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:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An anticoagulant isolated from salivary gland extracts of the ixodid tick Rhipicephalus appendiculatus was purified by gel filtration on Sephadex G-100, ion exchange on DEAE-cellulose, aprotinin-Sepharose, and by high-pressure-liquid size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the anticoagulant activity was associated with a protein of an apparent Mr of 65 kDa. The purified molecule had a pI in the range of 8.0-8.5 on chromatofocusing and was stable over a wide pH range, but was heat labile and susceptible to inactivation by trypsin and reductive alkylation. The anticoagulant did not inhibit thrombin-initiated fibrin formation and had no detectable fibrino(geno)lytic or phospholipase-like activities. Although it inhibited factor Xa-induced clotting of bovine plasma, it did not affect the amidase activity of factor Xa toward a synthetic substrate, suggesting that the anticoagulant acts at a site distinct from the active site of factor Xa or on other components of the prothrombinase complex.
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PMID:Purification and characterization of an anticoagulant from the salivary glands of the ixodid tick Rhipicephalus appendiculatus. 202 16

Hymenolepis diminuta liberated measurable amounts of 'Lowry-positive material' (LPM) and protein during incubation for 2 h in vitro. When tapeworms were incubated in the presence of bovine trypsin (BT), or when BT was added to the medium after removing the tapeworms, the enzyme's proteolytic activity was inhibited significantly. Centrifugation of the medium at 30,000 g yielded a pellet composed of tegumental elements, but this fraction did not inhibit BT. The 30,000 g supernatant fraction contained a chemical(s) that inhibited the proteolytic enzymes of the rodent host's intestinal contents (IC). The inhibitor(s) was stable following repeated freeze-thaw cycles, heat labile, and not degraded by BT or IC, and it inhibited the amidase activity of BT in a non-competitive manner.
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PMID:Hymenolepis diminuta (Cestoda) liberates an inhibitor of proteolytic enzymes during in vitro incubation. 209 1

This study, the first of its kind in a mosquito vector species, demonstrates the feasibility of studying prophenoloxidase activation in an insect containing not more than a few microliters of hemolymph. Mosquito phenoloxidase was found to be in an inactive proenzyme form, prophenoloxidase. Mosquito prophenoloxidase required bivalent cation for its activation; Ca2+ was found to be the most efficient for activation. Concomitant amidase activity was also observed prior to phenoloxidase activity. Through Western blotting, using a cross-reactive silkworm antiprophenoloxidase antibody, our results strongly suggest that mosquito prophenoloxidase activation resulted from limited proteolysis. Protease inhibitor studies reinforced this contention showing the involvement of (a) serine protease(s) with trypsin-like activity in the activation of mosquito prophenoloxidase.
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PMID:Studies on prophenoloxidase activation in the mosquito Aedes aegypti L. 211 57

A recombinant trypsin was designed whose catalytic activity can be regulated by varying the concentration of Cu2+ in solution. Substitution of Arg-96 with a His in rat trypsin (trypsin R96H) places a new imidazole group on the surface of the enzyme near the essential active-site His-57. The unique spatial orientation of these His side chains results in the formation of a stable, metal-binding site that chelates divalent first-row transition-metal ions. Occupancy of this site by a metal ion prevents the imidazole group of His-57 from participating as a general base in catalysis. As a consequence, the primary effect of the transition metal ion is to inhibit the esterase and amidase activities of trypsin R96H. The apparent Ki for this inhibition is in the micromolar range for copper, nickel, and zinc, the tightest binding being to Cu2+ at 21 microM. Trypsin R96H activity can be fully restored by removing the bound Cu2+ ion with EDTA. Multiple cycles of inhibition by Cu2+ ions and reactivation by EDTA demonstrate that reversible regulatory control has been introduced into the enzyme. These results describe a novel mode of inhibition of serine protease activity that may also prove applicable to other proteins.
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PMID:Regulation of serine protease activity by an engineered metal switch. 212 68

