278

Specialia

Such a n a l t e r a t i o n could lead to a pellagra-like s y n d r o m e ~4, b e h a v i o r a l d e p r e s s i o n 25, a n d c h a n g e s in sleep p a t t e r n s 26 in t h e i n f e c t e d host. Moreover, s o m e of t h e p a r a s i t e m e t a b o l i t e s , such as t r y p t o p h o l , m a y h a v e t o x i c or soporific effects of t h e i r own2~-29.

Rdsumd. L a t r a n s f o r m a t i o n du t r y p t o p h a n e en t r y p t o p h o l chez le t r y p a n o s o m e consiste en une t r a n s a m i n a t i o n suivie p a r une d 6 c a r b o x y l a t i o n ; une r 6 d u c t i o n s u b s 6 q u e n t e est p r o b a b l e m a i s n ' a p a s 6t6 prouv6e. L a t r a n s a m i n a t i o n

~ L. V. HANKES, J. ]~. LEKLEM, R. R. BROWN and R. C. P. M. MEKEL, Am. J. clin. Nutr. 2d, 730 (1971). 25 D. P. RosE, J. elin. Path. 25, 17 (1972). 26 M. JOUVET, Science 763, 32 (1969). ~7 A. FELDSTIZIN,F. H. CHANGand J. M. KUCHARSKI,Life Sci. 9, 323

(1970). 2s p. j. MO~tGANEand W. C. STERN, Brain Res. 50, 205 (1973).

EXPERIENTIA31/3

r e q u i e r t l'c~-ketoglutarate; la c o n v e r s i o n d u t r y p t o p h a n e en i n d o l e - l a c t a t e r e q u i e r t le N A D H . L ' i n d o l e - a c 6 t a t e est u n a u t r e p r o d u i t du m 6 t a b o l i s m e du t r y p t o p h a n e . H. H. STIBBS a0 a n d J. R. SEED

Laboratory o/ Parasitology, Department o/Biology, Tulane University, New Orleans (Louisiana 70178, USA), 77 August 1974.

.~9 Supported by research grants from the Research Corporation Brown-Hazen Fund and by NIH Research Grant No. 1-R01-NS 11393-01 from the National Institute of Neurological Diseases and Stroke. 80 Present address: Department of Tropical Public Health, Harvard School of Public Health, 665 HuntingtonAve., Boston, Mass. 02115, USA.

Creatine Phosphokinase Activity in Human Polymorphonuclear Leukocytes I t is generally a c c e p t e d t h a t t h e r e are m a n y similarities b e t w e e n t h e m e c h a n i s m of m u s c u l a r c o n t r a c t i o n a n d cell m o t i l i t y . I-IUXLEY 1 has r e c e n t l y p o i n t e d o u t t h a t t h e m o l e c u l a r s t r u c t u r e s a n d m e c h a n i s m s for cell m o v e m e n t r e s e m b l e t h o s e of muscle fibre c o n t r a c t i o n . This v i e w is f u r t h e r s u b s t a n t i a t e d b y our p r e s e n t f i n d i n g t h a t h u m a n 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 possess a c y t o p l a s m i c c r e a t i n e p h o s p h o k i n a s e , a n e n z y m e t h a t is essential for t h e n o r m a l c o n t r a c t i o n of t h e m u s c u l a r fibre. L e u k o c y t e s , h a v i n g c o n t r a c t i l e s t r u c t u r e s ~, could also be a convenient means to investigate hereditary muscular diseases, as for i n s t a n c e D u c h e n n e m u s c u l a r d y s t r o p h y . This disease, one of t h e m o s t severe h u m a n m y o p a t h i e s , shows sex i n h e r i t a n c e c h a r a c t e r i s t i c s 3 a n d is generally r e g a r d e d as a p r i m a r y m y o p a t h i c process 4, a l t h o u g h some authors have postulated a primary neuronal abnormalityS,% W e h a v e n o w s t u d i e d t h e p r o p e r t i e s of l e u k o c y t e c r e a t i n e p h o s p h o k i n a s e isolated f r o m b o t h n o r n l a l a n d d y s t r o p h i c s u b j e c t s : no s i g n i f i c a n t differences h a v e b e e n found, h o w e v e r , b e t w e e n t h e e n z y m e s f r o m t h e t w o sources Table 1. Specific activity of polymorphonuclear leukocyte-creatine phosphokinase from different subjects Spec. activity (Units/mg protein) Healthy volunteers

1 2 3 4

0.55 0.62 0.57 0.50

Dystrophic

L.C. T.C. R.R. P.G.

