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Biomedical subjects

J Larner

Publications and source records attributed to J Larner.

At least 145 records · Page 8Linked to original sources
PubMed1977

Activation of rat adipocyte glycogen synthase by insulins.

Incubation of fat cells with insulin increased glycogen synthase I activity without changing total synthase activity. This effect of insulin was dependent upon the particular lot of albumin present in the medium and was abolished by incubating cells with trypsin. Half-maximal activation of glycogen synthase was obtained with 8 microunits/ml of insulin, a concentration very similar to that which half-maximally stimulated 3-O-methylglucose uptake. The basal percentage of phosphorylase a activity was not detectably altered by insulin, although it was decreased by incubating cells with 5 mM glucose. Insulin (50 microunits/ml) markedly opposed actions of epinephrine (0.05 to 10 muM) to increase phosphorylase a activity and decrease glycogen synthase I activity, effects which were observed without glucose. Partial activation of glycogen synthase by insulin was seen after 1 min and complete activation after 4 min. Glucose alone produced a transient increase in synthase I activity. When cells were incubated with insulin plus glucose for 4 min, the increase in the percent synthase I activity was much greater than the additive effects of insulin and glucose alone. This potentiation of the effect of insulin on glucogen synthase I activity depended on the time of incubation with glucose and on the concentration of the hexose. If cells were incubated with cytochalasin B before insulin plus glucose, the effect of glucose was abolished. These results suggest that there are at least two mechanisms by which insulin can increase fat cell glycogen synthase I activity. One requires glucose and activation occurs secondary to an increase in glucose transport; where another mechanism(s) is operative even in the absence of glucose.

Adipose Tissue↗
PubMed1976

The influence of chain size and molecular weight on the kinetic constants for the span glucose to polysaccharide for rabbit muscle glycogen synthase.

The kinetic constants for the series of glucosyl acceptors for homogeneous rabbit muscle glycogen synthase I form free of glycogen were examined. The acceptors included glucose, maltose, G3, G4, G6, two hydrolyzed amyloses, amylodextrin and seven polysaccharides including amylopectin and glycogen. S0.5 and relative Vmax were estimated in each case. From these data a two site model of the enzyme is proposed, composed of a polysaccharide binding site and a separate catalytic site, the latter composed of several subsites.

Amylases↗
PubMed1976

Rabbit skeletal muscle glycogen synthase. II. Enzyme phosphorylation state and effector concentrations as interacting control parameters.

The effects of several inhibitors (ATP, ADP, AMP, UDP, and P1) and activators (Mg2+, glucose-6-P) of rabbit muscle glycogen synthase (UDP-glucose:glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11) were studied in relation to the phosphorylation state of the purified enzyme. All the modifiers had increasing effects with enzyme of increasing alkali-labile phosphate content. In experiments where combinations of effectors were present, it was apparent that (a) concentrations of modifiers in the physiological range could be significant in determining enzymic activity and (b) the sensitivity of the reaction rate to changes in phosphorylation state was critically dependent on the concentration of the small molecules. Changes in the phosphorylation of the enzyme corresponding to changes in the %I activity reported in the literature for studies in vivo were capable of producing large alterations in glycogen synthase activity. Because the magnitudes of such changes were dependent on the effector concentrations, there may be an integration of local cellular control, through small molecule effects, with hormonal control, through the phosphorylation state of glycogen synthase.

Animals↗
PubMed1976

Rabbit skeletal muscle glycogen synthase. I. Relationship between phosphorylation state and kinetic properties.