Incubation of alpha-chymotrypsin and alpha-lytic protease with chloro(2,2':6',2''-terpyridine)platinum(II), [Pt(trpy)Cl]+, results in attachment of Pt(trpy)2+ tags at both His 57 and His 40 in the former and His 57 in the latter. The [Pt(trpy)His]2+ chromophores are readily detected and quantitated owing to their characteristic, strong UV absorption. Although the tagging of His 57 modifies the catalytic triad (Ser 195, His 57, and Asp 102) and disrupts the charge relay, the platinated enzymes retain significant esterase and amidase activity for both specific and nonspecific substrates. Unlike suicide inhibitors, which inactivate the enzymes by filling the active site and imitating the tetrahedral intermediate, [Pt(trpy)Cl]+ reacts with a particular amino acid and permits binding of substrates. The kinetic constants for the following are reported: two esters and two amides with alpha-chymotrypsin and an amide with alpha-lytic protease. The kcat values are between 1 and 25% of, and the Km values are a little higher than, the corresponding values for the native enzymes. The catalytic activity is not due to the native enzymes, trypsin, or some zinc-containing protease. Activities of the native and of the platinated alpha-chymotrypsin depend similarly on pH although the pKa of His 57 is raised to 9.7 upon platination. The platinated enzymes undergo autodigestion slower than do the native enzymes. Because the Pt(trpy)2+ tags are noninvasive, stable, and yet easily removable by thiourea, [Pt(trpy)Cl]+ may be used to retard autodigestion of stored proteolytic enzymes.
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PMID:Catalytic activity of the serine proteases alpha-chymotrypsin and alpha-lytic protease tagged at the active site with a (terpyridine)platinum(II) chromophore. 222 78

The usage of substrate inhibitor analysis made it possible to estimate the levels of excretion of plasma proteinases, including plasma kallikrein in the urinary DValLeuArgpNA (S-2266)- and DProPheArgpNA (S-2302)-amidase activity in patients with latent and nephrotic types of chronic glomerulonephritis (CGN). The soya bean trypsin inhibitor, an inhibitor of plasma kallikrein and other plasma proteinases, such as that of the blood coagulative factors XIa and XIIa, and the high selective plasma kallikrein inhibitor DPhePheArgCH2Cl were used as those differentiating kallikreins of tissue and plasma origin. The S-2266 and S-2302-amidase activity of the urine from healthy subjects was shown to be determined by only tissue (renal) kallikrein. The urine from the patients with a latent CGN type displayed the activity of plasma proteinases, but plasma kallikrein made no significant contribution to the urine amidase activity in these patients. With a nephrotic CGN type, great quantities of trypsin-like proteinases were secreted from the plasma through the glomerular filter into the urine, the proportion of plasma kallikrein in the urinary S-2266 and S-2302-amidase activities being approximately 27%. The compensatory and pathogenetic role of plasma kallikrein is discussed if there is lower excretion of tissue (renal) kallikrein in CGN with the nephrotic syndrome.
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PMID:[A substrate inhibitor analysis of the urinary excretion of tissue and plasma kallikreins in patients with chronic glomerulonephritis]. 227 58

Guinea pig alpha-macroglobulin was purified to apparent homogeneity by sequential chromatography on Sephacryl S-300, DEAE-cellulose, and hydroxyapatite. A molecular weight of 780,000 was obtained by equilibrium sedimentation. The preparation migrated as a single band of Mr = 180,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Rabbit antiserum raised against the final preparation partially cross-reacted with human and rat alpha-2-macroglobulins but not with rat alpha-1-macroglobulin. Guinea pig alpha-macroglobulin stimulated the amidolytic activity of trypsin towards a small substrate, but inhibited the proteolytic activity of trypsin towards remazol brilliant blue hide powder. When treated with trypsin or methylamine, four thiol groups per molecule were newly generated. The reaction with trypsin proceeded with at least at two different rates: half of the thiol groups were generated in a fast reaction and the remaining half in a slower reaction. On the other hand, such a two-step reaction was not detected in the reaction with methylamine. The methylamine-treated alpha-macroglobulin retained half the capacity to bind trypsin and its mobility in polyacrylamide gel under nondenaturing conditions remained virtually unchanged. These properties are in marked contrast to those reported for human alpha-2-macroglobulin, but resemble those of rat alpha-2- and mouse alpha-macroglobulins. The amidase activity of trypsin bound to guinea pig alpha-macroglobulin was impaired by soybean trypsin inhibitor to a much greater degree than that of trypsin bound to human or rat alpha-2-macroglobulin.
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PMID:Isolation and characterization of alpha-macroglobulin from guinea pig plasma. 242 4

