15.8. 1976

Specialia

was d i r e c t l y i n v e s t i g a t e d in d i s r u p t e d e p i m a s t i g o t e s . Figure 2 shows, h o w e v e r , t h a t no c a t a l a s e a c t i v i t y could be d e t e c t e d in t h e e p i m a s t i g o t e h o m o g e n a t e . T h e possible e x i s t e n c e of cataIase i n h i b i t o r s in t h e h o m o g e n a t e m a y be e x c l u d e d b y c o m p a r i n g t h e a c t i v i t y of beef liver c a t a lase (Sigma Chemical C o m p a n y ; C-100) in h o m o g e n a t e containing and homogenate-free reaction mixtures, r e s p e c t i v e l y (Figure 2). I n c o n t r a s t to t h e s e n e g a t i v e results, p e r o x i d a s e a c t i v i t y could be d e m o n s t r a t e d in t h e e p i m a s t i g o t e h o m o g e n a t e (Figure 2) a n d also in t h e p a r t i c u l a t e f r a c t i o n s o b t a i n e d t h e r e f r o m (Table). P e r o x i dase a c t i v i t y was closely associated w i t h t h e p a r t i c u l a t e 480 • a n d 680 • fractions, w h i c h i n c l u d e d t h e microbodies (Figure 1B). The n o n - s e d i m e n t a b l e f r a c t i o n a t 105,000 • g s h o w e d a significant, t h o u g h lower, p e r o x i d a s e specific a c t i v i t y , w h i c h m i g h t reflect t h e release of e n z y m e f r o m m i c r o b o d i e s b r o k e n d u r i n g cell f r a c t i o n a t i o n . I n t h e a b s e n c e of ascorbate, t h e r a t e of h y d r o g e n p e r o x i d e decomposition by the microbody-containing fractions was negligible ( 0 - 3 % of t h e r a t e in t h e p r e s e n c e of ascorbate). Discussion. D A B p o s i t i v i t y is s t a n d a r d c y t o c h e m i c a I e v i d e n c e for t h e i d e n t i f i c a t i o n of c a t a l a s e - c o n t a i n i n g m i c r o b o d i e s ~ ,, 1,, 20 b u t t h e r e a c t i o n is also p o s i t i v e w i t h p e r o x i d a s e ~ - " a a n d c y t o c h r o m e oxidase~2,21,~. The p r e s e n c e of p e r o x i d a s e in T. cruzi m i c r o b o d i e s is in good a g r e e m e n t w i t h a) tile p r e f e r e n t d i s t r i b u t i o n of p e r o x i d a t i c a c t i v i t y in t h e p a r a s i t e h i g h d e n s i t y fractions

975

(Table); b) t h e f a c t t h a t p e r o x i d a s e s selectively oxidize d o n o r s h a v i n g t h e enediol s t r u c t u r e , s u c h as a s c o r b a t e ~s (Figure 2 a n d Table), a n d c) t h e c y t o c h e m i c a l d e m o n s t r a t i o n of p e r o x i d a s e in T. cruzi b y KALLINICOVA 25. P a r t i c i p a t i o n of c y t o c h r o m e oxidase in D A B p e r o x i d a t i o n m a y be e x c l u d e d b y t h e r e s t r i c t e d d i s t r i b u t i o n of t h e electrono p a q u e m a t e r i a l in F i g u r e 1. T h e a p p a r e n t a b s e n c e of c a t a l a s e in T. cruzi m i c r o b o d i e s recalls similar n e g a t i v e o b s e r v a t i o n s w i t h m i c r o b o d i e s f r o m Trichomonas/oetus 26. F u r t h e r m o r e , i n v e s t i g a t i o n of c a t a l a s e in o t h e r Trypanosomatidae 27- ~, a n d Trichomonas a0, w i t h m e t h o d s d i f f e r e n t f r o m t h e one e m p l o y e d b y us, failed t o d e m o n s t r a t e significant a m o u n t s of e n z y m e in t h e s e organisms. T. brucei has no catalase, b u t s h o w s p e r o x i d a s e a c t i v i t y ".

