a c t i v i t y a n d lower activities were f o u n d in m o s t o t h e r tissues including brain. W e h a v e b e e n i n t e r e s t e d in possible c h a n g e s in SOD in n e r v o u s tissue in c e r t a i n neurological diseases, a n d we r e p o r t here t h e d i s t r i b u t i o n of SOD in guinea-pig a n d h u m a n brain.

1 5 . 1 1 . 1976

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1 Acknowledgments. We thank the Kuntington's Chorea Foundation for financial s u p p o r t . A d d r e s s r e p r i n t r e q u e s t s to t h e Neuroc h e m i s t r y L a b . , V. A. H o s p i t a l , Seattle, W a s h . 98108, U S A .

Materials and methods. Guinea-pigs were killed by a blow to the neck followed by exsanguination. Brains were immediately excised and separated into gray and white matter with a dull spatula and were then homogenized and separated into subcellular fractions by a modification of the method of GRAY and WHITTAKER7 as previously d e s c r i b e d s. H u m a n brains were obtained at autopsy and tissue from various regions was either immediately homogenized and separated into subcellular fractions or was placed in vials, quickly frozen in dry ice-acetone and stored at --70~ In certain cases, analyses were carried out on unfrozen tissue, immediately after dissection. In most cases a 10% homogenate of t i s s u e i n 0 . 3 2 3/I s u c r o s e w a s p r e p a r e d for SOD assay. SOD was assayed according to the procedure of MISTRA a n d JPRIDOVICH s, w h i c h is b a s e d o n t h e a b i l i t y o f S O D

2 I. FRIDOWeH, A c c o u n t s Chem. Res. 5, 321 (1972). 3 W. BOHNENKAMP a n d U. WESER, H o p p e - S e y l e r ' s Z. physiol. C h e m . 356, 747 @975). 4 C, PEETERS-JORIS, A.-IVL VANDERVOORDE a n d B i o c h e m . J. 150, 31 (1975). s R. FRIED a n d P.

P. BAUDHU1N,

MANDEL,J. N e u r o c h e m . 2~, 433 (1975).

6 p. VAN HIEN, K. KOVACS a n d B. MATKOWCS, E n z y m e ]9, 1 (1975). 7 E. G. GRAY a n d V. P. WHITTAKER, J. A n a t . 96, 79 (1962). s p. D. SWANSON, F. H . HARVEY a n d W. L. STAHL, J. N e u r o c h e m . 20, 465 (1973). s H . P. MrSraX a n d I. FRIDOVICH, J . biol. Chem. 247, 3170 (1972).

T a b l e I. Regional d i s t r i b u t i o n of s u p e r o x i d e d i s m u t a s e in h u m a n b r a i n Specific a c t i v i t y (units S O D / m R protein) Region

Controls (~ = 3) ~

HuntingtonLq Chorea 1

Huntington's Chorea 2

Medulla Pone Cerebellar c o r t e x Cerebelhlm, d e n t a t e nucleus

32.9-40.4 32.7 38.0 46.4 61.3 35.8 38.9

-

-

P a r i e t a l Cortex Gray White

27.9 45.9 27.9 38.9

47.5 36.3

27.0

Frontal cortex Gray White

30.9-34.8 47.9-53.1

23.2 22.3

28.0 -

T e m p o r a l cortex, g r a y Hippocampus Substantia nigra Midbrain without sub. nigra Thalamus Hypothalamus Putamen C a u d a t e nucleus

27.8 39.5 41.6-46.8 42.4 64.1 39.2 52.6 33.2 60.5 37.2 55.5 34.7-47.8 36.6 51.4

39.9 39.9

37.4 37.4

-

Controls : 1. female, 71 y r (34 h) ; 2. male, 62 y r (29 h); 3. male, 86 y r (18 h). H u n t i n g t o n ' s C h o r e a : 1. female, 80 y r (7 h) ; 2. female, 33 y r (6 h). N u m b e r s in p a r e n t h e s e s refer to t i m e b e t w e e n d e a t h a n d a u t o p s y .

