215

Biochimica et Biophysica Acta, 568 (1979) 215--223 © Elsevier/North-Holland Biomedical Press

BBA 68720

PHOSPHORYLASE KINASE FROM HUMAN POLYMORPHONUCLEAR LEUKOCYTES

NIELS B O R R E G ~ R D SORENSEN

Department of Medicine, Marselisborg Hospital, DK-8000 ~rhus C. (Denmark) (Received September 22nd, 1978)

Key words: Phosphorylase kinase; Phosphorylation; Dephosphorylation; Activation; (Polymorphonuclear leukocyte)

Summary Phosphorylase kinase from human polymorphonuclear leukocytes was investigated in a gel filtered crude preparation (17 000 × g supernatant). It was found to exist in two forms, one (the phosphorylated form) more active than the other (the dephosphorylated form). Interconversion between the two forms was carried out by a cyclic AMP dependent protein kinase and phosphoprotein phosphatase, respectively. The ratio of activity measured at pH 8.0 and 6.0 was 0.36 for the non-activated and 0.83 for the activated form, which is in contrast to the behaviour of phosphorylase kinase from muscle. K~pp for the substrate phosphorylase b was 650 U/ml and 85 U/ml for the non-activated and activated form, respectively, whereas Ka~ p for ATP was 0.03 mM and identical for the two forms. The non-activated form of phosphorylase kinase was activated by Ca :+ in the range 10-7--5 • 10 -6 M, which may have physiological importance, whereas the activated form was insensitive to variations in Ca 2+ concentration between 10 -9 and 10 -3 M.

Introduction Phosphorylase kinase (EC 2.7.1.38) is the enzyme that converts phosphorylase b to a by a phosphorylation reaction and thereby facilitates glycogen breakdown. The enzyme from rabbit muscle has been extensively investigated by several groups and the action mechanism is known down to the molecular plan. The enzyme has been found to exist in activated and non-activated forms, Abbreviations used: E G T ~ . ethylene glycol bis03-aminoethyl ether)-N,N-tetraacetlc t . acid~ Pipes, piperazine-N,N'-bis(2 ethanesulfonic acid).

216 which are interconvertible by phosphorylation-dephosphorylation reactions [1--4]. Much less is known about the enzyme from liver tissue. Apart from being of interest from a pure biochemical point of view, a study of phosphorylase kinase from human tissues is particularly interesting, because it may enlighten some aspects of glycogen storage diseases in which phosphorylase kinase activity has been found low in liver and leukocytes [5--8]. It has also been suggested that the activation of glycogen breakdown in leukocytes [9] by phenylephrine is secondary to activation of nonactivated phosphorylase kinase by Ca 2+ released from intracellular pools. Since little is known about basic properties of phosphorylase kinase from leukocytes, this study was initiated to give knowledge of characteristics that may provide a safe basis for assaying the enzyme. Experimental procedures

