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

---

Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chronic treatment of 3T3-C2 fibroblasts with insulin causes the slow (t1/2 = 3-4 h) down-regulation of cellular insulin receptor to a new steady state level by accelerating receptor decay (Knutson, V.P., Ronnett, G.V., and Lane, M.D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2822-2826). In the present investigation, the synthesis and turnover of the receptor during the transition to the down-regulated state was examined by the heavy isotope density-shift method. It was observed that within two h after insulin addition, receptor decay increased abruptly for several hours then gradually declined until the "down-regulated" rate was achieved. The abrupt increase in receptor decay induced by insulin was preceded by a more rapid (t1/2 less than or equal to 10 min) translocation of cell surface receptor to an "intracellular" trypsin-resistant compartment. Thus, upon exposure to ligand, insulin receptor rapidly redistributes from the cell surface to an intracellular compartment, without an initial net loss of cellular receptors. The translocation process was rapidly reversed (t1/2 less than or equal to 20 min) upon removal of insulin. With prolonged exposure to insulin, the initial rapid translocation of receptor was followed by a slower inactivation of receptor apparently in the intracellular compartment. Cycloheximide, which lengthens receptor half-life by blocking a step in receptor inactivation, had no effect on receptor internalization. Internalization of insulin receptor and its bound ligand were, however, rapidly (less than 10 min) blocked by phenylarsine oxide. These results support the following sequence of events. Upon exposure to ligand, insulin receptors are translocated from the cell surface to an intracellular site which results in accelerated receptor decay and ultimately to a lower steady state cellular receptor level.
...
PMID:Rapid, reversible internalization of cell surface insulin receptors. Correlation with insulin-induced down-regulation. 635 82

We photolabeled and characterized insulin receptors in isolated adipocytes from normal human subjects and then studied the cellular fate of the labeled insulin-receptor complexes at physiologic temperatures. The biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)des-PheB1-insulin (NAPA-DP-insulin) was used to photoaffinity label the insulin receptors, and the specifically labeled cellular proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. At saturating concentrations, the binding of 125I-NAPA-DP-insulin to the isolated adipocytes at 16 degrees C was rapid (half-maximal in approximately 1 min and maximal in approximately 10 min) and approximately 25% of the specifically bound ligand was covalently linked to the cells by a 3-min exposure to long-wave (366 nm) ultraviolet light. Analysis of the photolabeled cellular proteins by PAGE in the absence of disulfide reductants revealed the specific labeling of a major protein band of Mr 330,000 and two less intense bands of Mr 295,000 and 260,000. Upon reduction of disulfide bonds with dithiothreitol, all three unreduced forms of the insulin receptor were converted into a major labeled Mr-125,000 band and a less intensely labeled Mr-90,000 band. The labeling of the Mr-125,000 receptor subunit was saturable and native porcine insulin effectively inhibited (half-maximal inhibition at 12 ng/ml) the photolabeling of this binding subunit by NAPA-DP insulin. When intact adipocytes photolabeled at 16 degrees C (a temperature that inhibits endocytosis) were immediately trypsinized, all of the labeled receptor bands were converted into small molecular weight tryptic fragments, indicating that at 16 degrees C all of the labeled insulin-receptor complexes remained on the cell surface. However, when the photolabeled cells were further incubated at 37 degrees C and then trypsinized, a proportion of the labeled receptors became trypsin insensitive, indicating that this fraction has been translocated to the cell interior and thus was inaccessible to the trypsin in the incubation medium. The intracellular translocation of the labeled receptors was observed within 2 min, became half-maximal by 10 min, and maximal by approximately 30 min of incubation at 37 degrees C. Cellular processing of the internalized insulin-receptor complexes also occurred, since incubation at 37 degrees C (but not 16 degrees C) resulted in the generation of a Mr-115,000 component from the labeled receptors. Inclusion of chloroquine, a drug with lysosomotropic properties, in the incubation media caused a time-dependent increase (maximal increase of 50% above control by 2 h at 37 degrees C) in the intracellular pool of labeled receptors. In contrast to these findings in human adipocytes, no appreciable internalization of insulin-receptor complexes and no chloroquine effect was observed in cultures human IM-9 lymphocytes during a 1-h incubation at 37 degrees C. We concluded that in isolated human adipocytes: (a) the subunit structure of insulin receptors is the same as that reported for several other tissues, (b) insulin-receptor complexes are rapidly internalized and processed at physiologic temperatures, and (c) the cellular processing of insulin-receptor complexes occurs at one or more chloroquine-sensitive intracellular site(s).
...
PMID:Insulin receptors in isolated human adipocytes. Characterization by photoaffinity labeling and evidence for internalization and cellular processing. 635 59

