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 conversion of proparathyroid hormone to parathyroid hormone (PTH) was studied in vitro employing pancreatic trypsin as a prototype converting enzyme. Digestion of intact radiolabeled bovine prohormone with trypsin (0.1%) (w/w) resulted in release of a peptide comigrating with intact hormone marker in systems resolving both on the basis of charge (urea polyacrylamide gels, pH 4.4) and size (sodium dodecyl sulfate-urea polyacrylamide gels, pH 7.2). Tryptic digestions of a synthetic analogue of bovine prohormone, ProPTH-(-6 + 34), consisting of the prohormone hexapeptide covalently bonded to the NIH2 terminus of the active fragment of the hormone, released in high yield the hexapeptide and the intact active hormone fragment before any other smaller fragments. Analyses of digestions were by: (i) thin-layer chromatography and amino acid analysis of digestion products; (ii) comparison of the biological activity of the prophormone substrate and the products of digestion; and (iii) peptide end-group analysis by the Edman method during progressive tryptic hydrolysis over 22 h. The latter experiments demonstrated cleavage of more than 75% of the hexapeptide-hormone peptide bond before cleavage of other trypsin-sensitive sites within the molecule. It is concluded that the specificity of cleavage at the hexapeptide-hormone bond in the process of intracellular hydrolysis of proparathyroid hormone resides primarily in the sequence and/or conformation of the precursor molecule; inasmuch as conversion of prohormone to hormone can be efficiently accomplished by pancreatic trypsin in vitro, there is, therefore, no need to postulate the existence of an intracellular converting enzyme within the parathyroid cell that possesses unique hydrolytic specificity.
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PMID:Conversion of proparathyroid hormone to parathyroid hormone: studies in vitro with trypsin. 99 Feb 67

Plasma membranes prepared from rat renal cortex contain both a parathyroid hormone-sensitive adenylate cyclase and a potent proteolytic activity which degrades the hormone into peptide fragments. The degree and pattern of degradation was determined by subjecting incubation mixtures to gel filtration and ion exchange chromatography. Estimation of the degree of degradation by acid precipitation of the intact hormone was inadequate since metabolism of the hormone apparently generated acid-insoluble fragments. When parathyroid hormone was incubated with membrane fraction, the capacity of its stimulatory effect on adenylate cyclase decreased steadily. This decrease of PTH activitiy could be closely related to the degradation of intact hormone by the same membrane preparation. The adenylate cyclase and degradative activity appeared to exist in similar membrane structures since they could not be separated by centrifugation through sucrose density gradients. The degradation of the hormone could not be inhibited by Trasylol and pancreatic or soybean trypsin inhibitors and was only slightly inhibited by ribonuclease and benzamidine. Histone (1 mg per ml), on the other hand, was able to decrease the degradation of the hormone and prevent the loss of its activity. Radioimmunoassay of the incubation mixtures showed that the rapid degradation of both amino- and carboxy-terminal regions of the hormone was prevented by histone. The oxidized, inactive hormone was also degraded to the same extent by the renal cortical membrane. Furthermore, the degradative activity was also found in plasma membrane preparations of renal medulla and liver. This lack of hormone and tissue specificity suggests that similar degradative activity exists in all tissues and that caution should be exercised in estimating hormonal potency based on activation of adenylate cyclase.
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PMID:Interaction of parathyroid hormone with membranes of kidney cortex: degradation of the hormone and activation of adenylate cyclase. 119 1

Recently, a parathyroid hypertensive factor was postulated to play a role in the pathogenesis of hypertension in genetically hypertensive rats. Therefore it was examined, whether in human parathyroid glands a vasopressor substance can be detected. For this purpose, homogenates of hyperplastic parathyroid glands from 20 patients with tertiary hyperparathyroidism were deproteinized and fractionated by gel chromatography. The fractions obtained were tested for vasopressor activity in isolated perfused rat kidneys. A vasopressor fraction containing substances of 0.6-2.5 kDa was identified in the parathyroid glands. The responsible product was heat sensitive, peptidase-, trypsin- and carboxypeptidase y- sensitive and hydrophilic, as it did not bind to hydrophobic reversed-phase gel. These results suggest that parathyroid glands contain a hydrophilic peptide-like vasopressor substance different from the parathyroid hormone.
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PMID:A vasopressor factor partially purified from human parathyroid glands. 141 52

