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Biomedical subjects

J Larner

Publications and source records attributed to J Larner.

At least 37 records · Page 2Linked to original sources
PubMed1978

Ca2+-stimulated phosphorylation of muscle glycogen synthase by phosphorylase b kinase.

Phosphorylase b kinase from rabbit muscle phosphorylates glycogen synthase purified from the same tissue. The reaction is markedly stimulated by Ca2+ and results in a decrease in the synthase %I activity. Phosphorylase b kinase action leads to the incorporation of phosphate (0.6 to 0.8 mol/mol of subunit) preferentially into a single cyanogen bromide fragment of synthase (fragment III). Cyclic AMP-independent synthase kinase also shows a specificity for the site(s) contained in fragment III whereas the cyclic AMP-dependent protein kinase exerts a preference for the site(s) located in a distinct cyanogen bromide fragment (fragment II). A Ca2+-stimulated endogenous kinase also results in the phosphorylation of fragment III and can be attributed to the presence of phosphorylase b kinase. The finding of a Ca2+-stimulated phosphorylation of glycogen synthase has important implications for the regulation of glycogen metabolism and particularly those processes thought to be controlled by cytoplasmic Ca2+ concentration.

Animals↗
PubMed1977

Activation of liver guanylate cyclase by bile salts and contaminants in crude secretin and pancreozymin preparations.

Crude preparations of secretin or pancreozymin increased and at higher concentrations decreased guanylate cyclase (GTP pyophosphate-lyase, EC 4.6.1.2) activity from soluble and particulate fractions of rat liver homogenates. Partially purified and synthetic secretin were without effect as was the biologically active octapeptide fragment of pancreozymin. The active contaminants in these preparations survived boiling, saponification, and treatment with phospholipase A, trypsin and neuraminidase C. The activity was extractable with chloroform/methanol and did not survive ashing. Eight bile salt contaminants in crude secretin were obtained with thin-layer chromatography. Two of the contaminating bile salts that increased liver particulate guanylate cyclase activity were identified as taurodeoxycholate and either glycochenodeoxycholate or glycodeoxycholate; taurocholate was inhibitory. The sodium salts of cholate, deoxycholate, chenodeoxycholate and their glycine-or taurine-conjugated forms either increased or decreased particulate and soluble rat liver guanylate cyclase activity depending upon their concentration. Thus, the previously reported stimulatory and inhibitory effects of secretin and pancreozymin preparations on guanylate cyclase activity are probable attributable to their bile salt contaminants.

Animals↗
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↗