Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have previously reported that rabbit skeletal muscle phosphorylase kinase is phosphorylated by glycogen synthase (casein) kinase-1 (CK-1) primarily on the beta subunit (beta = 1 mol of PO4; alpha = 0.2 mol of PO4) when the reaction was carried out in beta-glycerophosphate. The resultant enzyme activation was 16-fold (Singh, T. J., Akatsuka, A., and Huang, K.-P. (1982) J. Biol. Chem. 257, 13379-13384). In the present study we found that in Tris-Cl buffer CK-1 catalyzes the incorporation of greater than 2 mol of PO4/monomer into each of the alpha and beta subunits. Phosphorylase kinase activation resulting from the higher level of phosphorylation remained 16-fold. 32P-Labeled tryptic peptides from the alpha and beta subunits were analyzed by isoelectric focusing. Cyclic AMP-dependent protein kinase (A-kinase) phosphorylates a single major site in each of the alpha and beta subunits at 1.5 mM Mg2+. In addition to these two sites, A-kinase phosphorylates at least three other sites in the alpha subunit at 10 mM Mg2+. CK-1 also catalyzes the phosphorylation of multiple sites in both the alpha and beta subunits. Of the two major sites phosphorylated by CK-1 in the beta subunit, one of these sites is also recognized by A-kinase. At least three sites are phosphorylated by CK-1 in the alpha subunit. One of these sites is recognized by CK-1 only after a prior phosphorylation of phosphorylase kinase by A-kinase at a single site in each of the alpha and beta subunits at 1.5 mM Mg2+. The roles of the different phosphorylation sites in phosphorylase kinase activation are discussed.
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PMID:Comparison of the phosphorylation of rabbit skeletal muscle phosphorylase kinase by cAMP-dependent protein kinase and cAMP-independent glycogen synthase (casein) kinase-1. 609 48

Tyrosine hydroxylase (TH) in freshly prepared 45,000 g supernatant from rat striatum was fractionated by DEAE-cellulose chromatography. The elution was made with 2 vols. of buffer (50 mM Tris, pH 7.4; 2 mM dithiothreitol) followed by 4 vols. of a linear NaCl gradient (0 0.3 M) in the same buffer. TH activity was eluted in two distinct peaks: one at about 0.1 M salt (I), and the other at 0.2 M salt (II). The relationship between the two enzymes peaks was examined as follows. (1) Incubation of the supernatant in the presence of cAMP-dependent protein kinase, 1 mM ATP, 10 mM Mg2+, and 0.1 mM cAMP resulted in the elimination of peak I, with a concomitant increase of peak II. This shift of TH peaks was prevented when the protein kinase was blocked by the addition of its inhibitory modulator. (2) Incubation of the supernatant with alkaline phosphatase, an enzyme known to dephosphorylate a variety of phosphoproteins, resulted in the elimination of peak II, with a concomitant increase of peak I. (3) Only freshly prepared supernatants showed two distinct TH peaks from DEAE-cellulose. From supernatants held at 0 degrees C for 24 h. peak II was markedly reduced and peak I concomitantly increased. Since peak II appears to be readily convertible to peak I, no further fractionation was attempted. From the data obtained here, we believe that peaks I and II are respectively the nonphosphorylated and phosphorylated forms of TH. Furthermore, the endogenous distribution of the two TH forms in striatum was altered by the administration of haloperidol (2 mg/kg. i.p.), a neuroleptic drug known to activate the enzyme via a cAMP-dependent mechanism. At 90 min after the treatment, there was a marked increase of peak II, with a concomitant decrease of peak I. Thus, this procedure provides a simple means for estimating the degree of phosphorylation of TH in vivo in catecholaminergic neurons under various physiological and pharmacological conditions.
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PMID:Two forms of striatal tyrosine hydroxylase from DEAE-cellulose chromatography. 613 70

