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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new protease was isolated from an extract of leaves of Agave americana variegata. The protease (EC 3.4.-) was purified 565-fold with a yield of 39.5%. The 43.8 mg enzyme had a specific activity of 0.44 units/mg. According to electrophoretic, ultracentrifugal and other physical characterizations the enzyme was homogeneous. The enzyme had a MR of 57000, a S20,W-value of 4.37 S, a D20, W-value of 6.8-7.0 - 10(-7) cm2sec-1, a Stokes radius of 3.18 nm, a partial specific volume of 0.735 cm3g-1, a frictional ration of 1.25, a molecular absorbancy index at 280 nm of 5.773-10(4), an isoelectric point of 5.25 and contained 8-10% carbohydrate. The enzyme contained no cysteine. Agave protease could hydrolyze a variety of protein substrates although it did have a restricted specificity. It is not a sulphhydryl protease but seems to be an alkaline "serine" protease with an optimum pH of 7.8-8.0 Agave protease had marked esterolytic activity and with Cbz-Tyr-ONp had an apparent Michaelis constant of 0.0345 -10(-3) M and a V of 1.24 mol substrate/mol enzyme per sec. The enzyme did not need metal ions for optimal activity, monovalent cations did not influence its kinetic parameters, but it was inhibited by cobalt, pC1HgBzO- and TosPheCH2C1. With respect to its primary specificity, as well as its pH-dependence there was a resemblance with chymotrypsin, although the rate of hydrolysis of Agave protease is much lower.
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PMID:Isolation and partial characterization of a protease from Agave americana variegata. 0 46

The kinase activity of the threonine-sensitive aspartokinase-homoserine dehydrogenase enzyme complex of Escherichia coli was selectively inactivated by Co(III) incorporation. Incubation of the enzyme with Co(II) in the presence of oxygen or H2O2 resulted in incorporation of one Co(III) per subunit. The cobalt(III) bound to the enzyme was not removable by dialysis and presumably results from formation of "inert" coordination complexes with ligands contributed by the enzyme. Cobalt was released from the enzyme by incubation with dithiothreitol but not by metal chelating agents. The Co(III)-labeled enzyme was aspartokinase inactive but still retained 60% of its original homoserine dehydrogenase activity. Studies of the time course of inactivation showed aspartokinase inactivation paralleled Co(III) incorporation. The residual dehydrogenase activity of aspartokinase inactive enzyme was still inhibited by threonine Thus, Co(III) incorporation seems to result in a specific inactivation of kinase activity which permits enumeration of the number of aspartokinase sites. Limited alpha-chymotrypsin digestion of Co(III)-enzyme produced homoserine dehydrogenase-active fragments devoid of Co(III), further confirming the specificity of the labeling procedure. Aspartokinase inactivation obtained without concomitant desensitization of homoserine dehydrogenase to threonine inhibition suggests that kinase active site integrity is not required for threonine binding and inhibition of homoserine dehydrogenase.
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PMID:Cobalt(III) labeled aspartokinase-homoserine dehydrogenase of Escherichia coli. 110 Jan 5

Bacteriorhodopsin (bR) was regenerated from the cation-depleted blue membrane with pentaammineaquocobalt(III) tetrafluoroborate [( Co(NH3)5H2O]3+[BF4-]3). Illumination of the sample with orange light decreased the extinction at 568 nm concomitantly with a hypsochromic shift of the absorption maximum. The photocycle of this sample was inhibited, and the rate of proton pumping was reduced. Chymotryptic cleavage of the corresponding apomembrane into the two fragments C1 and C2 and their subsequent separation revealed that cobalt label is only attached to C1. The maximal incorporation of Co into this peptide was 0.3 Co/C1. After cleavage of C1 with cyanogen bromide and subsequent proteolysis with trypsin and chymotrypsin, this modification could be associated with peptides from cyanogen bromide fragments 6 and 9. The sequences were determined to be 101Val-Asp-Ala-Asp-Gln and 228Ala-Ile-Phe-Gly-Glu-Ala-Glu-Ala. These peptides contain the sequences Asp-Ala-Asp and Glu-Ala-Glu, respectively, which might be constituents of the same cation binding site. The observation that the incorporation of Co into bacteriorhodopsin is enhanced under illumination with orange light indicates that this site might be involved in the proton uptake.
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PMID:Modification of two peptides of bacteriorhodopsin with a pentaamminecobalt (III) complex. 277 14

