蛋白激酶C对胰岛素抵抗骨骼肌细胞的影响

目的探讨蛋白激酶C(Protein Kinase C,PKC)在棕榈酸(Palmitic Acid,PA)诱导的骨骼肌细胞胰岛素抵抗(Isulin Resistance,IR)中的作用。方法免疫荧光鉴定原代大鼠骨骼肌细胞,氧化酶-过氧化物酶偶联法(GOD-POD法)检测培养液中葡萄糖浓度。设立对照组、棕榈酸组(PA组)、罗格列酮组(Rosiglitazone,Ros组),每组一分为二,分别加PKC抑制剂白屈莱红碱(Chelerythrine Chloride,CC)与正常培养液作用1h,Western Blot检测PKB及P-Ser473 PKB表达水平。结果 90%以上的细胞-αsarcometric actin免疫荧光染色呈阳性反应,表明培养的细胞为骨骼肌细胞;0.6mmol/L的PA作用24h可诱导骨骼肌细胞产生胰岛素抵抗;PA组与对照组相比P-Ser473 PKB水平显著降低,与本组未加CC相比显著升高。同时,罗格列酮组及本组加CC中P-Ser473PKB水平均高于PA组。结论在PA诱导的骨骼肌细胞IR方面PKC起重要作用,罗格列酮与PKC抑制剂CC均能改善PA引起的IR。     

Methacholine sensitivity and cAMP protein kinase in tracheal smooth muscle

We studied regional variation in canine trachealis smooth muscle sensitivity and responsiveness to methacholine as well as basal and methacholine-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase activity. The trachea between the cricoid cartilage and the carina was divided into three segments of equal length (designated cervical, middle, and thoracic regions), each consisting of approximately 12-14 cartilage rings. Smooth muscle strips from each of the three regions were exposed to cumulative half-log increments of methacholine chloride. The sensitivity (-log EC50) and responsiveness (force per cross-sectional area and force per milligram protein) of the smooth muscle to methacholine in each region was determined from these data. Smooth muscle strips from cervical and thoracic regions were frozen before and after exposure to cumulative half-log increments of methacholine up to each region's previously determined EC50. Frozen samples were assayed for cAMP content or cAMP-dependent protein kinase activity. The relationship between resting tension and methacholine sensitivity and responsiveness were studied. For the size strips we used, 4 g resting tension set the average cervical and thoracic strips at 96 and 101% of their optimal length, respectively. The methacholine EC50 was not affected by a variation in resting tension. Sensitivity to methacholine was 7.1, 6.8, and 6.5 for cervical, middle, and thoracic regions, respectively. The responsiveness of the cervical and thoracic smooth muscle to methacholine was 16.4 and 16.3 g force/mm2, respectively, at an EC50 methacholine. Basal cAMP was lower in cervical smooth muscle than in thoracic. cAMP-dependent protein kinase activity ratios under both basal and EC50 methacholine-stimulated conditions were lower in cervical smooth muscle than in thoracic. We have observed in trachealis smooth muscle an inverse relationship between methacholine sensitivity and either cAMP or cAMP-dependent protein kinase activity. We suggest that cAMP and cAMP-dependent protein kinase play a role in the regulation of airway smooth muscle sensitivity to cholinergic agonists.     

Effects of adenosine 3':5'-monophosphate-dependent protein kinase on sarcoplasmic reticulum isolated from cardiac and slow and fast contracting skeletal muscles.

