scholarly journals Phosphorylation of partially purified 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen

1987 ◽  
Vol 245 (3) ◽  
pp. 893-897 ◽  
Author(s):  
J Gomez-Cambronero ◽  
J M Mato ◽  
F Vivanco ◽  
M Sanchez-Crespo
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Improved Method ◽  
Serine Residue ◽  
Dependent Protein Kinase ◽  
Single Protein ◽  
Dependent Protein

A new improved method for purification of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) from rat spleen is described. The catalytic subunit of cyclic AMP-dependent protein kinase in the presence of MgATP stimulated about 3-fold the activity of this partially purified enzyme activity. When [gamma-32P]ATP was included in the assay mixture, the analysis of phosphoprotein products by SDS/polyacrylamide-gel electrophoresis and autoradiography showed the incorporation of [32P]phosphate into a single protein band of about 30 kDa. Analysis of the phosphorylated amino acids indicated that the phosphate was incorporated into a serine residue. Activation of the acetylation reaction by the protein kinase was reversible. The reversal of the activation was coincident with the loss of the [32P]phosphate incorporated into the 30 kDa protein band, which suggests that the acetyltransferase is regulated by a phosphorylation-dephosphorylation mechanism dependent on cyclic AMP.

Enhanced phosphorylation of myocardial sarcoplasmic reticulum in experimental hyperthyroidism

1978 ◽  
Vol 234 (4) ◽  
pp. H426-H431
Author(s):  
C. J. Limas
Keyword(s):  
Protein Kinase ◽  
Sarcoplasmic Reticulum ◽  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Calcium Transport ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dependent Protein Kinase ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Phosphoprotein Phosphatase ◽  
Dependent Protein

Calcium transport by cardiac sarcoplasmic reticulum (SR) was compared in hyperthyroid (HT) and euthyroid (ET) rats. Both Ca2+ uptake (97 +/- 3.1 nmol/mg per min in HT vs. 63 +/- 2.9 nmol/mg per min in ET, P less than 0.01) and CA2+ -stimulated ATPase activity (61 +/- 4.1 vs. 37 +/- 1.6 nmol Pi/mg per min, P less than 0.01) were higher in the thyroxine-treated animals. These changes were accompanied by enhanced cyclic AMP-dependent phosphorylation of cardiac SR in hyperthyroid rats (180 +/- 4.3 pmol Pi/mg per min vs. 117 +/- 4.2 pmol Pi/mg per min, P less than 0.01). SDS-polyacrylamide gel electrophoresis of cardiac SR showed that phosphorylation of a 22,000-dalton protein (phospholamban) primarily accounted for the differences between the two groups. There was no difference in the rate of SR dephosphorylation by endogenous phosphoprotein phosphatase between HT and ET rats. Differences in cyclic AMP-dependent phosphorylation between the two groups were blunted in the presence of excess exogenous cyclic AMP-dependent protein kinase. These results suggest that increased levels or activity of endogenous cyclic AMP-dependent protein kinases may partially explain enhanced calcium transport by the cardiac SR of hyperthyroid animals.

scholarly journals Purification of phospholipid methyltransferase from rat liver microsomal fraction

1986 ◽  
Vol 237 (3) ◽  
pp. 699-705 ◽  
Author(s):  
M A Pajares ◽  
M Villalba ◽  
J M Mato
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Molecular Mass ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Microsomal Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Liver Microsomal Fraction

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.

Reversion of an S49 cell cyclic AMP-dependent protein kinase structural gene mutant occurs primarily by functional elimination of mutant gene expression

1983 ◽  
Vol 3 (2) ◽  
pp. 250-256
Author(s):  
T van Daalen Wetters ◽  
P Coffino
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Amino Acid ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Dibutyryl Camp ◽  
Dependent Protein Kinase ◽  
Type Function ◽  
Dependent Protein

The regulatory subunits of cyclic AMP (cAMP)-dependent protein kinase from a dibutyryl cAMP-resistant S49 mouse lymphoma cell mutant, clone U200/65.1, and its revertants were visualized by two-dimensional polyacrylamide gel electrophoresis. Clone U200/65.1 co-expressed electrophoretically distinguishable mutant and wild-type subunits (Steinberg et al., Cell 10:381-391, 1977). In all 48 clones examined, reversion of the mutant to dibutyryl cAMP sensitivity was accompanied by alterations in regulatory subunit labeling patterns. Some spontaneous (3 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (2 of 11) revertants retained mutant subunits, but these were altered in charge, degree of phosphorylation, or both. The charge alterations were consistent with single amino acid substitutions, suggesting that reversion was the result of second-site mutations in the mutant regulatory subunit allele that restored wild-type function, although not wild-type structure, to the gene product. The majority of spontaneous (8 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (9 of 11) revertants and all of the revertants induced by ethyl methane sulfonate (14 of 14) and ICR191 (12 of 12) displayed only wild-type subunits. Dibutyryl cAMP-resistant mutants isolated from several of these revertants displayed new mutant but not wild-type subunits, suggesting that the revertant parent expresses only a single, functional regulatory subunit allele. The mutant regulatory subunit allele can, therefore, be modified in two general ways to produce revertant phenotypes: (i) by mutations that restore its wild-type function, and (ii) by mutations that eliminate its function.

scholarly journals Comparison of Ca2+-dependent phosphorylation in viable dispersed brain cells with calmodulin-dependent protein kinase activity in cell-free preparations of rat brain

