15.6. 1975

Speeialia

S u c h a close a s s o c i a t i o n b e t w e e n I A A oxidase a n d peroxidase can be visualized from the evidence presented b y a n u m b e r of w o r k e r s ls-15. T h i s is also s u p p o r t e d b y t h e f a c t t h a t a u n i q u e I A A oxidase e n z y m e h a s n o t b e e n f o u n d , e x c e p t in t h e t o b a c c o r o o t e x t r a c t s 1% B. Z. SIEGEL and A. W. GALSTON, Science 157, 1557 (1967). R. E. STCTZ, P1. Physiol. 32, 31 (1957). 1~ p. N[. RAY, Arch. Biochem. Biophys. 87, 19 (1960). 15 B. I)ARBYSHIRE, Physiologia plant. 29, 293 (1973). 16 L. SEQUEIRAand L. I~{INEO, P1. Physiol. 41, 1200 (1966). n The research has been partly financed by a grant from the U.S. Department of Agriculture. One of us (RNC)'is thankful to the CSIR, India, for financial assistance. is

637

S u m m a r y . T h e I A A oxidase e n z y m e e x t r a c t e d f r o m Phaseolus mungo h y p o c o t y l s a p p e a r s to show allosteric b e h a v i o u r w i t h 2 sites, one r e p r e s e n t i n g t h e o x i d a s e (site I) a n d t h e o t h e r p e r o x i d a s e (site II). I t is c o n s i d e r e d t h a t t h e site I is c o n c e r n e d in t h e p r o d u c t i o n of a c t i v e I A A o x i d a t i o n p r o d u c t s , while site I I m e r e l y a c t s t o d e t o x i f y t h e excessive I A A .

is

K. K. NANDA, t~. GURUMURTI a n d

R. N. CHIBBAR 17 Botany Department, Pan]ab University, Chandigarh-760074 (India), 2 September 1974.

I n h i b i t i o n of C a t h e p s i n A A c t i v i t y by the P o t a t o P r o t e a s e I n h i b i t o r O n l y a few of t h e n a t u r a l i n h i b i t o r s of i n t r a c e l l u l a r p r o t e o l y t i c e n z y m e s h a v e so f a r b e e n discovered. N o t h i n g is k n o w n a b o u t t h e n a t u r a l i n h i b i t o r s of c a t h e p s i n A. I n h i b i t o r s of c a t h e p s i n t3 a n d C o c c u r in t h e c y t o p l a s m of l i v e r cells 1 a n d in t h e h e n ' s e g g - w h i t e 2. C a t h e p s i n t31 a c t i v i t y is i n h i b i t e d b y e 2 - m a c r o g l o b u l i n 3, t h e c a r b o h y d r a t e i n h i b i t o r a s s o c i a t e d w i t h h a p t o g l o b i n ~, a n d l e u p e p t i n 5. C a t h e p s i n s D a n d E are i n h i b i t e d b y p e p s t a t i n 5, 6. A specific s e r u m a n t i b o d y i n a c t i v a t e s c a t h e p s i n D 7 a n d c a t h e p s i n E is i n a c t i v a t e d b y a n i n h i b i t o r i s o l a t e d f r o m A scarfs lumbricoides ~. O u r p r e l i m i n a r y e x p e r i m e n t s s h o w e d t h a t acid cellular p r o t e a s e s of a spleen h o m o g e n a t e were p a r t l y i n h i b i t e d b y t h e p o t a t o p r o t e a s e i n h i b i t o r ( P P I ) w h e n t e s t s of t h e i r a c t i V i t y o n h a e m o g l o b i n a t p H 3.5 were madeg, 10. I n t h e s e i n v e s t i g a t i o n s , a n a t t e m p t was m a d e to d e t e r m i n e w h i c h of t h e s e e n z y m e s was i n h i b i t e d b y t h e P P I . P u r i f i e d p r e p a r a t i o n s of i n t r a c e l l u l a r p r o t e a s e s a n d specific s u b s t r a t e s were used for t h e s e e x p e r i m e n t s . Since, of all t h e i n t r a c e l l u l a r proteases, o n l y c a t h e p s i n A a n d p a r t i c u l a r l y c a t h e p s i n D b r i n g a b o u t t h e d e g r a d a t i o n of h a e m o g l o b i n n, p a r t i c u l a r a t t e n t i o n was p a i d t o t h o s e enzymes. T h e P P I was o b t a i n e d b y t h e m e t h o d p r e v i o u s l y described1% C a t h e p s i n A a n d D were o b t a i n e d f r o m h e n muscles a c c o r d i n g t o t h e m e t h o d g i v e n b y JODICE e t el. 11 T h e source of c a t h e p s i n B~ a n d C was 10% h o m o g e n a t e of ox spleen. T h e effect of P P I in v a r i o u s c o n c e n t r a t i o n (0.0062 to 0.1 m g / m l ) o n c a t h e p s i n A (0.6 m g / m l ) a c t i v i t y was t e s t e d a c c o r d i n g to t h e m e t h o d of J o D i c z et a1.11. T h e s u b s t r a t e was N - a - c a r h o b e n z o x y - L - g l u t a m y l - L - t y r o s i n e (6.2 m M ) in a 0.1 M a c e t a t e b u f f e r ( p H 6.0). I n c u b a t i o n