A primary culture method was established by comparing the different effects of four methods of enzymatic separation--trypsin, collagenase with and without trypsin pretreatment, and a trypsin-collagenase mixture--and five media: DMEM, DMEM and Ham's F 12 mixture, F 12, RPMI 1640 and Medium 199. The trypsin pretreatment/collagenase method was most preferable considering the high number of isolated cells, satisfactory adhesion, good growth and a single population at subconfluence. DMEM and the DMEM/F-12 mixture resulted in the best adhesion, cell growth and cell number at confluence. Primary cells separated by the trypsin pretreatment/collagenase method and cultured in DMEM were responsive to parathyroid hormone at the proliferating stage and had higher alkaline phosphatase activity than cells cultured from gingiva and mucosa after reaching confluence. The long-term cultured cells formed nodules that were slightly mineralized. These results indicate that the cultured pulp cells had properties characteristic of pulp cells in vivo. This enzymatic separation method may be useful in studies of the regulation of pulp metabolism and odontoblast differentiation.
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PMID:Establishment of primary cultures of pulp cells from bovine permanent incisors. 166 Feb 58

We observed that culture medium conditioned with fetal rat long bones contained peptides immunologically related to the parathyroid hormone-related peptide of malignancy (PTHrP) and stimulated cyclic AMP production in canine renal cortical membranes. Because the adenylate cyclase stimulating activity (CSA) of the medium increased when bone resorption was stimulated, it was suspected that these peptides were stored in the matrix and released during the resorption process. In this work, we extracted the noncollagenous proteins of fetal rat long bones and found that the extract contained significant amounts of CSA. The biologic activity of the extract was abolished after trypsin digestion and eluted at 24 and 37 kD on filtration HPLC. The CSA of bone extract and of both HPLC peaks could be inhibited by 3-34 and 7-34 parathyroid hormone analogs. It was not blocked by an antiserum directed against the N-terminal region of parathyroid hormone, but it was significantly inhibited after an overnight preincubation with an antiserum directed against the 1-11 fragment of PTHrP. One band migrating at 18 kD could be visualized after SDS-PAGE electrophoresis of bone extract and immunoblotting with the anti-PTHrP antiserum. We conclude that an adenylate cyclase stimulator immunologically similar to PTHrP is present in the matrix of fetal rat long bones. Adenylate cyclase stimulating peptides of lower molecular weight found in bone-conditioned medium could be active fragments formed by proteolysis during the resorption process.
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PMID:Adenylate cyclase stimulating activity immunologically similar to parathyroid hormone-related peptide can be extracted from fetal rat long bones. 166 3

We observed that culture medium conditioned with fetal rat long bones stimulated cyclic AMP production by canine renal cortical membranes. This cyclase-stimulating activity (CSA) was retained by an ultrafiltration membrane with a molecular weight cutoff of 5000; three biologically active peaks with an approximate molecular weight of 18,000-25,000, 9000-12,000, and 4000-6000 were separated by high-performance liquid chromatography. The biologic activity was destroyed by trypsin digestion. The stimulation of adenylate cyclase by the medium and by the three peaks was inhibited by [N-leu8,18,Tyr34]parathyroid hormone-(3-34)-amide and by [Tyr34]parathyroid hormone-(7-34)amide. Preincubation of the bone culture medium and of the three peaks with an antibody raised against human parathyroid hormone-(1-34) did not decrease the biologic activity more than incubation with nonimmune serum. However, the biologic activity of the three active peaks was significantly suppressed after preincubation with an antiserum directed against the N-terminal region of the parathyroid hormone-related peptide of malignancy. The release of CSA into the bone culture medium was enhanced by parathyroid hormone induction and by 1,25-dihydroxycholecalciferol. It was decreased by calcitonin. We conclude that fetal murine bones in culture release peptides that stimulate the adenylate cyclase of renal cortical membranes. These peptides are antigenically similar to the parathyroid hormone-related peptide of malignancy. Their release from bones is modulated by hormones that control bone resorption.
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PMID:Release of parathyroid hormonelike peptides by fetal rat long bones in culture. 239 1

