Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Insulin induces a rapid activation of p21ras in NIH 3T3 and Chinese hamster ovary cells that overexpress the insulin receptor. Previously, we suggested that p21ras may mediate insulin-induced gene expression. To test such a function of p21ras more directly, we studied the effect of different dominant inhibitory mutants of p21ras on the induction of gene expression in response to insulin. We transfected a collagenase promoter-chloramphenicol acetyltransferase (CAT) gene or a fos promoter-luciferase gene into NIH 3T3 cells that overexpressed the insulin receptor. The activities of both promoters were strongly induced after treatment with insulin. This induction could be suppressed by cotransfection of two inhibitory mutant ras genes, H-ras(Asn-17) or H-ras(Leu-61,Ser-186). In particular, insulin-induced activation of the fos promoter was inhibited completely by H-ras(Asn-17). These results show that p21ras functions as an intermediate in the insulin signal transduction route leading to the induction of gene expression.
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PMID:Two dominant inhibitory mutants of p21ras interfere with insulin-induced gene expression. 165 21

Photoaffinity labelling of hepatic insulin receptors revealed specifically-labelled bands of 130, 90 and 40 kDa. Endogenous protease activity in hepatic plasma membranes, as well as contaminating proteases present in preparations of clostridial collagenase, degraded some of the 130-kDa insulin-binding subunit to a 115-kDa form. However, a large proportion of the 130-kDa subunits were resistant to degradation, suggesting the presence of two classes of insulin receptor in hepatic plasma membranes. In one class the 130-kDa subunit was sensitive to proteolysis, while in the other it was not. In contrast, the 130-kDa receptor subunits of adipose tissue were all resistant to such degradation. Scatchard analysis of control and collagenase-treated plasma membranes demonstrated that conversion of the 130-kDa subunit to a 115-kDa form did not affect the insulin-binding characteristics of the receptor. It was also apparent that insulin binds to a single class of high-affinity sites in hepatic plasma membranes.
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PMID:Photoaffinity labelling of hepatic plasma membranes suggests two classes of hepatic insulin receptor. 299 14

Hepatocytes or hepatic plasma membranes were photoaffinity-labelled with radioiodinated N epsilon B29-monoazidobenzoyl-insulin. Analysis of the samples by SDS/polyacrylamide-gel electrophoresis and autoradiography revealed the insulin receptor as a predominant band of 450 kDa. When hepatic plasma membranes were first treated with clostridial collagenase and then photolabelled, the insulin receptor appeared as a predominant band of 360 kDa. This effect of collagenase treatment on the insulin receptor was due to Ca2+-dependent heat-labile proteinases contaminating the preparation of collagenase, and it could be mimicked by elastase. The decrease in size of the insulin receptor to 360 kDa resulted from the loss of a receptor component that was inaccessible to photolabelling. In contrast, the size of the insulin receptor of intact cells was not affected by collagenase treatment. This suggests that the site sensitive to proteolysis was located on the cytoplasmic side of the plasma membrane. In hepatic plasma membranes that were treated with collagenase or elastase, and contained the 360 kDa form of the insulin receptor, the binding affinity for insulin was increased by up to 2-fold. These findings support the concept that a component which is either a part of, or closely associated with, the insulin receptor may regulate its affinity for insulin.
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PMID:The structure of the hepatic insulin receptor and insulin binding. 302 65

It is the objective of the in vitro studies reported herein to further evaluate the role of insulin in the regulation of ovarian androgen biosynthesis, to assess its dose requirements, and to elucidate the cellular mechanism(s) underlying its high dose action. To this end, use was made of recently developed primary culture systems of ovarian androgen-producing cells, the differentiation of which is subject to regulation by gonadotropic and insulinotropic signaling. Treatment of collagenase-processed whole ovarian dispersates or highly enriched (greater than 90%) thecal-interstitial cells from immature rats with insulin (1 microgram/ml) or hCG (1 ng/ml), resulted in 1.5- and 2.6-fold increments in the accumulation of androsterone (3 alpha-hydroxy-5 alpha-androstan-17-one), the main androgenic steroid identified in culture medium by HPLC. However, combined treatment with both agents unmasked a synergistic interaction resulting in 5.7-fold amplification of hCG action, the increase in androsterone accumulation representing enhanced biosynthesis rather than diminished degradation. Unaccounted for by cellular growth and independent of the cellular density of plating (1 X 10(4)-1 X 10(6) viable cells/culture) or the hCG dose (0.1-10 ng/ml) employed, the insulin effect proved time and dose dependent with a minimal time requirement of 72 h. [125I-TyrA14]Iodoinsulin binding to untreated highly enriched thecal-interstitial cells proved highly specific, saturable, and reversible, displaying a single class (Hill coefficient = 0.93 +/- 0.07) of high affinity (Kd = 1.7 X 10(-10) M), low capacity (4746 +/- 283 sites/cell) binding sites. Treatment with physiological concentrations (10 ng/ml) of insulin produced limited, albeit measureable, down-regulation of the insulin receptor. In contrast, provision of relatively high concentrations (1 microgram/ml) of insulin resulted (despite marked adsorption/degradation) in substantial (greater than 60%) down-regulation of the insulin receptor, but not the type I insulin-like growth factor receptor, the ligand of which has also been shown to amplify hCG-supported androgen biosynthesis. These findings suggest that the thecal-interstitial cell is a site of insulin reception and action, that physiological concentrations of insulin are capable of participating in the regulation of ovarian androgen biosynthesis, and that this effect is probably mediated via high affinity insulin receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Insulin as a regulator of androgen biosynthesis by cultured rat ovarian cells: cellular mechanism(s) underlying physiological and pharmacological hormonal actions. 328 73