is H. D. I~'AHIMI, J. cell. Biol. d3, 275 (1969). 20 S. GOLDFISCHER, J. Histochem. 17, 681 (1969). 21 K. I. HIRAI, J. Histochem. Cytochem. 79, 434 (1971). 22 M. J. ]KARNOVSKY, J. Cell. Biol. 27, 4 9 A (1965).

ea R. C. GRAHAMJR. and M. J. KARNOVSKY,J. Histochem. Cytochem. 1~, 291 (1966). e4 S. GOLDFISCHER, J. cell. Biol. 34, 398 (1967). 25 V. D. KALLINIKOVA, A e t a protozool. 6, 87 (1968). 26 M. MOLLER, J. Histochem. Cytochem. 21, 955 (1973). 27 V~T. I. STRANGEWAYS, Ann. trop. Med. Parasit. 31, 405 (1937). 2s S. C. HARVEY, J. biol. Chem. 779, 435 (1949).

a9 j. F. RYLEY, Biocheln. J. 59, 353 (1955). 30 j. F. RYLEY, Bioehem. J. 59, 361 (1955).

Electron-Microscopic Mapping of the Hinge Re~ion of Myosin ~V~URRAY V E R N O N K I N G 1, 2

Departments o~ Biological Chemistry and Medicine, Harvard Medical School and the Massachusetts General Hospital, Boston (Massachusetts 0271d, USA), 1 March 7976. Summary. The t r y p s i n - s e n s i t i v e sites in t h e labile hinge region of t h e m y o s i n molecule are l o c a t e d w i t h h e i g h t e n e d a c c u r a c y ( 4- 2 nm) b y electron m i c r o s c o p y as lying a t 70, 85, 95, a n d 103 n m f r o m t h e C - t e r m i n u s of t h e rod section of t h e molecule. A r e s t r i c t e d region w i t h i n t h e rod section of t h e m y o s i n molecule lying a t 70-110 n m f r o m t h e C - t e r m i n u s shows special p r o p e r t i e s including h i g h s u s c e p t i b i l i t y to proteases a-7 a n d to t h e r m a l d e n a t u r a t i o n ~. Fc h a s b e e n t e r m e d t h e hinge region, a n d a role in muscle s h o r t e n i n g has b e e n p r o p o s e d 8-~~ The loci m u s t s u s c e p t i b l e t o t r y p t i c a t t a c k h a v e b e e n inferred f r o m t h e molecular w e i g h t s of digestion p r o d u c t s or d e t e r m i n e d in t h e e l e c t r o n m i c r o s c o p e 7, ~, ~2 I r e p o r t here a m o r e a c c u r a t e m a p p i n g of t h e sites of t r y p t i c a t t a c k on m y o s i n t h a t has b e e n a g g r e g a t e d into s e g m e n t s t r u c t u r e s or on similar arrays of l i g h t m e r o m y o s i n - C (LMM-C), w h i c h is a C - t e r m i n a l f r a g m e n t of m y o s i n t h a t is l i b e r a t e d b y digestion w i t h BrCN ~a, a n d w h i c h c o n t a i n s m o s t of t h e h i n g e region. A d v a n t a g e o u s l y , t h e molecules in t h e p l a n a r a r r a y s are held s t r a i g h t b y c o n t a c t w i t h neighbors, a n d positions of d i g e s t e d m a r g i n s can be a c c u r a t e l y m e a s u r e d in t h e e l e c t r o n microscope. Myosin a n d LMM-C were p r e p a r e d ~a-~ a n d a g g r e g a t e s were g r o w n b y dialysis-dilution ~6 to a final c o n c e n t r a t i o n of 0.22 ~ I calcium a c e t a t e . T h e r e s u l t i n g m y o s i n aggregates were aligned s e g m e n t s 16, a n d t h o s e w i t h LMM-C were a s e g m e n t s t r u c t u r e , p h a s e F', w i t h an o v e r l a p w i d t h of 88 • 4 n m a n d a fringe w i d t h of 12 • 2 n m (all limits of error are s t a n d a r d deviations). LMM-C was also a g g r e g a t e d b y dialysis-dilution f r o m 0.7 M t o 0.50.59 M calcium b u t y r a t e to give a n e w s e g m e n t s t r u c t u r e , p h a s e R, w i t h an o v e r l a p w i d t h of 64 • 2 n m a n d a fringe w i d t h of 30 • 1 nm.