Cell growth and synchronization, gene expression and regulation, maintenance of epithelial cell polarization are major functional aspects of the nephron which have been difficult to approach with conventional preparations. Certainly, without the introduction of tissue culture techniques, the opportunity to analyze these issues could not have been afforded. Several renal epithelial cell lines with differentiated characteristics of proximal and distal segments of the nephron are already available. LLC-PK1, derived from a normal Hampshire pig kidney, shows multiple differentiated characteristics of in vivo epithelia. Specifically, this cell line has Na+-dependent sugar, amino acid, and phosphate cotransport systems with similar characteristics as those present in the renal proximal tubule. The expression of the Na+-dependent sugar transport system in LLC-PK1 cells depends on the growing conditions of the cells. For instance, isolated cells obtained by trypsin-EDTA treatment of confluent monolayers or exponentially growing cells do not express the Na+-dependent sugar transport system. Full expression occurs after the monolayer reaches confluency. Expression of the transporter can also be modified by agents that affect the differentiation of other cellular systems, such as the Friend erythroleukemia cells. The expression of the Na-sugar cotransport system is also regulated by the concentration of glucose in the growth medium. Low glucose concentration increases the sugar influx through the Na+-coupled apical membrane transporter by increasing the number rather than the affinity of the transporter. This effect appears to be mediated through the sugar metabolism of the cell. The expression of the Na+-amino acid cotransport system in LLC-PK1 cells and the epithelial cell line MDCK derived from a normal dog kidney also responds to regulatory signals associated with cell growth or amino acid deprivation. LLC-PK1 cells and the cell line OK derived from an opposum kidney, shows a Na+-dependent phosphate cotransport system modulated by parathyroid hormone and cyclic nucleotides that will prove to be an excellent model not only to study the mechanisms of the action of the hormone, but to study the mechanisms involved in the expression and modulation of the Na+-dependent phosphate cotransport system.
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PMID:Sodium cotransport processes in renal epithelial cell lines. 242 Nov 46

Mixed bone cell cultures obtained by sequential collagenase-trypsin digestion of newborn chick, rat, and mouse calvaria responded to calcitonin gene-related peptide (CGRP) with a dose-dependent increase in cyclic AMP formation. The amplitude of response to CGRP in each species was less than that to parathyroid hormone (PTH). The CGRP effect was not the result of an action as a weak calcitonin agonist, since in most instances a calcitonin effect was not observed. Only in early digests of mouse calvarial cells were consistent stimulatory effects of calcitonin on cyclic AMP noted, and these were always considerably less in amplitude than those to CGRP. It is concluded that chick, rat, and mouse bones contain cells in osteoblast-rich populations that respond specifically to CGRP with a rise in cyclic AMP.
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PMID:Effects of calcitonin gene-related peptide on cyclic AMP formation in chicken, rat, and mouse bone cells. 254 86

The secretion of parathyroid hormone (PTH) is inversely related to the extracellular Ca2+ concentration (Ca2e+). To test the hypothesis that a Ca2+ sensor on the surface of parathyroid cells is involved in Ca2+-regulated PTH secretion, limited trypsinization of bovine parathyroid cells was carried out. Treatment with trypsin (1.1-10 mg/ml) inhibited, in a dose-dependent manner, PTH secretion stimulated by lowering Ca2e+ from 2.0 to 0.5 mmol/l. In control cells, activation of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced PTH secretion at 2.0 mmol Ca2e+/l but not at 0.5 mmol Ca2e+/l. In trypsinized cells, however, TPA enhanced PTH secretion at both 0.5 and 2.0 mmol Ca2e+/l. Isoproterenol-stimulated PTH secretion was maintained in trypsinized cells, but reduced cyclic AMP production revealed that some beta-adrenergic receptors were destroyed. The cytosolic free Ca2+ concentration (Ca2i+), as measured with fura-2, was raised within seconds in response to increasing Ca2e+ from 0.5 to 2.0 mmol/l and was then lowered within 1 min to a sustained plateau; the changes were the same in trypsinized and control cells. In conclusion, trypsinization of parathyroid cells abolished Ca2+-regulated PTH secretion without affecting Ca2i+.
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PMID:Trypsinization of bovine parathyroid cells abolishes Ca2+-regulated parathyroid hormone secretion. 254 48

We have partially purified a tumour factor capable of stimulating both bone resorption in vitro and cAMP accumulation in osteoblastic ROS 17/2 cells from three human tumours associated with humoral hypercalcaemia of malignancy. Purification of tumour factor by sequential acid urea extraction, gel filtration and cation-exchange chromatography, reverse-phase high performance liquid chromatography followed by analytical isoelectric focussing provided a basic protein (pI greater than 9.3) with a molecular weight of approximately 13,000 as a major component of the final preparation which retained both the two bioactivities. Bone resorbing activity and cAMP-increasing activity in purified factor correlated with each other. cAMP-increasing activity of the factor was heat- and acid-stable, but sensitive to alkaline ambient pH. Treatment with trypsin destroyed cAMP-increasing activity of the factor. Synthetic parathyroid hormone (PTH) antagonist, human PTH-(3-34) completely inhibited the cAMP-increasing activity of the factor. The results suggest that this protein factor, having its effects on both osteoclastic and osteoblastic functions, may be involved in development of enhanced bone resorption in some patients with humoral hypercalcaemia of malignancy.
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PMID:Co-purification of bone resorbing activity and adenylate cyclase stimulating activity from human tumours associated with the humoral hypercalcaemia of malignancy. 302 25


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