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

The ovarian granulosa cell has previously been shown to be a site of insulin-like growth factor (IGF) I production, reception, and action. It is the objective of this study to characterize in greater detail the soluble IGF binding activity released by this cell type. To this end, use was made of granulosa cells from immature diethylstilbestrol-treated rats. Serum-free media conditioned for 72 h by cultured untreated cells acquired polyethylene glycol (PEG)-precipitable [125I]IGF-I binding activity. The latter proved cell density-dependent, displaying a minimal inoculum requirement of less than or equal to 3 x 10(5) cells/culture. The daily elaboration of IGF-I binding activity appeared constant throughout the 72 h experimental period, the overall time-dependent accumulation of binding activity (over the same time period) proving virtually additive. Scatchard analysis of detailed competition studies with IGF-I suggests that the latter ligand binds to granulosa cell-derived IGF binding protein(s) (IGFBPs) with an apparent affinity of 3 x 10(-10) M. Qualitatively similar results were obtained when using [125I]IGF-II suggesting that the IGFBPs in question are not IGF-I-selective. In fact, specificity studies using either [125I]IGF-I or [125I]IGF-II revealed a rank order of competitive potencies compatible with that observed in other tissues so studied (IGF-II greater than IGF-I much greater than insulin). The proteinacious nature of the acid-stable IGF binding activity under study was indicated by its sensitivity to relatively low concentrations of cycloheximide, its apparent deactivation following repeated cycles of freezing and thawing, and its virtual elimination when subjected to boiling or trypsin treatment. Cycloheximide-induced blockade of protein biosynthesis also revealed that the IGF binding activity is subject to measurable turnover thereby suggesting that its accumulation represents the balance struck between synthetic and degradative processes. Western ligand blotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated media revealed a non-glycosylated major band doublet of 28-29 kDa. A single minor IGFBP species represented by a 23 kDa band was also appreciated in some but not all experiments. Taken together, these findings document the ability of ovarian granulosa cells to secrete a heterogenous mix of low molecular weight, high-affinity IGF-selective binding species. As such, these observations are in keeping with the concept of a complete intraovarian IGF system replete with ligands, receptors, and soluble binding activity.
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PMID:Ovarian granulosa cell-derived insulin-like growth factor (IGF) binding proteins: release of low molecular weight, high-affinity IGF-selective species. 171 Jan 90

This study examines the binding and degradation of IGF-II by the ovine liver. Binding and degradation of 125I-IGF-II to isolated hepatocytes was time, temperature and cell number dependent. Ovine and human IGF-II were 2-5 times more effective in inhibiting 125I-hIGF-II binding than were the IGF-I preparations. Insulin did not affect binding. Autoradiographs of 125I-hIGF-II affinity cross-linked to hepatocytes showed a major band of molecular weight 271,000 under reduced conditions. This band was eliminated by 100 nM hIGF-II or oIGF-II but not by excess hIGF-I, oIGF-I or insulin. The internalization of IGF-II was examined by treating the cells with trypsin or sodium acetate to remove surface-bound IGF-II. Both treatments showed that 20-25% of 125I-hIGF-II was internalized. Mannose-6-phosphate at 1, 2 and 4 mM enhanced the binding of 125I-hIGF-II to hepatocytes 3.5, 12.8 and 16.4%, respectively. The lysosomal inhibitors ammonium chloride, chloroquine and leupeptin had no effect on 125I-hIGF-II degradation or cell-associated radioactivity indicating a nonlysosomal pathway of degradation for 125I-hIGF-II in the ovine hepatocyte. The low molecular weight sheep serum IGF binding protein inhibited binding of 125I-hIGF-II in a dose-dependent manner but had no effect on degradation, which suggests that degradation of 125I-hIGF-II is independent of receptor interaction. These studies demonstrate that IGF-II binds to specific high affinity sites in sheep hepatocytes which display the characteristics of type II IGF receptors. A significant fraction of the receptor bound IGF-II is internalized but not degraded by these cells, which suggests that the biological actions of IGF-II may be exerted by an intracellular pathway in sheep hepatocytes.
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PMID:Interaction of insulin-like growth factors (IGF) with isolated sheep hepatocytes. II. Binding, internalization and degradation of IGF-II. 216 13

