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M H Cobb

Publications and source records attributed to M H Cobb.

122 records · Page 7Linked to original sources
PubMed1983

Phosphorylation activates the insulin receptor tyrosine protein kinase.

Preparations of insulin receptor from cultured 3T3-L1 adipocytes and human placenta previously was found to catalyze the phosphorylation of the 90,000-dalton component of the insulin receptor on tyrosine residues. This insulin-dependent phosphorylation has now been shown to coincide with the generation of an activated, insulin-independent, receptor protein kinase. Activation is dependent upon ATP, divalent cations (Mg2+ and Mn2+), and insulin (half-maximal activation occurs at 6-8 nM insulin). The time required for activation is consistent with that needed for insulin-dependent self-phosphorylation of the receptor present in eluates from wheat germ lectin-agarose columns and in preparations of affinity-purified placental receptor. Activation proceeds unabated in the presence of soybean trypsin inhibitor at 0.1 mg/ml and the activated, insulin-independent, protein kinase sediments in 5-20% sucrose gradients at the same position as the unmodified receptor. Under steady-state conditions, the phosphorylated receptor binds insulin in the same fashion as the unmodified receptor. It is proposed that the self-phosphorylated form of the receptor is the insulin-activated protein kinase that catalyzes the phosphorylation of exogenous protein and peptide substrates. A corollary of this hypothesis is that enzymatic dephosphorylation may be essential for reversibly terminating the activity of the insulin-receptor protein kinase.

Adenosine Triphosphate↗
PubMed1982

Mineralocorticoid-induced membrane proteins in MDCK cells.

The Madin-Darby canine kidney (MDCK) cell line, which exhibits properties indicative of a distal tubule origin, evidently binds and responds to mineralocorticoid hormones. We investigated the effects of aldosterone and deoxycorticosterone on protein synthesis in MDCK cells grown either in medium supplemented with serum or in a hormonally defined, serum-free medium. Aldosterone induced the synthesis of at least 2 membrane proteins with molecular weights of 35000 and 14000. The MDCK line may prove a useful model system for examining the mechanism of mineralocorticoid-regulated sodium transport and, in particular, the identification and study of hormone-induced proteins in a homogeneous cell population.

Aldosterone↗
PubMed1982

Insulin activates a tyrosine-specific protein kinase in extracts of 3T3-L1 adipocytes and human placenta.

Insulin activates a tyrosine-specific cAMP-independent protein kinase when added directly to detergent extracts of differentiated 3T3-L1 adipocytes and humal placental membranes. The kinase is also activated by antibody to the insulin receptor and, to a lesser extent, by proinsulin. It catalyzes the phosphorylation of the 92,000-dalton component of the insulin receptor, histone, and casein; in each case, tyrosine is the principal amino acid modified. Under the conditions used to activate the kinase, insulin does not affect the rate of dephosphorylation of the receptor or of histone. The insulin-activated kinase is copurified with the human placental insulin receptor until the final elution from insulin-Sepharose. It remains to be established whether the kinase and the insulin receptor are separate molecules.

Adipose Tissue↗
PubMed1982

Structural and conformational properties of peptides interacting with the glutathione receptor of hydra.

We have made a detailed investigation of structure-activity relationships of the glutathione-induced feeding response in the fresh water coelenterate, Hydra attenuata, to map structural and conformational properties of feeding response agonists. We find that the gamma-glutamyl residue of glutathione contains essential binding sites for receptor interaction, without which antagonistic as well as agonistic properties are lost. Any structural alteration which perturbs either the alpha-amino or the alpha-carboxyl group or their relative spatial orientations within the peptide has yielded an inactive derivative. An absolute requirement for activation of the receptor is a second-residue side chain of the appropriate size; analogues with second-residue side chains too large or too small are inhibitory. On the basis of the activity of conformational analogues of glutathione, torsional angles for the second residue equal to those of a right-handed alpha-helix are compatible with stimulus generation.

Animals↗
PubMed1981

Insulin-induced proteins in the toad urinary bladder.

Insulin increases active sodium transport by the toad urinary bladder within 15 min, an effect which persists for an least 20 h. In tissues pre-treated with inhibitors of transcription or translation, sodium transport briefly increases after insulin addition but returns to basal levels within 60-90 min. We have studied the effects of insulin on the incorporation of radioactive amino acids into mucosal cell proteins. Insulin had no detectable effect on the uptake of amino acids nor on their incorporation into total protein; however, using a dual label technique, we found that insulin increases the incorporation of amino acids into specific soluble and plasma membrane proteins of the granular mucosal cell. The time course and dose-response relationship of the induction of protein synthesis by insulin suggest that these proteins may play a role in the sustained elevation of sodium transport by insulin.

Amino Acids↗
PubMed1981

Insulin stimulates the phosphorylation of ribosomal protein S6 in a cell-free system derived from 3T3-L1 adipocytes.

