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PubMed1984

ATP analog specificity of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and phosphorylase kinase.

The ATP analog specificities of the homogeneous cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase have been compared by the ability of 27 analogs to compete with ATP in the protein kinase reaction. Although the data suggest general similarities between the ATP sites of the two homologous cyclic-nucleotide-dependent protein kinases, specific differences especially in the adenine binding pocket are indicated. These differences in affinity suggest potentially useful ATP analog inhibitors of each kinase. For example, apparent autophosphorylation of the purified regulatory subunit of the cAMP-dependent protein kinase is blocked by nebularin triphosphate, suggesting that the phosphorylation is catalyzed by trace contamination of cGMP-dependent protein kinase. Some of the ATP analogs have also been tested using phosphorylase b kinase in order to compare this enzyme with the cyclic-nucleotide-dependent enzymes. All three protein kinases have high specificity for the purine moiety of ATP, and lower specificity for the ribose or triphosphate. The similarity between the ATP site of phosphorylase b kinase to that of the cyclic-nucleotide-dependent protein kinases suggests that it is related to them. The ATP analog specificities of enzymes examined in this study are different from those reported for several unrelated ATP-utilizing enzymes.

Adenosine Triphosphate↗
PubMed1990

Phosphorylase kinase isozymes and phosphorylase in denervated skeletal muscles.

The effects of motor denervation on levels of phosphorylase kinase isozymes and phosphorylase were investigated in rat epitrochlearis, hemidiaphragm, and soleus muscles. Amounts of the proteins were measured after quantitative immunoprecipitation and found to be decreased by as much as 70% 2 weeks after denervation. Unexpectedly, denervation had little, if any, effect on the relative proportions of the two phosphorylase kinase isozymes. Phosphorylase and phosphorylase kinase were decreased by essentially the same extent after denervation, and the effects of denervation were comparable in all three muscles. The decreases in these enzymes explain, at least in part, the marked alterations in glycogen metabolism that occur after motor denervation.

Animals↗
PubMed1986

Evidence that phosphorylase kinase exhibits phosphatidylinositol kinase activity.

Phosphorylase kinase phosphorylates the pure phospholipid phosphatidylinositol. Furthermore, it catalyzed phosphatidylinositol 4-phosphate formation using as substrate phosphatidylinositol that is associated with an isolated trypsin-treated Ca2+-transport adenosinetriphosphatase (ATPase) preparation from skeletal muscle sarcoplasmic reticulum. On this basis a fast and easy assay was developed that allows one to follow the phosphatidylinositol kinase activity during a standard phosphorylase kinase preparation. Both activities are enriched in parallel approximately to the same degree. Neither chromatography on DEAE-cellulose nor that on hydroxyapatite in the presence of 1 M KCl separates phosphatidylinositol kinase from phosphorylase kinase. The presence of a lipid kinase, phosphatidylinositol kinase, in phosphorylase kinase is not a general phenomenon; diacylglycerol kinase can be easily separated from phosphorylase kinase. Polyclonal anti-phosphorylase kinase antibodies as well as a monoclonal antibody directed specifically against the alpha subunit of phosphorylase kinase immunoprecipitate both phosphorylase kinase and phosphatidylinositol kinase.

1-Phosphatidylinositol 4-Kinase↗
PubMed1986

Isozymes of phosphorylase kinase in rabbit skeletal muscle. Functional implications of differences in phosphorylase kinase and phosphorylase activities in individual muscle fibers.

