318

Specialia

P o l y a c r y l a m i d e is n o t a n t i g e n i c in itself, b u t w h e n conj u g a t e d to b o v i n e s e r u m a l b u m i n , a n t i b o d y c a p a b l e of specifically b i n d i n g r a d i o a c t i v e l y labeled P A A is elicitab!e. I t s a f f i n i t y a p p e a r s to be q u i t e small; h o w e v e r , m a t e r i a l of h i g h e r specific a c t i v i t y w o u l d increase t h e s e n s i t i v i t y , w h i c h in t u r n w o u l d increase t h e t i t e r of t h e a n t i b o d y p r o p o r t i o n a t e l y . A final d i l u t i o n of 1 : 5000 or m o r e w o u l d

EXPERIENTIA 34/3

be e n t i r e l y p r a c t i c a l for t h e r o u t i n e analysis of polya c r y l a m i d e in e f f l u e n t w a t e r s . T h e results i n d i c a t e t h a t R I A m a y p r o v e useful in analysis of o t h e r w a t e r soluble s y n t h e t i c p o l y m e r s t h a t e n t e r t h e e n v i r o n m e n t , such as e l e c t r o c o n d u c t i v e resins or p o l y e t h y l e n e i m i n e , a n d could be e x t e n d e d to a n a l y s i s of s a m p l e s of d o w n s t r e a m effluents.

Proteolysis in dystrophic hamster diaphragm and abdominal muscle K. N a k a t s u , J. Morison a n d J. E d m o n d s 1

Department o/ Pharmacology, Queen's University, Kingston (Canada K 7 L 3N6), 21 J u l y 1977 Summary. Proteolysis, as m e a s u r e d b y t y r o s i n e release, was e s t i m a t e d in a b d o m i n a l a n d d i a p h r a g m muscle of h a m s t e r s . T h e r e did n o t a p p e a r t o be a difference b e t w e e n d y s t r o p h i c a n d c o n t r o l h a m s t e r s . W a s t i n g of m u s c l e is one of t h e c h a r a c t e r i s t i c s of m u s c u l a r d y s t r o p h i e s . I n spite of r e s e a r c h i n t e r e s t in t h e s e diseases for several years, t h e etiologies are still u n k n o w n . E a r l y r e p o r t s b y W e i n s t o c k , E p s t e i n a n d M i l h o r a t 2, a n d o t h e r s led to t h e s u g g e s t i o n t h a t a b n o r m a l p r o t e i n b r e a k d o w n m i g h t be a n i m p o r t a n t f a c t o r in t h e p r o g r e s s of s o m e of t h e m u s c u l a r d y s t r o p h i e s . T h u s t h e o b j e c t i v e of t h i s s t u d y was t o d e t e r m i n e w h e t h e r increases in p r o t e o l y s i s could be d e t e c t e d p r i o r to d e t e c t i o n of o t h e r c h a n g e s d e s c r i b e d in t h e l i t e r a t u r e . I n t h i s c o m m u n i c a t i o n we r e p o r t t h e degrad a t i o n of p r o t e i n in muscle of y o u n g h a m s t e r s .

Table 1. Tyrosine release by abdominal and diaphragm muscles of dystrophic hamster ~tg Tyrosine release/mg noncollagen protein in 2 h Age Diaphragm Abdominal (days) Control Dystrophic Control 3 7 11 15

0.77 4- 0.24 (6) 1.04 4- 0.46

0.84 -t- 0.38 (10) 0.62 -q- 0.25

0.76 -t- 0.14 0.46 4- 0.32 (6) (6) 0.51 4- 0.13 0.63 4- 0.12

0.88 4(6) 0.89 4(6) 0.89 4(6) 0.53 i

(6)

(6)

(6)

(6)

(6)

0.17 0.09 0.14 0.02

Dystrophic 0.98 (10) 0.89 (6) 0.71 (6) 0.72

4- 0.20 4- 0.15 4- 0.27 4- 0.20

(6)

Incubations were conducted in Krebs-Ringer bicarbonate solution which contained 0.5 mM cycloheximide. Values given are means 4- SD; numbers in brackets indicate the number of animals.