We have analyzed the receptors for epidermal growth factor (urogastrone) (EGF-URO) and insulin in primary cultures of adult rat hepatocytes maintained for up to 3 weeks on human placental cell matrix in serum-free defined medium. Cross-link labeling experiments revealed that the insulin receptor, partially damaged by the collagenase isolation procedure, was rapidly regenerated to yield an intact receptor. In contrast, cross-link labeling of the EGF-URO receptor revealed that, upon prolonged culture, there was a progressive disappearance of the high molecular mass (175 kilodaltons (kDa)) receptor form, and an appearance of low molecular mass receptor species (130 and 105 kDa). After 3 weeks of culture, the low molecular mass receptor forms accounted for all of the labeled EGF-URO receptor present in the cells. Measurements of EGF-URO binding indicated that the number of EGF-URO binding sites per cell (2.0 x 10(5) +/- 0.3 x 10(5)) did not change during the 3 weeks of culture. However, there was a decrease in EGF-URO binding affinity, reflected by an increase in the KD from 0.6 to 3.0 nM. At zero time and after 3 weeks in culture, Scatchard plots of the binding data were linear; at intermediate time points, the plots were curvilinear. Despite the changes in the EGF-URO receptor that occurred, cells were still responsive to EGF-URO in terms of the inhibition of acetate incorporation into lipid. The ED50 for EGF-URO (about 0.2 nM) was the same for short-term cultures (48 h) as for cells maintained in culture for 3 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Receptors for epidermal growth factor (urogastrone) and insulin in primary cultures of rat hepatocytes maintained in serum-free medium. 349 Feb 67

A method has been developed to isolate skeletal muscle plasma membranes from mice in good yield without harsh extraction procedures. The method involves perfusion of mouse hindquarters with a calcium-deficient buffer containing collagenase and hyaluronidase. This is followed by gentle disruption, filtration, and differential centrifugations. The entire procedure takes about six hours and the yield is approximately 4 mg. protein from 10 g. equivalent of hindquarter muscle. The preparation contained predominantly plasma membranes based on specific activities of marker enzymes, electron microscopic data, and specific binding sites for insulin and a -adrenergic ligand. Studies using such preparations from lean, 4-5 week old and 12-20 week old db/db mice showed marked reduction in the phosphorylation of the 95 kDa subunit of the insulin receptor of the obese mice with no change in insulin binding. In addition, there was a progressive reduction in insulin sensitivity in stimulating receptor phosphorylation in the db/db mice.
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PMID:Impaired insulin receptor phosphorylation in skeletal muscle membranes of db/db mice: the use of a novel skeletal muscle plasma membrane preparation to compare insulin binding and stimulation of receptor phosphorylation. 352 5

Collagenase preparations (a mixture of enzymes including collagenase, clostripain, and a casein-degrading protease) degraded the beta subunit (Mr = 95,000) of the purified insulin receptor into fragments of Mr less than 15,000, without degrading the alpha subunit. The resulting beta-digested insulin receptor preparations were found to bind insulin as well as control insulin receptor, as assessed by either cross-linking of 125I-insulin to the digested receptor or by separating insulin bound to receptor from free insulin by high performance liquid chromatography. Moreover, the beta-digested insulin receptor preparations were still precipitated by a monoclonal antibody directed against the insulin-binding site. In contrast, the beta-digested insulin receptor lacked protein kinase activity since it no longer phosphorylated either itself, or an exogenous substrate, calf thymus histone. These results support the identification of the beta subunit of the insulin receptor as a protein kinase.
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PMID:Preferential degradation of the beta subunit of purified insulin receptor. Effect on insulin binding and protein kinase activities of the receptor. 631 28

