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PubMed1994

Calmodulin-dependent enzymes undergo a protein-induced conformational change that is associated with their interactions with calmodulin.

The anionic hydrophobic (amphipathic) fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sulfonate was used to investigate the surface hydrophobic properties of calmodulin (CaM)-dependent enzymes as follows: calcineurin, myosin light chain kinase, cyclic nucleotide phosphodiesterase, CaM-dependent protein kinase II, and the gamma-subunit of phosphorylase kinase. We found that certain domains of these enzymes that interacted with 2-(p-toluidinyl)-naphthalene-6-sulfonate were exposed by a transient proton (H+) increase within the neutral pH range. This H(+)-induced exposure, which could be caused either by direct addition of H+ or by the release of H+ from metal chelators upon their binding of Ca2+, seemed to be more closely linked with a change in pH value (i.e. transient H+ increase) than with the actual equilibrium pH value of the system. Unlike the case with CaM-dependent enzymes, the H(+)-induced conformational change was uncommon in CaM-independent enzymes. When CaM-binding domains were removed from calcineurin and smooth muscle myosin light chain kinase, the resultant enzymes no longer exposed new domains in response to H+ increase. Using dansylated CaM to monitor the formation of CaM-enzyme complexes, we found that complex formation occurred with an uptake of H+ from solution. When CaM-dependent enzymes were evaluated at suboptimal concentrations of Ca2+, addition of H+ enhanced both the formation of CaM-enzyme complexes and the CaM-dependent catalytic activities, but this synergistic H+ effect occurred within only a narrow range of Ca2+ concentrations. These findings suggest that the H(+)-exposed domains in CaM-dependent enzymes are involved in the binding of CaM and that both conformational changes in CaM and its enzyme targets are necessary for complex formation. Further, the findings are consistent with the notion that CaM-binding domains are masked in the nonactivated (uncomplexed) conformations of CaM-dependent enzymes. The interplay between H+ and Ca2+ is discussed in relation to other systems that display interdependent effects of these two ions.

Animals↗
PubMed2004

Human deoxycytidine kinase as a deoxyribonucleoside phosphorylase.

Human deoxycytidine kinase (dCK) is a key enzyme in the 5'-phosphorylation of purine and pyrimidine deoxynucleosides with deoxycytidine as the most efficient substrate. The ability of dCK to degrade 2'-deoxyribonucleosides to free nucleobases and 2-deoxy-alpha-d-ribofuranose-1-phosphate was demonstrated by 1H-31P correlation spectroscopy and by isotope enzyme kinetic methods. The reaction depended on inorganic phosphate, and dCK showed maximum cleavage activity between pH 7 and pH 8. In this pH range, [HPO4(2-)] is the dominant phosphate species, most likely being the phosphate donor. All natural deoxyribonucleosides could be cleaved and the Vmax of the phosphorylytic reaction compared to the kinase reaction was about 2-10%. The formation of free nucleobases occurred only with reduced dCK, because the reaction was highly dependent on the presence of reducing agents such as dithiotreitol. Thus, recombinant dCK can act as a phosphorylase, similar to the nucleoside phosphorylase family of enzymes. This catalytic activity is important for the design of in vitro experiments with dCK, such as crystallization and NMR spectroscopy.

Catalysis↗
PubMed1986

Apparent activation of the MgATP-dependent protein phosphatase by pp60v-src. Identification of an activity like that of glycogen synthase kinase 3 in immunoaffinity purified pp60v-src preparations.

Immunoaffinity purified pp60v-src was found to activate the MgATP-dependent protein phosphatase in the presence of MgATP. Although preliminary evidence suggested that phosphorylation of the inhibitor-2 subunit on tyrosine residues was responsible for the activation, preincubation of the pp60v-src preparation at 41 degrees C resulted in a rapid loss of its protein kinase activities towards both casein and inhibitor-2 while its ability to activate the protein phosphatase complex was relatively insensitive to this treatment. This result demonstrated that pp60v-src was not responsible for activation of the MgATP-dependent protein phosphatase. A protein kinase activity which phosphorylated glycogen synthase on serine residues was detected in the pp60v-src preparation. The protein kinase was active in the presence of inhibitors of phosphorylase kinase, glycogen synthase kinase 5/casein kinase II, and cAMP-dependent protein kinase. It is, therefore, likely that activation of the MgATP-dependent protein phosphatase resulted from the presence of a glycogen synthase kinase 3 like activity in the pp60v-src preparation. Our results illustrate the importance of applying multiple criteria to link the phosphorylation of a protein with an observed change in its activity.

Adenosine Triphosphate↗
PubMed1995

Purification and properties of glycogen phosphorylase from the fat body of larval Manduca sexta.