Buffers. The pH of buffers were adjusted at 22°C and the assay of phosphorylase kinase run at this temperature. Preparation of a gel filtered homogenate. Human polymorphonuclear leukocytes were prepared as previously described [10] and suspended in ice-cold 50 mM Tris-HC1 (pH 7.2)/50 mM mercaptoethanol/5 mM EDTA buffer, using 2 ml buffer per gram of leukocytes. It is essential to use freshly withdrawn blood, as phosphorylase kinase activity decreases with storage, as also noted by Hujing [7]. The cells were sonicated (20 kHz, 2 X 20 s), centrifuged at 17 000 × g for 15 min and the supernatant gel filtered on a Sephadex G-25 fine column equilibrated in 25 mM Tris-HC1 (pH 7.2), 25 mM mercaptoethanol and 0.2 mM EDTA, all procedures at 2°C. The gel-filtrate was used as enzyme source without further purification. Activation of phosphorylase kinase. Immediately after preparation of the gel filtrate, phosphorylase kinase was in its inactive form, presumably due to the action of endogenous protein phosphatase during preparation. However, routinely, and additional 20 min of incubation on a waterbath (22°C) was employed. The activation was then accomplished by endogenous protein kinase activity by adding (final concentrations) 10 pM cyclic AMP and 30 s later 0.3 mM ATP/6 mM MgC12 (pH 7.2) in a total volume of 0.1 ml to 0.9 ml of the enzyme preparation, and further phosphatase activity was prevented by 50 mM NaF, unless otherwise specified. After 15 min, the activation was completed and the reaction mixture placed at 0°C and assayed within 2 min for phosphorylase kinase activity. Assay of phosphorylase kinase. The assay was based on the rate of conversion of rabbit muscle phosphorylase b to a. Samples of non-activated or activated enzyme preparation were first diluted three times with 25 mM TrisHC1 (pH 7.2)/25 mM mercaptoethanol/0.2 mM EDTA, and 50 mM NaF. A 5 pl sample containing about 20 mg protein per ml was then added to 45 pl of an assay mixture containing 13.9 mM Tris-glycerophosphate buffer (pH 7.2)/ 110 U/ml rabbit muscle phosphorylase b/11.1 m g . m1-1 glycogen/0.33 mM ATP/6.6 mM MgC12/5.5 mg • m1-1 protein kinase inhibitor/55 mM NaF/1.1 mM EGTA, and 1.1 mM CaCl~. The assay was run at 22°C, and followed every 15 min for 45 min. The phosphorylase kinase reaction was stopped by diluting

217 10-pl samples with 500 pl ice-cold 50 mM Tris-HC1 (pH 7.2)/50 mM NaF/5 mM EGTA/1 mM DTT and the phosphorylase a and b activity measured. The progress curve of phosphorylase kinase activity was linear for 120 min provided less than 30% phosphorylase b was used. The activity of phosphorylase kinase was calculated from the slope of the progress curve. One unit of phosphorylase kinase is the activity, which converts 1 unit of phosphorylase b to a in one min at 22 ° C. For means of comparison to activities determined at 30 ° C, it should be noted that one unit of activity at 22°C corresponds to 2.5 units at 30°C, cf. Results. The concentration of ATP in the reaction was 0.3 mM, which is lower than used by most authors. The low concentration combined with the subsequent 51-fold dilution ensured that no carry-over effect of adenine nucleotides on phosphorylase b activity occurred. The inclusion of 1 mM EDTA/1 mM CaC12 in the assay mixture gives a minimum concentration of 2 • 10 -s M Ca 2÷ [28], which ensures full activity of the non-activated form of phosphorylase kinase. It was necessary to include a very high concentration of protein kinase inhibitor (5 mg/ml) in order to block completely any activation of phosphorylase kinase during the assay. Appropriate blanks for phosphorylase kinase activity were always included. The presence of phosphorylase phosphatase activity in the reaction mixture was investigated in a control experiment, where phosphorylase a was substituted for phosphorylase b and the mixture incubated for 45 min at 22°C with either activated or non-activated phosphorylase kinase. Under these conditions no phosphorylase phosphatase activity was observed. Assay o f phosphorylase. Phosphorylase was assayed in the direction of glycogen synthesis without or with 1 mM AMP, as previously described [11]. One unit incorporates one pmol glucose 1-phosphate into glycogen per min at 30 ° C. Protein was determined by the m e t h o d of Lowry et al. [12]. Chemicals. Phosphorylase a and b from rabbit muscle, protein kinase holoenzyme from rabbit muscle (P 3891), and protein kinase inhibitor (P 5015) were all products of Sigma and were gel filtered on Sephadex G-25 {fine) before use. Glycogen rabbit liver type III from Sigma was passed through a mixed ion exchange resin (Amberlite MB 3) before use. [U-14C]glucose 1-phosphate was from the Radiochemical Centre, Amersham, U.K. Results

