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 insulin or proinsulin response of the isolated rat adipocyte was destroyed by preincubation with trypsin. After 120 minutes, biological responsiveness partially regenerated. Similarly, the biological responsiveness of the isolated fat cell to non-suppressible insulin-like activity (NSILA) was only partially destroyed following trypsin digestion, and did not regenerate. In contrast to the above, cyclic AMP or dibutyryl cyclic AMP effects were unaltered by trypsin or neuraminidase digestion.
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PMID:Studies of the biological activity of insulin, cyclic nucleotides and concanavalin A in the isolated fat cell. 16 39

Familial hyperproinsulinemia is an autosomal dominant defect that is associated with strikingly elevated levels of serum proinsulin-like material. Our studies show that trypsin converts familial hyperproinsulinemia proinsulin to insulin more slowly than it converts a 131I-labeled porcine proinsulin marker. Molar yields of insulin indicated that the material may be an intermediate proinsulin. Studies with two human C-peptide antisera that differ in their relative immunoreactivity with human C-peptide and proinsulin showed that the two antisera reacted equally with familial hyperproinsulinemia proinsulin, suggesting that it is a partially cleaved proinsulin intermediate. Sulfitolysis of highly purified material to break the inter- and intra-chain disulfide bridges and subsequent adsorption on a specific B-chain antibody covalently bound to Sepharose beads showed that the C-peptide was still connected to the B-chain. These data indicate that familial hyperproinsulinemia proinsulin is normally cleaved at the C-peptide-A-chain linkage site. A structural abnormality appears to underlie familial hyperproinsulinemia proinsulin, which impairs its cleavage at the B-chain-C-peptide linkage site.
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PMID:Familial hyperproinsulinemia: partial characterization of circulating proinsulin-like material. 28 74

Poly(A)-containing mRNA isolated from the islets of Langerhans obtained from two species of fish, angler fish (Lophius americanus) and sea raven (Hemitripterus americanus), stimulated protein synthesis 16-fold in a wheat germ cell-free system. Characterization of the translation products by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed a major polypeptide weighing 11,500 daltons that was specifically precipitated by an antibody against angler fish insulin. Partial sequence analysis of the amino terminal revealed that this polypeptide is preproinsulin, in which the amino terminus of proinsulin is preceded by either 23 (angler fish) or 25 (sea raven) amino acid residues. Translation of fish islet mRNA in a wheat germ cell-free system in the presence of dog pancreas microsomal membranes led to the correct cleavage of the nascent preproinsulin, resulting in the synthesis of authentic fish proinsulin, as verified by partial sequence analysis. Moreover, the synthesized fish proinsulin was segregated, presumably into the luminal space of the dog pancreas microsomal vesicles, because it was found to be resistant to proteolysis by added trypsin and chymotrypsin. Our data thus suggest that the mechanisms and information for the transfer of secretory proteins across the microsomal membrane are highly conserved during evolution.
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PMID:Cell-free synthesis of fish preproinsulin, and processing by heterologous mammalian microsomal membranes. 32 65

1. Rabbit islets of Langerhans were disrupted by ultrasonic methods and the sonicated preparations were used to study proinsulin biosynthesis. 2. When [3h]leucine is incubated in such preparations, incorporation takes place into proinsulin, as evidenced by characterization on polyacrylamide gels, and by the conversion of this labelled material into insulin, by using trypsin. 3. The labelled proinsulin may also be purified by antiinsulin antibody bound to Sepharose. 4. With the broken-cell preparation it was shown that incorporation of leucine is accelerated by increasing the glucose content of the medium from 2mM to 16mM. However, 16mM-galactose or -sucrose did not stimulate incorporation significantly from basal values. This effect of glucose was abolished by cycloheximide. 5. The significance of these findings in relation to the mechanism of glucose stimulation of proinsulin biosynthesis is discussed.
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PMID:Proinsulin biosynthesis in broken-cell preparations of islets of Langerhans. 34 1

