Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Since peptide mapping with proteolytic enzymes such as trypsin and Staphylococcus aureus V8 protease is a powerful tool for the characterization of proteins, investigators should be cognizant of possible artifacts due to the technique itself. This article describes the identification of minor peaks found in the maps of recombinant human relaxin and insulin-like growth factor I as transpeptidation products. Both proteins have some homology to insulin with relaxin being composed of two chains designated A and B, while insulin-like growth factor I is composed of a single polypeptide chain. Digestion of relaxin with trypsin at pH 7.2 yields two peptides, T2,3(A10-18) and T7(B10-13), linked together by a disulfide bond. An unexpected component at a 10% level was identified to be the T2-T7 peptide pair where T3(ArgA18) has formed a peptide bond with the amino-terminal LeuB10 of the T7 peptide. It was also observed that the digestion of insulin-like growth factor I with V8 protease normally yields two peptides V4(13-20) and V9(59-70) linked by a disulfide bridge. A minor peak at a 1 to 2% level was identified to be a single polypeptide resulting from the formation of a peptide bond between the amino-terminal Met59 of V9 and the carboxyl-terminal Asp20 of V4, with the disulfide bond intact. These transpeptidation products were isolated by reversed-phase HPLC and identified using amino-terminal sequence and mass spectrometric analyses.
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PMID:Transpeptidation during the analytical proteolysis of proteins. 188 34

The effect of conditioned medium (CM) from rat calvaria (RC) cel cultures on the growth and differentiation of osteogenic cells in rat bone marrow stromal cell (BMSC) cultures was investigated. Control cultures received either CM from periodontal ligament fibroblast cultures or fresh medium. RCCM stimulated the formation of nodules of bonelike tissue in bone marrow stromal cell cultures in a dose-dependent manner,and the maximal stimulation was associated with the osteoblast-enriched cell populations of the RC cultures. Ultrafiltration demonstrated that activity was confined to a CM fraction of 10- to 30-kilodalton molecular size. The activity was sensitive to boiling and trypsin treatments, but was not affected by neutralizing antibodies to transforming growth factor beta or insulin-like growth factor I or II. RCCM was found to initially increase the number and proportion of cells that expressed alkaline phosphatase activity, although the proportion of alkaline phosphatase-positive cells subsequently declined. These data were consistent with an initial stimulation of proliferation of a subpopulation of osteoprogenitor cells within the cultures, followed by their differentiation. The results suggest that mature osteoblasts may produce a paracrine growth factor that can stimulate the differentiation of osteoblasts from precursor cells.
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PMID:Stimulation of the differentiation of osteogenic rat bone marrow stromal cells by osteoblast cultures. 203 May 75

Oxytocin initiates its insulin-like action in adipocytes through oxytocin-specific receptors. We have studied binding and structural properties of these receptors with the radioligand [3H]oxytocin. Steady-state binding was reached after 45 min, at 21 degrees C, and 10 min at 37 degrees C. Scatchard analyses of equilibrium binding data indicated a single class of oxytocin binding sites at 21 degrees C (KD = 3.3 nM, RT = 6 X 10(4) sites/cell) and 2 binding sites at 37 degrees C (KD = 1.5 nM, RT = 6 X 10(4) sites/cell; and KD = 20 nM, RT = 30 X 10(4) sites/cell). Insulin, insulin-like growth factor I, and epidermal growth factor increased oxytocin binding (approximately 20-40%), whereas adenosine, a regulator of oxytocin action, did not affect oxytocin binding. Binding activity of oxytocin was impaired by pretreatment of the hormone or adipocytes with dithiothreitol. Dithiothreitol treatment of adipocytes preferentially inactivated high-affinity binding sites. N-ethyl maleimide inhibited oxytocin binding in adipocytes more than dithiothreitol. In contrast to the inhibitory effects of dithiothreitol and N-ethyl maleimide, proteases (trypsin, chymotrypsin and papain) were not able to inhibit fat cell binding activity. These results suggested that in isolated adipocytes: there are high-affinity and low-affinity receptors, but the low-affinity receptors are absent at 21 degrees C; the binding of oxytocin can be regulated by insulin, and growth factors; and the oxytocin receptors contain disulfide bridges and free thiols that are essential for the maintenance of oxytocin binding.
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PMID:Binding and structural properties of oxytocin receptors in isolated rat epididymal adipocytes. 281 58

The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identities of the insulin and IGF I receptor beta-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the beta-subunit of the insulin receptor correlated with occupancy of the beta-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the beta-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the beta-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.
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PMID:Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells. 283 27

