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.

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 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.

scholarly journals Phosphorylation of high- and low-molecular-mass atrial natriuretic peptide analogs by cyclic AMP-dependent protein kinase

FEBS Letters ◽  
1987 ◽  
Vol 224 (2) ◽  
pp. 325-330 ◽  
Author(s):  
Gillian M. Olins ◽  
Pramod P. Mehta ◽  
Delores J. Blehm ◽  
Dennis R. Patton ◽  
Mark E. Zupec ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Atrial Natriuretic Peptide ◽  
Molecular Mass ◽  
Natriuretic Peptide ◽  
Dependent Protein Kinase ◽  
Peptide Analogs ◽  
Dependent Protein ◽  
Atrial Natriuretic

scholarly journals Antiserum against the catalytic subunit of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase. Reactivity towards various protein kinases

1980 ◽  
Vol 192 (1) ◽  
pp. 223-230 ◽  
Author(s):  
G Schwoch ◽  
A Hamann ◽  
H Hilz
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Rat Liver ◽  
Catalytic Subunit ◽  
Type Ii ◽  
Dependent Protein Kinase ◽  
Subunit C ◽  
Bovine Heart ◽  
Dependent Protein

An antiserum against the catalytic subunit C of cyclic AMP-dependent protein kinase, isolated from bovine heart type II protein kinase, was produced in rabbits. Reaction of the catalytic subunit with antiserum and separation of the immunoglobulin G fraction by Protein A-Sepharose quantitatively removed the enzyme from solutions. Comparative immunotitration of protein kinases showed that the amount of antiserum required to eliminate 50% of the enzymic activity was identical for pure catalytic subunit, and for holoenzymes type I and type II. The reactivity of the holoenzymes with the antiserum was identical in the absence or the presence of dissociating concentrations of cyclic AMP. Most of the holoenzyme (type II) remains intact when bound to the antibodies as shown by quantification of the regulatory subunit in the supernatant of the immunoprecipitate. Titration with the antibodies also revealed the presence of a cyclic AMP-independent histone kinase in bovine heart protein kinase I preparations obtained by DEAE-cellulose chromatography. Cyclic AMP-dependent protein kinase purified from the particulate fraction of bovine heart reacted with the antiserum to the same degree as the soluble enzyme, whereas two cyclic AMP-independent kinases separated from the particle fraction neither reacted with the antiserum nor influenced the reaction of the antibodies with the cyclic AMP-dependent protein kinase. Immunotitration of the protein kinase catalytic subunit C from rat liver revealed that the antibodies had rather similar reactivities towards the rat liver and the bovine heart enzyme. This points to a relatively high degree of homology of the catalytic subunit in mammalian tissues and species. Broad applicability of the antiserum to problems related to cyclic AMP-dependent protein kinases is thus indicated.

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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