In homogenates and subcellular fractions of pancreatic islets of Wistar rats we could demonstrate three groups of protein degrading enzymes. The proteinases of group 1 are characterized by both trypsin-like and carboxypeptidase B-like specificities with slightly acid pH optima (pH 5.5-6.5) and seem to play important roles in the conversion of proinsulin into insulin. The properties suggest that these enzymes localized in the secretion granule/mitochondria fraction are related to the tissue cathepsins. Group 2 enzymes are thiol-depending proteinases with a pH optimum at 7.0 occuring mainly in the cytosol and to a lesser extent in the fraction of nuclei and cell debris. Group 3 represents the thiol protein oxidoreductase with a pH optimum of 7.0. This enzyme degrading disulfide bonds could also be important in the formation of the disulfide bonds during protein folding after synthesis.
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PMID:Proteolytic and transhydrogenolytic activities in isolated pancreatic islets of rats. 35 9

This work addressed the problem of heterogeneity of immunoreactive insulin (IRI) in human plasma. Subjects with normal glucose tolerance were given 75g of an oral glucose solution, followed in 30 min by an intravenous infusion of 30g of arginine over 30 min. At the end of the infusion blood was withdrawn for analysis. IRI was extracted from plasma of individual subject by immunosorbent columns and was fractionated by gel filtration, disc gel electrophoresis and isoelectric focusing. Human IRI components were identified by molecular size, immunoreactivity with a human proinsulin antibody, sensitivity to trypsin, and by comparison of electrophoretic mobility and isoelectric point with porcine pancreatic products, after suitable correction for electric charge and molecular weight differences. The pattern of IRI heterogeneity was the same among six healthy subjects. Heterogeneity of proinsulin-size IRI in circulation was more marked than that of insulin-size material. Proinsulin and desdipeptide proinsulin were present in approximately equal amounts accompanied by minor amounts of split proinsulin and monodesamido-desdipeptide proinsulin. Insulin-size IRI contained over 80% insulin. Minor amounts of monodesamidoinsulin and diarginylinsulin were observed in some cases. The types of IRI components observed in plasma are evidence in support of a physiologic role of trypsin-and carboxypeptidase B-like enzymes in the conversion of proinsulin to insulin. Moreover, this study provides a base line for investigation of abnormalities in proinsulin-to-insulin conversion that may be associated with certain pathologic states.
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PMID:Characterization of proinsulin-insulin intermediates in human plasma. 35 97

A pancreatic endopeptidase localized to the beta-cells of the pancreas by immunohistochemical techniques has been purified to homogeneity by following its functional and antigenic characteristics as a glandular kallikrein (EC 3.4.21.8). The enzyme gave a single stained band on alkaline disc gel electrophoresis which corresponded in location with the kinin-generating activity eluted from a replicate gel, was of 54,000 molecular weight by gel filtration, was devoid of caseinolytic activity, elicited a monospecific antiserum in a rabbit, and gave a line of complete identity with a single constituent in pancreatic extract, crude urine, and purified urokallikrein when analyzed with monospecific antibody to urokallikrein. The pancreatic glandular kallikrein generated three cleavage products of increasing anodal mobility from bovine and porcine proinsulin, and the presence of pancreatic kininase or bovine carboxypeptidase B increased the quantity of these products. Although the conversion products did not correspond to diarginyl- and monoarginylinsulin, the product of intermediate mobility was also obtained when proinsulin was treated with a low concentration of trypsin in the presence of kininase. The most rapidly migrating product did correspond to desalanylinsulin in the reference standard. Kininase alone had no action on proinsulin, and aprotinin prevented cleavage by kallikrein alone or in combination with kininase. Although the chemical structure of the proinsulin cleavage products has not been established, human pancreatic kallikrein is considered a putative activator of proinsulin because of its location in the beta-cell, its preferential action on proinsulin and kininogen as compared to azocasein, and its capacity to generate insulin intermediate products that are further modified by human pancreatic kininase or bovine carboxypeptidase B.
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PMID:Sequential cleavage of proinsulin by human pancreatic kallikrein and a human pancreatic kininase. 38 42

