166

Specialia

p e r c e n t a g e of I F e x c r e t e d i n t o t h e urine w i t h i n 30 m i n (after e m p t y i n g a n d w a s h i n g t h e b l a d d e r ) were m e a s u r e d a n d results are r e p o r t e d in t a b l e 1 as m e a n 4- S . E . T h e s t a t i s t i c a l significance of t h e difference was e v a l u a t e d according to Student's t-test. I t a p p e a r s t h a t I F , once desialylated, r e m a i n s in t h e c i r c u l a t i o n for a v e r y s h o r t t i m e a n d i n t a c t its half-life is a l m o s t h a l v e d . Moreover, i t is i n t e r e s t i n g to n o t e t h a t o n l y a negligible a m o u n t h a s b e e n e x c r e t e d w i t h t h e urine, s u g g e s t i n g t h a t t h e liver u p t a k e m a y h a v e increased. F o r t h i s r e a s o n t h e role of t h e liver h a s b e e n t e s t e d b y u s i n g t h e i s o l a t e d p e r f u s e d r a b b i t liver p r e p a r a t i o n 25. T h e v i a b i l i t y of t h e liver h a s b e e n g r e a t l y i m p r o v e d in c o m p a r i s o n to o u r p r e v i o u s s t u d y ~~ b y u s i n g a n efficient o x y g e n a t o r a n d fresh r a b b i t blood as p e r f u s i o n m e d i u m . As s h o w n in t a b l e 2, o n l y 2 3 - 3 9 % of n a t i v e I F d i s a p p e a r s in 15 m i n f r o m t h e r e c i r c u l a t i n g p e r f u s a t e while t h e loss of d e s i a l y l a t e d I F is u p t o 74-850/o . This s t r i k i n g dif-

Isoenzymes

of creatine phosphokinase

EXPERIENTIA 33]2

terence suggests t h a t t h e f a s t e r d i s a p p e a r a n c e of desialyla t e d I F f r o m t h e p e r f u s a t e is a t t r i b u t a b l e to t h e b i n d i n g of t h e a s i a l o i n t e r f e r o n to t h e liver. B o t h t h e r e s u l t s in v i v o a n d in v i t r o s t r o n g l y suggest t h a t t h e m e c h a n i s m of I F c a t a b o l i s m is e q u i v a l e n t to t h a t r e p o r t e d for s e v e r a l c i r c u l a t i n g g l y c o p r o t e i n s 2s. D e s i a l y l a t i o n o f I F is t h e p r e l i m i n a r y essential s t e p 27 a n d m i g h t occur in m i c r o e n v i r o n m e n t s w i t h sluggish c i r c u l a t i o n or m o r e likely b y m e m b r a n e - b o u n d sialidase while I F is a t t a c h e d to t h e cell m e m b r a n e ~8. 25 L.L. Miller, C. G. Bly, M. L. Watson and W. F. Bale, J. exp. Med. 9 9d, 431 (1951). 26 A. G. Morell, G. Gregoriadis, I. H. Seheinberg, J. Hickman and G. Ashwell, J. biol. Chem. 246, 1461 (1971). 27 V. Bocci, Experientia.32, 135 (1976). 28 This work was supported by a grant from the Consiglio Nazionale delie Ricerehe, Roma (subproject on antiviral therapy), contract No. 76.00646.84).