An in-vitro test of degradation of haptocorrin, a cobalamin-binding glycoprotein, was used to diagnose exocrine pancreatic dysfunction. This radioisotopic test (TDH) required only 50 microliters duodenal juice collected during endoscopy after stimulation with 1 U/kg secretin intravenously. The initial reaction mixture, composed of salivary haptocorrin saturated with cobalt-57-labelled cyanocobalamin and unsaturated intrinsic factor, was incubated with 25 microliters duodenal juice. The percentage of degraded haptocorrin was estimated from the proportion of labelled cyanocobalamin that was transferred from haptocorrin to intrinsic factor. The TDH result was 41.6 +/- 31.7% (SD) in a group of chronic pancreatitis patients (n = 22) and 91.5 +/- 4.8% in the control group (n = 47). The sensitivity and specificity for exocrine pancreatic dysfunction were estimated as 0.91 and 0.96, respectively, for a lower limit of normal values of 81.7%. A hyperbolic relation was found between the TDH and the trypsin or chymotrypsin activity in duodenal juice (p less than 0.001). In this study, the N-benzoyl-tyrosyl-p-aminobenzoic acid test was less sensitive than the TDH, since its result was abnormal in only 64% of the patients. The TDH was easier to carry out and less time-consuming than the determination of pancreatic enzyme output in duodenal juice collected after hormonal stimulation.
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PMID:In-vitro test of haptocorrin degradation for biological diagnosis of exocrine pancreatic dysfunction using duodenal juice collected during endoscopy. 287 85

Proteases in preparations of carboxypeptidase A progressively inactivate solutions of the apoenzyme but not the metal-containing enzyme. Free amino acids generated by proteolysis interfere with spectral studies after reconstituting the apoenzyme with cobalt. Purification by affinity chromatography eliminates this effect. Affinity-purified apoenzyme is susceptible to digestion with chymotrypsin but the metalloenzyme is not.
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PMID:Protease susceptibility of zinc- and apo-carboxypeptidase A. 391 41

The effect of AG+, Cu2+, Cd2+, Co2+ and Ni2+ on the activity of alkaline mesentericopeptidase (EC 3.4.21.-) has been studied. Ag+, Cu2+ and Cd2+ were found to be reversible non-competitive inhibitors of the enzyme. The pH-dependence of Ki for Ag+-inhibition is sigmoidal with a pKa near 6. The Kilim values, calculated for the pH-independent region of the metal-enzyme inhibition, are close to the corresponding dissociation constants of metal-imidazole complexes, thus implying that the inhibitory effect of metal ions on enzyme activity is due to complex formation with the imidazole group of the active site histidine. The method of the two-component inhibition showed that Cu2+ and Ag+ bind to the same ligand of the enzyme molecule. The addition of Cu2+ decreases the rate of deacylation of the hydrolysis of p-nitrophenyl valerate, catalyzed by alkaline mesentericopeptidase in contrast to alpha-chymotrypsin where the acylation step is affected.
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PMID:Inhibitory effect of metal ions on alkaline mesentericopeptidase. 701 96