Trypsin-like and carboxypeptidase B-like proteinases are believed to play important roles in the conversion of proinsulin into insulin as well as in the intracellular processing of a variety of other precursor forms. To facilitate the study of these enzymes we have developed sensitive methods for their detection in tissue preparations and incubation media. Studies with rat islet homogenates indicate the presence of both trypsin-like and carboxypeptidase B-like activities with slightly acidic pH optima. The trypsin-like activity was activated by thiols and inhibited by several thiol reagents but the carboxypeptidase was inhibited only by chelating agents. These properties suggest that these enzymes are related to the tissue cathepsins. Additional experimental approaches to the problems of positively identifying and localizing converting enzymes at the subcellular level are briefly discussed.
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PMID:Carboxypeptidase B-like and trypsin-like activities in isolated rat pancreatic islets. 82 May 28

Plasma immunoreactive glucagon (IRG) concentrations were measured in 36 patients with chronic renal failure (CRF) and 32 normal subjects. In addition, the components of circulating IRG were analyzed by gel filtration in the fasting state and after physiological stimuli. Fasting IRG was elevated (P less than 0.001) in CRF patients (534 +/- 32 pg/ml) compared with the levels found in healthy subjects (113 +/- 9 pg/ml). Oral glucose suppressed plasma IRG in CRF patients from a basal level of 568 +/- 52 to a nadir of 354 +/- 57 pg/ml (120 min). This degree of suppression (38%) was comparable to that found in normal subjects (basal = 154 +/- 20 to 100 +/- 23 pg/ml) at 120 min (35%). Intravenous infusion of arginine (250 mg/kg) resulted in a 71% rise in IRG in CRF patients and a 166% increase in normal subjects. Gel filtration of fasting plasma from CRF patients showed three major peaks. The earliest (A) was found in the void volume (mol wt greater than 40,000) and constituted 16.5 +/- 4.7% of the elution profile. The middle peak (B) eluted just beyond the proinsulin marker (approximately 9,000 mol wt) and constituted the largest proportion of the elution profile (56.5 +/- 3.4%). The third peak (C) coincided with the standard glucagon and [125I]glucagon markers (3,485 mol wt) and comprised 27.0 +/- 4% of the IRG profile. In contrast, only peaks A and C were found in fasting plasma of normal subjects (53.6 +/- 10.4% in A and 46.4 +/- 10.4 in C). After oral glucose, glucagon immunoreactivity in the 3,500 mol wt peak (C) was markedly suppressed, while the B peak in patients with CRF declined to a lesser extent. The A peak in both groups was unchanged. After an arginine infusion only the C peak increased in both groups of subjects. Gel filtration of plasma in 3 M acetic acid gave similar profiles to those obtained in glycine albumin buffer. Exposure of serum to trypsin indicated that the B and C peaks were digestible, while the A peak was resistant to the action of the enzyme. In one sample, peak C increased after a 2-h exposure of serum to trypsin. We conclude that circulating IRG in normal subjects and patients with CRF is heterogenous. The hyperglucagonemia of renal failure is largely due to an increase in IRG material of approximately 9,000 mol wt, consistent with proglucagon, although the 3,500 mol wt component is also considerably elevated (threefold). The significance of circulating IRG levels should be interpreted with caution until the relative biological activity of the three components is established.
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PMID:Heterogeneity of plasma glucagon. Circulating components in normal subjects and patients with chronic renal failure. 95 99

Evidence is presented that proglucagon from anglefish islets is a single chain polypeptide with 78 amino acid residues and that the glucagon portion of it is liberated after tryptic cleavage. The most striking characteristic in the conversion of the anglerfish proglucagon to glucagon is that the cleaved peptide bonds display enormous sensitivity toward trypsin. Thus, conversion of the prohormone to glucagon occurs very rapidly within 3-10 min with a 1:500-1:1000 molar ratio of enzyme to substrate. Further, trypic cleavage of the anglerfish glucagon requires higher concentrations of trypsin (molar ratio 1:25 enzyme to substrate) and longer incubation time. The behavior of proglucagon and glucagon toward trypsin shows striking similarities with the tryptic conversion of anglerfish proinsulin to insulin.
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PMID:Isolation and partial characterization of anglefish proglucagon. 109 38


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