T a b l e II. S u b c e l l u l a r d i s t r i b u t i o n of s u p e r o x i d e d i s m u t a s e Fraction

G u i n e a pig g r a y m a t t e r (n = 5) Specific a c t i v i t y ~

ttomogenate Nuclear

24.30 -+- 5.44 b 24.26 - - 3.63

Crude mitochondrial Myelin Synaptosomal Purified mitochondrial

26.04 22.58 26.16 15.54

Microsomal Supernataut

19.48 q- 3.04 46.60 i 9.61

aSpecific a c t i v i t y = u n i t s / r a g p r o t e i n a t 30~

-b -~ •

(u = 2)

D i s t r i b u t i o n (%) 100 46.36 -:c 2.95

3.87 2.41 5.18 0.98

bMean v a l u e s

Human temporal cortex gray matter

17.00 15.80 31.58 11.70

-~ ~ ~ ~

4.30 4.69 ~ 3.96 ~ 2.51 ~

6.72 ~ 1.89 34.65 ~= 3.09

SD. ~

Specific a c t i v i t y

D i s t i b u t i o n (%)

27.40 23.80

100 54.2

24.70 -

6.9 -

19.00 68.90

4.4 25.6

to the c r u d e m i t o c h o n d r i a I f r a c t i o n .

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to i n h i b i t t h e s p o n t a n e o u s o x i d a t m n of e p i n e p h r i n e to a d r e n o c h r o m e a t p H 10.2. T h e t o t a l v o l u m e was 1.0 m l a n d a t 30~ t h e r a t e of a u t o o x i d a t i o n was f o u n d t o be 0.025 OD u n i t s / m i n a t 480 n m . P r o t e i n was a s s a y e d acc o r d i n g to LOWRY et al. *0 H u m a n b r a i n c o n t r o l s used here h a d n o k n o w n neuroIogical a b n o r m a l i t i e s . Results and discussion. T h e d i s t r i b u t i o n of SOD a c t i v i t y for v a r i o u s b r a i n regions is p r e s e n t e d in T a b l e I a n d shows a r e m a r k a b l y h o m o g e n e o u s p a t t e r n in h u m a n b r a i n . T h e a c t i v i t y was p r e s e n t in all areas e x a m i n e d ( m e a n v a l u e for all a r e a s : 40.69 • 9.69 (SD) u n i t s / m g protein), in b o t h g r a y a n d w h i t e m a t t e r , a n d t h e e n z y m e was s t a b l e for a t least 6 m o n t h s w h e n s t o r e d a t - - 7 0 ~ T h e d i s t r i b u t i o n of t h i s e n z y m e h a s also b e e n r e p o r t e d r e c e n t l y in r a t t e l e n c e p h a l o n , c e r e b e l l u m a n d m e d u l l a o b l o n g a t a , a n d similar a c t i v i t i e s were f o u n d in t h e s e regions in b o t h m a l e a n d female a n i m a l s a. T h e s e o b s e r v a t i o n s would suggest t h a t t h e e n z y m e is w i d e l y d i s t r i b u t e d in b r a i n a n d is v e r y likely p r e s e n t in b o t h n e u r o n s a n d glia. I t was p a r t i c u l a r l y i n t e r e s t i n g to n o t e t h a t b r a i n s t a k e n a t a u t o p s y f r o m p a t i e n t s afflicted w i t h H u n t i n g t o n ' s Chorea h a d a p p a r e n t l y n o r m a l levels in b o t h t h e c e r e b r a l cortex, c a u d a t e n u c l e u s a n d p u t a m e n (Table I). T h i s a u t o s o m a l d o m i n a n t disease of u n k n o w n etiology s t r i k e s t h e b a s a l ganglia ( c a u d a t e n u c l e u s a n d p u t a m e n ) h a r d e s t , leading t o loss of s m a l l e r i n t e r n e u r o n s a n d inf i l t r a t i o n of t h i s region b y glial cells. I n p a t i e n t s w i t h t h i s disease, t h i s region c a n be so a d v e r s e l y affected t h a t it is r e d u c e d to o n l y 10% of t h e gross w e i g h t of n o r m a l controls. I t h a s p r e v i o u s l y b e e n e s t a b l i s h e d n t h a t a n u m b e r of o t h e r e n z y m e s are d r a m a t i c a l l y r e d u c e d in H u n t i n g t o n ' s Chorea, i n c l u d i n g choline a c e t y l t r a n s f e r a s e a n d g l u t a m i c acid d e c a r b o x y l a s e , w h i c h are i n v o l v e d in t h e s y n t h e s i s of t h e n e u r o n a l p u t a t i v e t r a n s m i t t e r s ,