Interconversion o f activated and non-activated phosphorylase kinase After 20-min incubation of a gel filtered 17 000 X g supernatant, phosphorylase kinase from leukocytes is in its non-activated form (Fig. 1). Addition of cyclic AMP and MgATP leads to rapid activation of the enzyme, which is stable to gel filtration. However, in the absence of added NaF, which blocks the activity of endogenous phosphatases of the enzyme preparation, the activation was slowly reversed over a period of 40 min. The activation by cyclic AMP and MgATP is completely prevented by addition of protein kinase inhibitor. These results suggest that phosphorylase kinase of polymorphonuclear leukocytes exists in two forms, which are interconvertible by phosphorylationdephosphorylation reactions, the former being catalyzed by a cyclic AMPdependent protein kinase.

218 "E

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Fig. 1. 3 m l of a gel f i l t e r e d 17 0 0 0 X g s u p e r n a t a n t in 25 m M Tris-HCl ( p H 7.2), 25 m M m e r c a p t o e t h a n o l a n d 0.2 m M E D T A was p r e i n c u b a t e d for 20 rain in t h e a b s e n c e of N a F . A c t i v a t i o n was t h e n i n i t i a t e d b y 10 pM cyclic AMP, 0.3 m M A T P , 6 m M Mg 2+ ( a r r o w ) a n d the r e a c t i o n (A A), f o l l o w e d for 40 m i n by w i t h d r a w i n g 20-pl samples. T h e a c t i v a t i o n was s t o p p e d b y m i x i n g the s a m p l e s w i t h 10 m g / m l p r o t e i n kinase i n h i b i t o r a n d 50 m M N a F to a t o t a l v o l u m e of 25 pl a n d the s a m p l e s w e r e t h e n k e p t o n ice until assay for p h o s p h o r y l a s e kinase a c t i v i t y . D u r i n g t h e a c t i v a t i o n p e r i o d , t o t a l p h o s p h o r y l a s e kinase a c t i v i t y was d e t e r m i n e d b y w i t h d r a w i n g 1 0 0 pl s a m p l e s ( e o) w h i c h were m i x e d w i t h 50 m M N a F , 0.3 m M A T P , 6 m M MgCl2~ 5 pg p r o t e i n kinase a n d 10 ~uM cyclic AMP in a final v o l u m e of 1 2 5 pl a n d i n c u b a t e d for 10 rain at 2 2 ° C . P b o s p h o r y l a s e kinase a c t i v i t y was t h e n a s s a y e d .

Thermal stability and activation When activated and non-activated forms of phosphorylase kinase were incubated in the presence of 50 mM NaF, the enzym e activity was completely stable for 2 h, both at 22 ° C and 30 ° C, and thus not activated or inactivated by endogenous proteases. The thermal activation of both forms were studied at 22, 30 and 37°C. The Arrhenius plot gave straight lines in both cases and t e m p er atu r e coefficients of 2 . 0 6 . 1 0 4 cal/mol and 2 . 2 4 . 1 0 4 cal/mol were calculated for the activated and non-activated forms, respectively.

Effect of trypsin Similar to phosphorylase kinase from liver [13] and muscle [14], the nonactivated form o f the l e u k o c y t e e n z y m e is rapidly activated by trypsin, whereas the activated f o rm is n o t f ur t her activated (Fig. 2). Continued exposure to trypsin leads to inactivation of bot h enzym e forms.

Kapp for muscle phosphorylase b and A TP m When studied at saturating concentrations of MgATP and at pH 7.2 (Fig. 3a), the apparent Km for muscle phosphorylase b was 85 U/ml and 680 U/ml for the activated and non-activated forms of phosphorylase kinase, respectively, whereas the maximal velocity was the same for both forms. When the ATP concentration was varied and the Mg2+-concentration kept constant at 6 mM, the apparent K m for ATP was determined to 0.03 mM for both the activated and non-activated e n z y m e form at two constant concentrations of muscle phosphorylase b (Fig. 3b). It was n o t feasible to carry o u t these determinations at saturating concentrations of the substrate phosphorylase b, but the fact t hat the K app for MgATP was the same at the two concentrations of phosphorylase b suggests th at there is not allosteric interaction between the t w o substrates.