Cardiac sarcoplasmic reticulum contains an endogenous calcium-calmodulin-dependent protein kinase and a 22,000-Da substrate, phospholamban. This kinase is half-maximally activated (EC50) by 3.8 +/- 0.3 microM calcium and is absolutely dependent on exogenous calmodulin (EC50 = 49 nM). To determine the effect of this phosphorylation on calcium transport, sarcoplasmic reticulum vesicles (0.5 mg/ml) were preincubated under conditions for optimal phosphorylation (50 mM potassium phosphate, pH 7.0, 10 mM MgCl2, 0.5 mM EGTA, 0.478 mM CACl2, 0.1 microM calmodulin, 0.5 mM ATP). Control sarcoplasmic reticulum was preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both control and phosphorylated vesicles were centrifuged and resuspended in 0.3 M sucrose, 20 mM Tris-HCl, 100 mM KCl, pH 7.0, to remove calmodulin and subsequently assayed for calcium (45Ca) transport in the presence of 2.5 mM Tris-oxalate. Phosphorylation of sarcoplasmic reticulum vesicles by calcium-calmodulin-dependent protein kinase resulted in a significant increase (2- to 4-fold) in the rate of calcium transport at low calcium concentrations (less than 3 microM), while calcium transport was minimally affected at higher calcium. Hill coefficients (n) derived from Hill plots of transport data showed no difference between control and phosphorylated sarcoplasmic reticulum (n = 2.0), indicating that phosphorylation does not alter the cooperativity between calcium sites on the calcium pump. The EC50 for calcium activation of calcium transport by control vesicles was 0.86 +/- 0.1 microM calcium, and phosphorylation of phospholamban decreased this value to 0.61 +/- 0.07 microM calcium (n = 7, p less than 0.028), indicating an increase in the apparent affinity for calcium upon phosphorylation. These results were found to be specific for calcium-calmodulin-dependent phosphorylation of phospholamban. Control experiments on the effects of the reactants used in the phosphorylation assay and subsequent centrifugation of sarcoplasmic reticulum showed no alteration of the rate of calcium transport. Therefore, the calcium pump in cardiac sarcoplasmic reticulum appears to be regulated by an endogenous calcium-calmodulin-dependent protein kinase, and this may provide an important regulatory mechanism for the myocardium.
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PMID:Regulation of cardiac sarcoplasmic reticulum calcium transport by calcium-calmodulin-dependent phosphorylation. 622 13

Soluble TSH receptors were released into the medium when bovine thyroid plasma membranes were incubated in 0.01 M Tris-HCl, pH 7.5, at 0 or 20 C. This is a conventional hypotonic medium used in binding assays. The characteristics of binding of bovine [125I]TSH to the released TSH receptor were almost the same as those of binding to TSH receptors solubilized by lithium 3,5-diiodosalicylate or to the original plasma membrane. Released TSH receptor had two binding sites with Ka values of 0.7 x 10(10) and 0.1 x 10(8) M-1. T3, T4, KI, methimazole, and propylthiouracil had no effect on spontaneous TSH receptor release or on bovine [125I]TSH binding to solubilized TSH receptor. Hydrocortisone (10(-5)--10(-3) M) and d,l-propranolol (10(-3) M) inhibited receptor release. cAMP increased the release of TSH receptor. Hydrocortisone, d,l-propranolol, and cAMP had no effect on bovine [125I]TSH binding to solubilized or released TSH receptor. d,l-Propranolol and hydrocortisone may act as membrane-stabilizing agents. cAMP stimulation of release suggests that the release mechanism could depend upon a protein kinase-phosphoprotein system. Although these studies were conducted with membranes in an unphysiological medium, receptor release may occur normally and could be a source of circulating antigen related to production of antireceptor antibody in autoimmune thyroid diseases. Release of receptors during incubation in vitro may affect the results of studies of hormone-receptor interaction.
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PMID:Release of thyrotropin receptor from thyroid plasma membranes: effect of hydrocortisone, propranolol, and adenosine 3',5'-monophosphate. 624 31

The optimal assay conditions for rat myocardial cytosol protein kinases have been determined. The assay medium contained in 0.25 ml: 40 mM Tris/HCl pH 7.4, 5 mM MgCl2, 600 micrograms histone f1, 10--30 micrograms enzyme protein, 32 microM [gamma-32P]-ATP, with or without additions of 1 microM cAMP. The incubation time at 37 degrees C was 5 minutes. The protein kinase activity in the absence of cAMP decreased with development, while the activity in the presence of cAMP remained unchanged. The protein kinase activity of the left ventricle was higher than that in the right ventricle.
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PMID:Cyclic AMP-dependent protein kinases in the rat myocardial cytosol. 624 38