A periplasmic insulin-cleaving proteinase (ICP), purified to its electrophoretic homogeneity in the SDS-PAGE from the Gram-negative bacterium Acinetobacter calcoaceticus, was examined and compared in its properties with the protease III (protease Pi, pitrilysin, EC 3.4.99.44) of Escherichia coli and the insulin-destroying proteinase (IDE, insulinase, EC 3.4.99.45) from eucaryotes. The enzyme was proven to be a metalloprotease like protease III and IDE, as was shown by the inhibitory effects exerted by EDTA and o-phenanthroline. Furthermore, dialysis against EDTA and o-phenanthroline led to a complete loss of activity, which could be restored by addition of Co2+, and, to a lesser extent, but at a lower metal ion concentration by Zn2+. Similar to protease III and IDE, ICP prefers the cleavage of small polypeptides (insulin, insulin B-chain, glucagon) to the cleavage of proteins (casein, human serum albumin, globin) and was inactive against synthetic amino acid derivates (esters, p-nitranilides, and furoylacroleyl substrates) of subtilisin, thermolysin, trypsin, and chymotrypsin. The peptide-bond-specificity of the ICP in the cleavage of the oxidized insulin B-chain was investigated and the results were compared to the specificity of protease III of E. coli, IDE, protease-24,11, and thermolysin. Cleavage sites in the oxidized insulin B-chain generated by ICP are Asn3-Gln4, His10-Leu11, Ala14-Leu15, Leu17-Val18, Gly23-Phe24, Phe24-Phe25, and Phe25-Tyr26. Principally, ICP cleaves between hydrophobic amino acids and amides. The ICP shares one of the only two cleavage sites with the protease III and four sites with the IDE.
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PMID:A periplasmic insulin-cleaving proteinase (ICP) from Acinetobacter calcoaceticus sharing properties with protease III from Escherichia coli and IDE from eucaryotes. 773 84

The efficiency of cobalt(III)-ligated peptides as acceptor nucleophiles in acyl transfer reactions catalyzed by alpha-chymotrypsin was examined. A series of metallopeptides with the general formula [H-(Gly)n-OCo(NH3)5]2+ (1 < or = n < or = 4) was tested. The aminolysis rate of acyl-chymotrypsin was measured spectrophotometrically by monitoring the concentration of unreacted nucleophile. The rate of the competing hydrolysis of acyl-chymotrypsin was obtained by automatic titration with base, using a pH-stat. The main result was that the positively charged metallopeptides, in general, were more efficient nucleophiles than the corresponding amides and free peptides that were examined for comparison. A binding model that rationalizes the findings is given.
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PMID:Cobalt(III)-ligated peptides as acyl acceptors in peptide synthesis catalyzed by chymotrypsin. 777 80

The pattern and kinetics of partial proteolysis of Arthrobacter D-xylose isomerase tetramer was studied in order to determine the flexibility of surface loops that may control its stability. It was completely resistant to trypsin, chymotrypsin and elastase at 37 degrees C, but thermolysin cleaved specifically and quantitatively at Thr-347-Leu-348 between helices 10 and 11 to remove 47 residues from the C-terminus of each 43.3 kDa subunit. At high temperatures, helices 9 and 10 were removed from each 38 kDa subunit to give a 36 kDa tetramer. The kinetics of nicking by thermolysin indicated that the Thr-347-Leu-348 loop is locked at low temperatures, but 'melts' at 25 degrees C and is fully flexible above 34 degrees C. The flexibility appears to be associated with binding of Ca2+ ions at the active site, since Co2+, Mg2+ and xylitol protect in proportion to their ability to displace Ca2+. The missing C-terminal helices make many intersubunit contacts that appear in the structure to stabilize the tetramer, but the properties of the purified nicked proteins are almost indistinguishable from the native enzyme. Both the 38 kDa tetramer and the 36 kDa tetramer are identically active and dissociate similarly in urea or SDS to fully active dimers, but the nicked dimers are slightly less stable to urea at 62 degrees C. In the Mg2+ form the thermostability of the 38 kDa tetramer is identical with that of the native enzyme, but the 36 kDa tetramer has a slightly lower 'melting point' (70 degrees C versus 80 degrees C), which may be due to unravelling from the end of helix 8. Since elimination of all the C-terminal helices and many intersubunit contacts has so little effect, one can conclude that the 'weak point' that controls the protein's thermostability lies within the N-terminal beta-barrel domain.
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PMID:Arthrobacter D-xylose isomerase: partial proteolysis with thermolysin. 842 59

Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90-95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyrylcholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. The pH optimum of the peptidase was in the range of 7.5-9.5 and the divalent cations Co2+, Mn2+, and Zn2+ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co2+, Mn2+, and Zn2+ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited alpha-chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.
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PMID:The peptidase activity of human serum butyrylcholinesterase: studies using monoclonal antibodies and characterization of the peptidase. 842 27


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