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R A Jungmann

Publications and source records attributed to R A Jungmann.

At least 37 records · Page 2Linked to original sources
PubMed1985

Subcellular distribution of protein kinase C of GH3 cells: quantitation and characterization by polyacrylamide gel electrophoresis.

Quantitative estimation of cytosolic Ca2+- and phospholipid-dependent protein kinase (PKC) activity was performed by polyacrylamide gel electrophoresis under nondenaturating conditions (PAGE). With this method less than 50 micrograms of cytosol protein can be accurately quantitated for PKC activity. The amount of cytosolic PKC activity recovered after PAGE was comparable to the amount obtained by DEAE-cellulose chromatography. Homogenization of GH3 cells in the presence of 2 mM EGTA/EDTA revealed that 80% of the total cellular PKC activity resided in the cytosol. However, omission of the ion chelator during cell disruption followed by subcellular fractionation and extraction of subcellular fractions by EDTA/EGTA showed that 80% of the total PKC was found in the lysosomal-mitochondrial and microsomal extracts. Detailed analysis of PKC activities demonstrated that cytosolic PKC was identical with the PKC solubilized by EDTA/EGTA from subcellular fractions. In conclusion, GH3 cells appear to contain one species of PKC with an apparent molecular weight of 90,000 which seems to be associated with membranes via a calcium-dependent mechanism (or mechanisms).

Animals↗
PubMed1985

Isolation and characterization of a cDNA and a pseudogene for mouse lactate dehydrogenase-A isozyme.

A mouse lactate dehydrogenase-A cDNA was isolated and it was shown to contain the 393bp of the protein-coding sequence and 488bp of the 3' untranslated region. The amino acid sequence deduced from its open reading frame provided independent evidence for the sequence of residues 201-331 of mouse LDH-A subunit (muscle). This cDNA clone was used as a probe to isolate a mouse genomic clone containing a truncated, processed LDH-A pseudogene. This pseudogene showed 81.6% homology at 713 positions compared with the LDH-A cDNA sequence. The divergence of this pseudogene was estimated to have occurred 39 million years ago.

Animals↗
PubMed1985

The phosphoform of the regulatory subunit RII of cyclic AMP-dependent protein kinase possesses intrinsic topoisomerase activity.

The phosphoform of the type II regulatory subunit (phospho-RII-cAMP) of cAMP-dependent protein kinase from rat liver was found to possess intrinsic topoisomerase activity towards several DNA substrates such as phi X174, pBR322, SV40, and M13. Like the type I topoisomerases from several eukaryotic cells, phospho-RII X cAMP can relax both positive and negative superhelical turns of phi X174 DNA. Topological isomers with a decreasing number of superhelical turns can be identified as transient products. Conditions under which phospho-RII X cAMP relaxes superhelical phi X174 DNA lead to transient formation of a DNA-phospho-RII X cAMP complex via DNA strand breakage and covalent attachment of the DNA to a tyrosine residue of phospho-RII X cAMP via a phospho-RII X cAMP depends on the presence of cAMP and is altered by changes in the degree of phosphorylation of RII. Both dephosphorylation and removal of cAMP from phospho-RII X cAMP abolish its topoisomerase activity.

Animals↗
PubMed1985

Localization of nuclear subunits of cyclic AMP-dependent protein kinase by the immunocolloidal gold method.