EXPERIENTIA 32/11

a c e t y l c h o l i n e a n d y - a m i n o b u t y r i c acid. I t a p p e a r s likely t h e n t h a t t h e r e is no r e d u c t i o n in t h e level of SOD in t h e basal g a n g l i a in t h i s disease state. T h e s u b c e l l u l a r d i s t r i b u t i o n of SOD is g i v e n in T a b l e II. I n b o t h g u i n e a - p i g a n d h m n a n cortical g r a y m a t t e r , t h e h i g h e s t specific a c t i v i t y was f o u n d in t h e s u p e r n a t a n t fraction, w h e r e 2 6 - 3 5 % of t h e t o t a l a c t i v i t y was recovered a f t e r d i f f e r e n t i a l c e n t r i f u g a t i o n . A f t e r f r a c t i o n a t i o n of t h e c r u d e m i t o c h o n d r i a l f r a c t i o n o n a sucrose g r a d i e n t o v e r h a l f of t h e r e c o v e r e d SOD a c t i v i t y was in t h e s y n a p t o s o m a l fraction. T h e s e e x p e r i m e n t s would a p p e a r to show t h a t t h e b r a i n e n z y m e is w i d e l y d i s t r i b uted, b u t we could release u p t o 80 85% of t h e SOD in soluble f o r m b y h y p o o s m o t i c t r e a t m e n t or brief sonic i r r a d i a t i o n of t h e h o m o g e n a t e . I n liver u p to 84% of t h e SOD c a n b e r e c o v e r e d in soluble f o r m f r o m h o m o g e n a t e s w i t h o u t t h e l a t t e r t r e a t m e n t s % T h e liver a n d b r a i n e n z y m e s a p p e a r as 2 isozymes, A a n d B, a n d in s t u d i e s w i t h K B cells t h e f o r m e r was f o u n d in soluble form, while t h e l a t t e r was e n r i c h e d in m i t o c h o n d r i a l < H o w ever, u n l i k e t h e s i t u a t i o n in liver~, no c l e a r - c u t b i m o d a l d i s t r i b u t i o n of S O D was o b s e r v e d in b r a i n in t h e p r e s e n t studies. Moreover, t h e p u r i f i e d m i t o c h o n d r i a l f r a c t i o n f r o m g u i n e a - p i g b r a i n h a d r e l a t i v e l y low specific a c t i v i t y a n d we were n o t able to d e m o n s t r a t e e n z y m e l a t e n c y s u s c e p t i b l e to sonic i r r a d i a t i o n . T h e p r e s e n t w o r k suggests differences b e t w e e n liver a n d b r a i n tissue in b o t h t h e d i s t r i b u t i o n a n d association of SOD w i t h s u b c e l l u l a r components. to O. H. LOWRY, N. J. ROS~BROU(;H,A. L. FARR and R. ]. RANDALL, J. biol. Chem. 193, 265 (1951). 1I W. L. STAHL and P. D. SWANSON, Neurology 24, 813 (1974). 12 G. BECKMAN, E. LUNDGREN and A. TXRNVIK, H u m . H e r e d i t y 23, 338 (1973).

Allantoinase in the Marine Polychaete E u d i s t y l i a v a n c o u v e r i 1 DORA R. M~ PASSINO 2 a n d G. W. BROWN, JR.