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Effect of Ca2÷ Ca 2÷ p r o f o u n d l y affects the activity of p h o s p h o r y l a s e kinase in its nonactivated form, w h i c h is essentially inactive at Ca :+ c o n c e n t r a t i o n s b e l o w 3 . 10 -~ M and b e c o m e s fully active at c o n c e n t r a t i o n s above 5 • 10 -6 M (Fig. 4). The activated form is fully active over the range o f Ca 2÷ c o n c e n t r a t i o n s used. It was n o t possible to abolish the activity o f the activated e n z y m e form completely by adding E G T A to the assay mixture. However, in the presence o f 20 mM E G T A c o r r e s p o n d i n g to a calculated c o n c e n t r a t i o n o f Ca 2÷ b e l o w 1 0 -1° M, the activity of the activated e n z y m e decreased to 50% o f the maximal activity.

Effect of pH (Fig. 5) B o t h forms o f p h o s p h o r y l a s e kinase had maximal activity at pH 6.0. The non-activated form s h o w e d a steep decline in activity with a m i n i m u m at pH

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Fig. 3. a. R e c i p r o c a l p l o t o f a c t i v i t y of a c t i v a t e d ( e e) a n d n o n - a c t i v a t e d (A A) p h o s p h o r y l a s e kinase as a f u n c t i o n o f m u s c l e p h o s p h o r y l a s e b c o n c e n t r a t i o n a t c o n s t a n t c o n c e n t r a t i o n of 6 m M MgCl 2 a n d 0.3 m M A T P . b. R e c i p r o c a l p l o t of a c t i v i t y of a c t i v a t e d (circles) a n d n o n - a c t i v a t e d (triangles) phosp h o r y l a s e kinase a t v a r i e d c o n c e n t r a t i o n s o f A T P . M g 2 + c o n c e n t r a t i o n was k e p t c o n s t a n t at 6 raM. E x p e r i m e n t s w e r e c o n d u c t e d a t t w o c o n s t a n t c o n c e n t r a t i o n s o f m u s c l e p h o s p h o r y l a s e b. O p e n s y m b o l s , 96 U p h o s p h o r y l a s e b / m l , c l o s e d s y m b o l s , 167 U p h o s p h o r y l a s e b / m l .

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F i g . 5. T h e a c t i v i t y o f a c t i v a t e d ( c i r c l e s ) a n d n o n - a c t i v a t e d ( t r i a n g l e s ) p h o s p h o r y l a s e k i n a s e w a s a s s a y e d at different pH of the assay mixture described in Methods, however, with the omission of EGTA and CaC12 ( c l o s e d s y m b o l s ) , a n d a t d i f f e r e n t p H o f a n a s s a y m i x t u r e w h e r e 1 2 . 5 m M P i p e s / 1 0 m M m e r c a p t o ethanol was substituted for Tris-glycerophosphate and EGTA and CaCl 2 were omitted (open symbols).

8.0 as the pH increased, whereas the activated form showed a slight decrease in activity with no minimum as the pH increased. It has been shown that protein kinase inhibitor is equally effective at pH 6.0--9.0 (Juhl, H., personal communication). Discussion The present experiments have shown that phosphorylase kinase from human polymorphonuclear leukocytes, as has been found for the enzyme from muscle [1--4] and liver [13,15--18], exists in two forms, one more active than the other. The results are compatible with the assumption that activation is accomplished by a phosphorylation reaction by cyclic AMP dependent protein kinase since: (1) The activation is initiated by adding cyclic AMP and MgATP and can be completely blocked by protein kinase inhibitor. ( 2 ) T h e activation is stable to gel filtration, provided NaF is added. (3) The activation is reversible, and inactivation is completely prevented by the addition of NaF, which is known to block phosphorylase kinase phosphatase activity [20]. The fact that activation could take place in the absence of added cyclic AMP, is explained by the observation that cyclic AMP dependent protein kinase in a 17 000 X g supernatant from human polymorphonuclear leukocytes, is partially dissociated in its regulatory and catalytic subunits, even though no cyclic AMP has been added (Juhl, H., personal communication). It has been shown that, as for the muscle enzyme [ 14], activation results in a higher affinity towards the substrate phosphorylase b with no change in either maximal catalytic capacity or affinity towards the other substrate. The affinity towards the substrate MgATP was not influenced by varying the concentration of the other substrate, phosphorylase b (Fig. 3b). Similar information about the liver enzyme has, to our knowledge, not been published.