1 Present address: Electron Optics L a b o r a t o r y , Division of Laboratories and Research, N. Y. S t a t e D e p a r t m e n t of Health, Albany, New York 12201, USA. Memorial I n s t i t u t e , Buffalo, N. Y. 14263, USA. 2 I t h a n k Dr. MICHAEL YOUNG for v a l u a b l e advice and encourage-

nlent, Drs. JE~OM= Ggoas and ROMAISER. BRUNSfor use of their electron microscope, and Mrs. MURIEL H. BLANCHARDfor excellent technical assistance. This work was supported by the American Heart Association, The John A. Hartford Foundation, Inc., and the National Institutes of Health. a E. MIHALYI and A. G. SZENT-GY6RGYI, J. biol. Chem. 201, 189 (1953). r J. GERGELY, M. A. GOUVEA, and D. KARIBIAN, J. biol. Chem. 212, 165 (1955). 5 w. R. MIDDLEBROOK,Science IS0, 621 (1959). D. R. KOMINZ, E. R. MITCHELL, T. NInEI, and C. M. KAY, Biochemistry 4, 2373 (1965). 7 S. LowE% H. S. SLAVTER,A. G. WEEDS, and H. BAKER,J. molec. Biol. 42, 1 (1969). 8 M. BURKE, S. HllVIMELFARB, and W. F. HARRINGTON, B i o c h e m i s t r y 72, 701 (1973). 9 F. PEPE, J. molec. Biol. 27, 227 (1967). i0 H. HUXLEY, Science 164, 1356 (1969}. 1I E. F. WOODS, S. HIMMELFARB, and W. F. HARRINGTON, J. biol. Cheni. 238, 2374 (1963). 12 1). M. SEGAL, S. ~IMMELFARB, and W. F. HARRINC~TON, J. biol. Chem. 242, 1241 (1967).

13 M. YOUNG, M. H. BLANClIARD,and D. BROWN, Proc. narD. Acad. Sci., USA 61, 1087 (1968). 14 W . g . KIELLEYand L. B. BRADLEY, J, biol. Chem. 218, 653 (1956). la E. G. RICHARDS,C. S. C~UNG, D. B. MENZEL, and H. S. OLCOTT, Biochemistry 6, 528 (1967). 16 M. V. KING and M. YOUNG, J. molee. Biol. 63, 539 (1972).

976

Specialia

S a m p l e s of t h e a g g r e g a t e s s u s p e n d e d in t h e i r dialysis s o l u t i o n s were digested a t 20~ w i t h t r y p s i n in a 1:255 m a s s r a t i o t o m y o s i n or 1:92.2 t o LMM-C. A n o t h e r s a m p l e of m y o s i n s e g m e n t s was t r a n s f e r r e d to 0.5 M a m m o n i u m a c e t a t e p r i o r t o digestion t o s t u d y t h e effect of s a l t composition. A l i q u o t s r e m o v e d a f t e r v a r y i n g t i m e s were q u e n c h e d w i t h chilled s o l u t i o n s of s o y b e a n t r y p s i n inh i b i t o r , a n d s p e c i m e n s were m o u n t e d , n e g a t i v e l y s t a i n e d , a n d e x a m i n e d in t h e e l e c t r o n microscope ~6. T h e F i g u r e s u m m a r i z e s for t h e d i f f e r e n t digestions t h e d i s t a n c e s of scission loci f r o m t h e o v e r l a p margins, i.e., t h e l e n g t h s of t h e r e s i d u a l l i g h t - m e r o m y o s i n f r a g m e n t s .