Nine samples of purified rabbit skeletal muscle glycogen synthase (UDP-glucose:glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11) were obtained with alkali-labile phosphate contents ranging from 0.27 to 3.49 residues per 85,000 molecular weight subunit. The enzyme samples appeared essentially homogeneous when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and had relatively constant specific activity under standard conditions with saturating UDP-glucose and glucose-l-P concentrations (37.1 +/- 1.0 mumol of glucose incorporated/mg/min). When the UDP-glucose concentration was varied, deviations from Michaelis-Menten kinetics were observed for all samples (Hill slopes of 0.79 +/- 0.02), but these deviations were virtually abolished by the presence of 5 mM glucose-6-P. Glucose-6-P decreased the S0.5 (concentration required for half-maximal rate) for UDP-glucose. The plots of activity increase caused by glucose-6-P versus glucose-6-P concentration became progressively more sigmoid in shape with enzyme samples of higher phosphate content. Both the S0.5 for UDP-glucose and the M0.5 for glucose-6-P (concentration for half-maximal activation) were sensitive functions of the alkali-labile phosphate content of glycogen synthase. The M0.5 increased from 3.3 muM at 0.27 phosphate/subunit to 2.7 mM at 3.5 phosphates/subunit, and the S0.5 varied from 0.75 mM (0.27 phosphate/subunit) to at least 61 mM (2.3 phosphates/subunit). Both parameters increased continuously with phosphate content, with the greatest absolute changes occurring at values greater than 2 residues of phosphates/subunit. The effects of both phosphorylation and activation by glucose-6-P appeared to be mediated primarily through alteration of the apparent affinity for UDP-glucose. With the phosphorylating conditions used, the results suggested that phosphate could not be introduced into the glycogen synthase molecule without altering its kinetic properties. Further, either the different phosphorylation sites were not equivalent, or else identical sites interacted in determining the kinetic properties of glycogen synthase.

Animals↗
PubMed1976

Actions of insulin-potentiating peptides on glycogen synthesis.

The action of urinary and synthetic AcG (acceleratory factor from growth hormone) peptides was studied in vitro and in vivo. Both peptides were inactive alone and active only in the presence of insulin to enhance glucose uptake, glycogen synthesis, and glycogen synthase conversion to the active I form in vitro and in vivo. Responses were dependent on both peptide and insulin concentrations in a dose-dependent manner. No response was obtained with glucose alone, but the presence of glucose did enhance the response of insulin alone or insulin in the presence of peptide. It is concluded that both AcG peptides enhance either the effective concentration or the activity of insulin at its site of action.

Animals↗
PubMed1976

Mechanisms of regulation of glycogen synthesis and degradation.

The control of glycogen metabolism is discussed in terms of four principles which have emerged from molecular analysis of these systems; namely, separate enzymes for synthesis and degradation, covalent controls, noncovalent controls, and the cascade concept. The extension of some of these principles into other areas, including the control of inotropic action, is considered. Finally, how these controls function in the living cell physiologically is considered in two specific examples.

Animals↗
PubMed1976

In vivo studies on the mechanism of action of the tumor inhibitor vernolepin in the Walker 256 carcinosarcoma.

Rats bearing the Walker 256 intramuscular carcinosarcoma were treated intraperitoneally with tritium-labeled vernolepin or with its nontumor-inhibitory methanol adduct. Following treatment with 3H-vernolepin on several different dosage schedules, the tumors were found to contain significantly more radioactivity per gram wet weight than control tissue (muscle from the contralateral limb). After the administration of the nontumor-inhibitory methanol adduct, no such difference was observed. The distribution of radioactivity in various other organs (liver, kidney, spleen, intestine, lung, heat, fat, blood, and brain) was measured following treatment with the parent compound (3H-vernolepin). The implications of these data in terms of the suggested mechanism of action of sesquiterpene lactone tumor inhibitors is discussed.

Animals↗
PubMed1975

Structural studies on rabbit muscle glycogen synthase. I. Subunit composition.

Essentially glycogen-free, fully converted rabbit muscle glycogen synthase I and D forms were purified to a specific activity of 30 approximately 35 units/mg, higher than that previously reported. Polyacrylamide gel electrophoresis of the synthase I and D forms in the presence of sodium dodecyl sulfate revealed two species with molecular weights of 85,000 and 81,000 (I form) and one species with molecular weight of 85,000 (D form), respectively. The 81,000 subunit of the synthase I form is a product of proteolysis, since its proportion decreased when the enzyme was purified in the presence of a proteinase inhibitor and ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Two-dimensional chromatography and high voltage electrophoretic maps of the peptides produced by exhaustive tryptic digestion of synthase I and D forms gave 78 and 85 peptides, respectively. These values agreed with the expected theoretical number of peptides assuming that the molecular weight of the enzyme subunit is 85,000 based on the analytical data of arginine and lysine content. Pro-Leu- was found as the NH2-terminal dipeptide sequence of synthase D form by the dansyl Edman method. The automated sequential degradation of synthase I form by the method of Edman provided the following NH2-terminal octapeptide sequence: Pro-Leu-Ser-Ser-Thr-Leu-Ser-Val-. The molecular weight of the subunit was also determined to be 80,000 approximately 90,000 by the quantitative analysis of phenylthiohydantoinproline. The purified synthase I and D forms each show two protein bands on gel electrophoresis in the absence of sodium dodecyl sulfate, which were also found to be enzymatically active by employing an activity stain directly on the gels. The molecular species of the I and D forms have molecular weights of 155,000 and 340,000 (I form), and 278,000 and 350,000 (D form), respectively, as measured by gel electrophoresis. Thus, the native synthase I and D forms as visualized on gels under these conditions consist of dimer and tetramer, and trimer and tetramer, respectively.