221 Several workers have reported that in glycogenosis tp Via, which is characterized by deficiency of phosphorylase kinase in the liver, the activity of the corresponding leukocyte enzyme is very low [6,7,20,21]. Although it can be concluded, that the leukocyte enzyme is always affected in this disease, whether it is the X-linked or the autosomal recessive form, a residual activity of about 20% can, however, always be demonstrated, whereas in the liver the enzyme activity is almost completely absent [20]. This, however, does not justify the conclusion that the liver and leukocyte enzymes are different since the way of obtaining tissue for assay is different and the exposure to proteolytic activity during preparation may be different for the two tissues. Therefore, positive evidence should be presented before any conclusion about the identity of the liver and leukocyte enzymes is made. Huijing [21] has suggested that two forms of deficient phosphorylase kinase activity can be detected in leukocytes from patients with glycogen storage disease, one form with a very low maximal catalytic activity (deficient in enzyme) and one with a highly reduced affinity towards phosphorylase b (K~pp 1000--2000 U/ml), but normal V. In the present study Ka~ p for nonactivated phosphorylase kinase from normal leukocytes was determined to 680 U/ml, which probably is not significantly different from the value reported by Huijing [7]. Huijing did not distinguish between activated and non-activated phosphorylase kinase but from an evaluation of his experimental procedure we believe that he worked with the activated form, provided the enzyme can be maximally activated. It therefore remains:to be shown (1) whether the phosphorylase kinase in leukocytes from patients with glycogenosis tp Via exists in a non-activated and an activated form both of which have abnormal kinetic properties, but where, by coincidence, the kinetic properties of the activated form from these patients is similar to the kinetic properties of the nonactivated enzyme from normal subjects or (2) whether the enzyme from patients with glycogenosis tp Via, despite normal c o n t e n t of protein kinase [8], cannot be activated and therefore is the kinetically normal non-activated phosphorylase kinase. The fact that the non-activated form of phosphorylase kinase is activated by trypsin, similar to the enzyme from muscle [14] and liver [13], indirectly excludes that our enzyme preparation has been significantly modified by proteolysis. The effect of Ca 2÷ on phosphorylase kinase from muscle is well known. Both the activated and non-activated forms are totally dependent on Ca 2÷ for activity and both are stimulated in the range 10 -s to 10 -s M [22]. Also, the activated and non-activated forms of liver phosphorylase kinase are stimulated in the same range of Ca 2÷ concentrations [13,18,23]. We found that the activity of the non-activated form of leukocyte phosphorylase kinase was totally dependent on Ca 2÷ with activation occurring in the concentration range 10 -7-5 ° :10 - 6 M with half maximal activation at 1 0 - 6 M Ca 2÷. In contrast, the activity of activated phosphorylase kinase was independent of variations in Ca 2÷ concentrations between 10 -9 and 10 -3 M. It is therefore suggested that the activity of non-activated phosphorylase kinase in leukocytes fluctuates in response to fluctuations in intracellular Ca 2+ concentrations, whereas the activity of the activated form is insensitive to physiological variations in intra-