Physiological concentrations of insulin stimulate the incorporation of 32P into ribosomal protein S6 in intact 3T3-L1 adipocytes (Smith, C. J., Wejksnora, P. J., Warner, J. B., Rubin, C. S., and Rosen, O. M. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2725-2729). Extracts from cells treated with insulin also exhibit an increased ability to incorporate 32P from [gamma-32P]ATP into protein S6 in vitro (Smith, C. J., Rubin, C. S., and Rosen, O. M. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2641-2645). We now report that the direct addition of insulin to particulate preparations containing high affinity insulin receptors and ribosomes derived from insulin-sensitive cells (not previously exposed to insulin) stimulates incorporation of 32P from [gamma-32P]ATP into ribosomal protein S6 1.5- to 3.0-fold. The stimulation occurs at the same concentrations of insulin (0.1-1.0 nM) and within the same period of time as it does in intact cells.

Adipose Tissue↗
PubMed1981

Role of induced proteins in insulin-stimulated sodium transport.

Insulin increases active sodium transport by the toad urinary bladder, an effect that begins with 15 minutes and persists for at least 20 hours. Although pretreatment of bladders with inhibitors of RNA or protein synthesis has no effect on the response to insulin within the first hour, these agents block the long-term component of insulin-stimulated sodium transport. To examine the relationship of protein synthesis to the sustained increase in sodium transport elicited by insulin, we have studied the effects of the hormone upon the incorporation of radioactive amino acids into mucosal cell proteins. There is no detectable effect of insulin on the uptake of aminoisobutyric acid into mucosal cells or on the incorporation of labeled precursors into total protein; however, using a dual-label technique, we find that insulin increases the incorporation of amino acids into specific soluble and plasma-membrane proteins of granular mucosal cells. Insulin has no discernible effect upon the incorporation of amino acids into proteins of mitochondria-rich mucosal cells. Thus the effects of insulin upon sodium transport appear to be the result of two separate mechanisms, (1) a short-term response that is independent of protein synthesis and (2) a long-term response that is expressed after the first hour of hormone treatment and that requires the synthesis of one or more specific proteins in the granular cell.

Amino Acids↗
PubMed1981

Insulin-induced alterations in the lactoperoxidase-catalyzed radioiodination of membrane proteins of the toad bladder epithelium.

Insulin-stimulated sodium transport in the toad urinary bladder consists of two components, a brief element of rapid onset that is independent of protein synthesis, and a sustained increase, slower in onset, that is dependent upon RNA and protein synthesis. The mucosal epithelium of the toad bladder was labeled by lactoperoxidase-catalyzed radioiodination (125I) following 15 min and 3 h exposure to insulin. The membrane of "mitochondria-rich" and "granular" mucosal cells from these tissues were analyzed by electrophoresis in SDS-urea. Compared to untreated tissues, membranes of "granular" mucosal cells from tissues exposed to insulin for 15 min contained a band (Mr = 15,000) with significantly increased labeling. Bladders exposed to insulin for 3 h showed no consistent increase in labeling. These data suggest that there are differences in the conformation of apical membrane proteins during the two phases of hormone-induced sodium transport. The technique may also offer an opportunity to identify "effector" proteins mediating this and other insulin responses.

Animals↗
PubMed1981

Effects of insulin on ribonucleic acid synthesis in toad urinary bladder.

We previously found that the sustained rise in active sodium transport by the toad urinary bladder after exposure to insulin is associated with the synthesis of a membrane protein with a molecular weight of 25,000. Studying the effects of insulin on the incorporation of uridine into RNA of epithelial cells of the bladder, we now report that insulin causes a 39% increase in the incorporation of [3H]uridine into poly(A)-containing RNA extracted from granular cells, the predominant epithelial cell type. The increased accumulation of label in RNA, most notably species ranging from 5-10S, could be demonstrated whether cells were pulsed with [3H]uridine or continuously exposed to the label. Using a reticulocyte lysate system, we compared the translation products of RNA from untreated and insulin-treated granular mucosal cells. Exposure to insulin was associated with the expression of a mRNA coding for a protein with molecular weight of approximately 25,000, similar in size to the insulin-induced membrane protein we previously identified in the intact tissue.

Animals↗
PubMed1980

Transporting renal epithelium: culture in hormonally defined serum-free medium.

A hormonally defined medium was used to isolate a homogeneous epithelioid cell population from canine kidney. Monolayers of these cells form domes, an indication of active ion transport, and this process is inhibited by ouabain. This technique allows the isolation of primary cultures of renal epithelial cells, free of fibroblasts, for the characterization of biochemical and physiological properties related to renal function.

Animals↗
PubMed1978

Interaction of glutathione analogues with Hydra attenuata gamma-glutamyltransferase.

gamma-Glutamyltransferase activity was studied in extracts of the cnidarian Hydra attenuata. The binding of gamma-glutamyl peptide analogues to the enzyme was studied by observing their effects on heat denaturation and their inhibition of p-nitroaniline release from gamma-glutamyl p-nitroanilide. Neither position-1 analogues, in which the gamma-glutamyl moiety was changed to a beta-aspartyl (beta-Asp-Abu-Gly) or an alpha-glutamyl (Glu-Abu-Gly) linkage, nor glutamate protected the enzyme against inactivation at 58 degrees C. GSH (reduced glutathione), gamma-Glu-Abu-Gly and gamma-Glu-Met on the other hand did prevent heat denaturation. GSH and analogues of GSH were competitive inhibitors of p-nitroaniline release, but those analogues in which glycine was replaced by 2-aminoisobutyrate, phenylalanine, leucine or tyrosine had Ki values that were approximately five times those of analogues with the cysteine residue replaced.

Amino Acids↗