0.57 0.61 0.50 0.60

L. and T.C. R.R. P.G.

0.64 0.54 0.66

Mothers (carriers)

The specific activity of creatine phosphokinase from crude extracts of leukocyfes from healthy volunteers, 4 patients (two brothers) affected by Duehenne muscular distrophy, and their mothers, w a s determined as described under material and methods.

Materials and methods. V e n o u s b l o o d s a m p l e s of 4 n o r m a l , 4 d y s t r o p h i c s u b j e c t s a n d 3 h e a l t h y carriers of t h e D u c h e n n e d y s t r o p h y ( r e p r e s e n t e d b y t h e m o t h e r s of t h e 4 p a t i e n t s ) were used. The clinical diagnosis of D u c h e n n e m y o p a t h y for t h e 4 p a t i e n t s (R.R., T.C., L.C., a n d P.G., w h i c h were r e s p e c t i v e l y 7, 9, 10 a n d 14 y e a r s old) was c o n f i r m e d also b y t h e result of t h e e l e c t r o m y o g r a p h i c analysis. 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 were o b t a i n e d f r o m freshly d r a w n , h e p a r i n i z e d blood. S e p a r a t i o n of granuloc y t e s f r o m red ceils a n d l y m p h o c y t e s w a s carried o u t b y s e d i m e n t a t i o n ill gelatin followed b y 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 c c o r d i n g to t h e m e t h o d of YAM et al.~: 20 m l of b l o o d y i e l d e d a p p r o x i m a t e l y 11-12 • 10 ~ of 9 0 - 9 5 % p u r e p o l y m o r p h o n u c l e a r leukocytes, t h a t were s t o r e d a t --20~ u n t i l h a n d l i n g . The lysis of l e u k o c y t e s was o b t a i n e d b y freezing a t - - 4 0 ~ a n d t h a w i n g a t 37~ 3 t i m e s in tile p r e s e n c e of 1 m l of saline solution 7. Tile s u s p e n s i o n was c e n t r i f u g e d a t 20,000 • for 20 rain a n d to t h e clear s u p e r n a t a n t , c o n t a i n i n g c r e a t i n e p h o s p h o kinase a c t i v i t y , 4 m g / m l of a l b u m i n t o increase t h e p r o t e i n c o n c e n t r a t i o n were added. P r o t e i n was t h e n p r e c i p i t a t e d b y a d d i t i o n of 326 m g of a m m o n i u m sulfate p e r m l of solution. T h e p r e c i p i t a t e , collected b y c e n t r i f u g a t i o n , was dissolved in 0.5 m l of 0.05 M tris a c e t a t e c o n t a i n i n g 1 m M E D T A , p H 7.2. Tile a m m o n i u m sulfate f r a c t i o n w a s a l m o s t free of m y o k i n a s e , w h i c h i n t e r f e r e d in t h e d e t e r m i n a t i o n of 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 . Creatine p h o s p h o k i n a s e was a s s a y e d s p e c t r o p h o t o m e t r i c a l l y ac-

1 H. E. HUXLEY, Nature, Lond. 23d, 445 (1973). 2 N. K. WESSELLS, B. SPOONER, J. F. ASH, M. O. BRADLEY, M. A. LUDUENA, E. L. TAYLOR, J. T. WRENN and K. M. YAMADA,

Science 777, 135 (1971). 3 t?. E. STEPHENS and F. H. TYLER, Am. J. human Genet. 3, 111 (1951). 4 H. SUGITAand F. H. TYLER, Trans. Ass. Am. Physicians 76, 231 (1963). 5 A. J. McCOMASand R. E. P. SIGA, Lancet, 1, 1119 (1970). GA. J. McCoMAs, R. E. P. SICAand S. CURRIE, Nature, Lond. 226, 1263 (1970). T. L. YAM,G. L. CASTOLDIand W. J. MITUS,J. Lab. din. Med. 70, 699 (1967).