in white blood cells*

H. W. Cho, L. P r i t i p k o 1, H. ~1/-.Meltzer a n d C. Keller

Department o[ Psychiatry, University o/ Chicago, 950 East 59th Street, Chicago (Illinois 60637, USA), and Department o[ Biophysics, Institute o/ Psychiatry, Academy o/ Medical Sciences, Zagorodnoy Shossee 2, Moshwa (USSR), 10 March 7976 Summary. C r e a t i n e p h o s p h o k i n a s e a c t i v i t y was f o u n d in h u m a n l y m p h o c y t e s . O n l y MM t y p e of t h e e n z y m e was d e t e c t e d in l y m p h o c y t e s a n d p o l y m o r p h o n u c l e a r leukocytes. C r e a t i n e p h o s p h o k i n a s e (CPK) p l a y s a n i m p o r t a n t p h y s iological role in t h e e n e r g y m e t a b o l i s m of s k e l e t a l a n d cardiac muscle, a n d n e r v e cells. I t c a t a l y z e s t h e r e a c t i o n : c r e a t i n e p h o s p h a t e + A D P = A T P + c r e a t i n e 2. C P K exists in m u l t i p l e !orms. I t is a dimer, c o m p o s e d of 2 t y p e s of s u b u n i t s , d e s i g n a t e d M a n d B. These c o m b i n e to produce t h r e e isozymes, d e s i g n a t e d MI~, M B a n d B B 3. MM t y p e is m a i n l y f o u n d in s k e l e t a l muscle, B B t y p e p r e d o m i n a n t l y in t h e n e r v o u s s y s t e m a n d M B t y p e in c a r d i a c muscle 3. Similarities b e t w e e n c o n t r a c t i l e muscle a n d m o b i l e cells are w i d e l y recognized. H u x l e y 4 h a s recently pointed out that actin- and myosin-like proteins are p r e s e n t in m o t i l e n o n - m u s c u l a r ceils, s u c h as a m o e b a , a c a n t h a m o e b a castellanii, a n d blood platele'Ls. Traniello e t al.~ h a s e x a m i n e d h u m a n p o l y m o r p h o n u c l e a r leukoc y t e s (PMNL) a n d f o u n d t h a t t h e s e cells c o n t a i n cytop l a s m i c C P K . H o w e v e r , n o m e n t i o n was m a d e as t o t h e isozyme of C P K p r e s e n t in t h e leukocytes. M e l t z e r a n d G u s c h w a n h a v e p r e v i o u s l y r e p o r t e d B B - t y p e C P K in t h e p l a t e l e t s of r a t s a n d r a b b i t s , a l t h o u g h C P K a c t i v i t y was u n d e t e c t a b l e in h u m a n p l a t e l e t s 8. W e t h e r e f o r e i n v e s t i g a t e d w h i c h t y p e of C P K was p r e s e n t in l y m p h o c y t e s a n d leukocytes: W e w i s h t o r e p o r t t h a t C P K a c t i v i t y is also p r e s e n t in h u m a n l y m p h o c y t e s a n d t h a t o n l y M M t y p e of C P K is p r e s e n t in l y m p h o c y t e s a n d leukocytes. Materials and methods. L e u k o c y t e s a n d l y m p h o c y t e s were i s o l a t e d b y a m o d i f i c a t i o n of t h e m e t h o d s d e s c r i b e d b y M e n d e l s o h n et al. 7 a n d t 3 o y u m s. 20 m l of v e n o u s b l o o d were d r a w n f r o m e a c h of 4 n o r m a l s u b j e c t s in h e p a r i n i z e d n o n sterile V a c u t a i n e r s . W i t h t h e V a c u t a i n e r s o r i e n t e d a t a 45 ~ angle, b l o o d s a m p l e s were allowed t o s t a n d for 1 h a t 37 ~ T h e r e s u l t i n g u p p e r l a y e r of l e u k o c y t e - r i c h p l a s m a was collected a n d d i l u t e d to twice v o l u m e w i t h Ca-Mg-free (CMF) T y r o d e ' s solution, p H 7.4. 20 ml, a t m o s t , of t h i s

d i l u t e d p l a s m a were l a y e r e d over 9 m l of t h e following s o l u t i o n in a 50 m l conical p l a s t i c c e n t r i f u g e t u b e (Falcon): 1.8 m l of 50% s o d i u m d i a t r i a z o a t e ( H y p a q u e , "Winthrop Labs, N e w York, New York), 6.35 m l of 9 % a q u e o u s Ficoll (molecular weight, a p p r o x i m a t e l y 400,000, S i g m a C h e m i c a l Co., St. Louis, Mo.) a n d 0.85 m l of water. C e n t r i f u g a t i o n was p e r f o r m e d a t 4~ a t 885 g for 15 rain. T h e u p p e r m o s t p l a s m a l a y e r was r e m o v e d w i t h a P a s t e u r p i p e t t e . T h e w h i t e fluffy l y m p h o c y t e - r i c h i n t e r p h a s e l a y e r originally b e t w e e n t h e p l a s m a l a y e r a n d t h e Ficoll s o l u t i o n was t h e n collected b y a s p i r a t i o n in 15 m l conical, plastic c e n t r i f u g e tubes. N e x t , t h e r e m a i n i n g Ficoll layer was c a r e f u l l y r e m o v e d , l e a v i n g a l e u k o c y t e - r i c h Iayer a t t h e b o t t o m of t h e t u b e . T h i s l e u k o c y t e - r i c h l a y e r was r e s u s p e n d e d in 2 m l of C M F a n d collected b y a s p i r a t i o n . Each lymphocyte-rich fraction and each leukocyte-rich f r a c t i o n were w a s h e d t w i c e b y r e s u s p e n s i o n in 10 m l C M F a n d c e n t r i f u g a t i o n a t 110 • for 10 m i n in o r d e r to r e m o v e platelets, p l a s m a a n d Ficoll. A f t e r w a s h i n g , all l y m p h o c y t e - r i c h f r a c t i o n s were c o m b i n e d in one 15 m l