Potent insulin-like activity was found in the conditioned medium of human promyelocytic leukemia (HL-60) cells. The conditioned medium of HL-60 cells at high density stimulated [3H]glucose incorporation into lipids in rat adipocytes in a time- and dose-dependent manner. The dose-response curve for this factor was not parallel to that for insulin, and the maximal effect achieved was much greater than reached by insulin or multiplication-stimulating activity. Moreover, the maximal effect reached by either insulin or the conditioned medium was additive. The insulin-like activity was not suppressed in the presence of antiinsulin antibody. Insulin-like activity was not detectable by radioreceptor assay for insulin, suggesting that the factor does not act through the insulin receptor. The factor in the conditioned medium of HL-60 cells was heat stable and sensitive to trypsin. When the conditioned medium was subjected to gel filtration on a Sephadex G-100 column, the major part of insulin-like activity eluted in the position corresponding to an apparent molecular weight between RNAase and insulin markers. The remaining activity, approximately 10% of the total, appeared with a larger molecular weight species. On isoelectric focusing of the smaller molecular species, insulin-like activity was largely focused in the position corresponding to pI 7.8-8.2.
...
PMID:New powerful insulin-like protein from human promyelocytic leukemia cells. 636 2

Autophosphorylation of the insulin receptor was studied using a glycoprotein fraction solubilized and purified partially from the rat hepatoma cell line, Fao. Incubation of this receptor preparation with [gamma-32P] ATP, Mn2+, and insulin yielded a single insulin-stimulated phosphoprotein of Mr = 95,000 which corresponds to the beta-subunit of the insulin receptor. At 22 degrees C, incorporation of 32P was half-maximal at 30 s and about 90% complete after 2 min. At steady state, about 200 pmol of 32P were incorporated per mg of protein; this value corresponded to about 2 molecules of phosphate per insulin binding site estimated from Scatchard plots. Insulin increased the Vmax for autophosphorylation of the insulin receptor kinase nearly 20-fold with no effect on the Km for ATP. Mn2+ stimulated autophosphorylation by decreasing the Km of the kinase for ATP, whereas Mg2+ had no effect. Dilution of the insulin receptor over a 10-fold concentration range did not decrease the rate of autophosphorylation suggesting that it may occur by an intramolecular mechanism. When the phosphorylated beta-subunit of the insulin receptor was digested with trypsin, at least 5 phosphopeptides could be separated by high performance liquid chromatography on a mu Bondapak C18 reverse-phase column. Insulin stimulated the phosphorylation of all sites. These phosphate acceptor sites varied in their rate and degree of phosphorylation. Phosphopeptides pp4 and pp5 were phosphorylated very rapidly and reached steady state within 20 s, whereas phosphorylation of pp1 and pp2 required several minutes to reach steady state.
...
PMID:Kinetic properties and sites of autophosphorylation of the partially purified insulin receptor from hepatoma cells. 636 36

A procedure was developed for the immunoprecipitation of glycosylated and nonglycosylated forms of the insulin receptor and its precursors without prior purification using lectins. 3T3-L1 adipocytes were labeled with [35S]methionine after which 35S-labeled receptor polypeptides were specifically immunoprecipitated and characterized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The first 35S-polypeptide detected was a 190-kDa glycosylated proreceptor which was rapidly (t1/2 approximately equal to 15 min) processed to a 210-kDa intermediate. The latter precursor was more slowly (t1/2 approximately equal to 2 h) proteolytically processed to 125-kDa (alpha') and 83-kDa (beta') precursors of the mature alpha- and beta-receptor subunits. Immediately prior to insertion into the plasma membrane, i.e. about 3 h after translation, the alpha'- and beta'-precursor polypeptides were converted to the mature 135-kDa alpha- and 95-kDa beta-receptor subunits. The characteristics of the oligosaccharide moieties of the receptor precursors and products were investigated. The 210-kDa precursor and its two products, the 125-kDa alpha'- and 83-kDa beta'-species, and the mature alpha- and beta-receptor subunits bind tightly to wheat germ lectin, whereas the 190-kDa proreceptor species is not bound. Upon incubation with endoglycosidase H, both the 210- and 190-kDa species are converted to a 180-kDa species. The 125-kDa alpha'- and 83-kDa beta'-species are also cleaved by endoglycosidase H, being reduced in size to 97 and 79 kDa, respectively. Based on their sensitivity to endoglycosidase H and insensitivity to neuraminidase, the oligosaccharide chains of the receptor precursors (190, 210, 125, and 83 kDa) do not contain terminal sialic acid (or other capping sugars). However, near the time of insertion into the plasma membrane, capping of the alpha'- and beta'-species by sialic acid occurs, giving rise to the mature 135-kDa alpha- and 95-kDa beta-receptor subunits, which are partially endoglycosidase H-resistant and neuraminidase-sensitive. When 3T3-L1 adipocytes are treated with tunicamycin, a 180-kDa proreceptor aglycopolypeptide is synthesized which is incapable of undergoing further processing and proteolytic cleavage to the alpha- and beta (or alpha'- and beta'-)-subunits. The 180-kDa species, which appears to be the aglyco-form of hte 190-kDa proreceptor generated by endoglycosidase H, is resistant to trypsin in the intact cell and apparently has not reached the cell surface. Thus, the oligosaccharide moieties of the insulin receptor precursor are crucial for proper processing, intracellular translocation, and formation of functionally competent insulin re
...
PMID:Role of glycosylation in the processing of newly translated insulin proreceptor in 3T3-L1 adipocytes. 636 59

