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D Sarkar

Publications and source records attributed to D Sarkar.

At least 91 records · Page 5Linked to original sources
PubMed1986

Identification and regulation of tissue plasminogen activator activity in rat cumulus-oocyte complexes.

Plasminogen activators convert plasminogen into plasmin, a serine protease that initiates extracellular proteolysis. Two types of plasminogen activator activities have recently been demonstrated in granulosa cells, and the proteolysis-inducing enzymes are believed to be involved in ovulation. However, little attention has been paid to the presence of these enzymes in oocytes. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique, we studied plasminogen activator activity in oocytes. Denuded oocytes collected from ovaries of hypophysectomized, estrogen-treated immature rats contained a tissue-type plasminogen activator (tPA), but not urokinase (uPA). In contrast, oocyte-free granulosa cells in these preantral follicles contained uPA, but not tPA. The tPA activity found in oocytes was plasminogen-dependent; incubation with increasing numbers (25-200) of denuded oocytes resulted in a dose-dependent increase in fibrinolysis only in the presence of plasminogen. Cellular localization of tPA was studied in the preantral follicles using an immuno-cytochemical method. Positive tPA staining was detected in the cytoplasm, but not in the germinal vesicle or zona pellucida of the oocytes. Furthermore, analysis using a reverse fibrin-overlay method did not reveal the presence of a plasminogen activator inhibitor. Culturing of denuded oocytes for 24 h increased the cellular content of tPA, but the enzyme activity was not further enhanced by treatment with FSH or forskolin. Also, no tPA activity was detected in the medium. We further studied plasminogen activator activities in the cumulus-oocyte complexes. Although only tPA activity was detected in freshly obtained cumulus-oocyte complexes, incubation for 24 h increased both tPA and uPA activity. Furthermore, tPA, but not uPA, activity was stimulated by treatment with FSH or forskolin. This was accompanied by the secretion of tPA into the medium. The identity of tPA and uPA in the cumulus-oocyte complexes was further confirmed by immunoprecipitation with specific antibodies. Isolation of denuded oocytes and cumulus cells after hormonal stimulation of the cumulus-oocyte complexes suggested that tPA activity was stimulated in both cell types and that the cumulus cells may mediate the action of FSH and forskolin on oocytes. In conclusion, the detection and regulation of tPA activity in cumulus-oocyte complexes suggest possible involvement of this enzyme in ovulation or the process of cumulus cell expansion and dispersion. Changes in oocyte tPA content may also serve as an indicator of oocyte development.

Animals↗
PubMed1984

Identification of a calmodulin-binding protein that co-purifies with the regulatory subunit of brain protein kinase II.

Highly purified preparations of cytosolic brain RII contain a tightly associated polypeptide with a Mr of 75,000 (P75). When purified brain and heart RII were preincubated with Ca2+ and 125I-calmodulin and then were subjected to polyacrylamide gel gelectrophoresis under nondenaturing conditions a major calmodulin-binding component was found only in the brain RII sample. The calmodulin-binding activity exhibited a higher sedimentation coefficient (9 S) than free RII (5 S) indicating that it might be a complex of P75 and RII dimers. This possibility was investigated using two specific monoclonal antibodies. Western blot analyses revealed that monoclonal antibody 918 exclusively bound to P75 in the brain RII preparation while monoclonal antibody 107 complexed RII. The calmodulin-binding component was noncovalently labeled with 125I-calmodulin and separated from excess free RII by polyacrylamide gel electrophoresis under nondenaturing conditions. The identity of the polypeptides comprising the binding protein was subsequently established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Polypeptides with Mrs values of 55,000 and 75,000 that bound monoclonal antibodies 107 and 918, respectively, were observed. Thus the calmodulin-binding component in brain RII preparations is a complex containing an RII dimer and one or two molecules of P75. P75 was also present in high concentrations in Triton X-100 extracts prepared from cerebral cortex and liver membranes. P75 is phosphorylated by both cAMP-dependent and calcium-phospholipid-activated protein kinases.