15.3. 1975

279

Speeialia

/

4

! V 3

2

1

o12 d4

o

13.6

C).8

1

1.0

-3

CreatineI~hosphatex 10

Fig. 1. Reciprocal plot of initial velocity against creatine phosphate concentration at a series of different pH values. The samples (1 hal) contained 2.5 mUnits of creatine phosphokinase (ammonium sulfate fraction) from normal subjects, ereatine phosphate as indicated and 0.08 M glycylglycine buffer pH 6.0, (0); pH 7.0, (9 pH 7.35, (• and pH 8.0 (A). Other conditions were as reported under methods. Initial velocity is expressed as txmoles of NADPH formed per min.

2O

Km(mM)

V

15

6

I0

5!

o'6

0 pH

pH

Fig. 2. Effect of pH on the Km and V of creatine phosphokinasefrom normal subjects. Data were taken from Figure 1. Maximal velocity is expressed as mnoles of NADPH formed per min.

100 ~----.// %

c o r d i n g t o t h e p r o c e d u r e of NIELSEN a n d LUDVIGSEN s, w h i c h m e a s u r e d t h e f o r m a t i o n of A T P in t h e f o r w a r d r e a c t i o n b y following t h e r e d u c t i o n of N A D P , a t 340 nm. The r e a c t i o n m i x t u r e (1 ml) c o n t a i n e d 0.08 M glycylglycine buffer, p H 7.4 ; 0.1 m M E D T A ; 10 m M m a g n e s i u m a c e t a t e ; 2.5 m M A D P ; 5 m M glucose; 0.2 m M N A D P ; 2 ~g e a c h of glucose 6 - p h o s p h a t e d e h y d r o g e n a s e a n d hexokinase and 5 m M creatine phosphate. T h e i n t e r f e r e n c e of m y o k i n a s e was e l i m i n a t e d b y s u b s t r a c t i n g t h e a m o u n t of A T P p r o d u c e d in c o n t r o l samples, w i t h o u t c r e a t i n e p h o s p h a t e , f r o m t h a t p r o d u c e d in t h e c o m p l e t e a s s a y s y s t e m . 1 u n i t of t h e e n z y m e w a s d e f i n e d as t h e a m o u n t t h a t will p r o d u c e 1 ~mole of A T P p e r rain u n d e r t h e a b o v e c o n d i t i o n s . P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d b y t h e m e t h o d of LOWRY et al. 9. Results and discussion. Crude e x t r a c t s of c r e a t i n e ' phosphokinase from normal dystrophic subjects and h e a l t h y carriers of t h e D u c h e n e d y s t r o p h i c ( r e p r e s e n t e d b y t h e m o t h e r s of t h e 4 p a t i e n t s ) , s h o w e d a n i d e n t i c a l specific a c t i v i t y (Table I). A c o m p a r a t i v e k i n e t i c s t u d y of c r e a t i n e p h o s p h o k i n a s e obtained from the normal and the dystrophic subjects was carried out on t h e a m m o n i u m sulfate f r a c t i o n of t h e e n z y m e . The results w i t h t h e n o r m a l s u b j e c t s are s h o w n in F i g u r e s 1 and 2. p H o p t i m u m was a t p H 7.2 (Figure 2B). K m i n c r e a s e d f r o m 6.2 m M a t p H 6.0 to 20 m M a t p H 6.8 a n d d e c r e a s e d again to 6.5 m M a t p H 8.0 (Figure 2 B). Similar values of t h e K m were f o u n d for t h e e n z y m e f r o m d y s t r o p h i c subjects. This is s h o w n in T a b l e II. U n l i k e c r e a t i n e p h o s p h o k i l i a s e f r o m muscle, l e u k o c y t e creatine phosphokinase from both normal and dystrophic s u b j e c t s e x h i b i t e d a v e r y n a r r o w p i t r a n g e of s t a b i l i t y , f r o m p H 6.8 t o 7.3, w h e n s t o r e d a t 0 ~ Alkaline m e d i u m i n a c t i v a t e d v e r y r a p i d l y t h e e n z y m e f r o m leukocytes, while muscle kinase was c o m p l e t e l y stable 1~ L o w c o n c e n t r a t i o n s of E D T A were r e q u i r e d t o a v o i d f o r t u i t o u s loss in a c t i v i t y d u r i n g storage. L e u k o c y t e c r e a t i n e phosphokinases from normal and dystrophic subjects were also e q u a l l y s e n s i t i v e t o h e a t d e n a t u r a t i o n a t p H 7.0 (Figure 3). E x p o s u r e for 10 m i n to t e m p e r a t u r e up to 47 ~ d e c r e a s e d only s l i g h t l y t h e c a t a l y t i c a c t i v i t y (20%); e x p o s u r e to h i g h e r t e m p e r a t u r e s was on t h e c o n t r a r y m u c h m o r e effective, a n d c o m p l e t e i n a c t i v a t i o n was o b t a i n e d a t 60 ~ To our k n o w l e d g e this is t h e first r e p o r t on t h e presence of c r e a t i n e p h o s p h o k i n a s e in m o t i l e ceils. This

