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 rab3A gene product is a 25-kilodalton guanine nucleotide-binding protein that is expressed at high levels in neural tissue and has about 30% homology to the ras gene product. Recombinant Rab3A protein and p25rab3A purified from bovine brain membranes have been used as substrates to look for factors that regulate its biochemical activity. A factor in rat brain cytosol exists that accelerates, by approximately 10-fold, the release and subsequent rebinding of guanine nucleotides to both native and recombinant p25rab3A. We have partially purified this activity, termed Rab3A-GRF, and a GTPase-activating protein (Rab3A-GAP) reported previously. The two activities copurified through a variety of procedures but were separated by Mono Q anion-exchange chromatography, indicating that the activities arise from distinct polypeptides. Both factors were thermolabile, sensitive to trypsin, and specific for Rab3A, exhibiting little or no activity toward c-Ha-Ras or Rab2 proteins. By gel filtration chromatography and sucrose density ultracentrifugation, both Rab3A-GRF and Rab3A-GAP have Stokes radii of 79 A and sedimentation coefficients of 8.9 S. We calculate a molecular mass of 295,000 daltons and a frictional ratio of 1.80 for each factor.
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PMID:Characterization of a guanine nucleotide-releasing factor and a GTPase-activating protein that are specific for the ras-related protein p25rab3A. 131 Oct 81

Clinical and veterinary uses of growth hormone-releasing factor [GRF(1- 29)NH2] require the design of analogs that are resistant to proteolysis by serum and liver degrading enzymes. This study investigated rat GRF(1-29)NH2 processing in serum and liver homogenate by means of high pressure liquid chromatography (HPLC). Synthetic rGRF(1-29)NH2 (30 microM) was incubated (0-120 min, 37 degrees C) in serum (49 +/- 8 mg prot./ml). The rGRF(1-29)NH2 (10 microM) was also incubated (0-120 min, 37 degrees C) with liver homogenate (200 +/- 6 micrograms prot./ml). Time course studies of rGRF(1-29)NH2 disappearance showed apparent half-lives of 18 +/- 4 min and 13 +/- 3 min in serum and liver homogenate, respectively. This was accompanied by the appearance of degradation products that were all less hydrophobic than the native peptide. In the serum, two major metabolites were detected and isolated by preparative HPLC. Combined results of amino acid analysis, sequencing, and chromatography with synthetic homologs revealed the presence of rGRF(1-20)OH and (3-20)OH. A small amount of rGRF(12-29)NH2, coeluting with rGRF(3-20)OH, was also found by sequencing. In the liver, rGRF(1-18)OH, (3-18)OH, and (1-10)OH were identified. The peptide bond Ala2-Asp3 (DPP IV cleavage site) was hydrolyzed in both serum and liver. Other tissue-specific cleavage sites were Arg11-Arg12 and Arg20-Lys21 (trypsin-like cleavage site) in the serum, and Tyr10-Arg11 and Tyr18-Ala19 (chymotrypsin-like cleavage site) in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catabolism of rat growth hormone-releasing factor(1-29) amide in rat serum and liver. 143 11

Human growth hormone-releasing factor, GRF(1-44)-NH2, was synthesized by trypsin catalyzed coupling of Leu-NH2 to Arg43 of the precursor, GRF(1-43)-OH, prepared by solid phase peptide synthesis. The semisynthetic GRF(1-44)-NH2 was fully characterized and showed full potency in the rat pituitary in vitro bioassay. Conversion to GRF(1-44)-NH2 was limited to 60-70% in both 75% v:v N,N'-dimethylacetamide and 95% v:v 1,4-butanediol due to competing transpeptidations at Arg41 and Arg38 generating [Leu42]-GRF(1-42)-NH2 and [Leu39]-GRF(1-39)-NH2 side-products, respectively. The rates of formation and yields of GRF(1-44)-NH2 versus pH, Leu-NH2 concentration, and solvent composition were also studied.
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PMID:Semisynthesis of human growth hormone-releasing factor by trypsin catalyzed coupling of leucine amide to a C-terminal acid precursor. 147 84

