Carlos Villar-Palasi

Substrate specificity and some other properties of baker's yeast hexokinase

Abstract The substrate specificity of bakers yeast hexokinase has been characterized by the study of the Michaelis constants and the phosphorylation rates of a score of related compounds chosen so as to represent a wide range of possibilities. It has been found that the pattern of substrate specificity is broadly similar to that of brain hexokinase, although there are several significant differences. Bakers yeast hexokinase has maximal activity at pH 7.5, with half-maximal activity at ph 5.4 and 9.4. The enzyme is more stable within the range pH 5–8. It is rapidly inactivated at temperature of 55–60°. The effects of the concentration of ATP, Mg++ and ethylendiaminetetraacetate have been studied. The course of liberation of the enzyme during autolysis and its solubility in fresh homogenates have been studied. A simplified purification is described. A very simple method for the assay of hexokinase has been developed on the basis of the photometric utilization of glucose oxidase.

Glycogen Synthase and Its Control

Publisher Summary This chapter discusses the characteristics of glycogen synthase and its control mechanisms. Glycogenic steroids in mammalian liver appear to act via insulin, as do puromycin and thyroxine in tadpole liver. Testosterone has been shown to increase weight and glycogen content of rat perineal muscle of castrate animals. The rat levator ani muscle present only in the male is a completely white muscle, which is part of the male reproductive system that is trophically dependent on androgens. The action of injected estrogen has been studied in the rat levator ani of the castrate rat. The covalent conversions act to interchange the allosteric control mechanisms of the two forms of the enzyme. In liver, insulin has both an effect to increase total phosphatase activity and to stimulate conversion of an inactive form of phosphatase to an active form. Glycogen synthase has no pyridoxal phosphate and has much more phosphate than phosphorylase.

Insulin mediator stimulation of pyruvate dehydrogenase phosphatases

A two stage assay for detecting insulin mediator based upon its stimulation of soluble pyruvate dehydrogenase (PDH) phosphatase to activate soluble pyruvate dehydrogenase complex (PDC) has been developed. This coupled assay determines the activation of PDC by monitoring production of [14C]CO2 from [1-14C]pyruvic acid. In addition to being more sensitive than the rat liver mitoplast assay previously used, it allows for the separation and investigation of the effects of mediator on the PDH phosphatases individually. It has been previously shown that the insulin mediator stimulates the most abundant PDH phosphatase, the divalent cation dependent PDH phosphatase, by decreasing the phosphatases metal requirement (1). A metal independent PDH phosphatase has been found in bovine heart mitochondria. This phosphatase is not immunoprecipitated by antiphosphatase 2A antibody, it is not inhibited by okadaic acid, and it is not stimulated by spermine. However, it is stimulated (more than threefold) by insulin mediator prepared from isolated rat liver membranes. It is inhibited by Mg-ATP, with half-maximal inhibition at 0.3 mM; however, this inhibition is overcome by the insulin mediator.

Effect of glucose phosphorylation on the activation by insulin of skeletal muscle glycogen synthase

The effect of insulin injection on skeletal muscle glycogen synthase activation was studied in anesthetized, normal, fed rats. Insulin stimulated the conversion of glycogen synthase to the active I form, increased the concentration of glucose 6-phosphate, and activated glycogen synthase phosphatase. A close correlation between glucose 6-phosphate concentrations, per cent glycogen synthase in the active I form, and phosphatase activity was found. When boiled extracts of muscle from control and insulin-injected animals were added to glycogen pellets containing phosphatase 1G, the difference in phosphatase activity between muscle extracts from insulin-injected and control rats was restored, indicating that the phosphatase was activated by heat-stable factors. Deproteinized muscle extracts from control and insulin-injected rats, at concentrations equivalent to those present in muscle, were tested for the activation of glycogen synthase by purified protein phosphatases 1 and 2A. The activation with the insulin extracts was four-fold larger than with the control extracts. When the extracts from insulin-injected rats were treated with glucose 6-phosphatase, the difference in activation with the control rat extracts was canceled. It would appear that, as in other insulin sensitive tissues, in skeletal muscle the increase in glucose 6-phosphate subsequent to the activation of glucose transport by insulin contributes to the activation of glycogen synthase.

Long-term effects of insulin on the enzyme activity and messenger RNA of glycogen synthase in rat hepatoma H4 cells: an effect of insulin on glycogen synthase mRNA stability.

Insulin induced glycogen synthase activity and decreased glycogen synthase mRNA concentrations in rat hepatoma H4 cells. Total enzyme activity measured with glucose 6-phosphate gradually increased during a 24-h insulin incubation. The time course of glycogen synthase activation measured by the activity ratio (low G-6-P/high G-6-P) in response to insulin was biphasic with the first peak at 15 min and the second peak at 4 to 6 h. When cells were incubated with insulin and cycloheximide, the first peak persisted while the second peak was abolished. These data suggest that the first activation peak derives from the classic effect of insulin via dephosphorylation and the second peak from an insulin-induced protein synthesis of a glycogen synthase activator. Ribonuclease protection assays with a cloned rat liver glycogen synthase cDNA were used to quantitate glycogen synthase mRNA. Insulin unexpectedly decreased glycogen synthase mRNA in a time- and a dose-dependent manner. After incubation with the RNA synthesis inhibitor, 5, 6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB) without and with insulin, the half time of glycogen synthase mRNA decreased from 6.0 +/- 0.80 to 3.9 +/- 0.75 h, respectively. Nuclear run-off experiments with isolated nuclei showed no change of transcription of glycogen synthase mRNA. These data suggest that insulin in this system affects glycogen synthase mRNA stability rather than transcription.

