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)

Alpha1-Antitrypsin was isolated from rabbit plasma by salting out with (NH4)2SO4 followed by ion-exchange chromatography either on DEAE-Sephadex or DEAE-cellulose (each at pH8.8 and 6.5), and affinity chromatography on Sepharose-Cibacron Blue and Sepharose-concanavalin A. The protein thus obtained was homogeneous during crossed immunoelectrophoresis by using an antiserum to whole rabbit plasma, but it migrated as two broad bands when electrophoresed in alkaline polyacrylamide gels. Under optimal loading conditions, two or three subcomponents could be distinguished in each band. The two major forms of rabbit alpha1-antitrypsin, designated components F and S, were separated by preparative polyacrylamide-gel electrophoresis, and some of their physico-chemical properties were established. Both forms reacted with trypsin at a molar ratio of 1:1. Their elution volumes from a Sephadex G-200 column were identical, corresponding to a mol.wt. of 58000; however, some heterogeneity was observed after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing in polyacrylamide gel in a pH 4-6 gradient revealed a multiple-band pattern for each form in the range of pH4.4-4.9. The two forms of rabbit alpha1-antitrypsin possessed the same N-terminal amino acid (glutamic acid) and had very similar amino acid and carbohydrate compositions.
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PMID:Isolation and partial characterization of rabbit plasma alpha1-antitrypsin. 30 50

Rat prorenin was synthesized by Chinese-hamster ovary cells transfected with an expression vector containing rat preprorenin cDNA sequences, then purified by concanavalin A-Sepharose chromatography and h.p.l.c. on G3000SW. The molecular mass of purified prorenin was 46,000 Da, as determined by h.p.l.c. on G3000SW. Immunoblot analysis indicated that recombinant prorenin cross-reacted with anti-(mature renin) antibody and two kinds of antibodies recognizing the N-terminus and C-terminus of the prosegment of rat prorenin. Recombinant prorenin was bound to a Cibacron Blue-Sepharose column and eluted with 1.4 M-NaCl, but was not retained by an octapeptide renin inhibitor (H-77)-Sepharose column. Trypsin activation of prorenin increased the renin activity 110-fold, caused binding to an H-77-Sepharose column and nullified the reactivity to the above two kinds of anti-prosegment antibodies, findings indicating that the activation of prorenin with trypsin is due to the cleavage of the prosegment. Rat plasma inactive renin, partially purified by h.p.l.c. on G3000SW, had much the same physicochemical characteristics as the recombinant prorenin. These results provide evidence that rat plasma inactive renin is prorenin. Recombinant prorenin is a useful material for examining the physiological role of circulating prorenin.
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PMID:Similarity between physicochemical properties of recombinant rat prorenin and native inactive renin. 203 49

The presence of two distinct high-molecular-weight proteases with similar pH optima in the weakly alkaline region was shown in cytosol of the bovine brain cortex. They were separated by ammonium sulfate fractionation and each was further purified by DEAE-Sephacel, Sephacryl S-300, DEAE-Cibacron Blue 3GA-agarose, heparin-agarose, and Sepharose 6B chromatography. The larger enzyme (Mr 1,400 kDa), which precipitates at 0-38% ammonium sulfate saturation, seems to be active in ATP + ubiquitin (Ub)-dependent proteolysis; it has low basal caseinolytic activity that is stimulated 3-fold by ATP, and when Ub is present ATP causes a 4.5-fold stimulation. A second proteinase was also found to be present (Mr 700 kDa) that precipitates at 38-80% ammonium sulfate saturation, is composed of multiple subunits ranging in Mr from 18 to 30 kDa, and degrades both protein and peptide substrates, demonstrating trypsin-, chymotrypsin- and cucumisin-like activities. Catalytic, biochemical, and immunological characteristics of this proteinase indicate that it is a multicatalytic proteinase complex (MPC), whose enzyme activity, in contrast to that of MPC from bovine pituitaries (1-3), is stimulated 1.7-fold by addition of ATP in the absence of ubiquitin at the early steps of purification; this property is lost during the course of further purification. Both proteinases are present in the nerve cells, since the primary chicken embryonic telencephalon neuronal cell culture extracts contain both ATP + Ub-dependent proteinase and MPC activities.
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PMID:The presence of ATP + ubiquitin-dependent proteinase and multicatalytic proteinase complex in bovine brain. 255 23