An immunocolloidal gold electron microscopy method is described allowing the ultrastructural localization and quantitation of the regulatory subunits RI and RII and the catalytic subunit C of cAMP-dependent protein kinase. Using a postembedding indirect immunogold labeling procedure that employs specific antisera, the catalytic and regulatory subunits were localized in electron-dense regions of the nucleus and in cytoplasmic areas with a minimum of nonspecific staining. Antigenic domains were localized in regions of the heterochromatin, nucleolus, interchromatin granules, and in the endoplasmic reticulum of different cell types, such as rat hepatocytes, ovarian granulosa cells, and spermatogonia, as well as cultured H4IIE hepatoma cells. Morphometric quantitation of the relative staining density of nuclear antigens indicated a marked modulation of the number of subunits per unit area under various physiologic conditions. For instance, following partial hepatectomy in rats, the staining density of the nuclear RI and C subunits was markedly increased 16 h after surgery. Glucagon treatment of rats increased the staining density of only the nuclear catalytic subunit. Dibutyryl cAMP treatment of H4IIE hepatoma cells led to a marked increase in the nuclear staining density of all three subunits of cAMP-dependent protein kinase. These studies demonstrate that specific antisera against cAMP-dependent protein kinase subunits may be used in combination with immunogold electron microscopy to identify the ultrastructural location of the subunits and to provide a semi-quantitative estimate of their relative cellular density.

Animals↗
PubMed1985

Ultrastructural localization of cAMP-dependent protein kinase subunits in regenerating rat hepatocytes using immunogold electron microscopy.

We have used the indirect colloidal immunogold technique to examine the ultrastructural localization and distribution of the regulatory subunits (RI and RII) and catalytic subunit (C) of cAMP-dependent protein kinase (cA-PK) in hepatocyte nuclei during the prereplicative phase of rat liver regeneration. The technique allowed the localization of all three subunits of cA-PK in hepatocyte nuclei. Morphometric quantitation of the relative staining intensity of nuclear antigen indicated a marked increase of immunogold staining of nuclear RI and C 16 hours following partial hepatectomy. In contrast, staining of nuclear RII was not affected. These immunocytochemical observations correlate well with our previous data (Laks, M.S. et al. (1981) J. Biol. Chem. 256, 8775-8785) obtained by biochemical methods indicating a modulation of cA-PK activity after partial hepatectomy.

Animals↗
PubMed1985

cAMP-dependent protein kinases of rat pituitary GH3 cells.

Two isoenzymes (type I and type II) of cAMP-dependent protein kinases were found in GH3 cytosol, isozyme type II activity being the predominant form (approximately 90%). Photoaffinity labeling of GH3 cell extracts with 8-N3-[32P]cAMP revealed three cAMP-binding proteins exhibiting molecular weights of 53,000, 51,000 and 48,000, respectively. The latter represents the regulatory subunit of type I isoenzyme whereas the 53,000 and 51,000 cAMP-binding proteins correspond to two different molecular forms of the type II isoenzyme regulatory subunit which are phosphorylated by a cAMP-dependent mechanism.

Animals↗
PubMed1984

Phosphorylation of rat C6 glioma cell DNA-dependent RNA polymerase II in vivo. Identification of phosphorylated subunits and modulation of phosphorylation by isoproterenol and N6,O2'-dibutyryl cyclic AMP.

Evidence is presented that isoproterenol treatment of rat C6 glioma cells, under conditions that increase glioma cell cAMP levels, causes the phosphorylative modification of several RNA polymerase II subunits. RNA polymerase II in control and isoproterenol-stimulated 32Pi-labeled confluent glioma cells was immunoprecipitated from ribonuclease-treated nuclear extracts with hen anti-calf RNA polymerase II antiserum conjugated to Sepharose. The immunoprecipitated RNA polymerase II was analyzed for 32P-labeled subunits by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Using this technique, we have shown that isoproterenol causes a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 214,000, 180,000, 140,000, 35,000, 28,000, and 16,500 daltons. Phosphate incorporation occurred exclusively on serine in all of the six subunits. About 0.5-2 mol of phosphate/mol of RNA polymerase II subunit were incorporated. Dibutyryl cAMP (10(-3)M) mimics the stimulatory action of isoproterenol and mediates increased phosphate incorporation into the six subunits. (RS)-propranolol (10(-4)M) prevents the isoproterenol-mediated phosphorylative changes. These data indicate that isoproterenol, via cAMP, mediates a transient structural modification of RNA polymerase II subunits in rat C6 glioma cells which may possibly lead to a modulation of RNA polymerase II function(s).

