A family of polypeptide substrates and inhibitors of insulin receptor kinase

Previous studies have shown that reduced carbamoylmethylated lysozyme (RCAM-lysozyme, MW approximately 14.5K) is a substrate and inhibitor (Ki approximately 0.6 microM) of insulin receptor kinase (InsRK) autophosphorylation (Kohanski & Lane, 1986; Lane & Kohanski, 1986). In this study we have prepared a family of defined modified derivatives of RCAM-lysozyme and used them to probe the nature of the substrate and inhibitory sites of InsRK. All open-chain derivatives of lysozyme in which either the tryptophanyl, methionyl, cysteinyl, arginyl, or histidyl side chains were modified served as substrates and were potent inhibitors of InsRK autophosphorylation. This was true whether the substitutions were either hydrophilic or hydrophobic, although the hydrophilic derivatives had a higher inhibitory potency. Tryptic peptides derived from RCAM-lysozyme, however, were inactive as inhibitors, and a mixture of the three cyanogen bromide fragments (containing 12, 24, and 93 amino acids, respectively) was found to be less potent in inhibiting the receptor kinase. Derivatization of either tyrosyl or carboxyl side chains produced derivatives that were neither substrates nor capable of inhibiting receptor autophosphorylation. Derivatives with modified amino groups were substrates for InsRK but were not able to inhibit InsRK autophosphorylation. The present study suggests that (a) unphosphorylated InsRK has a large hydrophilic substrate binding domain and is effectively inhibited by long-chain polypeptides but not by short sequences, (b) some of the amino, carboxyl, and hydroxyphenyl side chains are essential to the inhibitory nature of these polypeptides, and (c) derivatives that fail to inhibit autophosphorylation can still be recognized and phosphorylated by active InsRK.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of thymine dimers on DNA:DNA hybridization

DNA from bacteriophage T7 was irradiated at long ultraviolet wavelengths in the presence of silver ions. Such treatment leads to selective production of thymine: thymine dimers in DNA. The DNA was melted and the renaturation rate was determined as a function of thymine dimer content and renaturation temperature. Under “normal” hybridization conditions little change in the renaturation rate was observed even when 30% of the thymine was dimerized. This result is consistent with the view that up to a 15% change in the primary sequence of DNA dose not appreciably change the renaturation rate.     

Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin

A factor (ARF) that is required for the cholera toxin-dependent ADP-ribosylation of the stimulatory, GTP-binding regulatory component (Gs) of adenylate cyclase has been purified about 2000-fold from cholate extracts of rabbit liver membranes. ARF is an intrinsic membrane protein with Mr = 21,000. The final product can be resolved into two polypeptides with very similar molecular weights; each of these has ARF activity. The ADP-ribosylation of Gs can now be studied with defined components. GTP and ARF are both necessary cofactors. The data imply that the substrates for the activated toxin are NAD and a GTP X Gs X ARF complex, and the reaction proceeds in a lipid environment. The apparent ability of ARF to bind to the alpha subunit of Gs suggests that it may play another, unknown role in the regulation of adenylate cyclase activity.     

The Visceral Adiposity Syndrome in Japanese-American Men

Japanese-Americans have an increased prevalence of non-insulin-dependent diabetes mellitus and coronary heart disease when compared to native Japanese. This increase has been associated with fasting hyperinsulinemia, hypertriglyceridemia, and low plasma levels of high-density lipoprotein (HDL) cholesterol. The purpose of this study was to examine the relationship of both visceral adiposity and insulin resistance to this metabolic syndrome and to the presence of a predominance of small, dense low-density lipoprotein (LDL) particles (LDL subclass phenotype B) that has been associated with increased atherogenic risk. Six Japanese-American men with non-insulin-dependent diabetes, each receiving an oral sulfonylurea, were selected. One or 2 nondiabetic Japanese-American men, matched by age and body mass index, were selected for each diabetic subject, giving a total of 9 nondiabetic men. Diabetic subjects had significantly higher fasting plasma glucose (p=0.0007) and lower insulin sensitivity (S_I, p=0.018) using the minimal model technique than nondiabetic subjects matched for body mass index. Six men (2 with diabetes) had LDL phenotype A and 8 (4 with diabetes) had phenotype B. One nondiabetic subject had an intermediate low-density lipoprotein pattern. Significantly greater amounts of intra-abdominal fat (p=0.045) measured by computed tomography were found in the men with phenotype B while fasting insulin (p=0.070) and triglycerides (p=0.051) tended to be higher. Intra-abdominal fat was significantly correlated with S_I (r=-0.559), plasma triglycerides (r=0.541), plasma free fatty acids (r=0.677), LDL density (relative flotation rate, r=-0.803), and plasma HDL-cholesterol (r=-0.717). S_I was significantly correlated only with plasma free fatty acids (r=-0.546) and tended to be correlated with hepatic lipase activity (r=-0.512, p=0.061). In conclusion, these observations indicate that in non-obese Japanese-American men, the metabolic features of the so-called insulin resistance syndrome, including LDL phenotype B, are more strongly correlated with visceral adiposity than with S_I. It may therefore be more appropriate to call this the visceral adiposity syndrome. Although questions concerning mechanisms still remain, we postulate that visceral adiposity plays a central role in the development of many of the metabolic abnormalities, including LDL subclass phenotype B, that occur in this metabolic syndrome.     

Human granulocyte 6 phosphogluconate dehydrogenase. Purification by elective elution with NADP+, immunological and kinetic properties.

Human granulocyte 6 phosphogluconate dehydrogenase has been totally purified from a single patient with chronic granulocytic leukaemia. 48 mg of protein, of specific activity 20 IU per mg of protein, have been obtained in the course of three different steps only. The overall yield was 30 p. cent and the purification was 100 folds. Purified 6 phosphogluconate dehydrogenase was homogeneous when tested in acrylamide and acrylamide SDS gel electrophoresis or in immunodiffusion. The enzyme was immunologically identical in red blood cells, blood platelets and normal leukocytes. The fixation of both substrates, NADP-+ and 6 phosphogluconate, seemed to proceed through a non ordered mechanism. NADPH was an inhibitor strictly competitive with respect to NADP-+ and non competitive with respect to 6 phosphogluconate. 2-3 Diphosphoglycerate seemed to be able to bind on both the fixation sites of NADP-+ and 6 phosphogluconate. The inhibition by ATP was competitive with 6 phosphogluconate and non competitive with NADP-+. 6 phosphogluconate dehydrogenase was inactivated by SH reagents and was partially protected against this inactivation by both substrates. Both substrates protected the enzyme against thermal inactivation. The influence of ionic strength, pH and ions have been studied, and the results have been compared to those reported by other authors for erythrocyte enzyme.     

Autoantibodies to the insulin receptor activate glycogen synthase in rat adipocytes

Summary Autoantibodies to the insulin receptor mimic the effects of insulin on glycogen synthase and phosphorylase. The interaction of antibodies with adipocyte cell surface insulin receptors seems sufficient to promote stable changes in the activities of these intracellular enzymes, suggesting that internalization or processing of insulin is not important in the generation of these biological responses.