Inhibition of eathepsin A activity by various concentrations of potato protease inhibitor (PPI) measured on N-g-carbobenzoxy-Lglutamyl-L-tyrosine PPI, final concentration (mg/ml

cr (mM/ml)

Activity (%)

0.100 0.050 0.025 0.012 0.00625 Control

2.3 5.8 61.0 120.0 150.0 225.0

1.0 2.5 27.1 53.3 66.4 100.0

Final concentration: of enzyme, 0.6 mg/ml; of substrate, 6.2 raM.

was c a r r i e d o u t for 1 h a t 37 ~ T h e r e a c t i o n was s t o p p e d b y a d d i n g T C A a n d t h e a - a m i n e - n i t r o g e n released was d e t e r m i n e d b y t h e n i n h i d r i n c o l o r i m e t r i c m e t h o d 12 T h e effect of P P I (0.1 m g / m l ) o n c a t h e p s i n B 1 a c t i v i t y was i n v e s t i g a t e d a c c o r d i n g to t h e m e t h o d d e s c r i b e d b y KEILOVA a n d TOMA~EK 2. T h e s u b s t r a t e was N - a - b e n z o y l D , L - a r g i n i n e - p - n i t r o a n f l i d e (1 m g / m l ) in a 0.1 M p h o s p h a t e b u f f e r ( p H 6.0) c o n t a i n i n g 1 m M E D T A a n d 25 m M cysteine-HC1 for a c t i v a t i o n . I n c u b a t i o n was carried o u t for 1 h a t 40 ~ T h e r e a c t i o n was s t o p p e d b y a d d i n g glacial acetic acid. T h e p - n i t r o a n i l i n e released was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y a t 405 nrn. T h e effect of P P I (0.1 m g / m l ) on c a t h e p s i n C a c t i v i t y was i n v e s t i g a t e d b y t h e s a m e p r o c e d u r e as t h a t used for c a t h e p s i n B 1 a c t i v i t y 2 e x c e p t t h a t glycyl-L-phenyla l a n i n e - p - n i t r o a n i l i d e (0.1 m g / m l ) was used as s u b s t r a t e . T h e effect of P P I (0.1 m g / m l ) on c a t h e p s i n D (0.4 m g / ml) a c t i v i t y was d e t e r m i n e d b y u s i n g as s u b s t r a t e uread e n a t u r a t e d h a e m o g l o b i n (10 m g / m l ) in 0.04 M B r i t t o n a n d R o b i n s o n u n i v e r s a l b u f f e r ( p H 3.5) 13. I n c u b a t i o n was carried o u t for 1 h a t 37 ~ T h e r e a c t i o n was s t o p p e d b y a d d i n g TCA, a n d t h e t y r o s i n e released was d e t e r m i n e d b y m e a n s of t h e ~'OLIN-CIOCALTEU r e a g e n t 14 T h e P P I i n h i b i t s c a t h e p s i n A a c t i v i t y (Table). T h e degree of c a t h e p s i n A a c t i v i t y i n h i b i t i o n d e p e n d s o n t h e P P t c o n c e n t r a t i o n . I n t h e p r e s e n c e of t h e P P I in a final c o n c e n t r a t i o n of 0.1 m g / m l , a p p r o x i m a t e l y 1 % of t h e a c t i v i t y of t h e e n z y m e r e m a i n e d . T h i s i n h i b i t o r does n o t i n h i b i t t h e a c t i v i t y of c a t h e p s i n B1, C a n d D. C a t h e p s i n A is i n h i b i t e d b y d i i s o p r o p y l o f l u o r p h o s p h a t e serine c a r b o x y -