Effects of cyclic adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase were studied in sarcoplasmic reticulum prepared from cardiac and slow and fast (white) skeletal muscle. Cyclic AMP-dependent protein kinase failed to catalyze phosphorylation of fast skeletal muscle microsomes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyclic AMP-dependent protein kinase was without effect on calcium uptake by these microsomes. Treatment of cardiac microsomes obtained from dog, cat, rabbit, and guinea pig with cyclic AMP-dependent protein kinase and ATP resulted in phosphorylation of a 22,000-dalton protein component in the amounts of 0.75, 0.25, 0.30, and 0.14 nmol of phosphorus/mg of microsomal protein, respectively. Calcium uptake by cardiac microsomes was stimulated 1.8- to 2.5-fold when microsomes were treated with cyclic AMP-dependent protein kinase. Protein kinases partially purified from bovine heart and rabbit skeletal muscle were both effective in mediating these effects on phosphorylation and calcium transport in dog cardiac sarcoplasmic reticulum. Slow skeletal muscle sarcoplasmic reticulum also contains a protein with a molecular weight of approximately 22,000 that can be phosphorylated by protein kinase. Phosphorylation of this component ranged from 0.005 to 0.016 nmol of phosphorous/mg of microsomal protein in dog biceps femoris. A statistically significant increase in calcium uptake by these membranes was produced by the protein kinase. Increases in protein kinase-catalyzed phosphorylation of a low molecular weight microsomal component and in calcium transport by sarcoplasmic reticulum of cardiac and slow skeletal muscle may be related to the relaxation-promoting effects of epinephrine seen in these types of muscle. Conversely, the absence of a relaxation-promoting effect of epinephrine in fast skeletal muscle may be associated with the lack of effect of cyclic AMP and protein kinase on calcium transport by the sarcoplasmic reticulum of this type of muscle.     

Myosin light chain kinase phosphorylation in tracheal smooth muscle

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+.     

Expression of a mutant regulatory subunit of cAMP-dependent protein kinase in the Caco-2 human colonic carcinoma cell line.

Caco-2 human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of RI (regulatory subunit of the type 1 cAMP-dependent protein kinase), driven by the metallothionein 1 promoter. A stable transformant was isolated that expressed the mutant RI gene in a Zn(2+)-inducible manner. The consequences of the RI mutation on cAMP-dependent protein kinase activity, cell division, and regulation of chloride efflux were examined. When grown in the absence of ZnSO4, protein kinase activity in the transformant was stimulated 2.5-fold by cAMP and approached the levels of cAMP-dependent protein kinase activity seen in parental Caco-2 cells; when treated with ZnSO4, cAMP-dependent protein kinase activity in the transformant was inhibited by 60%. In the absence of ZnSO4 the transformant grew with the same doubling time and to the same saturation density as the untransformed parent. In the presence of ZnSO4 the transformant exhibited a cAMP-reversible inhibition of cell division, indicating that a functional cAMP-dependent protein kinase was required for the growth of these cells in culture. Induction of the mutant RI gene also abolished forskolin-stimulated chloride efflux from these cells, suggesting obligatory roles for cAMP and cAMP-dependent protein kinase in forskolin's actions on chloride channel activity. We anticipate that this transformant will be useful for further studies on the roles of cAMP and cAMP-dependent protein kinase in the regulation of intestinal epithelial cells, including regulation of cell proliferation and differentiation, and regulation of chloride channel activity by neurohormones and neurotransmitters.     

Nonactivated phosphorylase kinase is a phosphoprotein: differentiation of two classes of endogenous phosphoserine residues by phosphorus-31 nuclear magnetic resonance spectroscopy and phosphatase sensitivity

A standard preparation of phosphorylase kinase from rabbit skeletal muscle contains 2 mol of phosphoserine/mol of alpha beta gamma delta. This basal stoichiometry is not influenced by application of propranolol and insulin in vivo; these serine phosphates could not be hydrolyzed by phosphatases of the muscle extract or by alkaline phosphatases. When the enzyme is purified in the presence of the protein phosphatase inhibitor sodium fluoride, it contains either 1 or 3 additional mol of phosphoserine/mol of alpha beta gamma delta, termed phosphatase-sensitive phosphates. Both classes of phosphates yield in formic acid one single 31P NMR signal of a narrow line width (approximately 3 Hz) very similar in chemical shift to free phosphoserine. Phosphoserine is also identified by its chemical shift when dissolved in 8 M guanidinium chloride and by its electrophoretic mobility after acid hydrolysis. By self-phosphorylation of phosphorylase kinase, 14 additional mol of phosphate/mol of alpha beta gamma delta was incorporated, and all were identified as phosphoserine by 31P NMR spectroscopy. In native phosphorylase kinase, the 31P NMR signals of both the basal and the phosphatase-sensitive phosphates are substantially broadened and reduced in intensity, indicating strong interactions of the phosphate groups with the protein. The basal and phosphatase-sensitive phosphates give in 8 M guanidinium chloride a homogeneous NMR signal above pH 6; it splits into a doublet below pH 6 and into a triplet below pH 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of calcium ion concentration on cell fusion,cell division and creatine kinase activity in muscle cell cultures

