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 conversion of proparathyroid hormone (proparathormone) to parathyroid hormone (parathormone) by subcellular fractions of the bovine parathyroid has been investigated. The identification of the conversion product as parathormone was established by its elution postion during ion exchange chromatography and gel filtration, and by partial amino acid sequence analysis of its NH2-terminal region. Total homogenates and derived subcellular fractions (600 X g pellet, 5,000 X g pellet, 20,000 X g pellet, 190,000 X g pellet, and 190,000 X g supernatant) all catalyzed the conversion of exogenous [3H]- or [14C]prohormone. Over 60% of the converting activity was in the particulate fractions; the 190,000 X g particulate fraction contained the highest specific converting activity. The converting activity appeared to be an integral component of the membranes since it could only be partially removed by extraction with Triton X-100. The production of parathormone by the particulate converting enzyme increased with time and the concentration of enzyme protein. The optimum pH range was between 7 and 9, and the enzyme was inactive below pH 6. Conversion by the particulate enzyme was inhibited by benzamidine or chloroquine, but not by pancreatic trypsin inhibitor, indicating its dissimilarity to trypsin. When a mixture of [14C]proparathormone and [3H]parathormone was used as substrate, the particulate enzyme did not metabolize the hormone despite over 70% conversion of the prohormone to hormone and other peptides. There was a close correlation between the subcellular distribution of converting activity and that of newly formed parathormone found in the membrane fraction. These data suggest that the particulate converting activity is that concerned with the formation of parathormone in vivo.
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PMID:Conversion of proparathyroid hormone to parathyroid hormone by a particulate enzyme of the parathyroid gland. 1 Mar 4

Six populations of bone cells (populations 1-6) were obtained by sequential digestion of mouse calvaria with collagenase and trypsin. After release from the tissue, each cell population was cultured for seven days. Parathormone, but not calcitonin, elicited an increase in intracellular cyclic AMP in the cells of populations 4, 5, and 6. In contrast, both hormones elicited increases in cyclic AMP in populations 2 and 3 but had no effect on population 1. When the cells of population 2 were exposed to a Falcontissue culture polystyrene surface for periods of time up to 5 min, many cells adhered. The nonadhering cell population contained a lesser proportion of cells responsive to calcitonin, whereas the adhering population contained a greater proportion responsive to this hormone. Conversely, when the cells of population 2 were exposed to an acid-treated nylon surface, the nonadhering cells contained a larger proportion of those responsive to calcitonin and a smaller proportion responsive to parathormone. When those cells that were enriched for calcitonin responsiveness were examined, we found an increased proportion that exhibited an asymmetric bipolar morphology. These differed from large amorphous, often binucleate, cells which predominated in those populations that responded exclusively to parathormone. These results establish that bone contains at least two types of target cells--one that responds to parathormone but not calcitonin, the other that responds predominantly to calcitonin.
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PMID:Target cells in bone for parathormone and calcitonin are different: enrichment for each cell type by sequential digestion of mouse calvaria and selective adhesion to polymeric surfaces. 17 56

Two metabolically distinct types of bone cell populations were isolated from mouse calvaria by a repetitive digestive procedure with a mixture of collagenase and trypsin. Cells released early in the digestion showed approximately two-fold increases in cAMP when treated with either parathormone or calcitonin. These populations were denoted CT type. Later eluting cells showed larger parathormone-induced increases in cAMP but did not respond to calcitonin. These populations were denoted PT type. Six metabolic and enzymatic activites were measured in the two types of populations: acid and alkaline phosphatases, hyaluronate synthesis, citrate decarboxylation, prolyl hydroxylase, and general protein synthesis. Although each of these activites was present in both cell types, the basal levels of acid phosphatase and hyaluronate synthesis were higher in the CT cells, whereas alkaline phosphatase, citrate decarboxylation, and prolyl hydroxylase were higher in te PT cells. Parathormone stimulated acid phosphatase and hyaluronate synthesis by 100-200% only in the CT cells; in inhibited alkaline phosphatase, citrate decarboxylation, and prolyl hydroxylase by 75-90% only in the PT cells. Calcitonin alone had no effect on any of these activities other than cAMP production, but in inhibited the action of parathormone in the CT cells. The sensitivities, time courses of development,and magnitudes of these hormonal effects were similar to those observed previously in intact calvaria, indicating that the isolated cell system is a reliable model for the study of bone metabolism. Based on the metabolic responses of the cells, we postulate that the CT type of populations is enriched in osteoclasts and, possibly, osteocytes, and the PT type of population is enriched in osteoblasts.
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PMID:Biochemical characterization with parathormone and calcitonin of isolated bone cells: provisional identification of osteoclasts and osteoblasts. 18 58

