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Adenylate cyclase activity was examined as a measure of inhibitory guanine nucleotide binding protein (Gi) function in liver plasma membranes from rats made chemically diabetic by streptozotocin (STZ) treatment. Clonidine activation of the alpha 2 adrenergic receptor, which activates Gi, inhibited forskolin--stimulated adenylate cyclase activity in control membranes. However, there was no effect on adenylate cyclase activity in membranes from STZ diabetic animals. Also, a polyclonal antipeptide antibody was raised to a highly conserved segment of the Gi alpha 2 subunit. This antibody specifically recognizes a 41 kilodalton protein, is blocked by an excess of peptide, does not recognize the alpha-subunit of transducin, and immunoprecipitates a 41 kilodalton protein which was ADP-ribosylated by pertussis toxin. Immunoblots using this antibody detect no difference between normal and STZ diabetic animals in the level of liver plasma membrane Gi expression. Therefore, STZ-induced diabetes altered the function of Gi but had no effect on Gi expression.

The apparent molecular size of circulating human plasma renin is 43,000 daltons. In this report, the Stokes' radius of renin extracted from human kidney cortex in low ionic strength buffer in the presence of protease inhibitors was shown to correspond to an apparent molecular weight of 58,000 +/- 3,000. If protease inhibitors are omitted, if the kidney tissue is frozen and thawed multiple times, or if the kidney extract is acidified to pH 2.8, renin activity of an apparent molecular size of 42,000 +/- 3,000 can be identified. Both species of renin bind to an affinity support bearing the substrate analog inhibitor His-Pro-Phe-His-Leu-D-Leu-Val-Tyr. Antibody raised to the higher molecular form of the enzyme inhibits the activity of both forms in an equivalent manner. These observations suggest that the larger form of renin may be a biosynthetic precursor of plasma renin, either in the form of a zymogen or an enzyme-binding protein complex.

A plasmid expression vector encoding human insulin-like growth factor I (hIGF-I) in the form of a 97-amino acid precursor protein containing the first 27 amino acids of prebovine GH and the 70 amino acids of hIGF-I has been used to transform mouse L cells. A stably transformed mouse L cell clone has been isolated which expresses and secretes hIGF-I. The secreted peptide comprises 3% of the protein in conditioned medium. IGF-I can be purified to homogeneity in 2 chromatographic steps. One liter of conditioned medium yields approximately 200 micrograms purified peptide. Amino-terminal sequence analysis confirms that the signal peptide has been proteolytically hydrolyzed from the precursor protein before secretion to form [Ala0]hIGF-I. The recombinant peptide and serum-derived hIGF-I are equipotent as inhibitors of the binding of [125I]IGF-I to the type 1 receptor of human placenta and to a crude preparation of acid-stable human serum binding proteins. The peptides are equipotent in 2 in vitro assays, the stimulation of the rate of 2-[1,2-N-3H]deoxyglucose transport in BC3H1 cells and the stimulation of [methyl-3-3H]thymidine incorporation into DNA in A10 cells. In contrast to a control mouse L cell line, DNA synthesis in the [Ala0]IGF-I-secreting line is completely unresponsive to [Thr59]IGF-I, while it responds normally to calf serum (10%). Thus, the [Ala0]IGF-I-secreting line is selectively desensitized to IGF-I. The binding of [125I]IGF-I to both lines is identical, indicating that the loss of responsiveness to IGF-I is not due to a loss of cell surface receptor. The ability to render mouse L cells unresponsive to IGF-I is transferred in the conditioned medium of the [Ala0]IGF-I-secreting cell line. In addition, pretreatment of control cells with [Thr59]IGF-I (10 nM) results in attenuation of the response to a subsequent dose of IGF-I. These data indicate that prolonged exposure to high levels of IGF-I may cause a postreceptor-mediated desensitization to IGF-I. Alternatively, IGF-I may promote secretion of an inhibitor of IGF-mediated DNA synthesis.

The in vivo administration of sodium orthovanadate stimulated the incorporation of [14C]glucose into [14C] glycogen, in a dose- and time-dependent manner, in mouse diaphragm. Activation of diaphragm insulin receptor was measured by exogenous tyrosine kinase activity and an antibody that recognizes a conformational change in the receptor beta-subunit upon autophosphorylation. Neither method detected insulin receptor activation by in vivo vanadate administration, suggesting that vanadate's insulin-mimetic effect on mouse diaphragm glycogenesis occurs at a site distal to the insulin receptor.