Table 2. Tyrosine release by dystrophic hamster muscle in the absence of cycloheximide Age (days) 3 7 11 15 20-40

~tg Tyosine release/mg noncollagen protein in 2 h Diaphragm Abdominal Control Dystrophic Control Dystrophic 1.36 • 0.83 (6) 0.58 4- 0.29 (7) 0.39 :[: 0.09 (7) 0.43 4- 0.03 (5) 0.43 -b 0.14 (7)

0.74 4- 0.36 (10) 0.38 4- 0.17 (7) 0.59 -t- 0.43 (6) 0.85 4-4-0.58 (8) 0.60 4-4-0.17 (6)

0.84 -t- 0.54 (6) 0.44 4- 0.09 (6) 0.40 4- 0.12 (7) 0.45 4- 0.02 (5) 0.56 4- 0.25 (8)

0.97 (10) 0.51 (5) 0.48 (6) 0.91 (8) 0.62 (7)

-t- 0.75 4- 0.10 4- 0.33 -t- 0.76 4- 0.18

Conditions were the same as for table 1 except that eyeloheximide was exeluded.

Materials and methods. T h e h a m s t e r s used for t h e s e experim e n t s were UM-X7.1 a n d t h e c o n t r o l s were Syrian Golden H a m s t e r s ; b o t h sexes were used. Because of t h e y o u n g age of t h e a n i m a l s required, t h e y were all b r e d a n d raised in t h e a n i m a l q u a r t e r s of Q u e e n ' s U r d v e r s i t y . H a m s t e r s were sacrificed b y d e c a p i t a t i o n a n d t h e i r d i a p h r a g m s a n d a b d o m i n a l muscles w e r e r e m o v e d as q u i c k l y as possible. The r a t e of t y r o s i n e release f r o m t h e muscles was t h e n e s t i m a t e d b y t h e m e t h o d of Fulks, Li a n d G o l d b e r g 8 t2o p r o v i d e an i n d e x of proteolysis. Briefly t h e tissues were p r e i n c u b a t e d (37 ~ in 3 ml of K r e b s - R i n g e r b i c a r b o n a t e solution ( K R B ) for 30 rain a n d t h e n i n c u b a t e d in a f u r t h e r 3 m l of K R B for 2 h. A f t e r r e m o v i n g t h e tissues t h e t y r o s i n e c o n t e n t s of t h e i n c u b a t i o n m e d i a were d e t e r m i n e d b y t h e m e t h o d of W a a l k e s a n d Udenfriend4. This p r o c e d u r e involves r e a c t i n g t y r o s i n e w i t h 2 - n i t r o s o n a p h t h o l a n d m e a s u r i n g fluorescence i n t e n s i t y . P r o t e i n as noncollagen p r o t e i n was m e a s u r e d b y t h e m e t h o d of Lilienthal e t a l ) , using b o v i n e s e r u m a l b u m i n as t h e reference. Results and discussion. H o m b u r g e r et al. ~ h a v e r e p o r t e d t h a t t h e earliest d e t e c t a b l e m o r p h o l o g i c a l lesion in dyst r o p h i c h a m s t e r s occurs a t a b o u t 20 d a y s of age. Since we w i s h e d to learn w h e t h e r a l t e r a t i o n s in p r o t e i n degrad a t i o n c o n t r i b u t e d to s u c h o b s e r v a t i o n s it was n e c e s s a r y t o e x a m i n e a n i m a l s y o u n g e r t h a n 20 days. Table 1 s h o w s t h e r a t e s of t y r o s i n e release f r o m d i a p h r a g m s of a n i m a l s a s y o u n g as 3 days. T h e s e were t h e y o u n g e s t a n i m a l s w h i c h we could h a n d l e b e c a u s e of t h e i r small size. A t a n y of t h e ages s t u d i e d t h e r e was no difference b e t w e e n t h e d y s t r o p h i c a n d c o n t r o l h a m s t e r s . T h e s e d a t a are c o n s i s t e n t w i t h t h o s e of G o l d s p i n k a n d G o l d s p i n k 7 a t t h e y o u n g e s t age t h e y used (30 days). I n t h e p r e s e n t s t u d i e s e x p e r i m e n t s were c o n d u c t e d in t h e p r e s e n c e of c y c l o h e x i m i d e to i n h i b i t r e - i n c o r p o r a t i o n of t y r o s i n e a n d also in its absence. Several r e p o r t s in t h e last 2 y e a r s h a v e i n d i c a t e d t h a t t h e a c t i o n of c y c l o h e x i m i d e m a y n o t be simple b l o c k a d e of p r o t e i n s y n t h e s i s as h a d been f o r m e r l y a s s u m e d . F o r e x a m p l e , W o o d s i d e s f o u n d 1 Supported by the Muscular Dystrophy Association of Canada. 2 I.M. Weinstock, S. Epstein and A. T. Milhorat, Proc. Soc. Expl Biol. Med. 99, 272 (1958). 3 R.M. Fulks, J. B. Li and A. L. Goldberg, J. biol. Chem. 250, 290 (1975). 4 T. P. Waalkes and S. Udenfriend, J. Lab. elin. Med. 50, 733 (1957). 5 J . L . Lilienthal, Jr, K. L. Zierler, ]3. P. Folk, R. 13uka and M. J. Riley, J. biol. Chem. 182, 501 (1950). 6 F. Homburger, J. R. Baker, C. W. Nixon, G. Wilgram and J. Harrop, J. Path. 13act. 89, 133 (1965). 7 D. F. Goldspink and G. Goldspink, Bioehem. J. 162, 191 (1977). 8 K. H. Woodside, Biochim. biophys. Acta d21, 70 (1976).