Immunological and microanalytical methods were used to investigate the two isozymes of phosphorylase kinase, enzyme w and enzyme r, in psoas major and tibialis anterior muscles. Peptide mapping experiments indicated that the alpha subunit of enzyme w and alpha' subunit of enzyme r were structurally very similar. Both subunits were completely immunoprecipitated from muscle extracts with an antibody specific for the beta subunit of the kinase, indicating that alpha and alpha' subunits are completely assembled with beta subunits in adult muscle fibers. The relative amounts of enzymes w and r in single fibers were determined from amounts of alpha and alpha' subunits, which were detected by immunoblotting. Phosphorylase kinase and phosphorylase activities were measured in the same fibers, as well as in individual fibers from diaphragm and soleus muscles. Slow oxidative fibers were found to contain low levels of enzyme r, but almost no enzyme w. Considerably more enzyme r was present in fast oxidative-glycolytic fibers. Fast glycolytic fibers contained the most enzyme w, and the highest levels of enzyme r were found in a subgroup of such fibers. Interestingly, more than half of the fast glycolytic fibers analyzed contained both isozymes. In these fibers phosphorylase was positively correlated with enzyme w, but negatively correlated with enzyme r. Total kinase activity ranged 30-fold from the highest in one of the psoas fibers to the lowest in one of the soleus fibers and was closely correlated with the phosphorylase levels. In psoas and soleus fibers, calculated absolute maximal rates for phosphorylase b to a conversion varied almost 2,500-fold.

Animals↗
PubMed1982

Inhibition by calmodulin of the cAMP-dependent protein kinase activation of phosphorylase kinase.

Calmodulin is shown to inhibit both the activation and phosphorylation of phosphorylase kinase by cAMP-dependent protein kinase. Maximal inhibition of both processes was approximately 66% at the highest calmodulin concentration tested (5.5 microM). It was found that the inhibition of phosphorylation was calcium-dependent, reversible by trifluoperazine, and specific for the beta subunit of phosphorylase kinase with no significant inhibition of phosphorylation of the alpha subunit. This inhibitory activity of calmodulin appears to be due to an interaction between calmodulin and the substrate, phosphorylase kinase. This finding implies either that the site of exogenous calmodulin interaction with phosphorylase kinase is at the beta subunit or that this interaction results in a conformational change of phosphorylase kinase that inhibits the interaction between cAMP-dependent protein kinase and the beta subunit of phosphorylase kinase. The beta subunit may contain a regulatory site that is recognized by either protein kinase or calmodulin. These findings further substantiate the role of the beta subunits in the activation of phosphorylase kinase and provide an additional example of substrate-directed control of phosphorylation.

Animals↗
PubMed1985

Lymphocyte phosphorylase kinase activities in the sex-linked form of liver phosphorylase kinase deficiency.

Lymphocyte phosphorylase kinase activities were measured in normal controls and in patients with the sex-linked form of liver phosphorylase kinase deficiency. The reaction due to phosphorylase kinase activity in normal lymphocytes (2.7 X 10(6) in the reaction tube) was found to be linear within 20-60 min at 30 degrees C. The reaction was directly proportional to the concentration of lymphocytes within 1.5 X 10(6)-9.0 X 10(6), at 30 degrees C for 60 min. The phosphorylase kinase activity in normal lymphocytes, which were pre-incubated at 50 degrees C or 95 degrees C for 1 min, decreased to 60% at 50 degrees C and 10% at 95 degrees C of that after pre-incubation at 0 degree C for 1 min. The activity of normal controls was 125 +/- 23.5 U/10(10) lymphocytes. Those of the patients with liver phosphorylase kinase deficiency due to the sex-linked form were 43.5 U in case 1, 54.5 U in case 2, and 51.3 U in case 3, respectively and those of the mothers were within the normal range. These results suggest that phosphorylase kinase in lymphocytes might be form intermediate between liver and muscle phosphorylase kinase.

Child↗
PubMed1997

Autosomal glycogenosis of liver and muscle due to phosphorylase kinase deficiency is caused by mutations in the phosphorylase kinase beta subunit (PHKB).