222 cellular Ca 2÷. In experiments with intact leukocytes suspended in a Ca2+-con taining buffer, it has recently been shown [9] that the activation of phosphorylase by phenylephrine is not associated with measurable changes in the concentration of cyclic AMP in the cells, but is associated with an increased efflux of Ca 2÷ from 4SCa prelabelled cells. The present finding supports the assumption, that an increase in intracellular Ca 2÷ concentration is responsible for the activation of phosphorylase through the action of non-activated Ca 2÷ dependent form of phosphorylase kinase. One characteristic feature of phosphorylase kinase from rabbit muscle is that the activity changes in response to variation in pH [24,25]. Both activated and non-activated phosphorylase kinase is more active at higher pH. For the nonactivated form the ratio of activity measured at pH 6.8 and 8.2 is 0.01, whereas the corresponding ratio for the activated form is 0.36 [2]. At all pH, the activated form being more active than the non-activated form. There has to our knowledge been nor report of the activity of the liver enzyme at different pH values. In leukocytes almost the inverse pH-dependency was found true. Both forms had maximum at the lowest pH investigated (pH 6.0). The non-activated form showed a steep fall in activity as the pH increased, whereas the activity of the activated form only decreased slightly. When measured in a Tris-glycerophosphate buffer, the pH 8.0/6.0 was determined to 0.36 + 0.014 (S.E.M., n = 5) for the non-activated form, whereas the corresponding ratio for activated phosphorylase kinase was 0.83 + 0.047 (S.E.M., n = 5). As shown in Fig. 5 the shape of the curve remains essentially the same, when using Pipes as buffer, but somewhat lower activity is obtained. The higher activity at low pH may have physiological importance since it is known that the intracellular pH in leukocytes decreases during phagocytosis [26], when at the same time glycogen breakdown is initiated [ 27 ]. Acknowledgements The author is greatly indebted to professor V. Esmann for critically revising the manuscript. The expert technical assistance of Mrs. Jonna Guldberg is acknowleged. The work was supported by grants from Statens Laegevidenskabelige Forskningsr~d Nos. 512-8127 and 512-10031. References 1 Delange, R.J., K e m p , R.G., Riley, D.W., C o o p e r , R.A. a n d Krebs, E.G. ( 1 9 6 8 ) J. Biol. Chem. 2 4 3 , 2200--2208 2 Cohen, P. ( 1 9 7 3 ) Eur. J. Bioehem. 34, 1 - - 1 4 3 Y e m a n , S.J. a n d Cohen, P. ( 1 9 7 5 ) Eur. J. Biochem. 51, 9 1 - - 1 0 4 4 A n t o n i w , J . F . a n d Cohen, P. ( 1 9 7 6 ) Eur. J. Biochem. 68, 4 5 - - 5 4 5 Hug, G., S c h u b e r t , W.K. a n d C h u c k , G. ( 1 9 6 6 ) Science 1 5 3 , 1 5 3 4 - - 1 5 3 5 6 Huijing, F. ( 1 9 6 7 ) Biochim. Biophys. A c t a 1 4 8 , 6 0 1 - - 6 0 3 7 Huijing, F. ( 1 9 7 0 ) Biochim. Biophys. A c t a 2 0 6 , 1 9 9 - - 2 0 7 8 Morishita, Y., Nishigama, K., Y a m a m u r a , H., K o d a m a , S., Negishi, H., Matsuo, M., Matsuo, T. a n d Nishizuka, Y. ( 1 9 7 3 ) Biochem. Biophys. Res. C o m m u n . 54, 8 3 3 - - 8 4 1 9 Herlin, T., Petersen, C.S. a n d E s m a n n , V. ( 1 9 7 8 ) Biochim. Biophys. A c t a 542, 6 3 - - 7 6 1 0 E s m a n n , V. ( 1 9 6 4 ) Metabolism 13, 3 4 4 11 Wang, P. a n d E s m a n n , V. ( 1 9 7 2 ) Anal. B i o c h e m . 4 7 , 4 9 5 - - 5 0 0 12 L o w r y , O.H., R o s e b r o u g h , N.J., Farr, A.L. a n d Randall, R.J. ( 1 9 5 1 ) J . Biol. Chem. 1 9 3 , 2 6 5 - - 2 7 5