Laboratory o/ Biochemical Ecology, College o[ Fisheries, University o/ Washington, Seattle (Washington 98195, USA), 70 May 1976. Summary. A l l a n t o i n a s e , a n e n z y m e in t h e p u r i n e - u r e a cycle, was f o u n d in EudistyHa vancouveri ( P o l y c h a e t a ) . T h e e n z y m e h a d a p H o p t i m u m a t 7.6. T h e K ~ was 0.012 M a i l a n t o i n , a n d t h e A r r h e n i u s e n e r g y of a c t i v a t i o n was I2.6 to 14.6 kcal/mol. A l l a n t o i n a s e ( a l l a n t o i n a m i d o h y d r o l a s e , E C 3.5.2.5) c a t a l y z e s t h e h y d r o l y s i s of a l l a n t o i n to a l l a n t o i c acid a, ~:

0 H I /C=O O=C'-.N/C'-. N' H H

?H2 ---~

/ H20

-

NH2

O=C COOH C=O I INH HN--C I I H

T h i s r e a c t i o n is p a r t of t h e p u r i n o l y t i c p a t h w a y for deg r a d a t i o n of uric acid to glyoxylic acid a n d urea. Allant o i n a s e is c o m m o n l y p r e s e n t in A m p h i b i a a,4, fish ~,5, g r e e n p l a n t s 6, a n d m i c r o o r g a n i s m s , e.g., Escherichia coli a n d Slreptocpccus allantoieusL B o t h t h e p r e s e n c e a n d a b s e n c e of a l l a n t o i n a s e h a v e b e e n r e p o r t e d for polyc h a e t e s s,9. B e c a u s e of o u r i n t e r e s t in t h e c o m p a r a t i v e b i o c h e m i s t r y of uricoIytic e n z y m e s a n d t h e i r e v o l u t i o n a r y significance for t h e p o l y c h a e t e s , we a s s a y e d p r e p a r a t i o n s of t h e c o m m o n sabellid w o r m Eudislylia vancouveui (Kinberg) for a l l a n t o i n a s e a c t i v i t y .

S p e c i m e n s were collected off d o c k s of San J u a n Island, P u g e t Sound, W a s h i n g t o n . H o m o g e n a t e s of e n t i r e w o r m s a n d also w a t e r e x t r a c t s of a n a c e t o n e p o w d e r 10 (mean = 5.4 m g p r o t e i n / m l ) p r e p a r e d f r o m a b d o m e n s of t h e polyc h a e t e were a s s a y e d a t 37~ b y e s t i m a t i n g t h e a m o u n t of a l l a n t o i c acid p r o d u c e d . A l l a n t o i c acid was c o n v e r t e d to glyoxylic acid w h i c h was m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y (470 rim) as t h e 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e in a l k a l i n e s o l u t i o n 11. T h e r e a c t i o n m i x t u r e c o n t a i n e d 28.6 ~*mole a l l a n t o i n ( a d j u s t e d t o p H 7.5 w i t h N a O H ) , 50 a m o l e Tris-HC1 buffer (pH 8.2), 0.4 m l of e x t r a c t or h o m o g e n a t e , a n d w a t e r to a final v o l u m e of 2.0 ml. T h e p H m e a s u r e d in situ was 7.6. P r o t e i n c o n c e n t r a t i o n s were determined12 w i t h purified b o v i n e s e r u m or purified egg a l b u m i n as s t a n d a r d s . T h e a c t i v i t y f o u n d for a l l a n t o i n a s e is s h o w n in t h e Table. T h e p H o p t i m u m of t h e e n z y m e w a s 7.6. T h e Michaetis c o n s t a n t , Kin, was f o u n d t o be 0.012 M for allantoin, as d e t e r m i n e d b y e i t h e r a L i n e w e a v e r - B u r k p l o t or b y a w e i g h t e d H o f s t e e p l o t ~a. A n A r r h e n i u s plot of log