A preliminary characterization of alkaline digestive proteases in the posterior midgut of the face fly, Musca autumnalis De Geer

1987 ◽  
Vol 65 (3) ◽  
pp. 635-639 ◽  
Author(s):  
F. C. Campbell ◽  
Jon G. Houseman ◽  
P. E. Morrison
Keyword(s):  
Aminopeptidase Activity ◽  
Tetraacetic Acid ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Posterior Midgut ◽  
The Face ◽  
Musca Autumnalis ◽  
Hydrolysis Of

Alkaline proteases in the posterior midgut of the face fly, Musca autumnalis De Geer, were identified using specific synthetic substrates and nonspecific substrate azocasein. Identification was confirmed with potential protease activators and inhibitors. Optimal azocasein hydrolysis occurred at pH 8.0 and substrate hydrolysis was inhibited by EGTA (ethyleneglycol-bis-(2-aminoethyl ether)-N,N-tetraacetic acid), tosyl-L-lysine chloromethyl ketone (TLCK), and soybean trypsin inhibitor (STI). Tryptic proteolysis was identified by maximal hydrolysis, at pH 8.0, of trypsin specific substrates BAPNA (benzoyl-DL-arginine-p-nitroanilide) and TAME (tosyl-L-arginine methyl ester). TAME hydrolysis was inhibited by PMSF (phenylmethylsulfonyl fluoride), STI, pepstatin A, and dithiothreitol (DTT), but BAPNA hydrolysis was inhibited only by STI and DTT. Chymotrypsin was demonstrated by maximal hydrolysis of BTEE (benzoyl-L-tyrosine ethyl ester) at pH 8.0 and DTT, PMSF, and STI inhibited hydrolysis of this substrate. Aminopeptidase activity was demonstrated by maximal hydrolysis of leucine-p-nitro-anilide at pH 8.0 and the peptidase was inhibited by o-phenanthroline. Carboxypeptidase A-like enzyme hydrolyzed hippuryl-DL-phenyllactic acid maximally at pH 7.5 and carboxypeptidase B showed maximum hydrolysis of hippuryl-L-arginine at pH 7.0. Both carboxypeptidases were inhibited by EDTA (ethylene diamine tetraacetic acid), EGTA, and DTT and carboxypeptidase A was also inhibited by PMSF.

Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

1975 ◽  
Vol 53 (7) ◽  
pp. 747-757 ◽  
Author(s):  
Graham J. Moore ◽  
N. Leo Benoiton
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Kinetic Behavior ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Basic Peptides ◽  
Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

Cathepsin B and aminopeptidase in the posterior midgut of Phymata wolffii Stål (Hemiptera: Phymatidae)

1985 ◽  
Vol 63 (6) ◽  
pp. 1288-1291 ◽  
Author(s):  
Jon G. Houseman ◽  
P. E. Morrison ◽  
A. E. R. Downe
Keyword(s):  
Molecular Weight ◽  
Trypsin Inhibitor ◽  
Cathepsin B ◽  
Ethylenediaminetetraacetic Acid ◽  
Soybean Trypsin Inhibitor ◽  
Aminopeptidase Activity ◽  
Chloromethyl Ketone ◽  
Posterior Midgut ◽  
Hydrolysis Of

The posterior midgut of the phymatid Phymata wolffii Stål contains cathepsin B and aminopeptidase activity. Identification of cathepsin B was based on maximal hydrolysis of benzoyl-DL-arginine-2-naphthylamide and benzoyl-DL-arginine-p-nitroanilide at pH 5.8 and 5.5, respectively. Cathepsin B hydrolysis of the tested substrates was activated by thiol chemicals and ethylenediaminetetraacetic acid (EDTA) and inhibited by tosyl-L-lysine chloromethyl ketone, iodoacetamide, and soybean trypsin inhibitor. Aminopeptidase hydrolyzed leucine-p-nitroanilide maximally at pH 7.8 and hydrolysis of the substrate was activated by magnesium and inhibited by EDTA, dithiothreitol, glutathione, and cysteine. The molecular weight of cathepsin B was 40 000 and was greater than 150 000 for aminopeptidase.

