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)

A method for the cryogenic storage of the cAMP-dependent protein kinase from bovine cardiac muscle is described. The catalytic parameters, kcat, KM, and kcat/KM are used to assess the activity of the enzyme both prior and subsequent to the freeze-thaw cycle. The enzyme is stored in cryogenic vials at -196 degrees C in liquid nitrogen. Complete retention of catalytic activity is dependent upon a rapid and efficient freeze-thaw cycle and the use of morpholinepropanesulfonic acid as the buffer. In addition, this buffer appears to eliminate the KCl- or NaCl-induced damage typically observed for enzymes stored at low temperature in phosphate buffer. As a result, morpholinepropanesulfonic acid may prove to be a more appropriate cryopreservation buffer than phosphate when the presence of salt is required for enzyme solubility or stability.
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PMID:Cryopreservation of the cyclic 3',5'-adenosine monophosphate-dependent protein kinase from bovine cardiac muscle. 273 19

A protein phosphatase which dephosphorylates phospholamban was purified from canine cardiac cytosol. Purification involved sequential chromatography on DEAE-Sephacel, polylysine-agarose, heparin-agarose, Mono Q HR 10/10, and Superose 6. The enzyme was composed of three subunits with Mr = 63,000, 55,000, and 38,000, and it could dephosphorylate the sites on phospholamban phosphorylated by either cAMP-dependent or calcium-calmodulin-dependent protein kinase. Phospholamban phosphatase activity was enhanced 12-, 9-, and 3-fold by the divalent cations Mg2+, Mn2+, and Ca2+, respectively. The phosphatase was inhibited by PPi, ATP, NaF, and Pi and the degree of inhibition was different with each compound. The substrate specificity of the purified phosphatase for cardiac phosphoproteins was determined using troponin I, phospholamban, and highly enriched sarcolemmal and sarcoplasmic reticulum preparations, phosphorylated by the cAMP-dependent protein kinase. The phosphatase exhibited the highest activity with phospholamban as substrate. Thus, dephosphorylation of phospholamban by this phosphatase may participate in regulation of sarcoplasmic reticulum function in cardiac muscle.
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PMID:Purification and characterization of phospholamban phosphatase from cardiac muscle. 284 19

We have purified putative L-type Ca2+ channels from chick heart by virtue of their associated high affinity receptors for the Ca2+ channel effectors, dihydropyridines (DHPs), and phenylalkylamines (PAAs). A peptide of 185,000-190,000 daltons was found to comigrate with the peak of DHP binding activity during purification through two successive cycles of lectin affinity chromatography and sucrose density gradient centrifugation. A previously described peptide of 140,000 daltons, whose Mr was increased to approximately 180,000 under nonreducing conditions, also copurified with the 185-kDa peptide and dihydropyridine binding activity. When cardiac membranes were photolabeled with either the dihydropyridine [3H]azidopine or the PAA [3H]azidopamil prior to purification, a single, specifically labeled component of 185,000-190,000 daltons was present in the purified fractions. The properties of this 185-kDa cardiac DHP/PAA receptor were compared to the smaller 165-kDa DHP/PAA receptor previously purified from skeletal muscle. Antibodies raised against the 165-kDa skeletal muscle DHP/PAA receptor reacted with both rabbit and chick skeletal muscle receptors, but only poorly recognized, if at all, the cardiac 185-190 kDa component. The 185-kDa peptide present in the purified fractions obtained from cardiac muscle did not undergo substantial phosphorylation by cAMP-dependent protein kinase, while the purified 165-kDa peptide from rabbit and chick skeletal muscle was a good substrate for this kinase. The results show that the DHP and PAA receptors in cardiac muscle are contained in a 185-190-kDa peptide that is significantly larger than, and structurally and immunologically different from, it skeletal muscle counterpart.
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PMID:Dihydropyridine and phenylalkylamine receptors associated with cardiac and skeletal muscle calcium channels are structurally different. 284 12

Phospholamban (PLB), an integral membrane protein of cardiac sarcoplasmic reticulum, was extracted from bovine cardiac muscle with an acidic chloroform/methanol mixture. A combination of gel permeation and ion-exchange chromatographies in organic solvents allowed the purification of PLB. The intensive use of organic solvents throughout the isolation yielded a highly purified and intact protein that can be phosphorylated by cAMP protein kinase. The ease of purification and the high yield obtained (2.5 mg/100 g of fresh tissue) show that organic solvents can be very useful in the extraction and purification of hydrophobic membrane proteins.
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PMID:Purification of phospholamban from bovine cardiac muscle with organic solvents. 291 87

We have examined the effects of added cAMP-dependent protein kinase and endogenous calmodulin-dependent kinase on Ca2+ transport in purified internal membranes from human platelets. Both Ca2+ uptake and Ca2+-ATPase activity were maximally stimulated about 2-fold by addition of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase inhibitor reduced both Ca2+ uptake and Ca2+-ATPase activities at concentrations which also inhibited cAMP-dependent protein phosphorylation. In addition, concerted stimulation of Ca2+-ATPase by exogenous calmodulin and added catalytic subunit of cAMP-dependent protein kinase was observed. A 22-kDa protein was phosphorylated by both cAMP-dependent and calmodulin-dependent kinases at the same rate as stimulation of the Ca2+-ATPase. Cyclic AMP-dependent phosphorylation of the 22-kDa polypeptide was inhibited by the protein kinase inhibitor and calmodulin-dependent phosphorylation was inhibited by chlorpromazine and EGTA. These results are consistent with the hypothesis that one mode of control of Ca2+ homeostasis in platelets may be similar to the phospholamban system in cardiac muscle.
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PMID:Regulation of human platelet membrane Ca2+ transport by cAMP- and calmodulin-dependent phosphorylation. 295 93