Cell fusion, cell number, soluble cell protein and creatine kinase activity have been measured simultaneously in chick muscle cell cultures exposed to various calcium ion concentrations for various periods of time, by adding either extra calcium chloride or the calcium-chelating agent, EGTA. Up to 0.75 mM EGTA cell fusion is not inhibited, but the specific activity of creatine kinase is reduced by 20–50%. Between 0.75 and 1.7 mM EGTA, cell fusion is gradually abolished and the increase in cell number prevented, but enzyme specific activity actually increases again and returns to control values. Adding extra Ca²⁺ produces small increases in cell fusion and soluble cell protein, but much greater increases in creatine kinase activity. EGTA stimulates thymidine incorporation into DNA at low concentrations and then inhibits again as its concentration is increased further. These effects of EGTA on cell division may be related to its effects on creatine kinase. The implications of these results are discussed in terms of current ideas about the inter-relationships between cell fusion, cell division and the accumulation of muscle proteins during differentiation. In particular they show that cell fusion is not essential for the attainment of normal levels of creatine kinase.     

Phosphorylation of muscle and non-muscle actins by casein kinase 1 in vitro

Skeletal muscle and Physarum actins were markedly phosphorylated by casein kinase 1, but not by casein kinase 2. The amount of radioactive phosphate incorporated into muscle actin with 110 units of casein kinase 1 was approx. 0.2 mol per mol of actin, which was significantly greater than those catalyzed using the same number of enzyme units of protein kinase A or protein kinase C. The Km values of casein kinase 1 for muscle and Physarum actins were 0.270 and 0.667 mg/ml, respectively.     

Purified rabbit brain protein kinase C relaxes skinned vascular smooth muscle and phosphorylates myosin light chain

To clarify the role of protein kinase C in the mechanical response, the effects of exogenous protein kinase C and its cofactors were investigated on skinned smooth muscle preparations of the rabbit mesenteric artery. Addition of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate (TPA) and phosphatidylserine (PS) caused slow inactivation of a maximal Ca2+ contraction of the muscle fiber and correspondingly increased protein kinase C phosphorylation of myosin light chain. Neither protein kinase C nor enzyme cofactors (PS and TPA) produced relaxation of this tissue and all three components caused significant relaxation. Furthermore, when the muscle fiber was activated by Ca2+-insensitive fragment of MLC-kinase, addition of protein kinase C with PS and TPA decreased the tension and increased protein kinase C phosphorylation of myosin light chain. This evidence suggests that protein kinase C phosphorylation of myosin light chain may play an inhibitory role in the contraction of vascular smooth muscle.     

Troglitazone inhibits angiotensin II-induced extracellular signal-regulated kinase 1/2 nuclear translocation and activation in vascular smooth muscle cells.

