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)

Harvesting MDCK cells with trypsin-EDTA reduces potassium currents (IK) to a mere 10%, presumably by hydrolysis of K+ channels, but replating at confluence restores them in 12-18 hr, through a process that requires transcription, translation and exocytic fusion of intracellular membrane vesicles to the plasma membrane (Ponce & Cereijido, 1991; Ponce et al., 1991a). In the present work we find that this restoration of IK also requires cell-cell contacts and the presence of 1.8 mM Ca2+. The role of extracellular Ca2+ may be substituted by 2.0 microM TRH, 10 nM PMA or 200 micrograms/ml DiC8. drugs that stimulate the system of phospholipase C (PLC) and protein kinase C (PKC). Conversely, the recovery of IK triggered by Ca-dependent contacts can be blocked by 110 microM neomycin, 2.0 microM H7, and 250 nM staurosporine, inhibitors of PLC and PKC. These results suggest that the expression of new K+ channels depends on Ca(2+)-activated contacts with neighboring cells and that the information is conveyed through PLC and PKC, a process in keeping with changes in its enzymatic activity and cellular distribution of PKC. Plasma membrane is also reduced and restored upon harvesting and replating, and depends on Ca(2+)-activated contracts. However, the effects of the chemicals tested on IK differ from the ones they elicit on the recovery of plasma membrane, suggesting that cells can independently regulate their population of K+ channels and the surface of their membrane.
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PMID:Expression of potassium channels in epithelial cells depends on calcium-activated cell-cell contacts. 776 7

This study was designed to construct the primary culture system to detect the change in TSH beta subunit (TSH beta) gene expression in individual cells. Adult, male Wistar rats were sacrificed by transcardial perfusion of 0.25% trypsin solution under pentobarbital anesthesia (50 mg/kg body weight). Their anterior pituitaries were removed, dispersed and cultured for 1, 2, 3, or 6 days with or without 1 nM triiodothyronine (T3) under the serum-free condition. In some cultures, TRH was added to a final concentration of 1 microM on 6, 12 or 24 h before fixation. Then the culture media were removed to measure TSH concentration. Cells were fixed with paraformaldehyde and hybridized with 35S-labeled RNA probe complementary to TSH beta mRNA. Emulsion autoradiography was subsequently performed. T3 treatment markedly suppressed relative cellular levels of TSH beta mRNA on 2, 3 and 6 days after the onset of culture (day 2, 3 and 6) and suppressed TSH secretion on day 3 and 6. TRH treatment increased TSH beta mRNA on 12 and 24 h after the treatment on day 2 and 3 but did not increase TSH beta mRNA on day 6. TSH concentration in the culture medium was increased by TRH treatment on 6, 12 and 24 h after the treatment on day 2, on 12 h and 24 h on day 3, and 24 h on day 6. On day 2 and 3, although T3 treatment suppressed basal level of TSH beta mRNA, TRH-induced increase in TSH beta mRNA was not suppressed by T3 treatment. These results show that the thyroid hormone and TRH regulate TSH beta gene expression independently. Our culture system may provide a useful model to examine the action of individual substances on a specific subpopulation of the anterior pituitary cells.
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PMID:In situ hybridization detection of TSH beta subunit gene expression in the serum-free primary culture of the adult rat pituitary. 782 27

The membrane-bound enzyme which catalyzes the degradation of thyrotropin-releasing hormone (TRH; Glp-His-Pro-NH2) could be released from membranes of rat and pig brain by treatment with trypsin under very mild incubation conditions. The solubilized enzyme was purified 200,000-fold, with an overall yield of 20%, by conventional chromatographic methods. The enzyme preparation appeared to be electrophoretically homogenous since SDS/PAGE analysis revealed a single band with a molecular mass of 116,000 Da. By gel-filtration chromatography, a molecular mass of 230,000 Da was estimated, suggesting that the enzyme consists of two identical subunits. The enzyme could be identified as a glycoprotein by lectin-binding analysis and by the reduction of the molecular mass to 97,000 Da upon treatment of the denatured enzyme with endoglycosidase-F/N-glycosidase F. In its native form, however, the enzyme was only partially deglycosylated and retained full enzymatic activity. In addition to TRH, the enzyme also hydrolyzed L-5-oxoprolyl-beta-naphthylamide, and thus a convenient fluorimetric assay could be established to determine high enzyme activities. The hydrolysis of both substrates was found to obey Michaelis-Menten kinetics, but considerable differences in the respective Km and Vmax values were noticed.
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PMID:Purification and characterization of the thyrotropin-releasing-hormone-degrading ectoenzyme. 792 52