15.3. 1978

Specialia

t h a t 18 p.M c y c l o h e x i m i d e r e d u c e d p r o t e i n s y n t h e s i s b y 9 3 % in p e r f u s e d r a t liver b u t t h i s c o n c e n t r a t i o n of d r u g also r e d u c e d p r o t e i n d e g r a d a t i o n b y 60~ Wildenthal a n d Griffen ~o b s e r v e d d e c r e a s e d c a t h e p s i n - D - a c t i v i t y d u e to c y c l o h e x i m i d e in c u l t u r e d f e t a l m o u s e h e a r t s . I t c a n be s e e n f r o m t a b l e 2 t h a t in t h e a b s e n c e of c y c l o h e x i m i d e t h e r e w a s still no d i f f e r e n c e b e t w e e n c o n t r o l a n d d y s t r o phic samples. I n a d d i t i o n t o d i a p h r a g m , we h a v e also s t u d i e d a b d o m i n a l m u s c l e b e c a u s e , like d i a p h r a g m , it is a flat, t h i n s h e e t a n d l e n d s itself t o d i f f u s i o n of s m a l l m o l e c u l e s i n t o a n d o u t of t h e t i s s u e . I t also h a d t h e a d v a n t a g e of b e i n g r e a d i l y rec o g n i z a b l e a n d r e m o v a b l e in t h e s e s m a l l a n i m a l s . T a b l e s 1 a n d 2 s h o w t h a t in t h i s m u s c l e also, t h e r a t e s of t y r o s i n e r e l e a s e were t h e s a m e in d y s t r o p h i c a n d c o n t r o l h a m s t e r s . T h e p r e s e n t d a t a are t e n t a t i v e l y i n t e r p r e t e d to m e a n t h a t i n c r e a s e d p r o t e o l y s i s m a y n o t be e s s e n t i a l to t h e d e v e l o p m e n t of t h e m u s c l e lesion b u t r a t h e r c o u l d be a r e s u l t .

319

T h i s i n t e r p r e t a t i o n is b a s e d on o u r o b s e r v a t i o n s t h a t m u s c l e s f r o m d y s t r o p h i c h a m s t e r s did n o t s h o w i n c r e a s e d t y r o s i n e release a t a g e s y o u n g e r t h a n t h o s e a t w h i c h t h e m o r p h o l o g i c a l lesion a p p e a r e d 6 . F u r t h e r m o r e G o l d s p i n k a n d Goldspink 7 d e m o n s t r a t e d increased tyrosine release a t 100 a n d 230 d a y s w h e n t h e d i s e a s e is well a d v a n c e d . I n m a k i n g a n i n t e r p r e t a t i o n it is well t o r e m e m b e r t h a t t h i s m e t h o d o n l y allows for e s t i m a t e s of o v e r a l l p r o t e i n d e g r a d a t i o n a n d d o e s n o t allow o n e to d e t e r m i n e t h e f a t e of a p a r t i c u l a r p r o t e i n w h i c h c o u l d be i n s t r u m e n t a l in t h e d e v e l o p m e n t of a lesion. W e also r e c o g n i z e t h a t it is n o t p o s s i b l e to r e l a t e d i r e c t l y t y r o s i n e release to a s s a y s of c a t h e p s i n a c t i v i t y b e c a u s e c a t h e p s i n a s s a y s are c o n d u c t e d u s i n g s y n t h e t i c or artificial s u b s t r a t e s .

