1376

specialia

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

15. 11. 1976

Specialia

a c t i v i t y v s . r e c i p r o c a l of a b s o l u t e t e m p e r a t u r e y i e l d e d e n e r g i e s of a c t i v a t i o n of 14.6 k c a l / m o l e a n d 12.6 k c a l / mole for the whole homogenate and acetone powder, r e s p e c t i v e l y . XYhile l i n e a r i t y of t h e A r r h e n i u s p l o t w a s o b t a i n e d o v e r t h e r a n g e of 1 4 - 3 8 ~ for t h e w h o l e h o m o g enate, the plot for the acetone extract deviated from a straight line above 22~ a feature noted previously 7 with some purified allantoinases from other sources. This is t h o u g h t n o t t o be a n e f f e c t o f d e n a t u r a t i o n o f t h e enzyme at the higher temperatures 7 but may represent a c o n f o r m a t i o n a l c h a n g e in t h e e n z y m e . T w o c l a s s e s of t h e p h y l u m A n n e l i d a , t h e O l i g o c h a e t a a n d H i r u d i n e a , a r e p u r i n o s t a t i c a n d l a c k e n z y m e s of u r i c o l y s i s ~ . F o r t h e t h i r d class, t h e P o l y c h a e t a , a11ant o i n a s e h a s b e e n f o u n d i n 4 of t h e 6 s p e c i e s of S e d e n t a r i a t e s t e d s, 9, b u t o n l y ' t r a c e s ' h a v e b e e n f o u n d in o n e of t h e 6 E r r a n t i a t e s t e d s. T h i s r e p o r t p r o v i d e s e v i d e n c e f o r t h e o c c u r r e n c e of a l l a n t o i n a s e in a n o t h e r s p e c i e s of S e d e n t a r i a . T h e s e d i s c o v e r i e s of p u r i n o l y t i c a c t i v i t y in t h e p o l y c h a e t e s g i v e e v i d e n c e of a b i o c h e m i c a l l i n k w i t h t h e

Allantoinase activity in extract of acetone powder of Eudistylia vancouveri for two typical experiments System

l:xperiment 1~ Complete Complete (boiled extract) Complete (zero time control) Experiment 2 u Complete Complete (boiled extract) Complete (zero time control)

Glyoxylic acid produced (gmloles)

0.245, 0.249 ~ 0.035 (/ 0.257, 0.266, 0.295 ~ 0.032 0

~30 rain incubation time, 7.70 mg protein/ml, b45 rain incubation time, 5.09 mg protein/ml, o Replicates.

1377

related phylum Sipunculida, which has a complete syst e m of p u r i n o l y s i s ~ . P o l y e h a e t e s of t h e S e d e n t a r i a m a y b e o n e of t h e f e w g r o u p s of o r g a n i s m s p o s s i b l y p o s s e s s i n g b o t h c o m p l e t e p u r i n o l y s i s a n d o r n i t h i n e - u r e a c y c l e e n z y m e s as m e c h a n i s m s f o r t h e p r o d u c t i o n of u r e a . I t w o u l d b e of i n t e r e s t t o d e t e r m i n e w h e t h e r or n o t L - o r n i t h i n e - k e t o a c i d a m i n o t r a n s f e r a s e ( E C 2.6.1.13) is p r e s e n t i n t h e S e d e n t a r i a since this enzyme provides a link between the ornithineu r e a c y c l e a n d t h e p u r i n e - u r e a c y c l e . T h e q u e s t i o n of w h y e r r a n t p o l y c h a e t e s l a c k e n z y m e s of u r i c o l y s i s a n d some sedentary polychaetes possess them might be ans w e r e d b y f u r t h e r e x a m i n a t i o n of t h e a d a p t i v e s i g n i f i c a n c e of t h i s p a t h w a y f o r p o l y c h a e t e s .