* Supported by U,S.P.H.S. MH 16 127. H.Y.M. is recipient of RCSA MH 47 808. 1 Department of Biophysics, Institute of Psychiatry, Moscow (USSR). 2 O. Meyerhoff and K. Lohmann, Biochem. Z. 253, 431 (1932). 3 H.M. Eppenberg, D. M. Dawson and N. O. Kaplan, J. biol. Chem. 242, 204 (1967). 4 H.E. Huxley, Nature, London 234, 445 (1973). 5 S. Traniello, M. G. D'Aloy and E. Grazi, Experientia 31, 278 (1975). 6 H.Y. Meltzer and A. Guschwan, Life Set. ll, 121 (1972). 7 J. Mendelsohn, A, Skinner and S. Kornfield, J. Clin. Invest. 50, 818 (1971). 8 A. Boyum, Seand. J. Clin. Lab. Invest. Suppl. 97, 77 (1968).

15. 2. 1977

Specialia

Activity of CPK in the extracts of human lymphocytes, polymorphonuclear leukoeytes, human skeletal muscle and brain CPK activity (mU/mg protein) Hmnan Human Human }{uman

lymphocytes PMN nmscle brain

11.3 11.4 68,000 6,100

CPK activity* (mU/108cells) 26 112 -

*CPK activity was estimated assuming 100% lysis after sonication.

conical plastic c e n t r i f u g e t u b e a n d all l e u k o c y t e - r i c h fractions in a n o t h e r . The t o t a l cell c o n t e n t a n d p u r i t y of e a c h s u s p e n s i o n were d e t e r m i n e d b y cell c o u n t s in a h a e m o c y t o m e t e r . Only cell s u s p e n s i o n of p u r i t y > 94% were a c c e p t e d for f u r t h e r s t u d y . The average p u r i t y of h a r v e s t e d cell s u s p e n s i o n s was 96%. T h e 2 s u s p e n s i o n s were c e n t r i f u g e d a t 250 • g for 10 rain a n d t h e CMF was r e m o v e d . Cells were t h e n r e s u s p e n d e d in 0.5 0.7 ml of cold G e l m a n H i g h R e s o l u t i o n Buffer ( i r i s - b a r b i t a l - s o d i u m barbital, pt-I 8.8) c o n t a i n i n g 4 mM m e r c a p t o e t h a n o l , a n d lysed b y s o n i c a t i o n for 20 sec w i t h a K o n t e s cell d i s r u p t e r . Cell debris w a s r e m o v e d b y cent r i f u g a t i o n a t 7700 • a n d 4~ for 10 rain. T o t a l C P K a c t i v i t y was m e a s u r e d b y t h e m e t h o d of Rosalki" w i t h r e a g e n t s o b t a i n e d f r o m Calbiochem. P r o t e i n c o n c e n t r a tion of t h e e x t r a c t s was a s s a y e d a c c o r d i n g to t h e m e t h o d of L o w r y et aI. *~. H u m a n b r a i n a n d skeletal muscle f r o m a u t o p s y were h o m o g e n i z e d (1:10) in cold isotonic saline solution b y m e a n s of T r i - R teflon tissue homogenizer. The h o m o g e n a t c was c e n t r i f u g e d a t 30,000 x g for 30 min to o b t a i n s u p e r n a t a n t . T h r e e 1 bd aliquots of the e x t r a c t s of w h i t e blood cells a n d t h e d i l u t e d tissue e x t r a c t s were e l e c t r o p h o r e s e d on agarose gels (Pfizer Pol-E-film) for

Electrophoretic mobilities of creatine phosphokinase in the extracts el polymorphonuelear Ieukocytes and lymphocytes. A A mixture of extracts of brain and cardiac muscle of humans. B Extracts of human lymphoeytes. C Extracts of human polymorphonuclear leukocytes.