Glycogen phosphorylase b has been purified to homogeneity from the fat body of larval Manduca sexta. The purification procedure involved ammonium sulfate precipitation, and chromatography of DEAE-cellulose, 5'-AMP-Sepharose and Q-Sepharose. The final product, which showed a single band on SDS-PAGE with a M(r) = 92,500, was purified 50-fold from the original homogenate in a yield of about 3%. The molecular mass of the native purified phosphorylase b was estimated to be 186,000 Da from gel filtration, suggesting that the native enzyme is a dimer. The apparent Km values for glycogen, phosphate and 5'-AMP were 1.4 mM, 82 mM and 1.1 mM, respectively. The enzyme had a pH optimum of 7.05, and was inhibited by ATP, ADP and glucose, but not by trehalose, even at high concentration. Conversion of phosphorylase b into the a form was achieved by incubation with rabbit phosphorylase kinase and Mg(2+)-ATP. The molecular mass of phosphorylase a was estimated to be 250,000 Da by gel filtration chromatography. The specific activity of the a form in the presence of 5'-AMP was 1.6-1.7-fold higher than the specific activity of the b form under the same conditions. Thus, 5'-AMP activates the a form by about 20%, whereas ATP has no effect on the phosphorylase a activity.

Animals↗
PubMed1982

Regulation of fructose-6-phosphate 2-kinase by phosphorylation and dephosphorylation: possible mechanism for coordinated control of glycolysis and glycogenolysis.

The kinetic properties and the control mechanism of fructose-6-phosphate 2-kinase (ATP: D-fructose-6-phosphate 2-phosphotransferase) were investigated. The molecular weight of the enzyme is approximately 100,000 as determined by gel filtration. The plot of initial velocity versus ATP concentration is hyperbolic with a Km of 1.2 mM. However, the plot of enzyme activity as a function of fructose-6-phosphate is sigmoidal. The apparent K0.5 for fructose-6-phosphate is 20 microM. Fructose-6-phosphate 2-kinase is inactivated by the catalytic subunit of cyclic AMP-dependent protein kinase, and the inactivation is closely correlated with phosphorylation. The enzyme is also inactivated by phosphorylase kinase in the presence of Ca2+ and calmodulin. The phosphorylated fructose-6-phosphate 2-kinase, which is inactive, is activated by phosphorylase phosphatase and alkaline phosphatase. The possible physiological significance of these observations in the coordinated control of glycogen metabolism and glycolysis is discussed.

Calcium↗
PubMed1975

Functional specificity of guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases from silkworm.

Adenosine 3':5'-monophosphate-dependent protein kinase partially purified from silkworm pupae shows identical functional activities with those of mammalian protein kinases; the insect and mammalian kinases are completely exchangeable in the phosphorylation of muscle glycogen phosphorylase kinase and glycogen synthetase resulting in the activation and inactivation of the respective enzymes. In contrast, guanosine 3':5'-monophosphate-dependent protein kinase obtained from the same organism is totally inactive in this role and phosphorylates different, mainly seryl and some threonyl, residues of acceptor proteins. Substrates of the latter kinase intimately involved in the regulation of biological processes have remained unknown.

Animals↗
PubMedDate not supplied

[Effect of a centrogenic fever on phosphorylase and pyruvate kinase activity in rabbit tissues].

Administration of pyrogenal into rabbits caused a decrease in the activity of phosphorylase A and pyruvate kinase in musculus gastrocnemius as well as pyruvate kinase -- in musculus soleus. The phosphorylase A activity was increased within 3 hrs after the drug administration but it was decreased after the treatment for three days. Administration of hydrocortisone decreased the activity of phosphorylase and pyruvate kinase in both muscles. Activities of total phosphorylase and phosphorylase A were decreased simultaneously with restoration of the pyruvate kinase activity in the both muscles after the complex administration of pyrogenal and hydrocortisone within 3 days. In liver tissue and kidney cortex activities of the enzymes did not differ from those of control animals under all the conditions studied. The various extent and direction in alterations of the same enzymes in dissimilar tissues of a single amnial after administration of pyrogenal and hydrocortisone were considered as the various adaptation of cells of different tissues to the effect of hydrocortisone and as the alteration in tissue reactivity in experimental fever animals to the hormonal effect.

Animals↗
PubMed1983

Characterization of a fluorescent substrate for the adenosine 3',5'-cyclic monophosphate-dependent protein kinase.

A synthetic tetradecapeptide derived from the phosphorylation site of the beta-subunit of phosphorylase kinase (Arg-Thr-Lys-Arg-Ser-Gly-Ser-Val-Tyr-Glu-Pro-Leu-Lys-Ile) is a highly efficient substrate for the cAMP-dependent protein kinase, exhibiting a 36% decrease in the intrinsic tyrosine fluorescence on phosphorylation. The fluorescence changes in continuous assays were monitored to demonstrate the roles of protein kinase effectors (cAMP, the type II regulatory subunit, and the 8000-Da heat-stable inhibitor) in the regulation of the enzyme and to determine Km and Vmax. The phosphorylation reaction requires 1 mol ATP/mol peptide. Amino acid analysis demonstrates the presence of phosphoserine in the phosphorylated peptide. Auxiliary experiments show that tyrosine phosphorylation can also be detected fluorometrically and distinguished from serine or threonine phosphorylation.