Amino Acids↗
PubMed1984

Localization of cyclic AMP-dependent protein kinase subunits in rat hepatocyte nuclei by immunogold electron microscopy.

We have applied the indirect colloidal immunogold technique to examine the ultrastructural localization of the catalytic subunit C and the regulatory subunits RI and RII of cyclic AMP-dependent protein kinase in rat hepatocyte nuclei before and after glucagon or dibutyryl cyclic AMP administration. The technique allowed the identification and localization of all three subunits in hepatocyte nuclei. Morphometric quantitation of the relative staining density of nuclear subunits indicated an increase of immunogold staining of nuclear catalytic subunit but not of the regulatory subunits after glucagon or dibutyryl cyclic AMP stimulation. The increase of catalytic subunit occurred in a biphasic manner with peak levels 2-30 min and 90-150 min after stimulation. Our experiments represent the first reported use of the immunogold procedure to identify and localize protein kinase subunits in the nucleus.

Animals↗
PubMed1984

Modulation of soluble ovarian adenosine 3',5'-monophosphate-dependent protein kinase activity during prepubertal development of the rat.

Studies were conducted to evaluate the ontogeny of cAMP-dependent protein kinases in the soluble fraction of rat ovaries obtained throughout prepubertal ovarian maturation (days 5-37). Protein kinase activity stimulated by cAMP and inhibited by the heat-stable protein kinase inhibitor, designated cAMP-dependent protein kinase activity, was already present in ovaries of 5-day-old rats. This kinase activity subsequently increased to reach maximal values between days 16 and 18, then plunged 4-fold to a nadir between days 21 and 23. cAMP-dependent protein kinase activity began to rise again by day 24, increasing 5-fold to a maximum on day 27. Experiments were conducted to evaluate the basis for the 80% reduction of cAMP-dependent protein kinase activity in the soluble extract of ovaries of 21- to 23-day-old rats. To determine whether the decrease in kinase activity was accompanied by a concomitant reduction of cAMP-binding activity, the ability of ovarian cytosol to bind [3H]cAMP was evaluated. Results showed that at the time of decreased cAMP-dependent protein kinase activity, total soluble cAMP-binding activity was not significantly reduced. Additionally, the cAMP-binding activity of the regulatory subunits RI and RII (regulatory subunits of the type I and II isoenzyme forms of cAMP-dependent protein kinase), as detected by photoaffinity labeling with 8-N3-[32P] cAMP, was not changed. To determine whether the decline in kinase activity was due to an actual disappearance of the catalytic subunit from the soluble ovarian extracts of ovaries of 21- to 23-day-old rats, the relative amounts of catalytic subunit were quantified by an enzyme-linked immunosorbent assay using an antiserum directed against bovine heart catalytic subunit. Results showed that the decrease in protein kinase catalytic activity was not due to a reduction in the amount of catalytic subunits. Experiments were conducted to determine whether the reduction of protein kinase catalytic activity in unfractionated cytosol was expressed after anion exchange chromatography. Results showed that the estimated total cAMP-stimulated protein kinase activity measured after DEAE-cellulose chromatography of the soluble ovarian extracts of 21- to 23-day-old rats was no longer depressed. Since the 80% reduction of catalytic kinase activity in ovarian extracts of 21- to 23-day-old rats was detectable in unfractionated cytosol before but not after DEAE-cellulose chromatography, we tested for the presence of an endogenous inhibitor of catalytic kinase activity in the cytosol fraction.

Aging↗
PubMed1983

Cyclic AMP regulation of lactate dehydrogenase. Isoproterenol and N6,O2-dibutyryl cyclic amp increase the rate of transcription and change the stability of lactate dehydrogenase a subunit messenger RNA in rat C6 glioma cells.