The incorporation of 33P into 54, 43 and 23 kDalton proteins of rabbit cardiac sarcolemma is demonstrated both in intact muscle and under isadrine myocarditis. The phosphorylation by cAMP-dependent protein kinase is shown only for the 23.5 kDalton protein. The level of this protein phosphorylation decreases under isadrine myocarditis in the presence of 10(-6) M cAMP and absence of "exogenic" protein kinase. In the preparations of the sarcolemma of the cardiac muscle under myocarditis the phosphorylation of the 23.5 kDalton protein is not stimulated by beta-agonists in the medium without "exogenic" protein kinase and cAMP. The latter being present, the phosphorylation reaches the control values.
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PMID:[Phosphorylation of cardiac sarcolemma proteins in isadrine myocarditis]. 299 68

Secondary structure of cAMP receptor protein of E. coli was predicted and compared to its crystal structure in the complex with cAMP solved by McKay and Steitz. The two conformations coincide in the DNA binding domain but strikingly differ in the other domain which binds cAMP and causes protein dimerization. The comparison indicates that cAMP destabilizes a very long helix instead of which sheets are formed creating a hydrophobic pocket where cAMP binds. Consequently, the helix-sheets isomerization and a resulting change in the relative monomer disposition in the dimer appears to be the origin of cAMP-induced allosteric activation of the protein. Extremely long helices were also predicted in the regions of the regulatory subunit of cAMP-dependent protein kinase from bovine cardiac muscle where cAMP binds. It is thus likely that the proposed mechanism of the effect of cAMP on protein structure has wider implications.
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PMID:Possible mechanism of the allosteric activation of cAMP receptor protein. 299 27

The divalent cation ionophore A23187 at a concentration of 1 nM produced an increased rate of oxalate-supported calcium uptake by isolated cardiac sarcoplasmic reticulum as determined by absorbance changes of the calcium-sensitive dye murexide. Addition of a higher concentration of A23187 (0.1 microM) produced a decreased rate of calcium uptake. Measurement of the time during which ATPase was activated by calcium addition also suggested an increased rate of calcium uptake in the presence of 1 nM A23187 and an inhibition of calcium uptake at a higher concentration of the ionophore (0.1 microM). Ca2+-stimulated ATPase activity and incorporation of 32Pi from [gamma-32P]ATP into sarcoplasmic reticular proteins were increased by A23187 at concentrations of 1 nM or greater. An increased coupling of calcium uptake to ATP hydrolysis was observed at 1 nM A23187, while concentrations of the ionophore greater than or equal to 10 nM produced a decreased coupling. Addition of an inhibitor of cyclic AMP-dependent protein kinase decreased the rate of calcium uptake, and this inhibition was reversed in a concentration-dependent manner by 0.01-1 nM A23187. These data suggest that A23187 can activate a mechanism involving the calcium-dependent phosphorylation of protein that may regulate the activity of the calcium uptake system of the sarcoplasmic reticulum. These observations appear to provide an explanation for some of the contractile effects of A23187 in intact cardiac muscle that suggest that treatment with the ionophore results in an increased sequestration of calcium from the cytoplasm.
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PMID:Low concentrations of A23187 increase calcium uptake by cardiac sarcoplasmic reticulum. 300 Jan 98

A cardiac muscle sarcolemmal preparation, enriched in adenylate cyclase, Na+, K+ -ATPase, beta, muscarinic and ouabain receptors, also contained endogenous protein kinase activity. Phosphorylation of sarcolemmal membrane proteins by the endogenous protein kinase occurred mainly on 22 000 and 12 000 Mr proteins. To determine the effect of this phosphorylation on sarcolemmal properties, sarcolemmal vesicles were preincubated under conditions for optimal phosphorylation while control vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both control and phosphorylated vesicles were centrifuged, resuspended in 10 mM Tris-Cl (pH 7.4) and subsequently assayed for ATPase activities and for binding of ouabain, dihydroalprenolol and quinuclidinyl benzilate to the membranes. Sarcolemmal phosphorylation was associated with an increase in Ca2+ -ATPase activity but had no effect on Mg2+ ATPase or Na+, K+ -ATPase activity or on ouabain binding. Muscarinic receptor and beta-adrenoreceptor binding also appeared to be unaffected.
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PMID:Influence of protein kinase phosphorylation on isolated sarcolemmal membranes. 300 20

Phosphoprotein phosphatase prepared from bovine cardiac muscle was used to study the roles of axonemal phosphoproteins in the flagellar motility of sea urchin spermatozoa. When isolated axonemes were incubated with cyclic AMP-dependent protein kinase, gamma-[32P]ATP and cyclic AMP, more than 15 polypeptides were phosphorylated. Most were dephosphorylated by treatment with phosphoprotein phosphatase. When Triton models of sea urchin spermatozoa were treated with phosphoprotein phosphatase followed by an addition of ATP, the flagellar motility of the models was drastically reduced in comparison with that of the untreated models. The motility of the phosphatase-treated Triton models was partially restored by an addition of cyclic AMP and cyclic AMP-dependent protein kinase. These data give strong support to the idea that the motility of eukaryotic flagella is controlled by a protein phosphorylation-dephosphorylation system.
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PMID:Phosphoprotein phosphatase inhibits flagellar movement of Triton models of sea urchin spermatozoa. 300 37


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