Five criteria were developed to validate the primary cell culture model for comparison of GRF-induced release of growth hormone in pituitary tissue from aging animals. Pituitaries from young (5-mo), middle-aged (14-mo), and old (24-mo) male Fischer 344 rats were dispersed using either trypsin/trypsin inhibitor or dispase and compared with respect to the number of pituitary cells recovered, cell viability, 3H-leucine incorporation into total protein, time course for recovery of optimal response to GRF, and the dose-relationship for GRF-induced release of growth hormone 2, 4, and 6 days after dispersal. Results indicated that direct comparison of cellular responses between tissues from young, middle-aged, and old rats in primary cell culture is confounded by variations in time for recovery of optimal responses, the effects of the enzymes used for dispersal, and the methods used to express the data.
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PMID:Effect of aging on GHRF-induced growth hormone release from anterior pituitary cells in primary culture. 199 67

The extent to which pituitary explants secrete GH requires clarification. In the present study GH release from ectopic pituitary tissue was assessed using the reverse hemolytic plaque assay. In addition, the effects of this tissue on eutopic GH and PRL release were simultaneously investigated. Anterior pituitary glands from adult female donor rats were grafted beneath the kidney capsule of adult male hosts. Four weeks later, this ectopic pituitary tissue and the eutopic pituitary glands of the host animals were removed, dispersed with trypsin, and subsequently assayed for GH and PRL release. Contrary to expectations, we found that 37% of the ectopic pituitary cells were GH secretors. Furthermore, these GH cells remained highly responsive to GRF (10(-7) M), which evoked a 7-fold increase (P less than 0.05) in mean GH plaque area. By comparison, only 28% of these ectopic cells secreted PRL, and this release was not consistently augmented by exposure to TRH (10(-7) M). In the second half of this study we found that the presence of ectopic pituitary tissue severely compromised the secretory capacity of the eutopic pituitary cells. More specifically, GH-releasing factor induced a 3-fold increase in GH secretion from the eutopic pituitary cells of sham-operated control animals, but it enhanced the release from cells of host animals by only 2-fold. Moreover, the response to TRH by the eutopic PRL cells from explant-bearing animals was curtailed to such an extent that it was not significantly greater (P greater than 0.05) than basal release in that group. We conclude that the potential of pituitary explant cells to release GH is far greater than previously believed and that this ectopic hormone production may inhibit hormone release from the eutopic pituitary cells.
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PMID:Pituitaries transplanted under the renal capsule contain functional growth hormone (GH) secretors and suppress GH and prolactin release from individual eutopic pituitary cells. 251 Sep 89

In this study we evaluated the quantitative influence of GRF and TRH on the rate of hormone secretion from single cells in cultures of male pituitaries. To accomplish this, we dispersed pituitaries from male rats with trypsin and cultured them for 24 or 48 h. Reverse hemolytic plaque assays for GH and prolactin were then performed on retrypsinized cultures to identify individual cells that secreted these hormones. Mammotropes and somatotropes were found to comprise 31.4 +/- 1.8 and 32.2 +/- 0.9% (mean +/- SE, n = 3 experiments), respectively, of all cells in 24-hour cultures. Immunocytochemical staining of different batches of cells from the same dispersions corroborated the proportions of these two cell types. Differences in the rate of basal hormone secretion were observed within each of these cell populations as evidenced by the gradual appearance of prolactin and GH plaques over a 4-hour period when incubations were conducted in the absence of stimulatory secretagogues. Addition of increasing concentrations of GRF (1 X 10(-10) -1 X 10(-7) M) or TRH (1 X 10(-9) -1 X 10(-6) M) to these incubations resulted in dose-related increases in the rate of GH and prolactin plaque formation, respectively. Maximal plaque development by somatotropes could be induced within 30 min of administering large doses of GRF, indicating that most, if not all somatotropes are responsive to this secretagogue. In contrast, approximately one third of all mammotropes could not be stimulated to form plaques acutely when subjected to similar treatment with TRH. This observation suggests that mammotropes are heterogeneous with respect to TRH responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Analysis by plaque assays of GH and prolactin release from individual cells in cultures of male pituitaries. Evidence for functional heterogeneity within rat mammotrope and somatotrope populations. 307 93

