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Biomedical subjects

R L Mellgren

Publications and source records attributed to R L Mellgren.

At least 55 records · Page 3Linked to original sources
PubMed1985

Subcellular localization of bovine heart calcium-dependent protease inhibitor.

Four monoclonal antibodies for a calcium-dependent protease inhibitor protein were produced by fusing Sp2/0 myeloma cells with spleen cells from a Balb/C mouse immunized with purified bovine heart inhibitor. Each of the monoclonal antibodies was highly specific for the inhibitory protein as revealed by electro-blot analysis. The antibodies recognized different antigenic sites on CNBr peptides prepared from the purified inhibitor protein. Immunofluorescent microscopy of sections from bovine heart ventricles treated with each of the antibodies demonstrated the same fluorescent pattern. Fluorescence was observed at or near the sarcolemma of the myocytes, and along the Z-discs of relaxed myofibrils within the myocytes. Contracted myofibrils did not appear to bind antibody. Immunostaining of glycerinated relaxed cardiac myofibrils revealed staining at the Z-discs. One of the antibodies could also stain the Z-disc region of bovine skeletal muscle myofibrils.

Animals↗
PubMed1984

Dephosphorylation of phosphorylase kinase by a histone H1-stimulated phosphoprotein phosphatase.

A histone H1-stimulated protein phosphatase isolated from rabbit skeletal muscle dephosphorylated [32P]phosphorylase kinase. The rate of dephosphorylation was markedly increased by 5-50 micrograms histone H1/ml. Only the alpha and alpha' subunits were dephosphorylated in the absence or presence of histone H1. This is consistent with previous results suggesting that the H1-stimulated phosphatase is a type-2 protein phosphatase. The present studies suggest that the histone H1-stimulation is the result of a direct interaction of the histone with the phosphatase.

Animals↗
PubMed1983

Histone H1-stimulated phosphorylase phosphatase from rabbit skeletal muscle.

A major rabbit skeletal muscle phosphorylase phosphatase activity which is markedly stimulated by histone H1 has been resolved from inhibitor-sensitive phosphorylase phosphatase (type-1 phosphatase), glycogen synthase kinase 3-activated phosphatase, phosphatase heat-stable inhibitor proteins, and alkaline phosphatase activity by various purification techniques. Evidence is presented that this phosphatase is a high-molecular weight form of a type-2 phosphatase. Our data suggest that this phosphatase may be regulated by histone H1, protamine or analogous polycationic compounds.

Animals↗
PubMed1983

Evidence that the heat-stable protein activator of phosphorylase phosphatase is histone H1.

The heat-stable protein activator of phosphorylase phosphatase [FEBS Lett. 146, 331-334 (1982)] was located in the nuclear fraction. The activator was isolated from the nuclear fraction of various tissues. The largest amount was obtained from calf thymus and swine renal cortex. The activator protein from swine renal cortex was purified to apparent homogeneity. The activator had the same mobility as authentic histone H1 on sodium dodecyl sulfate or acetic acid/urea polyacrylamide gel electrophoresis. Phosphorylase phosphatase was activated by histone H1 or purified activator in an identical manner. Other histones were without effect or inhibited phosphorylase phosphatase activity. It is concluded that the heat-stable protein activator of phosphorylase phosphatase is histone H1.

Animals↗
PubMed1983

Coordinated feedback regulation of muscle glycogen metabolism: inhibition of purified phosphorylase phosphatase by glycogen.

An assay method was devised for accurately measuring the activity of phosphorylase phosphatase in the presence of glycogen. Both oyster glycogen and rabbit liver glycogen inhibited the activity of purified phosphorylase phosphatase catalytic subunit in a concentration-dependent manner. In the presence of 120 mM KCl, micromolar AMP increased the sensitivity of the phosphatase to glycogen inhibition. The present studies suggest that glycogen feedback inhibition may be a coordinated mechanism producing a decrease in phosphorylase phosphatase activity as well as glycogen synthase phosphatase activity.

Adenosine Monophosphate↗
PubMed1983

The protein inhibitor of calcium-dependent proteases: purification from bovine heart and possible mechanisms of regulation.

A bovine heart protein which specifically inhibits calcium-dependent proteases has been purified to near homogeneity. The purified inhibitor had a Stokes radius of 6.8 nm estimated by gel filtration and a molecular weight of 145,000 estimated by sodium dodecyl sulfate-gel electrophoresis. There is evidence that it may be a glycoprotein. The inhibitor could be phosphorylated by bovine heart cyclic AMP-dependent protein kinase, and its inhibitory effect on Peak II (high-calcium-requiring) protease was modestly increased. However, no other phosphorylating or dephosphorylating conditions significantly influenced its activity. The inhibitor was not hydrolyzed by calcium-dependent proteases, but it was very sensitive to proteolytic inactivation by trypsin or proteases present in a lysosomal fraction from rat heart. Thus, proteolysis may represent a mechanism for decreasing the activity of the inhibitor in different physiologic or pathologic conditions.

Animals↗
PubMed1982

Rabbit skeletal muscle calcium-dependent protease requiring millimolar CA2+. Purification, subunit structure, and Ca2+-dependent autoproteolysis.