1 Supported by the U. S. National Science Foundation (Institutional Grant) and by the U. S. Environmental Protection Agency Grant No. T-900204 (WP 175). Publication No. 10 of the Laboratory of Biochemical Ecology; College of Fisheries Contribution No. 458. We thank Dr. ROBERT L. FERN_~LD for generously making available to us the facilities of the Friday Harbor Laboratories, Friday Harbor, Washington. .2 Present address: U. S. Fish and Wildlife Service, Great Lakes Fishery Laboratory, 1451 Green Road, Ann Arbor, Michigan 48105, USA. a S. J. PRZYLECKL Arch. intern. Physioh 24, 238 (1925). 4 A. BR~JNEL, Bull. Soc. Chim. biol. lO, 805 (1937). a A. BR~rNEL, Bull. Soc. Chim. biol. 19, 1027 (1937). 6 M. V. "I'RACEY, ill 11/Ioderne Methode#~ de~" P/lanzenanalyse (Eds. K. PAECH and M. V. TRACEV; Springer, Berlin 1955), vol. 4, p. 119. 7 G. D. VOOELS, F. TRIJBELS and A. UF>'INK, Biochiin. biophys. Aeta 122, 482 (1966). s p. RAZET and C. RETIf:RE, C. r. Acad. Sci., Paris 1) 26d, 356 (1967). ~ J. E. HUI.T, Comp. Biochem. Physiol. 31, 15 (1969). i0 G. \V. BROWN, JR., Arch. Biochem. Biophys. 114, 184 (1966). 11 G. W. BROWN, JR., Am. Zoo1. 4, 310 (1964). ~20. H. LOWRY, N. J. ROSEBROUO~I, A. L. FARR and R. J. RANDALL, J. biol. Chem. 193, 265 (1951). la G. N. WILKINSON, Biocheln. J. 80, 324 (1961). 1~ A. E. NI~:EDHAM,in Comparative Biochemistry o/ Nitrogen 3lctabolism (Ed. J. W. CA~aPBELI~; Academic Press, New York 1970), voI. 1, p. 207.

E v i d e n c e of a n E s s e n t i a l H i s t i d y l R e s i d u e i n A r y l s u l p h a t a s e B ~ A. A. FAROOQUI 2

Centre N a t i o n a l de la Recherche Scienli/ique, Centre de Neurochimie, l 1, rue H u m a n n , F - 6 7 0 8 5 Strasbourg Cedex (France), 30 A p r i l 1976. Summary. Diazotization and carbethoxylation e s s e n t i a l for a r y l s u l p h a t a s e B a c t i v i t y .

s t u d i e s of a r y l s u l p h a t a s e

A r y l s u l p h a t a s e B h a s r e c e n t l y b e e n o b t a i n e d in a h o m o g e n e o u s f o r m f r o m o x t i s s u e s a, ~ a n d h u m a n l i v e r 5. This enzyme differs from the corresponding arylsulphatase A both by its higher isoelectric point and lower m o l e c u l a r w e i g h t % A l t h o u g h s o m e i n f o r m a t i o n is a v a i l a b l e o n t h e f u n c t i o n a l g r o u p s in t h e a c t i v e s i t e , a n d o n t h e p h y s i o l o g i c a l role of a r y l s u l p h a t a s e A 7-10, v e r y l i t t l e is k n o w n a b o u t t h e a c t i v e s i t e a n d t h e p h y s i o l o g i c a l i m p o r t a n c e of a r y l s u l p h a t a s e B:U, 12. R e c e n t s t u d i e s o f AaociauA and WYXN 5 have indicated that probably a h i s t i d i n e r e s i d u e is i n v o l v e d i n t h e r e a c t i o n c a t a l y z e d b y a r y l s u l p h a t a s e B. T h e p r e s e n t s t u d y w a s u n d e r t a k e n t o g a i n m o r e i n f o r m a t i o n a b o u t t h e p a r t i c i p a t i o n of a h i s t i d i n e r e s i d u e in t h e h y d r o l y s i s of a r y l s u l p h a t e s b y a r y l s u l p h a t a s e t3. Materials and methods. T h e h o m o g e n e o u s p r e p a r a t i o n s of o x l i v e r a r y l s u l p h a t a s e s B I ~ a n d B l f w e r e p r e p a r e d

B h a v e i n d i c a t e d t h a t a h i s t i d i n e r e s i d u e is

1 The work was done at the Aligarh Muslim University, Aligarh (U. P.), India. 2 The author is grateful to Dr. A. N. KHAN Of I)epartment of Zoology for useful discussion. a W. S. BLSSZYNSKI and A. B. Roy, Biochim. biophys. Acta 317, 164 (1973). 4 A. A. FAROOQUI and A.B. Roy, Bioehim. biophys. Acta, in press (1976). 5 S. I. O. AGOGHUAand C. H. WYNN, Biochem. J. 153, 415 (1976). R. G. NIcttoLLs aud A. B. RoY, Enzymes 5, 21 (1971). 7 A. JERFY and A. B. RoY, Bioehim. biophys. Acts 371, 76 (1974}. s G. D. LEE and R. L. VAN-ETTEN, Arch. Bioehem. Biophys. 771, 424 (1975). 9 A. J'F-RFY and A. B. RoY, Biochim. biophys. Acta 293, 178 (1973). 10 A. A. FAROOQUI and B. K. BACHHAWAT,J. Neurochem. 20, 889 (1973). *~ A. L. I?LUHARTY, R. L. S'rEVE~S, D. FUNG, S. PEAK and H. KIHA~A, Biochem. biophys. Res. Commun. 6d, 955 (1.975). 1~ A. A. FAROOQUL Experientia, 32, in press (1976).