A Kinase Anchor Proteins↗
PubMed1984

Tryptic peptide mapping studies on the regulatory subunits of type II protein kinases from cerebral cortex and heart. Evidence for overall structural divergence and differences in the autophosphorylation and cAMP-binding domains.

Regulatory subunits of type II cAMP-dependent protein kinases (RII) (EC 2.7.1.37) from bovine brain and heart exhibit similar physicochemical and functional properties in vitro. However, the two forms of RII are markedly different in their (a) antigenic determinants, (b) cell and tissue distribution, and (c) subcellular localization. This suggests that each of these cAMP-binding proteins may possess some unique structural features. To assess the degree of overall divergence between the primary structures of brain RII and heart RII, tryptic peptides derived from the two proteins were mapped by reverse phase HPLC on a C18 column. When the column effluent was monitored at 280 nm, 15 peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. More complex HPLC profiles were observed by following peptide absorbance at 210 nm, but a similar level of diversity was apparent: 13 brain-RII-specific and 15 heart-RII-specific tryptic peptides were identified and resolved with a gradient (0-50%) of acetonitrile in 0.1% trifluoroacetic acid. In complementary experiments, classical two-dimensional mapping analyses revealed that several 32P-labeled tryptic fragments derived from autophosphorylated and photoaffinity-labeled brain RII were separate and distinct from the 32P-peptides isolated from similarly treated heart RII. The HPLC mapping data document a structural basis for the immunological disparity between brain RII and heart RII and suggest that the two cAMP-binding proteins are different proteins rather than interconvertible forms of a single protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Activation of cyclic AMP-dependent protein kinase isoenzymes: studies using specific antisera.

A novel method for rapidly determining the amount and degree of association-dissociation of the Type I and Type II cAMP-dependent protein kinases has been developed and validated. Antibodies directed against the regulatory subunits of Type I and Type II cAMP-dependent protein kinases were used. The antibodies formed complexes with holoenzymes and regulatory subunits which were precipitated by goat anti-rabbit IgG (immunoglobulin G). These complexes bound [3H]cAMP with an apparent Kb of 20 nM for protein kinase I and 80 nM for protein kinase II. Immunoprecipitated protein kinases I and II were catalytically active when incubated with cAMP, [gamma-32P]ATP, and histone H2B. When mixtures of the two kinase isoenzymes or cytosol were incubated with various amounts of [3H]cAMP and the isoenzymes were separated by precipitation with antisera specific for each isoenzyme, the amount of [3H]cAMP associated with immunoprecipitates was proportional to the concentration of [3H]cAMP. In contrast, the catalytic activity that was immunoprecipitated varied inversely with the concentration of [3H]cAMP, showing that the activation of protein kinase could be assessed by the disappearance of catalytic activity from the immunoprecipitates. In the absence of MgATP protein kinase I was activated by a 10-fold lower concentration of cAMP than protein kinase II. However, when MgATP was added to the incubation, there was no significant difference in the binding of [3H]cAMP or dissociation of catalytic subunits of the two isoenzymes. The anti-R antibodies were also used to rapidly quantitate the concentration of regulatory subunits and the relative ratio of protein kinases I and II in tissue cytosols.

Adenosine Triphosphate↗
PubMed1983

Theophylline concentrations in the emergency treatment of acute bronchial asthma.

Ninety-three patients presenting with acute asthma to the emergency department were studied to determine theophylline levels and their relationship to airway obstruction, history of prior medication use, and side effects of treatment. The mean pretreatment theophylline level was 6.4 micrograms/ml. Patients on long-acting preparations had significantly higher levels than those on short-acting medications (p less than 0.05). The mean post-treatment theophylline level was 16.7 micrograms/ml. Twenty-three patients had toxic levels post-treatment but none of these had a major adverse reaction. Twenty of these patients had been taking long-acting preparations. Fifty percent of the patients with symptoms of gastrointestinal toxicity had theophylline levels below 15 micrograms/ml. There was no correlation between the theophylline level or change in level and the degree of airway obstruction as measured by pulmonary function testing. Clinical findings are not reliable predictors of theophylline levels. Patients taking long-acting theophylline products should receive a lowered loading dose.

Acute Disease↗