The thiazolidinedione troglitazone inhibits angiotensin II-induced extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activity in vascular smooth muscle cells. Activation of extracellular signal-regulated kinase 1/2 by angiotensin II is a multistep process involving both its phosphorylation by mitogen-activated protein kinase extracellular signal-regulated kinase kinase in the cytoplasm and a subsequent translocation to the nucleus. The cytoplasmic activation of extracellular signal-regulated kinase 1/2 in vascular smooth muscle cells proceeds through the protein kinase Czeta --> mitogen-activated protein kinase extracellular signal-regulated kinase kinase --> extracellular signal-regulated kinase pathway. Troglitazone did not affect the angiotensin II-induced activation of protein kinase Czeta or its downstream signaling kinases extracellular signal-regulated kinase 1/2 in the cytosol. In contrast, angiotensin II-induced activation of protein kinase Czeta and extracellular signal-regulated kinase 1/2 in the nucleus were both inhibited by troglitazone. Nuclear translocation of extracellular signal-regulated kinase 1/2 induced by angiotensin II was completely blocked by troglitazone. Protein kinase Czeta, however, did not translocate upon angiotensin II stimulation. Troglitazone, therefore, inhibits both angiotensin II-induced nuclear translocation of extracellular signal-regulated kinase 1/2 and the nuclear activity of its upstream signaling kinase protein kinase Czeta. Since extracellular signal-regulated kinase 1/2 nuclear translocation may be a critical signaling step for multiple growth factors that stimulate vascular smooth muscle cells proliferation and migration, troglitazone may provide a new therapeutical approach for the prevention and treatment of atherosclerosis and restenosis.     

Phosphorylation of tyrosine hydroxylase by calmodulin-dependent multiprotein kinase

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated stoichiometrically by either cyclic AMP-dependent protein kinase or calmodulin-dependent multiprotein kinase from skeletal muscle, but not by five other protein kinases tested. The activity of tyrosine hydroxylase was elevated 3-fold by cyclic AMP-dependent protein kinase, but no activation was observed after phosphorylation by calmodulin-dependent multiprotein kinase. Phosphorylation produced by cyclic AMP-dependent protein kinase and calmodulin-dependent multiprotein kinase was additive, suggesting different sites of phosphorylation. This was confirmed by high-performance liquid chromatography analysis of tryptic phosphopeptides which demonstrated that the major sites phosphorylated by each protein kinase were distinct. A calmodulin-dependent multiprotein kinase that had identical properties and substrate specificity to the skeletal muscle enzyme was partially purified from rat pheochromocytoma. The possibility that this protein kinase is involved in the regulation of tyrosine hydroxylase activity in adrenergic tissue in vivo is discussed.     

Identification of two different phosphofructokinase-phosphorylating protein kinases from Ascaris suum muscle

Two different phosphofructokinase-phosphorylating protein kinases were separated from extracts of Ascaris suum muscle by chromatography on DEAE-Fractogel. They were tentatively designated phosphofructokinase kinase I and phosphofructokinase kinase II. Phosphofructokinase kinase I eluted from the chromatography column at an ionic strength of 0.07 and contained about 25% of the phosphofructokinase-phosphorylating activity assayed in crude extracts. The protein kinase activity was not stimulated by the addition of either cAMP or cGMP. It was inhibited by the heat-stable protein kinase inhibitory protein from rabbit muscle (Walsh inhibitor), by the regulatory subunit of cAMP-dependent protein kinase from beef heart, and by the cAMP-binding protein from Ascaris muscle. These properties suggest that phosphofructokinase kinase I is homologous to the catalytic subunit of cAMP-dependent protein kinases from mammals. This assumption is supported by the estimation of the Mr of 40,000 for the purified phosphofructokinase kinase I under denaturing conditions and by the fact that the presence of cAMP eliminated the inhibition by the cAMP binding proteins. The isoelectric point of the enzyme was 8.7. Phosphofructokinase kinase II was eluted from the DEAE-Fractogel column at an ionic strength of 0.16 and contained approximately 75% of the phosphofructokinase kinase activity measured in the extracts. The molecular and kinetic properties were significantly different from those of phosphofructokinase kinase I. The enzyme was not inhibited by the heat-stable inhibitor protein nor by cAMP-binding proteins. The Mr of the native enzyme was estimated as 220,000 by molecular sieve chromatography. The isoelectric point of the enzyme was pH 5.45.     

Effect of epinephrine and insulin on adenosine 3'5'-cyclic monophosphate--dependent protein kinase in human skeletal muscle in vivo.