Calmodulin prepared from red cell hemolysates was found to significantly increase Ca2+ uptake into cardiac microsomal preparations enriched in sarcoplasic reticulum in a dose-dependent manner. The stimulation of calcium uptake by calmodulin was additive to that stimulation produced by maximal stimulatory concentrations of adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase and cAMP, indicating separate mechanisms of action and potentially different modulatory roles for these two systems in the control of calcium transport. K+ significantly decreased calmodulin stimulation of calcium uptake, while in the absence of calmodulin, K+ increased Ca2+ uptake. In the absence of K+, calmodulin increased Ca2+ uptake to levels observed at maximal K+ concentrations without calmodulin present. Na+ produced effects similar to those of K+ in this preparation both in the presence and absence of calmodulin. The effect of calmodulin on the intermediate steps of the (Mg2+,Ca2+)ATPase in cardiac sarcoplasmic reticulum was also investigated. Calmodulin was found to reduce the steady-state level of the Ca2+-dependent phosphoprotein (ECaP) and increase the (Mg2+,Ca2+)ATPase activity of this preparation. Dephosphorylation of ECaP in the presence of Tris-ATP (0.5 mM) was significantly stimulated by calmodulin. These studies indicate that calmodulin stimulates Ca2+ transport in cardiac sarcoplasmic reticulum by increasing the turnover rate of the transport process.
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PMID:Characterization of calmodulin effects on calcium transport in cardiac microsomes enriched in sarcoplasmic reticulum. 625 83

Mutations in the lon (capR) gene result in multiple phenotypes, one of which is the failure to degrade abnormal and normal proteins (Deg-). Previous work with partially purified preparations showed that the lon (capR) gene product is a 94,000-dalton polypeptide with an affinity for nucleic acids. The lon (capR) protein has now been highly purified and is demonstrated to have an ATP-dependent protease activity. The enzyme hydrolyzed 3H-labeled alpha-casein into trichloroacetic acid-soluble forms in Tris buffer containing Mg2+ and ATP. The reaction has a pH optimum of 8.5 and ATP was the preferred nucleotide. CTP and UTP could substitute for ATP (75% and 67%, respectively) but GTP, ADP, AMP, cyclic AMP, and PPi could not. Proteolysis by the lon (capR) protein required ATP hydrolysis. Nonhydrolyzable analogs of ATP and CTP did not promote casein cleavage. When low concentrations of ATP were used, proteolysis stopped as the ATP pool was depleted. Casein stimulated lon (capR) ATPase activity, and the products were ADP and inorganic phosphate in equimolar amounts. No protein kinase activity was detected. The DNA-binding activity, present in partially pure preparations, was retained in the purified protein. The gene product purified from a lon nonsense mutant that exhibits the Deg- phenotype (capR9), lacked both the ATP-dependent protease and ATPase activities, though it retained DNA-binding activity. Absence of an ATP-dependent protease activity could account for many of the pleiotropic effects observed in lon mutants.
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PMID:ATP hydrolysis-dependent protease activity of the lon (capR) protein of Escherichia coli K-12. 645 36