Beating rat hearts were perfused with 125I-IGF-II alone or 125I-IGF-II and unlabeled IGF-II or insulin, then prepared for radioautography. Maximal 125I-IGF-II grain counts over capillaries were decreased in a dose-dependent manner by unlabeled IGF-II but were unaffected by coperfusion with insulin. To determine a potential role for capillary receptors in the transfer of circulating IGF to cardiac muscle, the effects of sequential loss of capillary IGF binding sites was determined. For IGF-I, loss of capillary binding sites by trypsin perfusion was accompanied by proportional decreases in the subsequent appearance of IGF-I in cardiac muscle. In contrast, similar decrements of capillary IGF-II binding did not affect muscle levels of IGF-II. We conclude that capillary endothelium of the intact heart possesses distinct IGF-I and IGF-II binding sites, with the capillary IGF-I binding sites being of potential importance in the transfer of vascular IGF-I to subendothelial cardiac muscle.
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PMID:IGF receptors in myocardial capillary endothelium: potential regulation of IGF-I transport to cardiac muscle. 296 74

Serine phosphorylation of insulin-like growth factor binding protein-1 (IGFBP-1) has been shown to alter its affinity for the insulin-like growth factors (IGF-I and IGF-II) and to modify its capacity to modulate cellular responses to the IGFs. Because of this, we determined the sites of serine phosphorylation. Purification of 32P-labeled IGFBP-1 was followed by digestion with trypsin and endoproteinase Glu-C and radiosequencing of labeled peptides. Three serines were found to be phosphorylated, with Ser101, Ser119, and Ser169 containing 70%, 5%, and 25% of the incorporated 32P, respectively. A mutated IGFBP-1, substituting alanine for serine at positions 98 and 101, was expressed in CHO cells. On nondenaturing gels, the wild type protein migrated as five isoforms (one non-phosphorylated and four phosphorylated). However, in the mutated protein, the most rapidly migrating band (a phosphorylated form) was not present. The cells containing the mutated cDNA incorporated 60% less 32P into immunoprecipitable IGFBP-1. The mutated protein had a 3-fold reduction in affinity for IGF-I compared to the wild type protein. We conclude that Ser101 represents the major site of phosphorylation containing 63% of the total 32P incorporated and that phosphorylation of Ser101 is important for maintenance of high affinity binding for this growth factor.
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PMID:Identification of the sites of phosphorylation in insulin-like growth factor binding protein-1. Regulation of its affinity by phosphorylation of serine 101. 767 48

Peptides such as somatostatin (SS14), epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and insulin-like growth factors (IGF-I and IGF-II) are present in breast milk from various species, and their significance in the developing gastrointestinal tract has been suggested. Our recent studies have indicated that rat milk soluble fraction (RMSF) protects SS14 in the gastrointestinal lumen by inhibiting in vitro the luminal peptidolysis. In the present studies, we have shown that RMSF inhibited in vitro degradation by midjejunal luminal flushings of suckling rats of 125I-labeled somatostatin 14[Tyr11], EGF, TGF alpha, IGF-I and IGF-II, as well as trypsin activity in vitro against benzoyl-L-arginyl-p-nitroanilide. The inhibitory factors present in the RMSF were further fractionated by gel filtration on Sephadex G100, ion-exchange chromatography on DEAE-Sephadex, and fast protein liquid chromatography (FPLC). Gel filtration of Sephadex G100 separated RMSF into three peaks of proteins: G1, G2, and G3; peptidase inhibitor activities were present exclusively in G1. Ion-exchange chromatography on DEAE-Sephadex column resolved peptidase inhibitory activity (G1) into three different peaks, D1, D2, and D3, eluted at sodium chloride concentrations of 0.05 M, 0.1 M, and 0.2 M, respectively. Further purification of D2 by FPLC resulted in a fraction rich in peptidase inhibitory activity, which was essentially free of trypsin inhibitory activity. Results indicate the presence of at least three peptidase inhibitors in rat milk, which may play a role in the protection of milk-borne peptides in the gastrointestinal lumen.
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PMID:Presence of multiple forms of peptidase inhibitors in rat milk. 814 98

Local metabolic balance between the bone formation and resorption is modulated by many growth factors present in the bone. The mechanism of this regulation has been investigated by many researchers but is not completely clarified yet. In this study, the author found the presence of several growth factors in the 1 M NaCl extract from the bovine bone and one of them was confirmed to be different from the factors previously reported in the bone tissue. This factor was purified by means of gel filtration and heparin-affinity chromatography and characterized in detail. The purified factor stimulated the proliferation of the fibroblast cell line, Balb/c3T3, but neither stimulated nor inhibited that of the osteoblast cell line, MC3T3-E1. The factor was stable against heat (65 degrees C), acid (2N HCl), 6M urea or 4M guanidine-HCl treatments. However, it was inactivated by trypsin. The molecular size of this factor was about 15,000 and had a high affinity for heparin. In contrast to aFGF or bFGF, the fibroblast-proliferating activity of this factor was suppressed dose-dependently by heparin addition. According to those properties, this factor was indicated to be different from TGF- beta, aFGF, bFGF, PDGF, IGF-I, IGF-II or BMP which were reported to be present in the bone tissue. It is suggested the presence of a new factor in the bone which may regulate the proliferation of fibroblasts in vivo.
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PMID:[Purification and characterization of growth factor extracted from bone]. 816 83