1 j . FINKENSTAEDT, Proc. Soc. exp. Biol. Med. 95, 302 (1957). 2 H. K~ILOVA and V. TOMA~EK, Bioehim. biophys. Acta 33g, 179 (1974). a p. M. STARKLy and A. J. BARRETT, Bioehem. J. 131, 823 (1973). 4 0 . SNELLMANand B. SYLVAN, Experientia 30, 1115 (1974). a H. IKEZAWA, T. AOVAGI, T. TAKEUCHI and H. UMEZAWA, J. Antibiot. 24, 488 (1971). 6 A. J. BARRETT and J. T. DINGLE, Biochem. J. 727, 439 (1972). 7 j. T. DINGLE, A. J. BARRETTand P. D. WESTO~r Bioehem. J. 123, 1 (1971). s H. KEILOVA and V. TOMA~EK, Biochim. biophys. Acta 28d, 461 (1972). 9 K. WOROWSKI and T. MARIAK, Aeta Pol. pharm. 30, 453 (1973). lo K. WOROWSKI, Thromb. Diath. haemorr. 32, 617 (1974). 11 A. JODICE, V. LXONGand J. WEnCSTOCK,Arch. Biochem. Biophys. 777, 477 (1966). 12 T. DEVENYI and T. GERGELY, Analytische Methoden zur Untersuchung yon Aminosduren, Peptiden und Proteinen (Akadcmiai Kiad6, Budapest 1968), p. 249. 13 M. L. Ai,~SON, J. gen. Physiol. 20, 265 (1937). la O. FOLIN and V. CIOCALTEU,J. biol. Chem. 73, 627 (1927).

638

Specialia

p e p t i d a s e 1L T h u s i t is p r o b a b l y s e n s i t i v e t o t h e a c t i o n of t h e P P I as are m a n y o t h e r p r o t e o l y t i c e n z y m e s w i t h serine a c t i v e centresZ~ C a t h e p s i n A as serine c a r b o x y p e p t i d a s e is n o t s e n s i t i v e t o t h e p o t a t o i n h i b i t o r of t h e p a n c r e a t i c c a r b o x y p e p t i d a s e s A a n d B w h i c h are m e t a l o p r o t e i n a s e s 16. C a t h e p s i n A a c t s chiefly o n t h e p r o d u c t s of p a r t i a l h a e m o g l o b i n d e g r a d a t i o n f o r m e d as a r e s u l t of t h e a c t i o n of c a t h e p s i n D tt. H a e m o g l o b i n d e g r a d a t i o n b y c a t h e p s i n A occurs m o s t r a p i d l y a t p H 3.5, w h e r e a s w h e n s y n t h e t i c s u b s t r a t e s are split b y t h i s e n z y m e , t h e o p t i m u m p H is 5.4 17. Since t h e P P I does n o t i n h i b i t c a t h e p s i n D a c t i v i t y , it would seem t h a t t h e p a r t i a l i n h i b i t i o n of h a e m o g l o b i n d e g r a d a t i o n b y t h e acid cellular p r o t e a s e s a t p H 3.5 in t h e p r e s e n c e of t h e p o t a t o i n h i b i t o r , o b s e r v e d p r e v i o u s l y 9, ~0, d e p e n d s o n t h e i n h i b i t i o n of t h i s c a t h e p s i n A i n h i b i t o r .

EXPERIENTIA 31/6

Summary. I t was f o u n d t h a t t h e p r o t e a s e i n h i b i t o r from the potato inhibits cathepsin A activity. This i n h i b i t o r does n o t i n h i b i t t h e a c t i v i t y of c a t h e p s i n B~, C a n d D. K. WOROXVSKI Department o/ Biochemistry, Institute o/Physiology and Biochemistry, Medical School, ul. Mickiewicza 2, Biatystok (Poland), 78 December 1975.

A. J. LOGUNOVand V. N. OREKHOWICH, Biochim. biophys. Res. Commun. 46, 1161 (1972). 16 C. A. RYA~, J. biol. Chem. 2d9, 2495 (1974). 17 j . S. FRUTON, in The Enzymes (Eds. P. D. BOYER, H. LARDY and K. ~[YRBACK; Academic Press, New York 1960), vol. 4, p. 233.

15

Differences in R N A Content Related to M a t i n g T y p e in A s c o b o l u s i m m e r s u s Several m e c h a n i s m s w h i c h r e d u c e self-fertilizing a n d e n c o u r a g e r e a s s o r t m e n t of g e n e t i c differences t h r o u g h o u t b r e e d i n g h a v e b e e n d e s c r i b e d in fungiX; b u t , u n t i l now, v e r y little is k n o w n of t h e physiological basis of m a t i n g t y p e d e t e r m i n a t i o n in t h e s e organisms. I n Ascobolus immersus, 2 alleles (A a n d a), a t a single locus, d e t e r m i n e m a t i n g t y p e . T h i s 2 allele s y s t e m , w h i c h r e p r e s e n t s t h e s i m p l e s t f o r m of heteroallelism, is v e r y w i d e s p r e a d b e i n g f o u n d in all g r o u p s of fungi. P r e v i o u s r e p o r t s f r o m t h i s l a b o r a t o r y 2-a i n d i c a t e d t h a t c e r t a i n v a r i a t i o n s in m o b i l i t y a n d R N A c o n t e n t are

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Migration

Fig. 1.2.4% polyacryMmide gel electrophoretic pattern of total RNA isolated from Ascobolus immersus wild-type strains S2A (A) and S2a (B). Abscissa: distance migrated in cm; ordinate: opticaI density at 260 nm.