223 1 3 V a n De Werve, G., H u e , L. a n d Hers, H . - G . ( 1 9 7 7 ) B i o c h e m . J. 1 6 2 , 1 3 5 - - 1 4 2 1 4 K r e b s , E . G . , L o v e , P.S., B r a t v o l d , G , E . , T r a y s e r , V . A . , M e y e r , W.L. a n d Fiske, E . H . ( 1 9 6 4 ) B i o c h e m i s t r y 3, 1 0 2 2 - - 1 0 3 3 1 5 V a n d e Werve, G., S t a l m a n s , W. a n d H e r s , H . - G . ( 1 9 7 7 ) B i o c h e m . J. 1 6 2 , 1 4 3 - - 1 4 6 16 V a n d e n h e e d e , J . R . , K e p p e n s , S. a n d De W u l f , H. ( 1 9 7 6 ) F E B S L e t t . 6 1 , 2 1 3 - - 2 1 7 17 V a n d e n h e e d e , J . R . , K h a n d e l w a l , R . L . a n d K r e b s , E . G . ( 1 9 7 7 ) J. Biol. C h e m . 2 5 2 , 7 4 8 8 - - 7 4 9 4 1 8 S h i m a z u , I. a n d A m a k a w a , A. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 3 8 5 , 2 4 2 - - 2 5 6 19 C o h e n , P., W a t s o n , D.C. a n d D i x o n , G . H . ( 1 9 7 5 ) E u r . J. B i o c h e m . 51, 7 9 - - 9 2 2 0 L e d e r e r , B., V a n H o o f , F., V a n d e n B e r g h e , G. a n d Hers, H . - G . ( 1 9 7 5 ) B i o c h e m . J. 1 4 7 , 2 3 - - 3 5 21 H u i j i n g , F. ( 1 9 7 5 ) P h y s i o l . Rev. 55, 6 0 9 - - 6 5 8 2 2 B r o s t r o m , C.D., H u n k e l e r , F . L . a n d K r e b s , E . G . ( 1 9 7 1 ) J. Biol. C h e m . 2 4 5 , 6 6 4 9 - - 6 6 5 6 23 K h o o , J . C . a n d S t e i n b e r g , D. ( 1 9 7 5 ) F E B S L e t t . 5 7 , 6 8 - - 7 2 2 4 K r e b s , E.G., G r a v e s , D . J . a n d F i s h e r , E . H . ( 1 9 5 9 ) J. Biol. C h e m . 2 3 4 , 2 8 6 7 - - 7 3 2 5 K r e b s , E . G . , D e L a n g e , R . J . , K e m p , R . G . a n d R i l e y , W.D. ( 1 9 6 6 ) P h a r m a c o l . R e v . 1 8 , 1 6 3 - - 1 7 1 26 J e n s e n , M.S. a n d B a i l e t o n , D . F . ( 1 9 7 3 ) J. Cell. Biol. 5 6 , 3 7 9 27 P e t e r s e n , C,S., H e r l i n , T. a n d E s m a n n , V. ( 1 9 7 8 ) B i o c h i m . B i o p h y s . A c t a 5 4 2 , 7 7 - - 8 7 2 6 P o r t z e h l , H., C a l d w e l l , P.C. a n d R(iegg, J . C . ( 1 9 6 4 ) B i o c h i m . B i o p h y s . A c t a 7 9 , 5 8 1 - - 5 9 1

Phosphorylase kinase from human polymorphonuclear leukocytes.

215 Biochimica et Biophysica Acta, 568 (1979) 215--223 © Elsevier/North-Holland Biomedical Press BBA 68720 PHOSPHORYLASE KINASE FROM HUMAN POLYMORP...
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