Cyclic AMP dependent protein kinase has beeen identified in human skeletal muscle tissue. In crude muscle extracts the enzyme was 3--5 fold activated by cyclic AMP. The cyclic AMP-dependent activity (corresponding to the inactive holoenzyme) was completely inhibited by the heat stable inhibitor of protein kinase. Reciprocal changes of the cyclic AMP-dependent activity in skeletal muscle were observed after administration of epinephrine and insulin in vivo. Infusion of epinephrine in healthy volunteers increased the level of cyclic AMP and decreased the activity of the cyclic AMP-depenent form (i.e. the inactive form) of protein kinase. These changes were reversible after cessation of epinephrine administration. The results are consistent with an activation of protein kinase in vivo due to an epinephrine mediated increase of the concentration of cyclic AMP. I.v. injection of insulin had the opposite effect on the enzyme in skeletal muscle, leading to increased activity of the cyclic AMP-dependent form of protein kinase. Insulin had no effect on the level of cyclic AMP, but promoted a transient increase of cyclic GMP 1 min. after insulin injection. The effect by insulin on protein kinase cannot be related to the level of cyclic AMP or cyclic GMP.     

Signaling pathways of kaempferol-3-neohesperidoside in glycogen synthesis in rat soleus muscle

Kaempferol 3-neohesperidoside is one of the several compounds that have been reported to have insulin-like properties in terms of glucose lowering. We studied the effect of kaempferol 3-neohesperidoside in glycogen synthesis in rat soleus muscle through the incorporation of ¹⁴C-d-glucose in glycogen. Kaempferol 3-neohesperidoside stimulates glycogen synthesis in rat soleus muscle by approximately 2.38-fold. Insulin at 100 nM showed a stimulatory effect on glycogen synthesis when compared with the control group. The stimulatory effect of kaempferol 3-neophesperidoside on glycogen synthesis was inhibited by wortmannin, the phosphatidylinositol 3-kinase (PI3K) inhibitor, and enhanced by lithium chloride, a glycogen synthase kinase 3 (GSK-3) inhibitor. Moreover, the stimulatory effect of kaempferol 3-neohesperidoside was also nullified by PD98059, a specific inhibitor of mitogen-activated protein kinase (MEK) and by calyculin A, an inhibitor of protein phosphatase 1 (PP1) activity. It was concluded that the PI3K – GSK-3 pathway and MAPK – PP1 pathway are involved in the stimulatory kaempferol 3-neohesperidoside effect on glycogen synthesis in rat soleus muscle.     

Comparison of enzyme activities on glycogen metabolism in rabbit slow and fast muscles

Activities of glycogen synthase (total) and branching enzyme in slow (soleus) muscle are higher than those in fast (vastus lateralis) muscle, while those of phosphorylase kinase (total), phosphorylase (total) and debranching enzyme are reversed. The active form ratio of glycogen synthase is higher in fast muscle, while those of phosphorylase kinase and phosphorylase are higher in slow muscle. Activities of cAMP-dependent protein kinase and protein phosphatase in slow muscle are higher than those in fast muscle. These results suggest that glycogen metabolizing enzymes in slow muscle, distinct from those in fast muscle, are regulated more strongly by cAMP-dependent protein kinase rather than by protein phosphatase.     

Phosphorylation of smooth muscle actin by the catalytic subunit of the cAMP-dependent protein kinase

Partially purified smooth muscle (chicken gizzard) actomyosin contains two major substrates of cAMP-dependent protein kinase: a protein of M_r = 130,000, identified as the calmodulin-dependent myosin light chain kinase, and a protein of M_r = 42,000. This latter protein was shown by a variety of electrophoretic procedures to be actin. Purified smooth muscle actin also was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. The rate of phosphorylation of smooth muscle actin was significantly enhanced by depolyjerization of actin. A maximum of 2.0 mol phosphate could be incorporated per mol G-actin. Skeletal muscle F-actin was not significantly phosphorylated by protein kinase; however, skeletal G-actin is a substrate for the protein kinase although its rate of phosphorylation was significantly slower than that of smooth muscle G-actin.     