The effect of insulin on glucose transport (2-deoxyglucose uptake) in adipocytes was measured in the absence and in the presence of 10 mM sodium-DL-beta-hydroxybutyrate. The ketone body had little or no effect on the basal or the maximally insulin-stimulated rate of transport. However, beta-hydroxybutyrate potentiated the effect of submaximal concentrations of insulin, i.e., it resulted in a leftward shift in the dose-response curve. The half-maximally effective concentration of insulin was decreased by approximately 30% from 0.58 ng/ml to 0.40 ng/ml. beta-Hydroxybutyrate caused a slight (approximately 10%) increase in 125I-insulin binding to the cells. To determine whether this small increase in insulin binding is responsible for the increased insulin sensitivity in the presence of the ketone, two mimickers of insulin action were used: a serum containing anti-insulin receptor antibodies and hydrogen peroxide. beta-Hydroxybutyrate increased the sensitivity of glucose transport to stimulation by the anti-receptor antibody, demonstrating that insulin itself does not have to be present. beta-Hydroxybutyrate also potentiated the effect of hydrogen peroxide (which acts at a level distal to the insulin receptor) even in cells that had been depleted of insulin receptors by trypsin treatment. Therefore, beta-hydroxybutyrate acts, at least partly, at a post-insulin receptor level. Adenosine increases the insulin sensitivity of adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:beta-Hydroxybutyrate increases the insulin sensitivity of adipocyte glucose transport at a postreceptor level. 638 23

We characterized insulin receptors on a human lymphoblastoid cell line (IM-9) and studied their regulation using anti-receptor antibodies and fluorescence flow cytometry. The fluorescence intensity distribution of insulin receptors on cells was determined by incubating the cells with one of three different anti-receptor antisera (human serum B-9 containing polyclonal autoantibodies, serum from a rabbit with polyclonal antibodies, and a monoclonal antibody to the receptor produced in mouse hybridomas), followed by incubation with an appropriate fluorescein isothiocyanate-labeled second antibody and analysis on an Epics-V flow cytometer. All three anti-receptor antibodies specifically labeled the insulin receptors. The monoclonal antibody showed the highest level of labeling. Treatment of cells with proteolytic enzymes, such as trypsin or chymotrypsin, produced a dose-dependent loss of 125I-labeled insulin (125I-insulin) binding but a relatively small decrease in the binding of anti-receptor antibodies, suggesting that most antibody binding occurred in domains other than the insulin binding site. Treatment with glycosidic enzymes, such as neuraminidase and beta-galactosidase did not affect the binding of 125I-insulin, and fluorescence was actually enhanced by about 20% in the beta-galactosidase-treated cells. Exposure of IM-9 cells to insulin resulted in a reduction in the number of insulin receptors. Analysis of the down-regulated cells by immunofluorescence revealed a complete correlation between the percent binding of 125I-insulin and percent peak fluorescence. In all cases, receptors were lost proportionally from all cells, yielding a single symmetrical peak by fluorescence analysis. Exposure of IM-9 cells to anti-receptor antibodies at 37 degrees C for 16 hr also produced a down-regulation in the number of insulin receptors. Incubation with human antiserum B-9 caused a 95% loss of both 125I-insulin binding and peak fluorescence, while the monoclonal antibody resulted in a 50% loss of receptors. Incubation of cells with anti-receptor antibodies for 2 hr at 4 degrees C did not produce any receptor loss; however, the human anti-receptor antisera B-2 and B-9 inhibited the binding of the monoclonal anti-receptor antibody by about 50%, suggesting that these antisera contained autoantibodies directed at the monoclonal antibody binding site. These data indicate that insulin receptors can be regulated by both insulin and anti-receptor antibody and demonstrate the utility of immunofluorescence and flow cytometry as a tool for the study of the insulin receptor.
...
PMID:Analysis of the insulin receptor by anti-receptor antibodies and flow cytometry. 639 Apr 39