Phase R of LMM-C

f

Phase F'of LMM-C

,/-

Myosin in calcium acetate

,

(

# .J

1

Myosin in ammoniumacetate_/---i

i

i

5O

i

i

100 nm

Cumulative frequency graphs of the distances from the C-termini of the molecules to scission sites in tryptic digestions of segment structures of myosin and LMM-C.

EXP~RIENTIA 32/8

To a v o i d t h e a r t i f a c t - p r o n e g r o u p i n g of m e a s u r e m e n t s i n t o ranges, t h e d a t a are p l o t t e d as c u m u l a t i v e f r e q u e n c y graphs, r a t h e r t h a n as h i s t o g r a m s . E a c h u n i t s t e p i n a b r o k e n line c o r r e s p o n d s t o one d a t u m , a n d t h e s t e p h e i g h t s are i n v e r s e l y p r o p o r t i o n a l t o t h e t o t a l n u m b e r of m e a s u r e m e n t s in each series, so t h a t a v e r a g e slopes reflect t h e densities of d a t a points. T h e digestions of m y o s i n s h o w a single site of pred o m i n a n t a t t a c k in e a c h s o l v e n t , b u t t h e sites differ: 103 s 2 n m f r o m t h e C - t e r m i n u s in a m m o n i u m a c e t a t e solution, b u t 95 =k 2 n m in c a l c i u m a c e t a t e . W h i l e v e r y close sites c a n n o t be d i s t i n g u i s h e d , S t u d e n t ' s t-test suggests t h a t t h e 95-rim a n d 103-rim sites differ signific a n t l y a t b e t t e r t h a n t h e 0.1% level. T h e r e m a i n i n g scission sites for m y o s i n m o s t l y c l u s t e r a b o u t 70 n m or 85 rim, w i t h one scission a t 103 n m in c a l c i u m a c e t a t e . All of t h i s fits w i t h t h e o b s e r v a t i o n 7 t h a t brief t r y p t i c digestion of dissolved m y o s i n yields l i g h t - m e r o m y o s i n molecules a b o u t 90 n m long, b u t 70 n m long u p o n f u r t h e r digestion. T h e d i g e s t i o n o5 p h a s e F ' of LMM-C in c a l c i u m a c e t a t e largely m a t c h e s t h a t of m y o s i n in t h e s a m e solvent, b u t w i t h less clearly d e m a r c a t e d scission a t t h e 9 5 - n m site, w h i c h lies n e a r t h e N - t e r m i n u s of LMM-C. T h u s t h e t y p e of s o l v e n t is t h e m a j o r f a c t o r g o v e r n i n g specificity of attack. I n g r e a t c o n t r a s t , the d i g e s t i o n of p h a s e R of LMM-C show-s a g r e a t loss of specificity of a t t a c k . A wide r a n g e of sites t h r o u g h o u t t h e h i n g e region is r a p i d l y cleaved. Thus, c a l c i u m b u t y r a t e h a s r e n d e r e d s u s c e p t i b l e m a n y a d d i t i o n a l sites w i t h i n t h e h i n g e region. T h i s fact, comb i n e d w i t h t h e o b s e r v a t i o n t h a t LMM-C yields ordered, t h o u g h a b e r r a n t a g g r e g a t e s in c a l c i u m b u t y r a t e solution, suggests t h a t t h i s salt i n d u c e s a n a l t e r a t i o n in t h e LMM-C molecule t h a t is l i m i t e d to t h e h i n g e region.

A n a e r o b e A k t i v i e r u n g y o n Katalase (EC 1.11.1.6) in G e g e n w a r t y o n M o n o d e h y d r o - L ( + ) - a s c o r b a t A n a e r o b i c Inactivation of Catalase (EC 1.11.1.6) in the P r e s e n c e of M o n o d e h y d r o - L ( + ) - a s c o r b a t e ~V. VOGT u n d W . g E l S