3-Oxo-5 alpha-steroid: NAD+ delta 4-oxidoreductase ("NADH-5 alpha-reductase", EC 1.3.1.?) is rapidly inactivated in the presence of 17 beta-hydroxy-4-androsten-3-one (testosterone). This activation is prevented by increasing the phosphate concentration. When the enzyme assay is carried out in Tris-HCl, only a small activity (1.7 nmol X min-1 X mg-1) is observed which may be further decreased by addition of phosphatases. Addition of the phosphatase inhibitor dextran sulphate or ATP, Mg++ and c-AMP results in a significant increase of activity (228% and 273%, respectively) compared with the Tris-HCl control. Glycerol 2-phosphate and glycerol 3-phosphate have a stabilizing effect on 3-oxo-5 alpha-steroid: NAD+ delta 4-oxidoreductase by decreasing the Km towards the substrate testosterone from 1.2 X 10(-5) mol/l to 3.3 X 10(-6) mol/l. V remains unchanged. Half maximal velocity (testosterone reduction) is achieved with 20 mumol/l glycerol 2-phosphate and glycerol 3-phosphate. Addition of c-AMP dependent protein kinase (EC 2.7.1.37) to a microsomal preparation pretreated with alkaline phosphatase (EC 3.1.3.1) results in a significant increase of 3-oxo-5 alpha-steroid: NAD+ delta 4-oxidoreductase activity compared with the control.
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PMID:Rat liver 3-oxo-5 alpha-steroid delta 4-dehydrogenase. Modulation of enzyme activity by changes in phosphorylation state. 652 91

Normal thymocyte and bone marrow terminal deoxynucleotidyl transferase (TdT) have distinguishing characteristics by phosphocellulose chromatography in Tris buffer: marrow TdT elutes as a single peak at 0.3 M salt, whereas thymocyte TdT separates into two forms, one at 0.3 M salt and one at 0.4 M salt. Since the majority of TdT-positive acute leukemias are anatomically bone marrow-derived, one would have predicted the presence of a bone marrow TdT-phosphocellulose chromatographic pattern in such patients. However, in 376 consecutive, untreated TdT-positive acute lymphoblastic leukemias (ALL) studied by us we have invariably encountered the two-peak thymocyte-type phosphocellulose pattern. The TdT patterns in the thymic-dependent, TdT-positive lymphoma of AKR mice, and the TdT-positive bone marrow-derived, thymic-independent Abelson virus leukemia of Balb/C mice duplicate the situation in human ALL: a thymocyte pattern is seen in both the marrow-derived and thymus-derived diseases. This chromatographic difference between leukemia-associated and normal marrow-associated TdT in both murine and human leukemia suggested that phosphocellulose-TdT patterns might be useful for monitoring residual marrow tumour cell burden in TdT-positive leukemia. This has not turned out to be the case: in eight patients studied in early relapse the blast cell TdT pattern was the single-peak 0.3 M species. Therefore, leukemic cell TdT cannot reliably be distinguished from normal marrow cell TdT. The chromatographic behaviour of TdT may be regulated by phosphorylation-dephosphorylation, the 0.3 M salt peak can be converted to the 0.4 M salt species by treatment with protein kinase and ATP, and the 0.4 M species can be converted to the 0.3 M form by exposure to alkaline phosphatase. Thus, apparently anatomic compartment-specific forms of TdT may only reflect differing cellular metabolic activity.
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PMID:Chromatographic forms of terminal deoxynucleotidyl transferase in normal lymphoid cells and in leukemia cells at presentation and relapse. 698 13

The native form of ATP citrate lyase (2 mol of phosphate/tetramer) and the dephospho-ATP citrate lyase (phosphate-free) purified to homogeneity from rat liver, are phosphorylated by ATP and by the catalytic subunit of cAMP-dependent protein kinase from rabbit muscle. A total of 2 mol of phosphate/tetramer were incorporated into native enzyme, while with the dephospho form, 4 mol of phosphate were incorporated. The phosphopeptides resulting from trypsin treatment which were isolated from phosphorylated forms of both native enzyme and the dephospho enzyme were similar. The ATP citrate lyase, phosphorylated to an extent of 4 mol of phosphate/tetramer, has the same Vmax as the native enzyme (2 mol of phosphate/tetramer). Native ATP citrate lyase, trypsin-treated to remove the phosphopeptide, could not be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase from rabbit muscle, suggesting a common trypsin-sensitive specific phosphorylation site. The phosphorylation rate varied with pH in potassium phosphate, imidazole/HCl, and Tris/HCl buffers. Divalent cations were essential for the activity of the protein kinase. The apparent Km value for ATP was found to be 50 microM.
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PMID:Phosphorylation of dephospho-ATP citrate lyase by the catalytic subunit of cAMP-dependent protein kinase. 705 76


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