Rabbit skeletal muscle calcium-dependent protease which requires millimolar Ca2+ concentration for activity is a dimer composed of Mr = 73,000 and 30,000 subunits. The subunit structure has been confirmed by co-elution of the two polypeptide bands on Sephadex G-200 chromatography, and by cross-linking the enzyme with dimethyl suberimidate followed by sodium dodecyl sulfate-disc gel electrophoresis. Preincubation of the enzyme with p-chloromercuribenzoate prior to nondenaturing electrophoresis resulted in dissociation of the subunits. The rabbit muscle calcium-dependent protease was rapidly inactivated when incubated in the presence of 6 mM Ca2+. The half-life of protease activity at 30 degrees C was independent of protease concentrations over the range of 0.005 to 0.272 mg/ml. The rate of inactivation was not affected by a 270-fold molar excess of a substrate protein succinylated lysozyme. Protease activity also rapidly decreased (t1/2 = 8.4 min) during the assay at 37 degrees C as determined by a decrease in linearity of the time course when substrate was limiting. The rate of protease inactivation during the assay was essentially the same as that observed when the protease was incubated at 37 degrees C in the absence of substrate (t1/2 = 7.2 min). The addition of either leupeptin or the calcium-dependent protease inhibitor protein from dog heart prevented protease inactivation. The protease displayed an increase in activity during the time course of autoproteolysis at 30 degrees C when activity was measured in the presence of 0.2 mM Ca2+ instead of 5 mM Ca2+.

Animals↗
PubMed1982

Foraging in a simulated natural environment: There's a rat loose in the lab.

Rats were required to earn their food in a large room having nine boxes placed in it, each of which contained food buried in sand. In different phases of the experiment the amount of time allowed for foraging, the amount of food available in each food patch, and the location of the different available amounts were varied. The rats exhaustively sampled all patches each session but seemed to have fairly strong preferences for certain locations over others. If position preferences were for patches containing small amounts of food, the sensitivity to amount available was increased so that when location was compensated for, a pattern of optimal foraging was evident. The importance of environmental constraints in producing optimal behavior and the relation of the observed behavior to laboratory findings are discussed.

Journal Article↗
PubMed1982

Rabbit kidney cortex phosphorylase phosphatases: evidence for complexes between high molecular weight forms and heat-stable inhibitor proteins.

Two forms of high molecular weight phosphorylase phosphatase have been partially resolved by gel filtration chromatography of rabbit kidney cortex extracts. Two heat-stable inhibitor proteins co-eluted with the phosphatase peaks. Phosphorylase phosphatase and heat-stable inhibitor activity also co-migrated on gel electrophoresis of cortex extracts. When extracts were heated to 95 degrees for 5 minutes prior to gel filtration or electrophoresis, phosphorylase phosphatase inhibitor activity eluted at a lower molecular weight and a higher mobility, respectively. Storing cortex extracts at -20 degrees for 6 months resulted in partial conversion of both phosphatase and inhibitor activities to lower molecular weight forms which co-eluted on gel filtration. The two inhibitor peaks from gel filtration chromatography were heat-treated and characterized. Both inhibitor peaks had molecular weight of 25,000 to 35,000. The inhibitory activity of one of the peaks was increased about 3.5-fold by incubation with cyclic AMP-dependent protein kinase and ATP, and required higher concentrations of TCA to be precipitated. Hence, one of the inhibitor peaks resembled rabbit muscle inhibitor -1, while the other peak may represent an inhibitor similar to rabbit muscle inhibitor -2. These results represent the first indication that low molecular weight heat-stable inhibitor proteins may be bound to high molecular weight phosphorylase phosphatases in the cell.

Animals↗
PubMed1980

Phosphorylation of heart glycogen synthase by cAMP-dependent protein kinase. Regulatory effects of ATP.

Glycogen synthase I, purified from bovine heart, had a specific activity of 33 units/mg and gave a single band on sodium dodecyl sulfate gel electrophoresis with a subunit molecular weight of 86,000. The enzyme was phosphorylated with cAMP-dependent protein kinase catalytic subunit, also isolated from heart. With 10 microM ATP, only one phosphate group was incorporated per subunit of glycogen synthase. The phosphorylation decreased the per cent of glycogen synthase I from 0.95 to 0.50 when activity was determined by assays with Na2SO4 and glucose 6-phosphate. Glycogen synthase containing one phosphate per subunit was designated GS-1. One additional phosphate was incorporated per synthase subunit when ATP was increased to 0.5 mM and the percent glycogen synthase I decreased from 0.50 to < 0.05. This enzyme form was designated GS-1,2. Conversion of GS-1 to Gs-1,2 gave cooperative kinetics with ATP concentration and a half-maximal stimulation at approximately 40 microM. Phosphorylation of GS-1 could also be achieved by adding other non-substrate nucleotide triphosphates such as ITP and UTP along with 10 microM ATP. Glucose-6-P and Na2SO4 were without effect on this phosphorylation reaction. Two separate peptides were obtained after CNBr cleavage of 32P-labeled GS-1,2 and only one from GS-1. Both enzyme forms contained a single phosphorylated peptide in common. Thus, heart glycogen synthase may be phosphorylated specifically in either of two different sites using appropriate concentrations of ATP. ATP acts as a substrate for the protein kinase and also affects the availability of a second site to phosphorylation by cAMP-dependent protein kinase.

Adenosine Triphosphate↗