The insulin binding properties and the molecular weights of the insulin receptor and its insulin binding subunit were studied in omental and subcutaneous adipocytes prepared from obese- and normal-weight subjects. Insulin binding by such adipocytes was decreased in obesity when the binding activity was expressed per unit of cell surface area. No significant difference from the lean controls was evident, however, when binding was calculated on a per cell basis, indicating that the total receptor content of the cells from the obese subjects was not altered. In addition, the normal difference in the receptor binding affinities previously reported between omental and subcutaneous cells from lean individuals was unaffected by the obese condition. Studies of the molecular weight of the non-reduced insulin receptor in fat cell membranes prepared from pieces of omental and subcutaneous fat demonstrated a major receptor species of 390-425K Mr. In contrast, adipocytes isolated by collagenase treatment of the fat had heterogeneous non-reduced receptor species of Mr 355K, 285K and small amounts of 427K and 182K. Although different non-reduced receptor species were evident depending on the adipocyte receptor preparation (e.g. isolated adipocytes or fat cell membranes), no differences were found between obese and lean controls or between subcutaneous and omental receptors when the appropriate comparisons were made. Upon sulphydryl reduction, all receptor preparations had a major binding subunit of 125K Mr. In conclusion, obesity is characterized by a dilution of the insulin receptor over the adipocyte cell surface in the absence of a change in total cellular content of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding and molecular weight properties of the insulin receptor from omental and subcutaneous adipocytes in human obesity. 639 88

Insulin and IGF-I receptor binding were characterized in cardiac muscle cells isolated from the brown trout, Salmo trutta fario. Cardiomyocyte suspensions obtained by perfusion of ventricles with collagenase showed a high degree of viability as judged by trypan blue exclusion, LDH leakage, and morphology. Specific insulin binding was 2.88 +/- 0.28%/10 mg cells after overnight incubation at 4 degrees. Scatchard analysis indicated the presence of high affinity insulin binding sites with an apparent dissociation constant (Kd) of 0.285 +/- 0.043 nM and a binding site density of 1. 61 +/- 0.19 x 10(8)/mg cells. Specificity of insulin binding was determined by displacing labeled insulin with increasing concentrations of IGF-I, and the Kd value obtained was 4.77 +/- 2.82 nM, 17-fold higher than Kd values for displacement of insulin tracer by nonlabeled insulin. The percentage of IGF-I specific binding (6.70 +/- 1.42%/10 mg cells), affinity (Kd = 0.163 +/- 0.023 nM), and binding site density (4.00 +/- 1.13 x 10(8)/mg cells) were higher than those of insulin. Displacement curves of labeled IGF-I with nonlabeled insulin (Kd = 33.6 +/- 9.9 nM), indicated a high specificity of the IGF-I binding site. High concentrations of cold insulin and IGF-I were able to decrease markedly the specific binding to their own receptor. Incubation with cold IGF-I also induced a diminution in insulin binding in agreement with the lower specificity of the insulin receptor. These data suggest that insulin and IGF-I are able to down-regulate their own receptor number in cardiac muscle cells. The present results demonstrate that the isolated cardiac myocyte preparation from brown trout is a useful model for studying insulin and IGF-I binding in fish heart tissue.
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PMID:Insulin and IGF-I binding in isolated trout cardiomyocytes. 881 93

We examined the effects of phosphatidylinositol 3-kinase (PI3K) inhibition by wortmannin or LY294002 on glucose-induced secretion from mouse islets. Islets were collagenase isolated and perifused or subjected to Western blot analyses and probed for insulin receptor-signaling components. In agreement with previous studies, mouse islets, when compared with rat islets, were minimally responsive to 10 mM glucose stimulation. The inclusion of 50 nM wortmannin or 10 microM LY294002 significantly amplified 10 mM glucose-induced release from mouse islets. The effect of wortmannin was abolished by the calcium channel antagonist nitrendipine or by lowering the glucose level to 3 mM. Wortmannin had no effect on 10 mM alpha-ketoisocaproate-induced secretion. In contrast to its potentiating effect on islets from CD-1 mice, wortmannin had no effect on 10 mM glucose-induced release from ob/ob mouse islets. Western blot analyses revealed the presence of the insulin receptor, insulin receptor substrate proteins 1 and 2 and PI3K in CD-1 islets. These results support the concept that a PI3K-dependent signaling pathway exists in beta-cells and that it may function to restrain glucose-induced insulin secretion from beta-cells. They also suggest that, as insulin resistance develops in peripheral tissues, a potential result of impaired PI3K activation, the same biochemical anomaly in beta-cells promotes a linked increase in insulin secretion to maintain glucose homeostasis.
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PMID:Inhibitors of phosphatidylinositol 3-kinase amplify insulin release from islets of lean but not obese mice. 1217 63


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