Phosphorylation of smooth myosin light chain kinase by smooth muscle Ca2+/calmodulin-dependent multifunctional protein kinase

Smooth muscle myosin light chain kinase (MLC kinase) was phosphorylated by smooth muscle calmodulin-dependent protein kinase II (CaM protein kinase II). When MLC kinase was free from calmodulin, two sites were phosphorylated. The phosphorylation at the one site was much faster than the other site; however, the phosphorylation at the first site was completely blocked by calmodulin binding to MLC kinase. Phosphorylation of MLC kinase by CaM protein kinase II increased the dissociation constant of MLC kinase for calmodulin about 10 times without changing the Vmax. The location of the phosphorylation sites was identified by isolating and sequencing the tryptic phosphopeptides of MLC kinase. The preferred site was identified as serine 512 and the second site as serine 525. These sites are the same as the sites phosphorylated by cAMP-dependent protein kinase.     

Purification and characterization of calmodulin-dependent multifunctional protein kinase from smooth muscle: isolation of caldesmon kinase

Previously, it was reported that smooth muscle caldesmon is a protein kinase and is autophosphorylated [Scott-Woo, G.C., & Walsh, M.P. (1988) Biochem. J. 252, 463-472]. We separated a Ca2+/calmodulin-dependent protein kinase from caldesmon in the presence of 15 mM MgCl2. The Ca2+/calmodulin-dependent caldesmon kinase was purified by using a series of liquid chromatography steps and was characterized. The subunit molecular weight (MW) of the kinase was 56K by SDS gel electrophoresis and was autophosphorylated. After the autophosphorylation, the kinase became active even in the absence of Ca2+/calmodulin. The substrate specificity of caldesmon kinase was similar to the rat brain calmodulin-dependent multifunctional protein kinase II (CaM PK-II) and phosphorylated brain synapsin and smooth muscle 20-kDa myosin light chain. The purified kinase bound to caldesmon, and the binding was abolished in the presence of high MgCl2. Enzymological parameters were measured for smooth muscle caldesmon kinase, and these were KCaM = 32 nM, KATP = 12 microM, Kcaldesmon = 4.9 microM, and KMg2+ = 1.1 mM. Optimum pH was 7.5-9.5. The observed properties were similar to brain CaM PK-II, and, therefore, it was concluded that smooth muscle caldesmon kinase is the isozyme of CaM PK-II in smooth muscle.     

Comparison of glycogen phosphorylase kinases of various rat tissues

Glycogen phosphorylase kinases in soluble fractions of various rat tissues were examined for the pH 6.8/8.5 activity ratio, Ca2+-dependency, activation by cyclic AMP-dependent protein kinase (protein kinase A), and reactivity with anti-skeletal muscle phosphorylase kinase serum. The enzymes could be divided into at least two major groups; muscle and liver types. The muscle type, that has a low value of pH 6.8/8.5 activity ratio, is highly dependent on Ca2+, markedly activated by protein kinase A, and strongly inhibited by the antiserum. Inversely, the liver type, that has a high value of pH 6.8/8.5 activity ratio, is poorly dependent on Ca2+, not activated by protein kinase A, and weakly inhibited by the antiserum. The enzymes from heart and skeletal muscle were similar and belonged to the former entity. Whereas, the enzymes from liver, kidney, spleen, lung, and testis appeared to belong to the latter entity. The enzyme from brain apparently differs from these entities, and seems to be an intermediate type or a hybrid of the two.     

Pyruvate kinase and creatine phosphokinase during development of the chicken with muscular dystrophy

Pyruvate kinase and creatine phosphokinase activities in breast muscle extracts and in serum, and protein content of the muscle extracts were determined during the first eight weeks of development of control and dystrophic chickens. In the dystrophic chicken serum enzyme levels were significantly greater than, and muscle protein content and enzyme activities on a gram wet weight basis were significantly less than control values, by the second week after hatching and thereafter. For both muscle and serum the relative differences between control and dystrophic groups was greater for pyruvate kinase than crearine phosphokinase. On a specific activity basis only pyruvate kinase levels in dystrophic muscle were significantly less than control values in 2–8-week-old chickens.