A novel leukemia cell growth-promoting activity has been identified in the culture supernatant from a human B cell leukemia cell line, BALL-1. The supernatant from unstimulated cultures of the BALL-1 cells significantly promoted the growth of 16 out of 24 leukemia/lymphoma cell lines of different lineages (T, B and non-lymphoid) in a minimal concentration of fetal bovine serum (FBS), and of 5 out of 12 cases of fresh leukemia cells in FBS-free medium. The growth-promoting activity in the BALL-1 supernatant has been further characterized using FPLC chromatography, molecular weight (MW) sieve filtration and dialysis. The MW of the factor was less than 10 kDa. The growth-promoting activity was heat and acid stable and resistant to trypsin treatment. The factor isolated from the BALL-1 supernatant was distinct from known polypeptide growth factors with MW below 10 kDa, such as epidermal growth factor, transforming growth factor alpha, insulin-like growth factor I (IGF-I), IGF-II and insulin, as determined by specific antibodies and by cell growth-promoting tests. The factor in the BALL-1 supernatant did not promote the proliferation of normal human fresh peripheral blood lymphocytes or mouse fibroblast cell line, BALB/c3T3. In addition to the BALL-1 supernatant, a similar growth-promoting activity was found in the culture supernatants from 13 of 17 leukemia/lymphoma cell lines tested. The activity in these culture supernatants promoted the growth of leukemia/lymphoma cell lines in autocrine and/or paracrine fashions. These observations suggest that the low MW cell growth-promoting activity found in the BALL-1 culture supernatant is mediated by a novel factor which may well be responsible for the clonal expansion of particular leukemic clones.
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PMID:A novel cell growth-promoting factor identified in a B cell leukemia cell line, BALL-1. 827 54

Using specific substrates, benzyloxycarbonyl-Gly-Gly-Leu-p-nitroanilide, benzyloxycarbonyl-Gly-Gly-Arg-2-naphthylamide and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide, cytosolic chymotrypsin-like, trypsin-like and cucumsin-like activities were determined, respectively, in rat epithelial tissues and differentiated human Caco-2 cells. The cytosolic fractions of rat colonic, rectal, nasal, and alveolar epithelial cells and differentiated human Caco-2 cells contained these three distinct enzyme activities. However, effects of enzyme inhibitors revealed that these three distinctive activities were not extensively involved in cytosolic or homogenate degradation of insulin and insulin-like growth factor I (IGF-I). It is concluded that proteasome-like activities may not significantly limit nonparenteral absorption of peptide and protein drugs such as insulin and IGF-I.
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PMID:The involvement of cytosolic chymotrypsin-like, trypsin-like, and cucumsin-like activities in degradation of insulin and insulin-like growth factor I by epithelial tissues. 858 71

The purpose of this study was to determine whether carbopol polymers, polyacrylic acid polymers, can inhibit lumenal degradation of insulin, calcitonin and insulin-like growth factor I (IGF-I) by trypsin and chymotrypsin and to understand whether reducing the pH of the incubation medium by these polymers results in inhibition. Further, the effects of carbopol polymers on the in-situ absorption of insulin were studied in rats. In saline, carbopol polymers at 1% and 4% (w/v%) inhibited close to 100% of trypsin and chymotrypsin activities against insulin. In 50 mM Tris buffer, carbopol polymers, including 934P, 974P and 971P, at 0.1% only weakly inhibited degradation of calcitonin and insulin by both enzymes; however, as the polymer concentration increased to 0.4%, degradation of insulin, calcitonin, and IGF-I by both enzymes was complete or almost complete. When the Tris buffer was increased to 100 mM, no inhibition was observed at 0.1%. Determination of the final pH of the incubation medium in the presence of polymers revealed that the inhibitory effects of carbopol polymers correlated with the final pH. When the incubation medium has no or low buffer capacity to buffer the protons released by carbopol polymers, these polymers are able to reduce the pH much lower than the optimum pH for the enzyme activities, and thus inhibit proteolytic degradation. When the buffer capacity of the incubation medium increases, the inhibitory effects of carbopol polymers weaken. In-situ absorption of insulin revealed that carbopol polymers improved insulin absorption and induced a significantly greater decline in blood glucose levels. It is concluded that carbopol polymers with strong bioadhesive properties also can inhibit lumenal degradation of peptide hormones, offering multiple advantages for their uses in oral drug delivery.
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PMID:Effects of polyacrylic polymers on the degradation of insulin and peptide drugs by chymotrypsin and trypsin. 872 88