Glycogen storage disease due to phosphorylase kinase deficiency occurs in several variants that differ in mode of inheritance and tissue-specificity. This heterogeneity is suspected to be largely due to mutations affecting different subunits and isoforms of phosphorylase kinase. The gene of the ubiquitously expressed beta subunit, PHKB, was a candidate for involvement in autosomally transmitted phosphorylase kinase deficiency of liver and muscle. To identify such mutations, the complete PHKB coding sequence was amplified by RT-PCR of RNA isolated from blood samples of patients and analyzed by direct sequencing of PCR products. The characterization of mutations was complemented by PCR of genomic DNA. In one female and four male patients, we identified five independent nonsense mutations (Y418ter; R428ter; Y974H+E975ter; Q656ter in two cases), one single-base insertion in codon N421, one splice-site mutation affecting exon 31, and a large deletion involving the loss of exon 8. Although these severe translation-disrupting mutations occur in constitutively expressed sequences of the only known beta subunit gene of phosphorylase kinase, PHKB, they are associated with a surprisingly mild clinical phenotype, affecting virtually only the liver, and relatively high residual enzyme activity of approximately 10%.

Adolescent↗
PubMed1977

Effect of Mg2+ concentration on the cAMP-dependent protein kinase-catalyzed activation of rabbit skeletal muscle phosphorylase kinase.

Phosphorylase kinase was found to be activated and phosphorylated at 10mM Mg2+ by the cAMP-dependent protein kinase-catalyzed reaction ot much higher levels than observed previously when reactions were carried out in 1 to 2 mM Mg2+ (Cohen, P. (1973) Eur. J. Biochem. 34, 1; Hayakawa, T., Perkin, J.P., and Krebs, E.G. (1973) Biochemistry 12, 574). That the reaction at 10 mM Mg2+ is protein kinase-catalyzed is supported by several observations: (a) the reaction is facilitated by the addition of protein kinase; (b) the reaction depends on cAMP when protein kinase holoenzyme is uded; (c) the reaction is not inhibited by 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetate which is known to inhibit autoactivation and autophosphorylation of phosphorylase kinase; and (d) the protein inhibitor of protein kinase inhibits this reaction. The phosphorylation and activation of phosphorylase kinase seem to occur in two phases. At low Mg2+ only the first phase is manifested and involves the incorporation of 2 mol of phosphate, 1 mol into each of Subunits A and B. At high Mg2+ additional sites are phosphorylated almost exclusively on Subunit A, with phosphate incorporation approaching the final level of 7 to 9 mol. Enzyme activity at high Mg2+ is 2 to 3 times higher than that observed when activation is studied at low Mg2+. The observation that both casein and type II histone are phosphorylated to the same extent at 1 mM and 10 mM Mg2+ suggested that high Mg2+ may be altering the conformation of phosphorylase kinase thus rendering more phosphorylation sites accessible to protein kinase. Since the phosphorylation of phosphorylase kinase by either the protein kinase-catalyzed or autocatalytic reaction can result in the incorporation of 7 to 9 mol of phosphate, the finding that only about seven sites become phosphorylated by both mechanisms acting together suggest that activation by these two mechanisms may involve common phosphorylation sites.

Animals↗
PubMed1995

[Kinetics of action of phosphorylase kinase in a cascade enzymatic system. III. Hysteretic properties of phosphorylase kinase from skeletal muscles].

The kinetic behaviour of rabbit skeletal muscle phosphorylase kinase at variable concentrations of the enzyme and the substrate (glycogen phosphorylase b) has been studied. The kinetic curves reveal a lag period whose duration decreases with a rise in the phosphorylase kinase concentration (when the reaction is initiated by an addition of the ATP + MgCl2 mixture to the enzyme preincubated with phosphorylase b, CaCl2, glycogen and glucose-1-phosphate or inorganic phosphate). A decrease of the phosphorylase b concentration eliminates the lag period. Under these conditions the specific activity of phosphorylase kinase decreases with a rise in the enzyme concentration. The kinetic behaviour of phosphorylase kinase is interpreted in terms of a model of a linearly associating system, such as M reversible M2 reversible M3 reversible ...Mi, where M is the dexadecameric molecule of phosphorylase kinase. Acceleration of the phosphorylase kinase-catalyzed reaction in the course of the enzymatic process seems to be due to the breakdown of inactive enzyme associates (Mi) caused by phosphorylase b. The short gamma-subunit of phosphorylase kinase devoid of the calmodulin-binding domain does not display any hysteretic properties.

Animals↗
PubMed1982

Liver phosphorylase kinase: characterization of two interconvertible forms and partial purification of phosphorylase kinase a.