Distributions of parathyroid hormone (PTH), proparathyroid hormone (ProPTH), preproparathyroid hormone (PreProPTH), and parathyroid secretory protein (PSP) were analyzed in subcellular fractions prepared from homogenates of bovine parathyroid glands. Slices of bovine parathyroid glands were incubated with radiolabeled amino acids for 3--30 min to selectively label newly synthesized proteins. Subcellular fractions were prepared from homogenates of the gland slices by differential centrifugation. Newly synthesized labeled hormonal polypeptides in the fractions were analyzed by electrophoresis on polyacrylamide gels, and total amounts of PTH and ProPTH (previously formed and newly synthesized) were determined by immunoassay. Ninety percent of total immunoreactive, 70--80% of newly synthesized PTH, ProPTH, and PreProPTH, and 50% of PSP were found in sedimentable particulate fractions. The low speed (800 X g) pellet, which consisted predominantly of cell debris and nuclei with adherent remnants of cytoplasm, contained 30--50% of the ProPTH and PTH. The intermediate speed (10,000 X g) pellet, which contained granules, was relatively enriched in PTH. Most particulate-associated hormone could be solubilized by treatment with deoxycholate (DOC) 98% and 97% of radiolabeled and 93% and 83% of immunoreactive ProPTH and PTH, respectively, in particulates sedimenting at 10,000 and 105,000 X g were rendered DOC-soluble. Approximately 50% of the PTH and ProPTH in the particulates resisted digestion by combined trypsin and chymotrypsin, whereas PreProPTH was completely susceptible to proteolysis. Up to 50% of the radiolabeled PTH and ProPTH added exogenously to parathyroid gland slices before homogenization became associated with the particulate fractions, and 70--80% or radiolabeled PreProPTH added to the subcellular fractions readily associated with the sedimentable material. The results indicate that in homogenates of parathyroid glands, PTH, ProPTH, PreProPTH, and PSP are associated with particulate structures. Furthermore, up to 50% of the association of ProPTH, PTH, and PSP with particulate fractions seems to be nonsepcific and occurs during the disruption of the tissues. The remaining 50% or more of hormonal protein is presumably sequestered within membrane-limited structures, such as microsomal vesicles. The complete susceptibility in particulate fractions of newly synthesized PreProPTH, but not of ProPTH, to limited proteolysis indicates that the two precursors are located in different subcellular compartments and suggests that PreProPTH is converted to ProPTH before its entry into the intracisternal space of the endoplasmic reticulum. Alternatively, the PreProPTH identified in parathyroid gland slices may represent polypeptide chains synthesized in the cell sol on polyribosomes that are not attached to endoplasmic reticulum but are adsorbed nonspecifically to the particulate fraction of the cell during the process of tissue homogenization.
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PMID:Subcellular distributions of parathyroid hormone, hormonal precursors, and parathyroid secretory protein. 44 53

The cleavage products from the conversion of proparathormone to parathormone by a bovine and porcine parathyroid microsomal converting activity have been analyzed. In the conversion reaction, the first 6 amino acid residues of the prohormone (Lys-Ser-Val-Lys-Lys-Arg-) are released as an intact hexapeptide. This is rapidly converted to a pentapeptide by removal of the NH2-terminal lysine and then to a tetrapeptide by removal of the COOH-terminal arginine. In order to test for the presence of a postulated COOH-terminal extension of the parathormone sequence in proparathormone, mixtures of 14C-proparathormone and 3H-parathormone were subjected to digestion by trypsin or Staphylococcus aureus protease. The resulting radioactive peptides from the hormone and its precursor were compared. There was no evidence that any fragments different from those from the hormone were released from the prohormone except those accounted for by the NH2-terminal hexapeptide adduct on proparathormone. Thus, the conversion of the prohormone to the hormone catalyzed by the microsomal membrane activity requires only the cleavage of this hexapeptide.
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PMID:The mode of conversion of proparathormone to parathormone by a particulate converting enzymic activity of the parathyroid gland. 63 52