04,,

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r e l a t e d to t h e d i f f e r e n t i a l r e s i d u a l g e n o t y p e or genetic b a c k g r o u n d of i n d i v i d u a l s ; w h i c h s u g g e s t e d t h a t A scobolus might be a most interesting system to investigate this p r o b l e m of s e x u a l d i f f e r e n t i a t i o n for w h i c h a d e a r t h of i n f o r m a t i o n exists. T h e p r e s e n t p a p e r r e p o r t s d a t a s h o w i n g real differences in t h e r e l a t i v e p r o p o r t i o n s of s t a b I e R N A c o m p o n e n t s in wild t y p e s t r a i n s of o p p o s i t e m a t i n g t y p e s w h i c h are p r o b a b l y r e l a t e d to t h e genetic c o n t r o l of s e x u a l d e v e l o p m e n t . Materials and methods. T h e locally collected w i l d - t y p e s t r a i n $2 of A. immersus was k i n d l y supplied b y Dr. J. R. BEAUDRY f r o m t h i s i n s t i t u t i o n . R N A was i s o l a t e d f r o m t h e m y c e l i u m of t h e d i f f e r e n t s t r a i n s b y cold p h e n o l i c d e p r o t e i n i z a t i o n ; a n d t h e R N A profiles were o b t a i n e d a f t e r p o l y a c r y l a m i d e gel electrophoresis, as p r e v i o u s l y described 3. Results. F i g u r e s 1 a n d 2 s h o w t h e e l e c t r o p h o r e t i c p a t t e r n s of global R N A o b t a i n e d f r o m S2A (A) a n d S2a (B) in 2.4% a n d 7.5% p o l y a c r y l a m i d e gels, respectively. One s h o u l d e x p e c t t h a t t h e r e l a t i v e p r o p o r t i o n s of s t a b l e R N A c o m p o n e n t s to r e m a i n c o n s t a n t i n b o t h w i l d - t y p e s t r a i n s of o p p o s i t e m a t i n g t y p e s , b u t it does n o t (Table). T h e 4S a n d 5S p e a k s are s i g n i f i c a n t l y h i g h e r for S2A t h a n S2a, while all t h e o t h e r R N A p o p u l a t i o n s are p r e s e n t in e q u i v a l e n t p r o p o r t i o n s . 5 d i f f e r e n t e x t r a c t s f r o m t h e same strains confirmed this apparent relationship b e t w e e n t h e m a t i n g t y p e allele a n d t h e r e l a t i v e R N A distribution. So as to get m o r e i n f o r m a t i o n a b o u t such a r e l a t i o n s h i p , one ascus p r e s e n t i n g 8 w i l d - t y p e ascospores was isolated in t h e p r o g e n y of t h e w i l d - t y p e c u l t u r e S2A X S2a. E a c h spore of t h e selected ascus yielded a s t r a i n w h i c h was b a c k c r o s s e d t o t h e 2 p a r e n t a l s t r a i n s so as to d e t e r mine the exact mating type; and the RNA populations f o u n d in t h e m y c e l i u m p r o d u c e d b y e a c h of t h e 8 segreg a n t s were studied. S u r p r i z i n g l y , e l e c t r o p h o r e t i c R N A profiles of w i l d - t y p e s t r a i n s w h i c h g e n o t y p e is A are p r a c t i c a l l y i d e n t i c a l to t h o s e of t h e p a r e n t a l s t r a i n S2A (Figures 1A a n d 2A) a n d vice versa. S t a t i s t i c a l

0. 1 j . R. S. FINCItAM and P. R. DAy, Fungal Genetics (Blackwell 0

1

2

3 4

5

6

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2 5 4

5 6

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Migration

Fig. 2. 7.5% polyacrylamide gei eleetrophoretic pattern of total RNA isolated from Ascobolus immersus wild-type strains S2A (A) and S2a (B). Abscissa: distance migrated in am; ordinate: optical density at 260 mn.

Scientific Publications, Oxford 1971). 2 C. HAMELIN and G. H. COUSI~EAU, Revue Can. Biol. 33, 243 (1974). 3 C. HAMELIN and G. H. COIISlNEAU, Can. J. Genet. CytoL 16, 39 (1974). 4 C. HAMZLIN and G. H. COUSlN~AU, Revue Can. Biol. 33, 249 (1974).

Inhibition of cathepsin A activity by the potato protease inhibitor.

15.6. 1975 Speeialia S u c h a close a s s o c i a t i o n b e t w e e n I A A oxidase a n d peroxidase can be visualized from the evidence presente...
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