The presence of two interconvertible forms of phosphorylase kinase has been confirmed in rat liver extracts. The pH optimum of the nonactivated form (PhK b) was lower than the pH optimum of the activated form (PhK a) as reported by others (2). In the absence of calcium the Km of PhK b for phosphorylase b was 53 +/- 10 U/ml with a Vm of 17 +/- 1 U/gm of tissue. The Km of PhK a for phosphorylase b was 20 +/- 2 U/ml with a Vm of 65 U/gm. Calcium stimulated both forms of phosphorylase kinase (A0.5 approximately 0.03 micro). In the presence of 0.1 microM calcium the Km for phosphorylase b of both forms of the enzyme was reduced. In addition, calcium increased the Vm of both forms, but the effect was greater for PhK b than for PhK a. The Km of both forms of phosphorylase kinase for ATP was 0.05 mM and was unaffected by calcium. All of these studies were done using liver phosphorylase b as substrate. Conditions for assaying PhK a activity virtually independent of PhK b activity also are indicated. This will enable the monitoring of interconversion reactions in tissue extracts. Phosphorylase kinase a was purified to near homogeneity using DEAE-cellulose, Sepharose 4B gel filtration and ATP affinity chromatography. The molecular weight was approximately 1 x 10(6). The pH profile, calcium requirements and kinetic constants were the same as those for PhK a in the crude extract.

Animals↗
PubMed2000

Site-directed mutants of glycogen phosphorylase are altered in their interaction with phosphorylase kinase.

Glycogen phosphorylase is found in resting muscle as phosphorylase b, which is inactive without AMP. Phosphorylation by phosphorylase kinase (PhK) produces phosphorylase a, which is active in the absence of AMP. PhK is the only kinase that can phosphorylate phosphorylase b, which in turn is the only physiological substrate for PhK. We have explored the reasons for this specificity and how these two enzymes recognize each other by studying site-directed mutants of glycogen phosphorylase. All mutants were assayed for changes in their interaction with a truncated form of the catalytic subunit of phosphorylase kinase, gamma(1-300). Five mutations (R69K, R69E, R43E, R43E/R69E, and E501A), made at sites that interact with the amino terminus in either phosphorylase b or a, showed little difference in phosphorylation by gamma(1-300) compared to wild-type phosphorylase b. Five mutations, made at three sites in the amino-terminal tail of phosphorylase (K11A, K11E, I13G, R16A, and R16E), however, produced decreases in catalytic efficiency for gamma(1-300), compared to that for phosphorylase b. R16E was the poorest substrate for gamma(1-300), giving a 47-fold decrease in catalytic efficiency. The amino terminus, and especially Arg 16, are very important factors for recognition of phosphorylase by gamma(1-300). A specific interaction between Lys 11 of phosphorylase and Glu 110 of gamma(1-300) was also confirmed. In addition, I13G and R16A were able to be phosphorylated by protein kinase A, which does not recognize native phosphorylase.

Amino Acid Substitution↗
PubMed1986

Involvement of cyclic AMP-dependent protein kinase on the phosphorylase kinase inhibition by glucose-6-phosphate in adipose tissue extracts.

In order to achieve further clarification of the regulation of glycogenolysis in adipose tissue, we studied the effect of glucose-6-phosphate on phosphorylase activation in Sephadex G-25 filtrate of adipose tissue. The activity of phosphorylase kinase was decreased by 50% and by 75% in the presence of 0.5 mM and 2 mM of glucose-6-phosphate, respectively. This inhibition could be partially prevented by 0.5 mM AMP. Furthermore, we investigated the influence of glucose-6-phosphate on the effect of cyclic-AMP-dependent protein kinase on the activation of phosphorylase. The addition of cyclic-AMP and cyclic-AMP-dependent protein kinase caused a decrease in the inhibition of the phosphorylase activation by glucose-6-phosphate. Also, the glucose-6-phosphate at physiological concentration, decreased adipose tissue cyclic-AMP-dependent protein kinase activity.

Adenosine Monophosphate↗