Our previous work has shown that pure hog renin, when injected into one-kidney, one-clip hypertensive rabbits elicits not only antirenin antibodies but also antibodies to what appears to be an altered form of renin (antigen M). Antiantigen M stains the cytoplasm of smooth muscle cells and certain other cells in tissues of normal and hypertensive rabbits. We now report studies in which pure 125I hog and rabbit renin have been infused into hypertensive rabbits for seven-day periods and the tissues subsequently examined for nondegraded radioactive components. The concentration and the distribution of radioactivity found in different tissues of six rabbits that received hog renin and six that received rabbit renin showed enormous variation, for which there is no reasonable explanation. A very significant amount of radioactivity was found to be incorporated into a high molecular weight or insoluble form that may be antigen M. A major portion of the radioactivity has a molecular weight of about 40,000 and has been assumed to be unaltered 125I renin. In plasma there was a very high molecular weight radioactive component that was capable of binding to antirenin or antiantigen M antibodies to a limited degree. In addition there was a major component with a molecular weight of 68,000 that is not formed in vitro but is produced in vivo. It resembles prorenin in human plasma in that it does not adsorb on pepstatin-Sepharose and does adsorb on Cibacron Blue. However, it differs from prorenin in that it does not bind to antirenin antibodies nor can it be activated by trypsin.
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PMID:Incorporation of labeled renin into the tissues of the rabbit. 267 72

Collagenase has been purified from the culture medium of a human myometrial smooth muscle cell line, and the properties of the pure enzyme compared to those of collagenase from another human mesenchymal cell, the fibroblast. The smooth muscle collagenase was purified using a new, rapid, and convenient three-step purification procedure consisting of chromatography on iminodiacetate-agarose chelated with zinc and on Cibacron Blue-agarose followed by gel filtration on Ultrogel AcA-44. The resultant pure collagenase is secreted as a zymogen indistinguishable from that of the fibroblast enzyme in molecular weight, amino acid composition, and in the nature of its conversion to active enzyme by trypsin. The amino acid sequence of the two enzymes at the trypsin cleavage site is the same. The two collagenases are also indistinguishable immunologically and display essentially identical kinetic behavior on a variety of collagen substrates. Although the two collagenases appear to be identical proteins, the mechanisms which regulate their production appear to be very different. Glucocorticosteroids, which inhibit collagenase production in human skin fibroblasts are without effect in the uterine smooth muscle cell. In contrast, the smooth muscle cell appears to require a component present in fetal bovine serum in order to produce the enzyme.
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PMID:Purification and characterization of human myometrial smooth muscle collagenase. 283 76

A membrane-associated O2-.-generating oxidase has been purified from activated bovine polymorphonuclear neutrophils (PMN). The oxidase was extracted with Triton X-100 from a PMN membrane fraction largely devoid of lysosomal granules. The Triton extract was purified by a series of steps, including ion-exchange chromatography on DE-52 cellulose, gel filtration on Sephadex G-200, and isoelectric focusing. The O2-.-generating oxidase activity was assayed as a superoxide dismutase inhibitable cytochrome c reductase. The activity of the purified enzyme was strictly dependent on NADPH as electron donor. The purification factor with respect to the phorbol myristate acetate activated PMN was 75, and the recovery was about 6%. The reactivity of the purified oxidase was increased by 3-4-fold after incubation with asolectin. The minimum molecular weight of the oxidase, deduced from migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 65 000 +/- 3000. The optimum pH of the oxidase was 7.5, its KM,NADPH was congruent to 30 microM, and its isoelectric point was at pH 5.0. The enzyme was inhibited by low concentrations of mersalyl (half-inhibition congruent to 10 microM) and Cibacron Blue (half-inhibition less than 10 microM). It was insensitive to 1 mM cyanide. Rapid loss of activity occurred at 0-2 degrees C, concomitantly with a decrease in sensitivity to superoxide dismutase: both activity and sensitivity to superoxide dismutase could be restored by addition of asolectin. The purified oxidase contained no spectrophotometrically detectable cytochrome b, and enzymatic assay failed to detect FAD in oxidase preparations subjected to heat treatment or trypsin digestion.
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PMID:Purification and properties of an O2-.-generating oxidase from bovine polymorphonuclear neutrophils. 300 51

Latent human leukocyte collagenase was isolated to apparent homogeneity by a simple and rapid method. Isolation was accomplished by gel filtration on Sepharose 6B and ion exchange chromatography on QAE Sephadex A-50 followed by affinity chromatography on Cibacron Blue Sepharose. The purified latent enzyme exhibits an apparent molecular weight of 70 kD as estimated by SDS-polyacrylamide gel electrophoresis. Reduction with dithiothreitol does not change the mobility of the latent human leukocyte collagenase on SDS-polyacrylamide gel electrophoresis, indicating that the enzyme consists of a single polypeptide chain. The enzyme could be activated by trypsin and thiol reagents such as phenylmercuric chloride and N-ethylmaleimide. Upon activation by trypsin a 54 kD polypeptide was formed from the 70 kD latent enzyme. Concomitant with the activation by thiol reagents, no loss of molecular weight was detected. Inactivated trypsin, i.e. phenylmethyl sulfonyl-trypsin or soybean trypsin inhibitor treated trypsin, was not able to activate latent human leukocyte collagenase. The results support the concept that latent human leukocyte collagenase exists as a proenzyme and thiol-dependent activation occurs through conformational perturbation in the proenzyme molecule.
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PMID:Activation of latent collagenase purified from human leukocytes. 303 86