9

K. Wildenthal and E. E. Griffen, Biochim. biophys. Aeta 44d, 519 (1976).

Studies on substrate specificity of X-prolyl dipeptidyl-aminopeptidase using new chromogenic substrates, X-Y-p-nitroanilides T. K a t o , T. N a g a t s u , T. K i m u r a a n d S. S a k a k i b a r a

Laboratory o/ Cell Physiology, Department o/ Li/e Chemistry, Graduate School at JVagatsuta, Tokyo Institute o/ Technology, Yokohama 227 (Japan); arm Peptide Institute, Protein Research Foundation, Minoh, Osaka 562 (Japan), 9 August 7977 Summary. S u b s t r a t e s p e c i f i c i t y of X - p r o l y l d i p e p t i d y l - a m i n o p e p t i d a s e ( d i p e p t i d y l a m i n o p e p t i d a s e IV) w a s e x a m i n e d by using newly synthesized 8 chromogenic substrates, X-Y-p-nitroanilides. Homogeneous enzyme from human subm a x i l l a r y g l a n d h y d r o l y z e d g l y c y l p r o l i n e p - n i t r o a n i l i d e a l m o s t specifically, e x c e p t a l a n y l a l a n i n e p - n i t r o a n i l i d e w h i c h h a d 11% a c t i v i t y . X - P r o l y l d i p e p t i d y l - a m i n o p e p t i d a s e 1 is a n e n z y m e w h i c h cleaves N-terminal X-proline from peptides. The enzyme w a s p u r i f i e d f r o m e i t h e r p o r c i n e k i d n e y 2 4 or h u m a n s u b m a x i l l a r y g l a n d ~. W e s y n t h e s i z e d s e v e r a l n e w c h r o m o genic s u b s t r a t e s , p - n i t r o a n i l i d e s of t h e d i p e p t i d e s , glycylproline, alanylproline, lysylproline, arginylproline, g l u t a m y l p r o l i n e , a n d a s p a r t y l p r o l i n e for X - p r o l y l dipeptidyl-aminopeptictase purified from h u m a n s u b m a x i i -

1 2

V.K. Hopsu-Havu and G. G. Glenner, Histochemie 3, 197 (1966). V. K. Hopsu-Havu, P. Rintola and G. G. Glenner, Aeta chem. scand. 22, 299 (1968). 3 A. Barth, H. Schulz and K. Neubert, Acta biol. med. germ. 32, 157 (1974). 4 A.J. Kenny, A. G. Booth, S. G. George, J. lngram, D. Kershaw, E. J. Wood and A. R. Young, Bioehem. J. 155, 169 (1976). 5 H. Oya, 1. Nagatsu and T. Nagatsu, Bioehim. biophys. Acta 258, 59t (1972).

Table 1. Analytical data for X-Y-p-nitroanilide 9 tosylates Compound

Gly-Pro-pNA 9TosOH

Melting point (decomp) ~

223 ~ 225

Gly-Leu-pNA 9TosOH 124 ~ 140

Optical rotation [~ D

Temperature (~

Molecular formula

Found calculated (%) C H

N

81.0 (C1 MeOH)

30

C2oHe407N4S

51.82 51.72

5.19 5.21

12.24 12.06

12.3 (C1 DMF)

25

C21H2sOrN4S 9 H20

50.73 50.59

5.90 6.06

11.01 11.24

Gly-Sar-pNA - TosOH

183 ~ 185

C18Hze.OTN4S

48.92 49.29

4.98 5.07

13.00 12.78

GIy-GIy-pNA. TosOH

220 ~ 222

C17H2oOTN4S 9 H20

45.92 46.15

4.95 5.01

13.01 12.66

Gly-Hyp-pNA. TosOH 142 ~ 146

36.3 (C1 DMF)

25

C2oH~4OsN4S - H~O

48.35

5.24

10.98

48.19

5.26

11.24

Gly-Ala-pNA. TosOH

239 ~ 241

-31.1 (el DMF)

25

C18H2aOTN4S 9 1/4H20

48.88 48.81

4.93 5.12

12.82 12.65

AIa-GIy-pNA. TosOH

244 ~ 247

16.3 (el DMF)

25

ClsH~2OTN4S 9 1/4H20

48.88 48.81

4..98 5.12

12.48 12.65

Ala-Ala-pNA 9TosOH

122 ~ 141

-11,1 (C1 DMF)

25

C~gH~4OTN4S 9 1/2H~O

49.07 49.45

5.21 5.46

11.95 12.14