167

30 min. T h e n 1 m l of C P K r e a g e n t (E.I. du P o n t de N e m o u r s & Co., I n s t r u m e n t P r o d u c t s Division, Wilm i n g t o n , Delaware) was s p r e a d o v e r t h e gel, followed b y i n c u b a t i o n of t h e gel a t 37~ for 30 rain. T h e b a n d s of i s o e n z y m e s of C P K were d e t e c t e d u n d e r U V light. Results and discussion. As s h o w n in t h e figure o n l y a single b a n d was p r e s e n t in t h e e x t r a c t s of b o t h polymorphonuclear leukocytes and lymphocytes. This band is l o c a t e d a t t h e s a m e p o s i t i o n as M M - t y p e C P K . Some non-specific r e a c t i o n p r o b a b l y occurred at t h e origin. The l y m p h o c y t e a n d l e u k o c y t e b a n d also p r o b a b l y includes s o m e non-specific reaction. H o w e v e r , since ' C P K a c t i v i t y ' in t h e a b s e n c e of s u b s t r a t e b u t w i t h t h e r e s t of t h e Rosalki r e a g e n t s was less t h a n 3% of t h e C P K a c t i v i t y of l y m p h o c y t e s a n d l e u k o c y t e s w h e n m e a s u r e d s p e c t r e p h o t o m e t r i c a l l y b y t h e c o m p l e t e Rosalki m e t h o d a n d since n e i t h e r B B b a n d or MB b a n d was d e t e c t e d a f t e r electrophoresis, we conclude t h a t t h e C P K of l y m p h o c y t e s a n d l e u k o c y t e s is MM. Q u a n t i t a t i v e e s t i m a t i o n of CPIK a c t i v i t y in h u m a n l y m p h o c y t e s a n d p o l y m o r p h o n u c l e a r l e u k o c y t e s in c o m p a r i son w i t h t h a t of h u m a n b r a i n a n d skeletal muscle was carried out. T h e C P K a c t i v i t y per 108 cells was 4.3Iold g r e a t e r in l e u k o c y t e s t h a n in l y m p h o c y t e s (table). This is probably related to the fact t h a t the relative cytoplasmic space a n d t h e a v e r a g e size of l e u k o c y t e s are g r e a t e r t h a n t h o s e of l y m p h o c y t e s ~ L T h e C P K activities per m g p r o t e i n of t h e c y t o p l a s m of l e u k o c y t e s a n d l y m p h o c y t e s were c o m p a r a b l e . T h e C P K a c t i v i t y of b o t h of t h e s e cell t y p e s were e x t r e m e l y low c o m p a r e d w i t h h u m a n b r a i n a n d muscle (table). This does n o t necessarily rule o u t t h a t C P K has s i g n i f i c a n t f u n c t i o n in t h e e n e r g y m e t a b o l i s m of l e u k o c y t e s a n d l y m p h o c y t e s . C o n t r a c t i o n of skeletal muscle involves c h a n g e s in its l e n g t h of 20% or m o r e in 50 milliseconds is. C o n d u c t i o n v e l o c i t y of m y e l i n a t e d n e r v e fibre ranges b e t w e e n 5 a n d 120 m e t e r p e r second ~a In c o m p a r i s o n with t h e s e rapid p h y s i c a l a n d electrochemical activities of muscle and n e r v e fibres, w h i c h require r a p i d r e p l e n i s h m e n t of A T P # o r e c r e a t i n e p h o s p h a t e , l e u k o c y t e s a n d l y m p h o c y t e s m o v e b y t h e process of chem o t a x i s t h r o u g h tissue spaces only 3 t i m e s t h e i r own length, each m i n u t e . F u r t h e r m o r e , t h e m o v e m e n t of t h e leukocytes to t h e t a r g e t site largely d e p e n d s on t h e c i r c u l a t i o n of b l o o d r a t h e r t h a n t h e i r own locomotion. Thus, t h e s e cells do n o t require a large pool of reserve e n e r g y for m o v e m e n t a n d t h e low C P K a c t i v i t y in t h e c y t o p l a s m of l e u k o c y t e s m a y be a d e q u a t e for t h e lowe n e r g y r e q u i r e m e n t s of t h e s e cells.

9 S. 13. Rosalki, J. l,ab. Clin. Med. 69, 696 (1967). 10 O.H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. biol. Chem. 193, 265 (1951). 11 J . H . Ferguson, in: Blood and Body Functions, p. 246. F. A. Davis Company 1965. 12 A.C. Guyton, in : Textbook of Medical Physiology, p. 85. W. B. Saunders Company 1971. 13 R.G. Clark, in: Clinical Neuroanaton3y and Neurophysiology, p. 12. F. A. Davis Company 1975.

Isoenzymes of creatine phosphokinase in white blood cells.

166 Specialia p e r c e n t a g e of I F e x c r e t e d i n t o t h e urine w i t h i n 30 m i n (after e m p t y i n g a n d w a s h i n g t h e b...
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