Pancreatitis was induced in 11 miniature pigs by infusing a bile salt-trypsin solution into the pancreatic duct. Seven animals served as sham-operated controls. Serum ionized calcium, total calcium, albumin, total protein, inorganic phosphorus, urea nitrogen, magnesium, insulin, glucagon, and hematocrit were determined every six to 12 h over a period of one week in both test and control animals. We observed significant decreases in ionized and total calcium, modest decreases in albumin, and significant increases in the inorganic phosphorus, urea nitrogen, and hematocrit in the pancreatitic pigs. The latter two findings were consistent with early acute hypovolemia. Glucagon and insulin appeared to play no role in the hypocalcemia. Glucagon concentrations increased to the same degree in both test and control animals, probably as a result of the stress of being handled and operated on. The highest concentrations of inorganic phosphorus and the lowest concentrations of both ionized and total calcium were seen 18 h after the induction of pancreatitis in the test animals. These findings suggest that parathyrin (parathormone) was not being secreted in adequate amounts, or that the target organs were unresponsive to parathyrin.
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PMID:Biochemical changes in a porcine model of acute pancreatitis. 65 76

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

The metabolism of natural and synthetic analogues of bovine proparathyroid hormone (Pro-bPTH) and the biological activity of the synthetic fragments were evaluated in an in vitro assay employing renal cortical membranes (adenylyl cyclase bioassay). Apparent biological activity of the prohormone analogue was correlated with the cleavage of prohormone to hormone by the membranes. Analysis by electrophoresis on polyacrylamide gels and by ion-exchange chromatography on carboxymethyl cellulose (CMC) of the synthetic analogue Pro-bPTH-(-6 leads to +34) labelled with 125I, after incubation with renal cortical membranes, revealed conversion of the analogue to a fragment co-migrating or co-eluting with bPTH (1-34). Similar electrophoretic analyses using biosynthetic Pro-bPTH-(-6 leads to +84) internally labelled with [3H] leucine revealed degradation of Pro-bPTH-(-6 leads to +84) to smaller fragments. Proteolysis of both native prohormone and prohormone analogue was markedly reduced in incubations performed in the presence of benzamidine, a competitive inhibitor of trypsin and trypsin-like enzymes. Inclusion of benzamidine in incubations with purified renal cortical membranes from rat or dog in the in vitro renal adenylyl cyclase bioassay resulted in a ten-fold lowering of the potency of the fragment Pro-bPTH-(-6 leads to +34) relative to that of bPTH-(1-34); the potency of Pro-bPTH-(-6 leads to +34) was reduced from 5.4-3.6% to 0.5-0.3%, on a molar basis, of the potency of bPTH-(1-34). There was no effect of benzamidine on the intrinsic activity of bPTH-(1-34). These studies indicate that most if not all of the apparent biological activity of Pro-bPTH-(-6 leads to +34), at least in vitro, is dependent upon prior enzymic conversion to bPTH-(1-34).
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PMID:Metabolism and biological activity of proparathyroid hormone and synthetic analogues in renal cortical membranes. 118 11

A unique calcium-dependent endopeptidase specifically cleaving on the carboxyl side of paired basic residues was partially purified from Golgi membrane fractions of rat liver. The enzyme, with optimal pH at around 6.0, hydrolyzes synthetic peptides corresponding to the amino-terminal sequences of proalbumin and proparathyroid hormone at the carboxyl sides of paired basic residues (Arg-Arg and Lys-Arg), but peptides corresponding to the amino-terminal sequences of proalbumin variants, in which Arg-Arg at the site of cleavage is replaced by Arg-Gln or His-Arg, are not affected by the enzyme. From its strict substrate specificity and inhibitory spectrum, this enzyme appears to be a novel endopeptidase distinct from trypsin and cathepsin B and may be physiologically involved in proprotein processing.
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PMID:A unique membrane-bound, calcium-dependent endopeptidase with specificity toward paired basic residues in rat liver Golgi fractions. 281 87

The concentrations of calcitonin and parathormone were studied in 63 patients with chronic pancreatitis during exacerbation. The results obtained were analyzed with relation to the state of pancreatic exocrine and endocrine function, gravity of disease and the blood level of calcium. The concentration of calcitonin was considerably raised, the most noticeable elevation was observed in patients with a severe course. The level of calcitonin also rose frequently in patients with high activity of blood pancreatic enzymes (amylase and radioimmune trypsin) and hyperglucagonemia. The level of parathormone did not undergo marked changes.
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PMID:[Calcium-regulating hormones of the blood in patients with chronic pancreatitis]. 336 66


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