8 L. NIELSEN and B. LUDVlGSEN,J. Lab. clin. Med. 62, 159 (1963). O. H. LowRY, N. J. ROSEBROUGH,A. L. FARRand R. J. RANDALL, J. biol. Chem. 793, 265 (1951). i0 S. A. KUBY,L. NODAand H. LARDY,J. biol. Chem. 210, 65 (1954).

o

>

50

TaMe If. Michaelis constant, for creatine phosphate, of leukocyte creatine phosphokinase from normal and dystrophic subjects

f2r

//

,/

Km for creatine phosphate

i

3'0

4o

50

60 ~

Temperature Fig. 3. Heat denaturation of leukocyte ereatine phosphokinase from normal and dystrophic subjects. The samples (0.5 ml) contained 0.036 Unit of creatine phosphokinase (ammonium sulfate fraction) from the 2 source, dissolved in 0.05 M tris-acetate, containing i mM EDTA, pH 7.0. Protein concentration was 1 mg/ml. After heating for 10 rain at the temperatures indicated, the samples were immediately chilled, and assayed for the catalytic activity as described under methods. Normal (9 and dystropie (O) subjects.

pH

Normal

Dystrophic

6.0

6.3 mM 18.0 mM 10.0 mM 6.6 mM

6.0 mM 17.0 mM 11.0 mM 6.5 mM

7.0 7.35 8.0

Experimental conditions were as described ill Figure 1.

280

Specialia

observation further emphasizes the functionaU and s t r u c t u r a l 2 s i m i l a r i t i e s b e t w e e n m o t i l e a n d m u s c u l a r cells. B e c a u s e of t h e s e analogies, cells w i t h m o t i l e a c t i v i t y could b e a s u i t a b l e s y s t e m t o i n v e s t i g a t e a b n o r m a l i t i e s a f f e c t i n g t h e m u s c u l a r fibre, s u c h as D u c h e n n e m u s c u l a r dystrophy. I n o u r i n v e s t i g a t i o n s , however, no differences b e t w e e n c r e a t i n e p h o s p h o k i n a s e f r o m l e u k o c y t e s of n o r m a l a n d d y s t r o p h i c s u b j e c t s h a s been found. T h e specific a c t i v i t y of t h e e n z y m e , t h e K m a t d i f f e r e n t p H , t h e p H o p t i m u m , a n d t h e r e s i s t a n c e t o h e a t d e n a t u r a t i o n were s i m i l a r in b o t h cases, t h u s i n d i c a t i n g t h a t d y s t r o p h i c l e u k o c y t e s

ll J . C . DR]EYFUS, G. SCttAPIRA and F. SCHAPIRA, Ann. N.Y. Acad.