Amino Acids↗
PubMed1975

Structural studies on rabbit muscle glycogen synthase. II. Limited proteolysis.

Limited tryptic digestion of either synthase I or D forms resulted in the appearance of a new glucose 6-phosphate-dependent form which was composed of 75,000 molecular weight subunits. Early in tryptic digestion, an intermediate 78,000 subunit was also observed with both forms of the enzyme. The NH2-terminal dipeptide sequence of the 75,000 subunit of both forms was the same as that of the original 85,000 subunit (Pro-Leu-) indicating degradation at or near the COOH-terminal end without affecting the NH2-terminal end. Studies interrelating loss of organic phosphate, increase in glucose 6-phosphate dependency, and retention of the NH2-terminal sequence during limited tryptic digestion suggest that there are phosphorylation sites at or near the COOH-terminal, as well as sites within the core of the subunit, which are of importance in the synthase I to D form interconversion reaction via phosphorylation. The pathway of limited tryptic proteolysis of either synthase I or D forms was the same as judged by the molecular weights of the subunit intermediates: 85,000 leads to 78,000 leads to 75,000. A Ca2+-stimulated proteinase activity was also detected in some highly purified preparations of the synthase D form, which led to the appearance of subunits of molecular weight 78,000 and 75,000 together with phosphopeptide(s). These findings suggest that the pathway of proteolysis of the Ca2+-stimulated proteinase is similar to that of trypsin.

Amino Acid Sequence↗
PubMed1975

Kinetic studies on muscle glycogen synthase.

Using the I form of rabbit muscle glycogen synthase essentially free of glycogen, the kinetics and mechanism of action was investigated. No evidence for an exchange between [14C]UDP and UDP-glucose was found. The bisubstrate kinetics of the enzyme for UDP-glucose and glycogen, as well as for UDP-glucose and maltose, was determined. An intersecting pattern in the double reciprocal plot (velocity versus substrate concentration) suggestive of a sequential mechanism (ordered or random) was found in all cases. The K-m for UDP-glucose (45 to 48 mM) was the same with either maltose or glycogen as acceptor. The K-m for maltose (230 mM) and for glycogen (1.5 mug/ml) differed.

Animals↗
PubMed1975

Glycogen synthase can use glucose as an acceptor.

Glycogen synthase purified to homogeneity from rabbit skeletal muscle is essentially free of carbohydrate and shows no activity in the absence of added acceptor. It can use glucose as a substrate converting it to a glucose disaccharide in the presence of UDP-glucose as cosubstrate. The reaction is dependent on time, and on UDP-glucose, glucose, and enzyme concentrations. The product of the single step reaction co-chromatographs in two solvent systems with maltose. The glucose disaccharide produced in the reaction with UDP-[14-C]glucose and nonradioactive glucose as well as with nonradioactive UDP-glucoes and [14-C]glucose is labeled asymmetrically. The linkage is characterized as alpha-1,4 and therefore the disaccharide is identified as maltose.

Animals↗
PubMed1975

Effects of group-selective reagents on rabbit muscle glycogen synthase.

A series of amino acid reagents was tested on the glucose-6-P dependent D, and independent I forms of glycogen synthase (UDPG: glycogen alpha-4-glucosyltransferase, EC 2.4.I. II) from rabbit skeletal muscle, at two levels of purification. Whereas blocking of aliphatic hydroxyl groups did not result in any inhibition of the enzyme(s), blocking of aromatic hydroxyl groups resulted in a gradual and complete inhibition. Under the stated assay conditions both forms of the enzyme were similarly affected in terms of activity, but the tyrosines of the D form were found to react more readily chemically. Tyrosine appears to be "essential" for catalysis. No desensitization to the allosteric modulator glucose-6-P was detected.

Affinity Labels↗