The mechanism of isoproterenol and N6,O2-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cAMP) induction of lactate dehydrogenase A subunit mRNA (mRNALDH) was investigated in the rat C6 glioma cell line. During the induction phase the concentration of nuclear mRNALDH sequences increased about 2.5-fold 4 h after the addition of isoproterenol or dibutyryl cAMP. Analysis of nuclear 32P-labeled mRNALDH sequences showed that isoproterenol or dibutyryl cAMP increased the basal rate of in vitro mRNALDH transcription about 3.6-fold within 4 h. The relative rates of in vivo mRNALDH synthesis were additionally measured by pulse-labeling of glioma cells for 15 min with [3H]uridine. The induction of mRNALDH in intact glioma cells by isoproterenol and dibutyryl cAMP was quantitatively comparable to that observed in isolated nuclei and the relative rate of [3H]uridine incorporation into mRNALDH was maximal 4 to 5 h after the initial induction stimulus. Increased synthesis of mRNALDH in vivo as well as in isolated nuclei occurred only at isoproterenol concentrations that caused elevated levels of glioma cell cAMP. Analysis of the kinetics of decay of [3H]uridine-labeled mRNALDH showed a linear rate of decay of non-induced mRNALDH with a t1/2 of 45 min. After isoproterenol stimulation mRNALDH decayed as two populations, one with a t1/2 of 50 min and the other one with a t1/2 of 2.5 h. These results indicate that both isoproterenol and dibutyrl cAMP regulate not only the rate of transcription of mRNALDH but that the stability of mRNALDH is increased during the induction phase.

Animals↗
PubMed1983

Ca2+-calmodulin-dependent phosphorylation of soluble and nuclear proteins in the rat ovary.

Studies were undertaken to determine if calmodulin-regulated Ca2+-dependent protein kinase system(s) exist in the prepubertal rat ovary. Phosphorylation studies were performed with ovarian cytosol, calmodulin-depleted cytosol, and nuclear extracts. In vitro phosphorylation of endogenous substrate proteins was accomplished by incubation of tissue fractions with [gamma-32P]ATP followed by electrophoretic separation and autoradiographic demonstration of phosphorylated proteins. Calmodulin-dependent phosphorylations of a cytosol protein, mol wt, 95,000, and of three nuclear proteins in the mol wt range of 50,000-60,000, were established by demonstrating: Ca2+ requirement; inhibition by the phenothiazine derivative chlorpromazine; and dependence upon addition of exogenous calmodulin to the calmodulin-depleted cytosol, or to the nuclear extract. These findings demonstrate the presence of rat ovarian cytosol and nuclear Ca2+-calmodulin-dependent protein kinase activities capable of recognizing endogenous substrate proteins.

Animals↗
PubMed1982

Characterization of cyclic 3':5'-amp-dependent protein kinase in sarcoplasmic reticulum and cytosol of canine myocardium.

Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic cAMP-dependent and Ca2+ -calmodulin-dependent protein kinase (EC 2.7.1.37) activities and a common substrate, phospholamban, for these enzymes. Cyclic AMP-dependent protein kinase associated with sarcoplasmic reticulum membranes was solubilized with Triton X-100. Solubilization of the sarcoplasmic reticulum protein kinase did not affect its dependency on cAMP or its substrate specificity. The solubilized cAMP-dependent protein kinase was purified by DEAE-cellulose chromatography and was characterized as a type II enzyme on the basis of its elution at high ionic strength. DEAE-purified cAMP-dependent protein kinase exhibited no Ca2+ -calmodulin-dependent protein kinase activity. Cytosol from canine cardiac muscle cells, chromatographed on DEAE-cellulose under conditions identical to those used with sarcoplasmic reticulum, exhibited the presence of both type I and type II cAMP-dependent protein kinase isozymes. The properties of the DEAE-cellulose purified type II protein kinases from sarcoplasmic reticulum and cytosol were similar. We conclude that cardiac sarcoplasmic reticulum contains primarily type II cAMP-dependent protein kinase and this is probably the enzyme which phosphorylates sarcoplasmic reticulum in vivo and regulates Ca2+ transport.

Affinity Labels↗