Pyroglutamyl aminopeptidase type-II is reported to be a highly specific, membrane-bound neuropeptidase, which has the ability to hydrolytically remove the L-pyroglutamyl residue (pGlu) from the N-terminus of thyrotropin releasing hormone (TRH, pGlu-His-Pro-NH2) and closely related tripeptides or tripeptide amides. The primary aim of this study was to purify this enzyme from bovine brain and to compare its characteristics with those previously reported. Following solubilization from the membrane fraction by limited proteolysis with trypsin, the enzyme was purified approximately 3000-fold with a 24% recovery of activity. A native molecular mass of 214,000 Da was determined for the purified enzyme by gel-filtration chromatography. A pH optimum of 6.8-7.6 was observed for the enzyme, with rapid inactivation occurring below pH 4.0 and above pH 9.2. Optimal enzyme activity was observed at 45 degrees C. On the basis of its inhibition, in a time-dependent manner, by metal complexing agents and its subsequent reactivation in the presence of metal ions, the enzyme was identified as a metallopeptidase. Substrate specificity studies revealed that, with the exception of pGlu-Phe-Pro-NH2, pGlu-7-amino-4-methyl-coumarin and pGlu-beta-naphthylamide, the purified enzyme removes N-terminal pGlu from only tri- and tetrapeptides with a histidine residue in the penultimate position. A number of N-terminal pyroglutamyl peptides of varying length were shown to competitively inhibit the enzyme. Of these, luteinizing hormone releasing hormone (LHRH) and LHRH 1-5, although not substrates for the enzyme, were found to be potent inhibitors, with Ki values of 8 and 11 microM, respectively. The study shows that while bovine brain PAPII shares many of the characteristics of PAPII from other mammalian tissues, its substrate specificity is not as narrow as previously reported.
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PMID:A study of a highly specific pyroglutamyl aminopeptidase type-II from the membrane fraction of bovine brain. 959 58

RT-PCR followed by 5'- and 3'- rapid amplification of cDNA ends was used to clone and sequence ovine prolactin-releasing peptide (PrRP). The cDNA was characterised by short 5'- and 3'-untranslated regions and a GC-rich (71%) coding region. The nucleotide and deduced amino acid sequences for the coding region showed 95.6 and 94.9% identity with bovine PrRP but the amino acid sequence of PrRP31 was conserved between these species. Northern blot analysis and RT-PCR showed that, as in the rat, the peptide was more abundantly expressed in the brainstem than the hypothalamus. However, in the ovine hypothalamus, PrRP mRNA expression was more widespread than in the rat, with expression detected in both rostral and caudal parts of the mediobasal hypothalamus. The effects of synthetic ovine PrRP on prolactin secretion both in vitro and in vivo were also examined. In primary cultures of sheep pituitary cells, PrRP significantly (P<0.01) increased prolactin concentrations in the culture medium but the response was not observed in every experiment and was only seen when pituitary glands were dispersed with collagenase rather than trypsin. PrRP was much less potent than TRH which caused a significant (P<0.01) two- to threefold increase in prolactin concentrations in every experiment. Intravenous (10 and 50 nmol) or intracerebroventricular (10 and 50 nmol) injection of PrRP had no significant effect on either plasma prolactin concentration or pulsatile LH secretion whereas intravenous injection of TRH (10 nmol) produced a highly significant (P<0.01) and more than sevenfold stimulation of plasma prolactin concentrations. In conclusion, these results suggest that PrRP is unlikely to be an important prolactin-releasing factor in this species.
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PMID:Prolactin-releasing peptide in the ewe: cDNA cloning, mRNA distribution and effects on prolactin secretion in vitro and in vivo. 1209 62

The TRH-like peptides pGlu-Glu-Pro amide, pGlu-Phe-Pro amide, and pGlu-Gln-Pro amide were isolated and identified some years ago, and these peptides have been proven to be present in many tissues and fluids. The presence of TRH-like immunoreactivity distinct from TRH in blood has been observed previously. In the present study, the presence of N-extended forms of TRH-like peptides in plasma has been investigated. Peripheral blood samples of human, rat, and rabbit were obtained and plasma was extracted. The peptides were separated in several steps of chromatography, including gel filtration, cation and anion exchange, and HPLC. The concentrations of the TRH-like peptides in the column fractions were measured by RIA with TRH antibody. The N-extended forms of TRH-like peptides were determined by RIA after trypsin digestion. In human plasma it was observed an N-extended form of TRH-like peptides in substantial concentration. After trypsin and heating, the N-extended forms of TRH-like peptides were rechromatographed on Sephadex G-50. This showed that the TRH-like peptides released have a similar size to TRH. The peptides were then separated by cation exchange chromatography, and the major fraction was unretained, indicating a neutral or acidic nature. Part of the unretained fraction was then chromatographed on anion exchange column in which the major fraction was retained, demonstrating the acidic nature of the peptides. Similar results have been observed in rat and rabbit. The other part of the unretained fraction from cation exchange chromatography of human plasma was purified on HPLC. The results demonstrated that the major component observed by HPLC cochromatographed with synthetic pGlu-Glu-Pro amide. This study represents the first demonstration of a circulating N-extended form of any TRH-like peptide.
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PMID:Characterization of a circulating N-extended form of the thyrotropin-releasing hormone-like peptide pGlu-Glu-Pro amide in human plasma. 1467 Dec 3


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