Sci. 75, 235 (1958). 13 j. M. S. P]EARCE, R. J. PEXNINGTOXand J. N. WOLTON, J. Neur. Neurosurg. Psycbiat. 27, 96 (1964). is j. C. DREYFUS and G. SCHAPIRA, Biochemistry o/ Hereditary Myopathies (Charles Thomas, Springfield, Ili. 1962). 14 R. PALMIERI, H. KENT]EL, H. JAGOBS, K. OKAB]E, R. YulE, F. ZIT]ER, F. TYL]ER and S. A. Ku~Y, Fedn. Proc. Abstr. 30, 1255 (1971). 15 This work was supported by the grant No. CT72.00768.04 of the Italian Consiglio Nazionale delle Ricerehe.

EXPERI]ENTIA31[3

c o n t a i n n o r m a l a m o u n t s of n o r m a l c r e a t i n e p h o s p h o k i n a s e molecules. T h i s last p o i n t seems t o e x c l u d e t h e l e a k i n g of c r e a t i n e p h o s p h o k i n a s e f r o m t h e leukocytes, a t v a r i a n c e w i t h t h e d y s t r o p h i c muscle n - ~ . T h u s t h e p o s t u l a t e d a l t e r a t i o n of m e m b r a n e p e r m e a b i l i t y to c r e a t i n e p h o s p h o k i n a s e seems to b e r e s t r i c t e d to t h e d y s t r o p h i c m u s c u l a r fibre a n d n o t e x t e n d e d t o t h e leukocyte. Similarly, t h e r e c e n t l y r e p o r t e d s t r u c t u r a l a l t e r a t i o n of t h e c r e a t i n e molecule i n h u m a n d y s t r o p h y 14 seems t o b e r e s t r i c t e d t o t h e m u s c l e fibre. M u s c u l a r a n d l e u k o c y t e p h o s p h o k i n a s e s m u s t t h e r e f o r e b e u n d e r i n d e p e n d e n t g e n e t i c control~5. Riassunto. Nei leucociti p o l i m o r f o n u c l e a t i u m a n i , cellule e a p a c i di m o v i m e n t i d u r a n t e la fagocitosi, g s t a t a o s s e r v a t a la p r e s e n z a d e l l ' e n z i m a c r e a t i n a fosfocinasi. Q u e s t a g u n ' a l t r a a n a l o g i a t r a cellule f o r n i t e di m o v J m e n t i e fibre muscolari. I1 c o n f r o n t o di a l c u n e c a r a t t e r i s t i c h e d e l l ' e n z i m a isolato d a leucociti p r o v e n i e n t i d a s o g g e t t i sani e distrofici n o n r i v e l a a l c u n a differenza s i g n i f i c a t i v a . SERENA TRANIELLO, MARIA G. D'ALoYA a n d E. GRAZI

Istituto di Chimica Biologica dell' Universit& Via Fossato di Mortara 25, 1-44700 Ferrara (Italy), 23 October 797d.