The presence of Val2 in mGRF(1-42)OH is unique and, as shown in this study, renders this GRF resistant to plasma DPP-IV, the main enzyme responsible for rapid hydrolysis and inactivation of Ala2-containing GRFs from other species via cleavages between Ala2-Asp3. The presence of DPP-IV activity in mouse serum, and mouse and bovine plasma has been demonstrated with Gly-Pro-p-nitroanilide and/or with two DPP-IV-sensitive bGRF analogs, [Leu27]bGRF(1-29)NH2 and [Ala15,Leu27]bGRF(1-29)NH2, which were effectively converted to their respective (3-29) fragments. During incubations of mGRF(1-42)OH in mouse serum or plasma, as well as in bovine plasma in vitro, no major fragments were detectable, except for small amounts of metabolites with HPLC retention times corresponding to those of mGRF(12-42)OH and mGRF(21-42)OH, indicative of possible trypsin-like cleavages between Arg11-Lys12 and Arg20-Lys21. Both mGRF(1-42)OH (t1/2 52-78.5 min) and [Val2,Ala15,Leu27]-bGRF(1-29)NH2 (t1/2 78.5 min) disappeared 5 to 7 times faster in mouse than in bovine plasma, indicating much higher activity of various degrading enzymes in mouse plasma. In summary, our data provide evidence that mGRF(1-42)OH, despite its resistance to plasma DPP-IV, is degraded relatively fast in mouse plasma or serum because of trypsin-like and other, non-DPP-IV-related, proteolytic cleavages.
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PMID:Metabolism of mouse growth hormone-releasing factor, mGRF(1-42)OH, and selected analogs from the bovine GRF series in mouse and bovine plasma in vitro. 791 20

The identification of peptide bonds vulnerable to tissue peptidases is a valuable approach to design peptide agonists which exhibit a longer duration of action than the native molecules. Therefore, the kinetic of disappearance of rat growth hormone-releasing factor (rGRF(1-29)NH2) and the identification of its metabolites were studied in rat pituitary and hypothalamus. Synthetic rGRF(1-29)NH2 (10 microM) was incubated (0-120 min, 37 degrees C) in the presence of a pituitary (237 +/- 51 micrograms protein/ml) or hypothalamus homogenate (576 +/- 27 micrograms protein/ml). Using analytical high pressure liquid chromatography (HPLC), apparent half-lives of 22 +/- 3 min and 25 +/- 4 min were found in pituitary and hypothalamus, respectively. In both tissues, three degradation products, all less hydrophobic than the native peptide, were detected and isolated by preparative HPLC. The identification of the purified metabolites was ascertained by amino acid analysis, sequencing and chromatography with synthetic homologs. These results indicate that the main sites of cleavage in the pituitary and hypothalamus are Lys21-Leu22 (trypsin-like cleavage site), Leu14-Gly15 and Tyr10-Arg11 (chymotrypsin-like cleavage sites). TLCK and leupeptin did not affect the formation of fragment (1-21)OH while TPCK blocked the cleavage of Leu14-Gly15. The low affinity of fragment (1-21)NH2 for pituitary GRF binding sites suggests that hydrolysis of the Lys21-Leu22 bond inactivates rGRF(1-29)NH2 in this target tissue.
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PMID:Proteolytic degradation of rat growth hormone-releasing factor(1-29) amide in rat pituitary and hypothalamus. 839 7