1. Pig alpha-fetoprotein (AFP) and albumin were isolated from fetal serum by DEAE-Sephadex ion exchange chromatography combined with Cibacron Blue-Sepharose and trypsin-Sepharose adsorptions. 2. AFP, fetal albumin and adult albumin carried 2.6, 2.4, and 1.9 moles of fatty acids per mole of protein, respectively. 3. Most of fatty acids bound to AFP were polyunsaturated: mainly arachidonic (20:4, n-6) and docosahexaenoic (22:6, n-3) acids, which accounted respectively for 21.7 and 18.8% of the total fatty acids. 4. By contrast, the fatty acids found in the albumins (fetal and adult) were preferentially saturated and monounsaturated. 5. Arachidonic acid was a minor component in both albumins, and no docosahexaenoic acid was detected.
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PMID:Long-chain fatty acids bound to alpha-fetoprotein and to serum albumin from fetal and adult pig. 618 69

Inactive renin comprises well over half the total renin in normal human plasma. There is a direct relationship between active and inactive renin levels in normal and hypertensive populations, but the proportion of inactive renin varies inversely with the active renin level; as much as 98% of plasma renin is inactive in patients with low renin, whereas the proportion is consistently lower (usually 20-60%) in high-renin states. Two hypertensive patients with proven renin-secreting carcinomas of non-renal origin (pancreas and ovary) had high plasma active renin (119 and 138 ng/h per ml) and the highest inactive renin levels we have ever observed (5,200 and 14,300 ng/h per ml; normal range 3-50). The proportion of inactive renin (98-99%) far exceeded that found in other patients with high active renin levels. A third hypertensive patient with a probable renin-secreting ovarian carcinoma exhibited a similar pattern. Inactive renins isolated from plasma and tumors of these patients were biochemically similar to semipurified inactive renins from normal plasma or cadaver kidney. All were bound by Cibacron Blue-agarose, were not retained by pepstatin-Sepharose, and had greater apparent molecular weights (Mr) than the corresponding active forms. Plasma and tumor inactive renins from the three patients were similar in size (Mr 52,000-54,000), whereas normal plasma inactive renin had a slightly larger Mr than that from kidney (56,000 vs. 50,000). Inactive renin from each source was activated irreversibly by trypsin and reversibly by dialysis to pH 3.3 at 4 degrees C; the reversal process followed the kinetics of a first-order reaction in each instance. The trypsin-activated inactive renins were all identical to semipurified active renal renin in terms of pH optimum (pH 5.5-6.0) and kinetics with homologous angiotensinogen (Michaelis constants, 0.8-1.3 microM) and inhibition by pepstatin or by serial dilutions of renin-specific antibody. These results indicate that a markedly elevated plasma inactive renin level distinguishes patients with ectopic renin production from other high-renin hypertensive states. The co-production of inactive and active renin by extrarenal neoplasms provides strong presumptive evidence that inactive renin is a biosynthetic precursor of active renin. The unusually high proportion of inactive renin in plasma and tumor extracts from such patients is consistent with ineffective precursor processing by neoplastic tissue, suggesting that if activation of "prorenin" is involved in the normal regulation of active renin levels it more likely occurs in the tissue of origin (e.g., kidney) than in the circulation.
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PMID:Characterization of inactive renin ("prorenin") from renin-secreting tumors of nonrenal origin. Similarity to inactive renin from kidney and normal plasma. 636 74

A heme-controlled inhibitor of translation was isolated from the S-100 of rabbit reticulocytes by a novel procedure including chromatography on double-stranded ribonucleic acid (dsRNA)-cellulose. The inhibitor thus purified is extremely active and functionally resembles previously studied heme-controlled inhibitor preparations in terms of kinetics and extent of inhibition of translation, relief of inhibition by eukaryotic initiation factor 2 (eIF-2), relief of inhibition by 2-aminopurine, and preferential inhibition of alpha-over beta-globin synthesis. The action of this inhibitor on translation is resistant to treatment with bacterial alkaline phosphatase, micrococcal nuclease, or trypsin and to incubation at 95 degrees C, pH 2 or pH 12. The inhibitor not only is retained on DEAE-cellulose, phosphocellulose, and dsRNA-cellulose but also exhibits a high affinity for the dye Cibacron Blue, properties that suggest that it may be a protein. Unlike previously described heme-controlled inhibitor preparations, or preparations that did not pass over dsRNA-cellulose, the inhibitor recovered upon dsRNA-cellulose chromatography does not exhibit eIF-2 kinase activity. The inhibitor does not block ternary complex formation between eIF-2, methionyl-tRNAfMet, and GTP but inhibits the ability of eIF-2 to form a complex with labeled globin mRNA. In the presence of inhibitor, the formation of mRNA/eIF-2 complexes can be restored effectively by an excess of eIF-2 but not by an excess of mRNA. The inhibitor thus appears to block the interaction between eIF-2 and mRNA not by competing with eIF-2 for a binding site on mRNA but, instead, by acting on eIF-2 itself.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation of a heme-controlled inhibitor of translation that blocks the interaction between messenger rna and eukaryotic initiation factor 2. 647 77


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