Guillermo Romero

Insulin mediators and the control of pyruvate dehydrogenase complex.

Just a year after insulin was discovered in Toronto by virtue of its hypoglycemic effect, its discoverers found that this hormone increased both glycogen and fat in the organs of animals injected with it. We now know at the molecular level that glycogen synthase and pyruvate dehydrogenase complex (PDC), the first rate-limiting enzymes in the respective metabolic pathways involved in these effects, are each activated by insulin-induced dephosphorylation.2 The discovery by Linn, Pettit, and Reed3 that PDC was controlled by covalent phosphorylation in a manner analogous to glycogen synthase had two important effects: ( 1 ) it broadened the significance of covalent phosphorylation beyond the boundaries of glycogen metabolism, where it had been contained up to that time, to include lipid metabolism, and (2) it was the second example, following our work on glycogen synthase? of a biosynthetic enzyme activated by dephosphorylation and inactivated by phosphorylation. These early examples, together with the prior examples of the activation of phosphorylase and phosphorylase b kinase by covalent phosphorylation, led to the current generalization that biosynthetic enzymes are activated by dephosphorylation, but degradative enzymes are activated by phosphorylation. This mechanism provides simultaneous fail-safe control of both synthesis and degradation.

The generation of inositolglycan mediators from rat liver plasma membranes: The role of guanine nucleotide binding proteins

The guanine nucleotide dependence for the generation of inositolglycan second messengers from rat liver plasma membranes has been investigated. Plasma membranes, when treated with insulin release a soluble mediator substance which activates pyruvate dehydrogenase (PDH). Guanosine 5-[3-thio]triphosphate (GTP gamma S) was found to be as potent as insulin in stimulating mediator release. The stimulatory effects of GTP gamma S required the presence of magnesium and following preincubation of membranes with guanosine 5-[2-thio]diphosphate (GDP beta S) the stimulation of mediator release by either insulin or GTP gamma S was blocked. The activation of PDH by mediator fractions produced in response to either insulin or GTP gamma S was abolished following treatment of the fractions with anti-inositolglycan antibodies. The significance of these observations with respect to the possible involvement of a regulatory guanine-nucleotide binding protein (G-protein) in the generation of insulin mediators is discussed.

A simple procedure for the preparation and purification of the oligosaccharide components of the glycosyl-phosphatidylinositol anchor of membrane proteins

The oligosaccharide components of the glycosylphosphatidylinositol anchors of Trypanosoma brucei variant surface glycoproteins have been prepared and purified by treatment with hydrolytic enzymes and solvent extraction procedures followed by HPLC purification using a specific oligosaccharide binding matrix (Glyco-Pak N, by Waters). Three oligosaccharide peaks (peaks I, II and III) were resolved by a single isocratic HPLC step (70% acetonitrile in water). The material from these peaks was hydrolyzed in acid and analyzed by GC/MS. GC/MS analysis of the material obtained from each peak demonstrated the presence of inositol, glucosamine, and mannose in a 1:1:3 ratio. A variable number of galactose residues were detected in each peak. The galactose:inositol ratios of the purified components were 1:1, 2:1, and 3:1 for peaks I, II and III, respectively, suggesting that the separation obtained depends primarily on the number of sugar residues present in each fraction.