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

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

The actions of insulin-like growth factors (IGFs) are modulated by a family of high-affinity binding proteins (IGFBPs), including IGFBP-6, which preferentially binds IGF-II and is O-glycosylated. Glycosylated and nonglycosylated recombinant human IGFBP-6, expressed in Chinese hamster ovary cells and Escherichia coli, respectively, were purified using IGF-II affinity chromatography and reverse-phase medium-pressure chromatography. Electrospray ionization mass spectrometry (ESMS) of glycosylated IGFBP-6 revealed considerable heterogeneity of carbohydrate composition. Major glycoforms contained 8-16 monosaccharides, including N-acetylhexosamine, hexose, and N-acetylneuraminic acid. Glycosylation sites of IGFBP-6 were identified as Thr126, Ser144, Thr145, Thr146, and Ser152 by using a combination of ESMS and Edman sequencing of tryptic fragments separated by reverse-phase high-pressure liquid chromatography. One oligosaccharide chain contained 5-6 monosaccharides, whereas the others contained 2-4 monosaccharides. Glycosylated IGFBP-6 exhibited greater resistance to proteolysis by chymotrypsin and trypsin than nonglycosylated IGFBP-6. Native disulfide bond positions in IGFBP-6 were localized by means of observed disulfide-linked tryptic fragments, revealing that there are two disulfide-linked subdomains within each of the N- and C-terminal regions and confirming a previous suggestion that the latter regions are not interconnected. A model of IGFBP-6 is developed in which these distinct domains are separated by a central region which is O-glycosylated.
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PMID:Identification of O-glycosylation sites and partial characterization of carbohydrate structure and disulfide linkages of human insulin-like growth factor binding protein 6. 957 75

To investigate endoproteolytic processing of the type I insulin-like growth factor receptor (IGF-IR), we have examined its structure and activity in the furin-deficient LoVo-C5 cell line. Immunoprecipitation experiments using the monoclonal anti-IGF-IR antibody (alpha-IR3) showed that LoVo-C5 cells expressed a major high molecular mass receptor (200 kDa) corresponding to the unprocessed alpha/beta pro-receptor. A small amount of successfully cleaved alpha/beta heterodimers was also produced, indicating a residual endoproteolytic cleavage activity in these cells. In vitro, a soluble form of recombinant furin was able to cleave the pro-IGF-IR (200 kDa) into alpha-subunit (130 kDa) and beta-subunit (97 kDa). Measurement of IGF binding parameters in LoVo-C5 cells indicated a low number of typical type I IGF-binding sites (binding capacity, 5 x 10(3) sites/cell; Kd, 1.9 nM for IGF-I and 7.0 nM for IGF-II). These findings in LoVo-C5 contrast with those in HT29-D4 cells, which have active furin, and where IGF-IR (2.8 x 10(4) sites/cell) was fully processed. Moreover, the 200-kDa pro-IGF-IR of LoVo-C5 was unable to induce intracellular signaling, such as beta-subunit tyrosine autophosphorylation and insulin-related substrate-1 tyrosine phosphorylation. Flow immunocytometry analysis using alpha-IR3 antibody indicated that LoVo-C5 cells expressed 40% more receptors than HT29-D4 cells, suggesting that in LoVo-C5 cells only the small amount of mature type I IGF-IR binds IGFs with high affinity. To provide evidence for this idea, we showed that mild trypsin treatment of living LoVo-C5 cells partially restored alpha/beta cleavage of IGF-IR, and greatly enhanced (6-fold) the IGF-I binding capacity of LoVo-C5 cells, but did not restore IGF-IR signaling activity. Moreover, LoVo-C5 cells were totally unresponsive to IGF-I in terms of cell migration, in contrast to fully processed IGF-IR-HT29-D4 cells. Our data indicate that furin is involved in the endoproteolytic processing of the IGF-IR and suggest that this posttranslational event might be crucial for its ligand binding and signaling activities. However, our data do not exclude that other proprotein convertases could participate to IGF-IR maturation.
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PMID:Deficient processing and activity of type I insulin-like growth factor receptor in the furin-deficient LoVo-C5 cells. 972 28


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