We have compared the reactions of trypsin with human alpha 2-macroglobulin (alpha 2M), and three rat plasma protease inhibitors, alpha 1-macroglobulin (alpha 1M), alpha 1-inhibitor III (alpha 1I3), and alpha 2M. All four of these proteins appear to contain reactive thiol esters. The electrophoretic mobility in agarose gels of human and rat alpha 2M is increased by 1 mol of trypsin, while the mobility of alpha 1M and alpha 1I3 is decreased. Treatment with methylamine causes similar mobility changes, except in the case of rat alpha 2M. Titration of human and rat macroglobulins by repeated small additions of trypsin and by assay of liberated SH groups or enhanced ligand fluorescence revealed a stoichiometry of about 1 mol of trypsin/mol of inhibitor. In contrast, addition of macroglobulin to a fixed amount of trypsin and detection of residual amidase or protease activity revealed a stoichiometry of about 2 mol of trypsin for 1 mol of human alpha 2M, about 1.4 mol for rat alpha 1M, and about 1 mol for rat alpha 2M. One mol of trypsin reacted with 2 or more mol of alpha 1I3 by the criteria of SH groups liberated or protease inhibition. Methylamine-treated rat alpha 2M binds a significant amount of trypsin releasing about 2 mol of SH. Radioactive beta-trypsin was covalently bound to subunits of the purified plasma inhibitors. The Mr of the labeled products with rat and human alpha 2M had molecular weights which suggested trypsin was bound to intact as well as cleaved subunit chains and also to multiple chains via cross-linking. Rat alpha 1M also produced a product which may be an intact subunit alpha chain plus trypsin. Greater than 80% of the trypsin was bound covalently to these inhibitors at low molar ratios.
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PMID:Three high molecular weight protease inhibitors of rat plasma. Reactions with trypsin. 243 19

We have studied the differential susceptibility to N-glycanase (peptide-N4-[N-acetyl-beta-glucosaminyl]asparagine amidase) of oligosaccharides at the individual glycosylation sites of mouse TSH and free alpha-subunits. Mouse thyrotropic tumor tissue or hypothyroid pituitary tissue were incubated with D-[2-3H]mannose for 6 h. [3H]Mannose-labeled TSH or free alpha-subunits were obtained from homogenates using specific antisera and were digested with N-glycanase in their native state or after heat denaturation and reduction in the absence or presence of detergents. Tryptic fragments of the digestion products were then analyzed by reverse phase HPLC so that the effects of N-glycanase at the individual glycosylation sites could be determined. N-Glycanase treatment of native molecules did not cleave oligosaccharides efficiently at Asn56 of alpha-subunits and Asn23 of TSH beta, whereas oligosaccharides at Asn82 of alpha-subunits were more susceptible regardless of whether the alpha-subunits were combined with TSH beta. Heat denaturation, reduction, and the presence of detergents did not substantially increase the cleavage by N-glycanase of the protected oligosaccharides, suggesting that the primary structures of the TSH subunits influenced efficiency at specific sites. Pretreatment of free alpha-subunits with trypsin failed to enable N-glycanase to work fully, as oligosaccharides at Asn56 were cleaved less effectively than those at Asn82. Thus, the susceptibility to N-glycanase differs at the individual glycosylation sites of mouse TSH and free alpha-subunits, and these differences may result from effects of the primary structures of the TSH subunits.
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PMID:Differential susceptibility to N-glycanase at the individual glycosylation sites of mouse thyrotropin and free alpha-subunits. 245 9

Reactions of rabbit alpha-2-macroglobulin with methylamine and trypsin were studied and the results were compared with those obtained for previously described 2-macroglobulins from other species. Rabbit alpha-2-macroglobulin was cleaved by trypsin at a number of sites, whereas the human homologue was split essentially only in the "bait" region into two fragments of similar sizes. Reaction of native or methylamine-treated rabbit alpha-2-macroglobulin with trypsin resulted in a substantial decrease in the intensity of fluorescence induced by binding of 6-(p-toluidino)-2-naphthalenesulfonate or bis(8-anilino-1-naphthalenesulfonate). Under the same conditions, the fluorescence of the human protein increased. The time course of the reaction of rabbit alpha-2-macroglobulin with methylamine was studied by measuring (i) the generation of thiol groups, (ii) the decrease in trypsin-inhibiting activity with remazol brilliant blue hide powder as the substrate, and (iii) the decrease in trypsin-protein amidase activity. The thiol appearance reaction exhibited a multiphasic time course. The initial phase was found to follow second-order kinetics with an apparent rate constant of 1.2 M-1.s-1. Under the same conditions, the human protein showed monophasic kinetics with a rate constant of 12 M-1.s-1. Both the trypsin-inhibiting activity and the trypsin-protein amidase activity concurrently decreased at a slower rate than the thiol appearance. These results indicate that rabbit alpha-2-macroglobulin is more stable to nucleophilic attack by methylamine but less resistant to proteolysis by trypsin than the human homologue, and that the final conformation induced by methylamine differs considerably from that induced by trypsin.
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PMID:Changes in trypsin-binding properties and conformation of rabbit alpha-2-macroglobulin on reaction with methylamine. 247 83


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