Amino Acid Sequence↗
PubMed1999

Inhibition of glycogenolysis in primary rat hepatocytes by 1, 4-dideoxy-1,4-imino-D-arabinitol.

1,4-Dideoxy-1,4-imino-d-arabinitol (DAB) was identified previously as a potent inhibitor of both the phosphorylated and non-phosphorylated forms of glycogen phosphorylase (EC 2.4.1.1). In the present study, the effects of DAB were investigated in primary cultured rat hepatocytes. The transport of DAB into hepatocytes was dependent on time and DAB concentration. The rate of DAB transport was 192 pmol/min per mg of protein per mM DAB(medium-concentration). In hepatocytes, DAB inhibited basal and glucagon-stimulated glycogenolysis with IC(50) values of 1.0+/-0.3 and 1.1+/-0.2 microM, respectively. The primary inhibitory effect of DAB on glycogenolysis was shown to be due to inhibition of glycogen phosphorylase but, at higher concentrations of DAB, inhibition of the debranching enzyme (4-alpha-glucanotransferase, EC 2.4.1.25) may have an effect. No effects on glycogen synthesis were observed, demonstrating that glycogen recycling does not occur in cultured hepatocytes under the conditions tested. Furthermore, DAB had no effects on phosphorylase kinase, the enzyme responsible for phosphorylation and thereby activation of glycogen phosphorylase, or on protein phosphatase 1, the enzyme responsible for inactivation of glycogen phosphorylase through dephosphorylation.

Animals↗
PubMed1979

Glucocorticoids and hepatic glycogen metabolism.

The steady accumulation of glycogen in fetal rat liver during the last fifth of gestation is elicited by a transient rise in the level of circulating corticosterone. One effect of glucocorticoids is to induce glycogen synthase. The actual deposition of glycogen, however, depends on the appearance of a small amount of glycogen synthase in the active, dephosphorylated form. Induction of glycogen synthase phosphatase by glucocorticoids may explain the latter crucial process. Insulin enhances further the rate of glycogen deposition. The effect of insulin requires a previous exposure of the fetal liver to glucocorticoids. It is exerted on the enzyme interconversion system and appears not to involve new protein synthesis. Administration of glucocorticoids to adult fed or fasted animals causes within 3 h an intensive deposition of glycogen in the liver. This phenomenon is ultimately explained by both an activation of glycogen synthase and an inactivation of glycogen phosphorylase. The latter process may be due to an enhanced activity of phosphorylase phosphatase, or possibly of phosphorylase kinase phosphatase. The activation of glycogen synthase is explained by an enhanced activity of glycogen synthase phosphatase. The latter enzyme is normally profoundly inhibited by phosphorylase a; glucocorticoids cause the appearance in the liver of a protein factor that decreases and eventually cancels this inhibitory effect of phosphorylase a. It remains to be established whether or not some part of the glucocorticoid effect on adult liver is mediated by insulin.

Animals↗
PubMed1987

Homology probing: identification of cDNA clones encoding members of the protein-serine kinase family.

Mixed oligonucleotide probes were used to screen a HeLa cDNA library for clones encoding amino acid contiguities whose conservation is characteristic of the protein-serine kinase family. Eighty thousand clones were screened, from which 19 were identified as showing strong hybridization to two distinct probes. Four clones were chosen for characterization by partial DNA sequence analysis and 3 of these were found to encode amino acid sequences typical of protein-serine kinases. One deduced amino acid sequence shares 72% identity with rabbit skeletal muscle phosphorylase kinase gamma-subunit, while another is closely related to the yeast protein-serine kinases CDC2 in Schizosaccharomyces pombe and CDC28 in Saccharomyces cerevisiae. This screening approach should have applications in the identification of clones encoding previously unknown or poorly characterized members of other protein families.

Amino Acid Sequence↗
PubMed1998

Binding of 9-anthroylcholine monitors the interactions of adenosine cyclic 3',5'-phosphate-dependent protein kinase with MgATP, substrates, and regulatory subunits.

The isolated catalytic subunit of cAMP-dependent protein kinase and smooth muscle myosin light chain kinase undergo interactions with the fluorescent dye 9-anthroylcholine (9AC) that are responsive to the two enzymes' associations with substrates and effectors. Additionally, the binding of 9AC is highly sensitive to subtle structural or functional differences among closely related protein kinases. Skeletal muscle myosin light chain kinase and the catalytically active chymotryptic fragment of the gamma-subunit of phosphorylase kinase do not associate with 9AC. The 1:1 fluorescent complex of the isolated catalytic subunit of cAMP-dependent protein kinase with 9AC exhibits a dissociation constant of 21 microM. The association of the catalytic subunit with either of the regulatory subunits, RI and RII, results in decreases in the observed 9AC fluorescence that are reversed upon the addition of cAMP. The effects of MgATP and of polypeptide substrates (Kemptide, troponin I, protamine) on the 9AC-catalytic subunit complex are consistent with a general noncompetitive model in which the interactions of 9AC and the other ligands with the enzyme are mutually antagonistic but not purely competitive.

Adenosine Triphosphate↗