C o p p e r a n d Zinc L e v e l s in the B l o o d S e r u m and U r i n e of B i l h a r z i a l H e p a t i c F i b r o s i s I n o u r p r e v i o u s w o r k o n bilharziasis, t h e - u r i n a r y p o r p h y r i n s ; u r o a n d c o p r o p o r p h y r i n s 1, as well as t h e u r i n a r y a m i n o acids 2, were f o u n d t o b e e x c r e t e d in large a m o u n t , e x p e c i a l l y in a d v a n c e d cases of h e p a t o splenom e g a l y p a t i e n t s . Moreover, it a p p e a r s f r o m t h e w o r k of WATSON a n d SCHWARTZ 3 t h a t t h e u r i n a r y as well as feacal p o r p h y r i n s nro- a n d c o p r o p o r p h y r i n s are e x c r e t e d as zinc complexes. W h i l e w o r k i n g o n rats, VAN CAMPEN et al. 4 f o u n d t h a t t h e r e is a n i n t e r r e l a t i o n s h i p b e t w e e n zinc c o n c e n t r a t i o n a n d c o p p e r a b s o r p t i o n . Therefore, i t a p p e a r s of i n t e r e s t t o s t u d y t h e s e r u m a n d u r i n a r y level of b o t h zinc a n d c o p p e r in b i l h a r z i a l h e p a t i c fibrosis, a n d t o c o m p a r e w i t h t h e results o b t a i n e d f r o m n o r m a l healthy individuals. Materials and methods. 25 p a t i e n t s , 17 a d u l t m a l e s a n d 8 a d u l t females, in d i f f e r e n t stages of b i l h a r z i a l h e p a t i c fibrosis were selected for t h i s s t u d y . T h e s e cases were s u b j e c t e d t o t h o r o u g h clinical e x a m i n a t i o n , l i v e r f u n c t i o n tests, t o t a l p r o t e i n as well as d i f f e r e n t i a l b l o o d s e r u m p r o t e i n s were e s t i m a t e d b y electroptloresis. S e r u m a n d u r i n a r y zinc a n d c o p p e r levels were d e t e r m i n e d i n t h e patients studied and compared with the results obtained f r o m 10 n o r m a l i n d i v i d u a l s .

D e t e r m i n a t i o n of s e r u m a n d u r i n a r y zinc a n d c o p p e r was p e r f o r m e d a c c o r d i n g t o t h e m e t h o d of WILLIS 5, u s i n g t h e a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e r S P 90 A. Results. T h e T a b l e i n d i c a t e s t h e r a n g e a n d a v e r a g e v a l u e s of s e r u m a n d u r i n a r y zinc a n d c o p p e r of t h e s t u d i e d bilharzial patients, compared with the results obtained f r o m 10 n o r m a l h e a l t h y i n d i v i d u a l s . N o r m a l s e r u m zinc r a n g e s f r o m 105 t o 208 a n d t h e a v e r a g e f o u n d was 185 ~xg/100 ml. I n u r i n e t h e r a n g e lies b e t w e e n 0.185 a n d 0.610 m g / 2 4 h, while t h e m e a n u r i n a r y zinc r e c o r d e d was 0.400 rag/24 h. N o r m a l S e r u m copper, h o w e v e r , r a n g e s f r o m 65-190 ~g/100 m l a n d t h e m e a n v a l u e 125 ~xg/100 ml. U r i n a r y copper, h o w e v e r , v a r i e d f r o m t r a c e s t o 0.027 w i t h a m e a n v a l u e of 0.016 m g p e r day.

1 M. KHATTAB, A. M. EL MOFTI, L. SOLIMAN, M. ABD]EL AAL and S. EMARA, J. trop. Med. Hyg. 70, 259 (1967). L. SOLIMAN,M. M. ABD]EL KAD]ER,A. ATA, V. ABD]ELWAHAB and S. •MARA, J. trop. Med. Hyg. 72, 166 (1969). 3 C. S. WATSOX and S. SCHWARTZ, J. olin. Invest. 20, 440 (1941). 4 D. R. VAX CAMP]EN and P. SCAFI]E, J. Nutrition 91, 473 (1967). 5 j. B. WILLIS, Clin. Chem. J1, 251 (1965).

Range and average values of urinary and blood serum zinc and copper of normal controls and patients studied Cases

Zinc

Copper

Serum (ttg/100 ml)

Urine (mg/24 h)

Serum (~xg/100ml)

Urine (mg/24 h)

Normal control Range Mean 4- S.E.

105 208 185 ~ 9

0.185-0.610 0.400 d= 0.05

65-190 125 4- 15

0.000-0.027 0.016 • 0.002

Bilharzia hepatosplenomegaly Range Mean 4- S.E.

112 890 341 4- 38

0.200-1.800 1.113 4- 0.2

90-800 338 4- 31

0.000-0.070 0.021 4- 0.003