Zentrum [#r Biochemie am Kli*zikum der .[ustus Liebig-Universit~it, Friedrichstrasse 24, D-63 Giessen (Bundesrepublik Deutschland, BRD), 8. Miirz 7976. Summary. Catalase is p a r t i a l l y i n a c t i v a t e d i r r e v e r s i b l y in a n a e r o b i c s o l u t i o n s c o n t a i n i n g t o g e t h e r L ( + ) - a s c o r b a t e , m o n o d e h y d r o - L ( + ) - a s c o r b a t e , a n d d i d e h y d r o - L ( + ) - a s c o r b a t e . Tile e x p e r i m e n t s i n d i c a t e t h a t m o n o d e h y d r o - L ( + ) a s c o r b a t e i n a c t i v a t e s catalase. ORR 1-4 h a t die lange b e k a n n t e u n t e r aerobe~z B e d i n gungen zur Inaktivierung yon Katalase durch z(+)Ascorbat ftihrende Reaktion auf molekularer Ebene unt e r s u c h t u n d zuletzt2, a b e s c h r i e b e n , dass u n t e r a e r o b e r Inkubation der Katalase mit c(+)-Ascorbat oder z(+)A s c o r b a t u n d K u p f e r i o n e n (pH 7,0) e n t s t e h e n d e H y d r o xyl- o d e r P e r h y d r o x y l r a d i k a l e d a s A p o p r o t e i n d e r K a t a lase je n a c h B e d i n g u n g e n u n t e r s c h i e d l i c h ver&ndern. Die L i t e r a t u r zu diesem P r o b l e m ist in d e n A r b e i t e n 1-4 a u s f i i h r l i c h dargestellt. B i s l a n g ist n o c h n i c h t u n t e r s u c h t worden, ob nicht z(+)-Ascorbat, Monodehydro-L(-F)a s c o r b a t oder D i d e h y d r o - L ( + ) - a s c o r b a t , S u b s t a n z e n , die bei d e n ill d e r L i t e r a t u r m i t g e t e i l t e n E x p e r i m e n t e n imm e r n e b e n e i n a n d e r vorliegen, u n t e r a*zaeroben B e d i n g u n gen K a t a l a s e i n a k t i v i e r e n . Material and Methode~. R e i n e k r i s t a l l i n e D i d e h y d r o L(+)-ascorbinstture wurde nach dem Verfahren yon STAUDINOZER u n d ~VEIs s, M o n o d e h y d r o - L ( + ) - a s c o r b a t

dutch Komproportionierung yon L(+)-Ascorbat und D i d e h y d r o - L ( + ) - a s c o r b a t , wie bei YON FOERSTER e t al. 6 b e s c h r i e b e n , hergesteltt. E x t r e m r e i n e r Stickstoff d e r F a . Messer, G r i e s h e i m , w u r d e m i t d e m O x i s o r b s y s t e m d e r gleiehen F i r m s n a e h g e r e i n i g t . W a s s e r w u r d e deionisiert u n d zweimal in einer Q u a r z a p p a r a t u r destilliert. Die iibrigen C h e m i k a l i e n w a r e n a n a l y s e n r e i n e H a n d e l s p r o d u k t e . Die V o r i n k u b a t i o n d e r K a t a l a s e erfolgte bei 25~ in e i n e r S t i c k s t o f f a t m o s p h & r e . Das S c h u t z g a s s t r 6 m t e m i t 120 m l 9 m i n -1 fiber die ill e i n e m fOberdruckventil verschlossenen Kolben befindliche Reaktionsl6sung. Z u r F e s t s t e l l u n g der k a t a l a t i s c h e n Aktivit~it der K a t a lase b e s t i m m t e n wir die anfS.ngliche R e a k t i o n s g e s c h w i n digkeit der Zersetzung yon Wasserstoffperoxid durch K a t a l a s e bei 25 ~C. Ausser bei d e r U n t e r s u c h u n g d e r p H A b h ~ u g i g k e i t d e r R e a k t i o n w u r d e b e i p H 7,0 g e a r b e i t e t . E s w u r d e die A b n a h m e d e r E x t i n k t i o n bei 240 n m Wellenl~nge in e i n e m U V - S p e k t r a l p h o t o m e t e r S P 800 A m i t