The insulin-receptor cycle was investigated in cultured foetal rat hepatocytes by determining the variations in insulin-binding sites at the cell surface after short exposure to the hormone. Binding of 125I-insulin was measured at 4 degrees C after dissociation of prebound native insulin. Two protocols were used: exchange binding assay and binding after acid treatment; both gave the same results. Cell-surface 125I-insulin-receptor binding decreased sharply (by 40%) during the first 5 min of 10 nM-insulin exposure (t1/2 = 2 min) and remained practically constant thereafter; subsequent removal of the hormone restored the initial binding within 10 min. This fall-rise sequence corresponded to variations in the number of insulin receptors at the cell surface, with no detectable change in receptor affinity. The reversible translocation of insulin receptors from the cell surface to a compartment not accessible to insulin at 4 degrees C was hormone-concentration- and temperature-dependent. SDS/polyacrylamide-gel electrophoresis after cross-linking of bound 125I-insulin to cell-surface proteins with disuccinimidyl suberate showed that these variations were not associated with changes in Mr of binding components, in particular for the major labelled band of Mr 130,000. The insulin-receptor cycle could be repeated after intermittent exposure to insulin. Continuous or intermittent exposure to the hormone gave a similar glycogenic response, contrary to the partial effect of a unique short (5-20 min) exposure. A relationship could be established between the repetitive character of the rapid insulin-receptor cycle and the maximal expression of the biological effect in cultured foetal hepatocytes.
RICHARD^Strain CIP 82.01 of Vibrio alginolyticus and strain 1.029 (previously designated "Achromobacter iophagus") were compared. Strain 1.029 produces a collagenase of high specific activity (Achrornobacter collagenase; EC 3.4.24.8). Collagenase production is induced in strain CIP 82.01 by collagen or macromolecular fragments of collagen in a manner similar to collagenase induction in strain 1.029; caseinolytic proteinase is constitutive. In this study we demonstrate that both strains also produce a constitutive extracellular endonuclease. Collagenases from both strains cleave either native collagen in its helical region or a similar synthetic peptide; both enzymes are inhibited by ethylenediamine tetraacetate, but not by diisopropyl fluorophosphate. The collagenase subunit (molecular weight, 35,000) of strain CIP 82.01 is similar in amino acid composition to the subunit of the strain 1.029 enzyme, although some of the aspartic and threonine residues in strain CIP 82.01 are replaced by glutamic and serine residues in strain 1.029. Surface radioiodination followed by two-dimensional electrophoresis showed that there are quantitative differences in the major outer membrane proteins of the two strains. Strains CIP 82.01 and 1.029 differ qualitatively in resistance to ampicillin and carbenicillin, in cellobiose fermentation, in ornithine decarboxylase activity, and in halophilism. We propose that strain 1.029, which was originally designated A . iophagus, be included within the species V. alginolyticus, but that this organism be distinguished from other strains of this species by the designation Vibrio alginolyticus chemovar iophagus, with the corresponding collagenase designated "iophagus collagenase" (EC 3.4.24.8).In 1973, Welton and Woods isolated and described the gram-negative aerobic bacterium Achromobacter iophagus (34). In the presence of collagen or macromolecular fragments of collagen (15), a subculture of this organism (strain 1.029) produces a collagenase having a very high specific activity (12,20). The structural and functional properties of this Achromobacter collagenase (EC 3.4.24.8), as well as the metabolism of strain 1.029, have been described in a series of papers by Woods et al. (21,26,27,29,35) and by workers in our laboratory (4,10,11,13,14,19,30).In a recent study (27), which confirmed the original observation of collagenase induction by macromolecular collagen fragments (15), Achromobacter iophagus was reclassified as a strain of Vibrio alginolyticus; however, a direct experimental comparison of strain 1.029 with a welldefined wild-type strain of V . alginolyticus has been lacking. It is well established that the other strains of the Vibrionaceae (23, 31) can be induced (by collagen and fragments of collagen) to produce collagenolytic and gelatinolytic enzymes.We undertook a comparative study to determine the differences between strain 1.029 isolated from a random selection of cured hides in South Africa (34) and a wild-type strain (strain CIP 82.01) of V . alginolyticus from a different ge...
Sequential changes in the numbers of cell-surface receptors induced by a transitory exposure to insulin in cultured 18-day foetal-rat hepatocytes were investigated in the presence of drugs and at a temperature of 22 degrees C, which inhibit cellular insulin degradation. Chloroquine (70 microM) and monensin (3 microM) did not greatly change the initial rate of internalization of cell-surface receptor sites after exposure to 10 nM-insulin, but led to a steady state after 20 min, which represented 40% of the initial binding, compared with 5 min and 60% in the absence of the drug. Moreover, these drugs strongly decreased the proportion of receptor sites recovered at the cell surface after subsequent removal of the hormone. They were ineffective when insulin was not present. The removal of monensin together with the hormone allowed partial restoration of cell-surface receptor sites and degradation of cell-associated insulin to start again at the initial speed, indicating a reversible effect of the drug. During this phase, the drug concentration-dependence for the two effects showed that receptor recycling was restored with concentrations of monensin not as low as for insulin degradation. The effect of vinblastine (50-100 microM) was similar to that of chloroquine and monensin, whereas no modification in the internalization and recovery processes was observed in the presence of bacitracin concentrations (1-3 mM) that inhibit insulin degradation by 70%. A temperature of 22 degrees C did not prevent the receptor internalization, but had a slowing effect on the recycling process, which appeared to vary in experiments where insulin degradation remained inhibited. The present study shows that the process of insulin degradation mediated by receptor endocytosis is not a prerequisite for insulin-receptor recycling in cultured foetal hepatocytes.
Over the past 10 years, more than 40 potentially oncogenic genes, termed protooncogenes, have been identified in the human genome. Little is known of the physiological role of the proteins encoded by these genes, but they seem to be involved in the reception and transmission of hormonal and other environmental information from the cell membrane to the nucleus. These proteins may acquire transforming properties when over-expressed or if structurally altered following partial deletions or point mutations. Cytogenetic analysis shows loss of genetic material from specific chromosomal loci in many human tumors, suggesting that the absence of a functional gene at these loci may permit tumor development. The genes involved have been termed "anti-oncogenes". Understanding the control mechanisms of cell proliferation is essential in order to understand how cancer cells escape from this control. To this end, numerous oncogenes have been cloned, permitting the production of modified forms of oncogenic proteins and identification of the regions essential for their biological activity. Availability of large amounts of protein also allows the production of specific antibody which can be used to verify whether blockage of a given protein results in reversion of the transformed phenotype. If it can be shown that the expression of an oncogenic protein is essential for transformation, it should be possible to search for molecules that inhibit its action or which mimic the effects of an anti-oncogene. This type of research is already well advanced for the oncogenic ras proteins, and models have been established that permit both screening for potential inhibitors and design of specific antagonists.
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