To evaluate the protecting effect of camostat mesylate, N,N-dimethylcarbamoylmethyl-p-(p-guanidinobenzoyloxy)phenylacetate methanesulfonate, one of the synthetic trypsin inhibitors, on diabetic nephropathy, urinary albumin excretion was measured in streptozotocin-induced (50 mg/kg, i.p.) diabetic rats treated with oral camostat mesylate for 12 weeks. The rats were divided into three groups: (1) nondiabetic control rats; (2) diabetic rats, and (3) diabetic rats received rat chow containing 0.1% camostat mesylate (PI rats). After induction of diabetes, the ratio of kidney weight to body weight and urinary albumin excretion (UAE) were significantly increased. However, the ratio of kidney weight to body weight in PI rats was significantly lower than that in diabetic rats, and UAE in PI rats was also significantly lower than that in diabetic rats at 4, 8 and 12 weeks. Kidney tissue insulin-like growth factor I (IGF-I) contents were significantly reduced in diabetic rats, and there were no significant differences in kidney tissue IGF-I contents between diabetic and PI rats. These results suggest that camostat mesylate reduces the UAE probably through an inhibitory effect on initial diabetic renal hypertrophy and that camostat mesylate is available for diabetic nephropathy.
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PMID:An inhibition of urinary albumin excretion by protease inhibitor in streptozotocin-diabetic rats. 895 6

Insulin-degrading enzyme (IDE) is a component of a cytosolic complex that includes multicatalytic proteinase (MCP), the major cytoplasmic proteolytic activity. Insulin, the primary substrate for IDE, inhibits the proteolytic activity of the IDE-MCP complex but not of purified MCP. This provides a regulatory role for IDE in cellular proteolysis and a potential mechanism for intracellular insulin action. To examine the specificity and to explore the mechanisms for the IDE-MCP interaction, we studied the functional interaction of a variety of peptides with the complex. Atrial natriuretic peptide (ANP), relaxin, glucagon, proinsulin, and insulin-like growth factor II (IGF-II) bind to and are degraded by IDE. These peptides have significant inhibitory effects on the chymotrypsin-like and trypsin-like MCP catalytic activities but not the peptidyl-glutamyl hydrolyzing activity. A panel of peptides that are not ligands of IDE had no effect. To explore the potential mechanism for the IDE control of MCP activity, dose response curves for insulin-like growth factor I (IGF-I) and IGF-II effects on MCP chymotrypsin-like activity were determined. IGF-II, which (similar to insulin) is a good substrate for IDE, had a substantial inhibitory effect, whereas IGF-I, which is bound but poorly degraded, had little inhibitory activity on MCP. Proinsulin, another ligand of IDE that is tightly bound but poorly degraded, had a partial effect on MCP activity, but inhibited the full insulin effect. These data suggest a requirement for both the binding and degradation of IDE ligands for the full inhibition of MCP. Insulin-sized degradation products, substrates of IDE, also inhibited MCP activity. Further examination of the insulin effect on MCP included kinetic studies. Insulin produced a noncompetitive inhibition of both the chymotrypsin-like and trypsin-like activities of MCP. These data suggest that the insulin-IDE effect on MCP is due to conformational changes in the IDE-MCP complex and provide an intracellular mechanism of action for insulin.
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PMID:Characterization of the insulin inhibition of the peptidolytic activities of the insulin-degrading enzyme-proteasome complex. 900 Jun 94

We established a retinal pigment epithelium-derived cell line from transgenic mouse harboring temperature-sensitive simian virus 40 T-antigen gene (tsSV40T) and examined its characteristics. We enucleated both eyes from a 2-month-old transgenic mouse and removed the retinal pigment epithelial (RPE) cells and neuroretinal cells. After cloning the RPE cells, we obtained a cell line (RPET). RPET cells grew well at 33 degrees C but not at 37 degrees C, expressing on the temperature-sensitive character of tsSV40T, and maintained characters of RPE cells such as T1-tyrosinase production, phagocytosis of rod outer segments, and presence of cytokeratin, microvilli on the cell surface and lysosome-like granules around the Golgi apparatus in the cytoplasm. Conditioned medium (CM) from a culture of neuroretinal cells harboring tsSV40T was essentially required for growth. The factor(s) in CM was heat-and acid labile, but was resistant to trypsin digestion. In the presence of 3% CM, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and strong effects on RPET cells, whereas insulin, insulin-like growth factor I (IGF-I), and IGF-II had moderate growth effects. Interestingly, none of these growth factors stimulated the RPET cells in the absence of CM. EHS-Matrix had growth effect, whereas laminin, collagen types I and IV, and fibronectin had no marked growth effects on RPET cells. RPET cells were morphologically changed on a laminin-coated dish. They could not spread on the coated dish, and the majority of the cells floated. But when the floating cells were transferred to non-coated dishes, they immediately attached themselves. These results suggest that RPET cells are a good model for for finding novel growth factor(s) and for investigating the mechanism of cell-laminin attachment.
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PMID:A retinal pigment epithelium-derived cell line from transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene. 907 3


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