1985 ◽  
Vol 232 (3) ◽  
pp. 629-635 ◽  
Author(s):  
L L Norling ◽  
M Landt
Keyword(s):  
Protein Kinase ◽  
Gel Electrophoresis ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Brain Cells ◽  
Synapsin I ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Using two depolarizing agents, veratrine and high concentrations of extracellular KCl, we studied depolarization-stimulated phosphorylations in 32P-labelled dispersed brain tissue in order to identify phosphoprotein substrates for Ca2+ - and calmodulin-dependent protein kinase activity at the cellular level, for comparison with findings in cell-free preparations. In intact brain cells, the only prominent depolarization-stimulated phosphorylation was a 77 kDa protein separated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This phosphorylation was dependent on external Ca2+, since chelation of Ca2+ in media with 6 mM-EGTA or the presence of verapamil (a Ca2+ -channel blocker) in the incubation media inhibited depolarization-stimulated phosphorylation of the 77 kDa protein. Phosphorylation of the 77 kDa protein also appeared to be dependent on calmodulin, because depolarization-stimulated phosphorylation was significantly decreased (P less than 0.05) when 100 microM-trifluoperazine was present in the incubation media. Polymyxin B, an inhibitor of Ca2+- and phospholipid-dependent phosphorylation, and 12-O-tetradecanoylphorbol 13-acetate, the phorbol ester enhancing Ca2+- and phospholipid-dependent phosphorylation, had no effect on the phosphorylation of the 77 kDa protein. The 77 kDa phosphoprotein was identified as a protein previously named synapsin I [Ueda, Maeno & Greengard (1973) J. Biol. Chem 248, 8295-8305] on the basis of similar migration of native and proteolytic fragments of the 77 kDa protein with those of authentic synapsin I on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Whereas several studies with cell-free preparations showed that 57 kDa and 54 kDa endogenous phosphoproteins were the most prominent species phosphorylated in a Ca2+ and calmodulin-dependent manner, these results indicate that synapsin is the most prominent Ca2+-and calmodulin-dependent phosphorylation in intact cells. The phosphorylations of 54 kDa and 57 kDa proteins may not be as important in vivo, but instead occur as a result of the disruption of cellular integrity inherent in preparation of cell-free subfractions of brain tissue.

Phosphorylation of a bovine cardiac actin complex

1979 ◽  
Vol 236 (1) ◽  
pp. C41-C46 ◽  
Author(s):  
G. Bailin
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Atpase Activity ◽  
Gel Electrophoresis ◽  
Cardiac Myosin ◽  
Dependent Protein Kinase ◽  
Cardiac Actin ◽  
Troponin Complex ◽  
Dependent Protein ◽  
Binding Component

A bovine cardiac actin-tropomyosin-troponin complex was phosphorylated in the presence of [gamma-32P]ATP, Mg2+, adenosine 3',5'-monophosphate (cyclic AMP), and bovine cardiac cyclic-AMP-dependent protein kinase. Approximately 81% of the [32P]phosphate incorporated was identified as phosphoserine and phosphothreonine. Gel electrophoresis studies showed that 55% of the [32P]phosphate was associated with the inhibitory component of troponin (Tn-I) and 24% with a protein resembling the tropomyosin-binding component of troponin in the actin complex, respectively. The phosphorylation of Tn-I in the actin complex was inhibited 30% when Ca2+ was increased from 0.1 to 50 muM, but phosphorylation of other components was not affected by increasing Ca2+ concentration. Half-maximal calcium activation of the ATPase activity of reconstituted actomyosins made with the [32P]phosphorylated cardiac actin complex and cardiac myosin was shifted to Ca2+ values higher than those of actomyosins made with the nonphosphorylated actin complex.

scholarly journals Reversion of an S49 cell cyclic AMP-dependent protein kinase structural gene mutant occurs primarily by functional elimination of mutant gene expression.

1983 ◽  
Vol 3 (2) ◽  
pp. 250-256 ◽  
Author(s):  
T van Daalen Wetters ◽  
P Coffino
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Amino Acid ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Dibutyryl Camp ◽  
Dependent Protein Kinase ◽  
Type Function ◽  
Dependent Protein

The regulatory subunits of cyclic AMP (cAMP)-dependent protein kinase from a dibutyryl cAMP-resistant S49 mouse lymphoma cell mutant, clone U200/65.1, and its revertants were visualized by two-dimensional polyacrylamide gel electrophoresis. Clone U200/65.1 co-expressed electrophoretically distinguishable mutant and wild-type subunits (Steinberg et al., Cell 10:381-391, 1977). In all 48 clones examined, reversion of the mutant to dibutyryl cAMP sensitivity was accompanied by alterations in regulatory subunit labeling patterns. Some spontaneous (3 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (2 of 11) revertants retained mutant subunits, but these were altered in charge, degree of phosphorylation, or both. The charge alterations were consistent with single amino acid substitutions, suggesting that reversion was the result of second-site mutations in the mutant regulatory subunit allele that restored wild-type function, although not wild-type structure, to the gene product. The majority of spontaneous (8 of 11) and N-methyl-N'-nitro-N-nitrosoguanidine-induced (9 of 11) revertants and all of the revertants induced by ethyl methane sulfonate (14 of 14) and ICR191 (12 of 12) displayed only wild-type subunits. Dibutyryl cAMP-resistant mutants isolated from several of these revertants displayed new mutant but not wild-type subunits, suggesting that the revertant parent expresses only a single, functional regulatory subunit allele. The mutant regulatory subunit allele can, therefore, be modified in two general ways to produce revertant phenotypes: (i) by mutations that restore its wild-type function, and (ii) by mutations that eliminate its function.

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