Kinetic mechanism of skeletal muscle cyclic AMP-dependent protein kinase.

Despite the importance of cyclic AMP-dependent protein kinase in the hormonal regulation of metabolism in skeletal muscle [l] , comparatively little is known about the reaction mechanism of this enzyme. We present here the results of initial velocity experiments and product inhibition studies on skeletal muscle cyclic AMP-dependent protein kinase, Peak I, or its isolated catalytic subunit. The results obtained are consistent with a random Bi Bi kinetic mechanism.

Stimulation of purified muscle protein kinase by cyclic AMP and its butyrated derivatives

Abstract 1. 1. N6,O2′-Dibutyryl AMP, N6-monobutyryl cyclic AMP and O2′-monobutyryl cyclic AMP and O2′-monobutyryl cyclic AMP are able to activate to the same maximal velocity purified mucle cyclic AMP-dependent protein kinase. The concentrations required for this maximal stimulation, however, are widely different, the most effective being the N6-monobutyryl derivative. 2. 2. The relatively low Ka for N6-monobutyryl cyclic AMP (2·10−7–3·10−7 M), Ka for cyclic AMP 8·10−8 M), the observed similarity wich cyclic AMP in its ability to dissociate the protein kinase, and the reported formation of N6-monobutyryl cyclic AMP from dibutyryl cyclic AMP in many cells strongly suggests a direct effect of the N6 derivative on the protein kinase, not involving formation of free cyclic AMP or inhibition of phosphodiesterase.

Purification and properties of troponin T kinase from rabbit skeletal muscle.

A protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) which catalyzes the phosphorylation of troponin T, phosvitin and casein has been purified over 2000 fold from rabbit skeletal muscle. The partial purification of this new enzyme, designated troponin T kinase, involves precipitation of contaminating proteins at pH 6.1, fractionation of the supernatant with (NH4)2SO4 and successive column chromatographies on DEAE-cellulose, hydroxyapatite and Sepharose 6B. The chromatographic patterns on DEAE-cellulose and hydroxyapatite columns show two peaks of troponin T kinase activity. Gel filtration experiments indicate the existence of multiple, possibly aggregated, forms of the enzyme. The purified enzyme does not catalyze the phosphorylation of phosphorylase b, troponin I, troponin C, tropomyosin, protamine, or myosin light chain 2 nor does it catalyze the interconversion of glycogen synthase I into the D form. Troponin T kinase is not affected by the addition of cyclic nucleotides or AMP to the reaction mixture. Divalent cations (other than Mg2+, required for the reaction) do not stimulate the enzyme, and several are inhibitory. Other characteristics of the reaction catalyzed by troponin T kinase, such as Km values for ATP and substrate proteins, pH optima, effect of the concentration of Mg2+, substitution of ATP for GTP have also been studied.

Inhibition by glucose 6-phosphate of cyclic AMP-dependent protein kinase phosphorylation of glycogen synthase.

Cyclic AMP-dependent protein kinase phosphorylates and inactivates glycogen synthase. In the absence of cyclic AMP, glycogen synthase is able to partially activate cyclic AMP-dependent protein kinase, probably by inducing the dissociation of the catalytic and regulatory subunits. The activation of cyclic AMP-dependent protein kinase by glycogen synthase is greatly reduced by the addition of low, physiological concentrations of the allosteric activator of glycogen synthase, glucose 6-phosphate. This effect appears to be specific for both glycogen synthase as substrate of the kinase and for cyclic AMP-dependent protein kinase as glycogen synthase phosphorylating enzyme. The result is an apparent, although not real effect of glucose 6-phosphate as an inhibitor competing with cyclic AMP. The reported inhibition by insulin of the activity of cyclic AMP-dependent protein kinase in skeletal muscle may be explained by the increased intracellular levels of glucose 6-phosphate resulting from the action of the hormone on glucose transport.

Studies on Glycogen Synthase and Its Control by Hormones

Our purpose is to present evidence on four points: 1. The multiple forms of glycogen synthase from rabbit skeletal muscle are composed of dimers, trimers and tetramers of a multiple phos-phorylated single chain of molecular weight 85,000 daltons. 2. The enzyme is converted proteolytically to a 75,000 dalton subunit by removal of peptides exclusively from the C-terminal end of the molecule. The converted enzyme is a glucose 6-phosphate-dependent (D1) form, irrespective of whether the phosphorylated or dephosphorylated form of the enzyme is degraded. 3. There is a nonlinear effect of the multiple phosphorylation of the enzyme on the conversion from I to D form resulting in a new control mechanism differing from that of phosphorylase. 4. In addition to inactivating the protein kinase by conversion from a cyclic AMP-independent to a cyclic AMP-dependent form, insulin also generates a chemical signal by forming a novel chemical inhibitor of the kinase rapidly and specifically. Together these two mechanisms constitute a fail-safe mechanism to insure synthase activation and glycogen synthesis. These latter two discoveries of our laboratory constitute at least in part the molecular mechanism of altered cyclic AMP sensitivity brought on by insulin.