Trypsin treatment of a partially purified insulin receptor preparation from rat adipocytes stimulated the phosphorylation of 90,000- and 72,000-Da polypeptides immunoprecipitated by anti-insulin receptor antibody. The phosphorylation of tyrosine residues alone was observed in both polypeptides. Trypsin concentrations which stimulated insulin receptor phosphorylation were the same as those previously shown to activate rat adipocyte glycogen synthase. Trypsin treatment of the insulin receptor fraction also stimulated the phosphorylation of an exogenous substrate of tyrosine kinase similarly to insulin treatment. Trypsin treatment of a highly purified insulin receptor from human placenta also activated the phosphorylation of the receptor-derived peptides. These results suggest that the insulin-stimulated protein kinase, a component of the insulin receptor, was activated by tryptic digestion to phosphorylate polypeptides derived from the insulin receptor itself. Thus, it is suggested that stimulation by trypsin of phosphorylation of the insulin receptor may be related to the insulin-like metabolic actions of trypsin observed in rat adipocytes.
...
PMID:Insulin-like effect of trypsin on the phosphorylation of rat adipocyte insulin receptor. 641 37

We have followed the fate of cell surface insulin receptors in isolated rat hepatocytes by both a biochemical and a morphological approach. Hepatocytes were labeled with the photoreactive and biologically active 125I-labeled insulin analogue, [2-nitro-4-azidophenylacetylB2]des-PheB1-insulin, under conditions that allow for minimal internalization (2 hr at 15 degrees C). Analysis of the cell-associated radioactivity by NaDodSO4/polyacrylamide gel electrophoresis under reducing conditions followed by autoradiography revealed the specific labeling of a major insulin receptor subunit with Mr 130,000 and a minor degradation product with Mr 125,000. When the cells were exposed at 15 degrees C to trypsin at the end of the association period, these two bands were no longer observed, indicating that the labeled receptors were at the cell surface. This trypsin sensitivity of the receptor disappeared within 30-60 min of incubation of the cells at 37 degrees C, reflecting the internalization of the hormone-receptor complexes. Over the subsequent 4 hr of incubation, this was followed by a progressive reappearance of the receptor complexes at the cell surface, as indicated by the recovery of trypsin sensitivity of the labeled insulin receptors. An identical (both chronologically and quantitatively) journey of the insulin receptors was observed when the labeled material was studied by quantitative electron microscopic autoradiography. Thus, when the cells were incubated at 37 degrees C there was a rapid decrease (30-60 min) in the percentage of autoradiographic grains associated with the plasma membrane, followed by a progressive increase in this percentage over the subsequent 4 hr of incubation. In conclusion, using a biochemical and morphological approach to trace the photoaffinity-labeled insulin receptor, we have shown that the internalized hormone-receptor complex is recycled back to the cell surface.
...
PMID:Internalized insulin receptors are recycled to the cell surface in rat hepatocytes. 676 33

The effect of alterations to the insulin receptor on the insulin sensitivity of isolated adipocytes was studied. Receptor changes were induced by treatment of adipocytes with either phospholipase C or trypsin. After enzyme treatment, binding of insulin to insulin receptors and insulin-mediated glucose metabolism were examined. Exposure of adipocytes to phospholipase C (2 units/ml) significantly increased insulin binding to the cells, but destroyed the ability of the cells to oxidize glucose. After treatment with trypsin (500 micrograms/ml) for 5 min, insulin binding to the adipocytes was significantly increased. This was shown to be due to an increase in insulin-receptor affinity. Metabolic studies showed that trypsin treatment led to an increase in basal glucose transport but markedly decreased the response to insulin at all concentrations tested. Adipocytes treated with trypsin showed no significant difference in basal glucose oxidation rates when compared with controls, but were less sensitive to insulin at low insulin concentrations, and showed a decreased maximum response at high insulin concentrations. In conclusion, these findings indicate a dissociation between induced changes in binding of insulin to insulin receptors and subsequent hormone action. The importance of post-receptor events in the biological action of insulin is highlighted.
...
PMID:The effects of trypsin and phospholipase C on insulin binding and action in the isolated adipocyte. 699 Sep 21


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---