Characterization of lactococci and lactobacilli isolated from semihard goats' cheese

1991 ◽  
Vol 58 (1) ◽  
pp. 137-145 ◽  
Author(s):  
Teresa Requena ◽  
Carmen Peláez ◽  
Michel J. Desmazeaud
Keyword(s):  
Leucine Aminopeptidase ◽  
Sodium Dodecyl ◽  
Skim Milk ◽  
Titratable Acidity ◽  
Aminopeptidase Activity ◽  
Whole Cells ◽  
Alanine Aminopeptidase ◽  
Triton X ◽  
Hydrolysis Of

SummarySeveral strains ofLactococcus lactissubsp.lactis, Lactobacillus caseiandLactobacillus plantarumisolated from traditional goats' cheese have been studied for titratable acidity, proteolysis in milk and enzymic activities. Aminopeptidasc activities were measured with whole cells and cells permeabilized with Triton X-100. Caseinolytic activity was investigated using electrophoresis in polyacrylamide gel with sodium dodecyl sulphate.Lc. lactissubsp.lactishad a level of proteolytic activity in skim milk greater than that ofLb. casei, while this activity inLb. plantarumwas very low. Alanine aminopeptidase activity was almost non-existent for all strains tested, while lysine aminopeptidase activity appeared to be of fundamentally intracellular origin. Leucine aminopeptidase activity was also greater in cells that had been permeabilized than in whole cells forLb. caseiandLb. plantarum. Lc. lactissubsp.lactisleucine aminopeptidase activity was greater in whole cells. No significant hydrolysis of casein was found withLb. caseiI FPL 725 andLb. plantarumIFPL 722 permeabilized with Triton X-100 after 24 h incubation with whole bovine casein.

Reversible Inhibition of Carboxypeptidase A. II. Inhibition of Esterase Activity by Dicarboxylic Acid Anions

1974 ◽  
Vol 52 (11) ◽  
pp. 2053-2063 ◽  
Author(s):  
John W. Bunting ◽  
Chester D. Myers
Keyword(s):  
Dicarboxylic Acid ◽  
Competitive Inhibition ◽  
Dicarboxylic Acids ◽  
Competitive Inhibitor ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Monocarboxylic Acids ◽  
Reversible Inhibition ◽  
Competitive Inhibitors ◽  
Hydrolysis Of

Reversible inhibition of the hydrolysis of O-hippuryl-L-3-phenyllactic acid by carboxypeptidase A has been studied for a series of decarboxylic acids at 25°, pH 7.5, and ionic strength 0.2. All inhibitors studied displayed either strictly competitive or partially competitive inhibition kinetics. For the series CO2H(CH2)nCO2H, strictly competitive inhibition was observed for n = 1, 3, 4, 8, 10, whereas partially competitive inhibition occurs for n = 2, 5, 6, 7. A series of 11 alkyl- and aryl-substituted malonic acids were all strictly competitive inhibitors; for a series of six alkylmalonic acids the inhibition constants are correlated with the Hansch π-parameter by the equation –log K1 = 2.257π + 1.75; arylmalonic acids are poorer inhibitors than expected on the basis of their π-parameters, in accord with a similar observation for monocarboxylic acids. Phthalic acid is a strictly competitive inhibitor (K1 = 1.7 mM), whereas the isomeric isophthalic and terephthalic acids cause relatively little inhibition even at 0.1 M; maleic acid is a partially competitive inhibitor, whereas the isomeric fumaric acid gives only 15% inhibition at 0.1 M. Homophthalic acid and 2,2-dimethyl- and 3,3-dimethylglutaric acids were also investigated.The characteristics of partially competitive inhibition displayed by all dicarboxylic acids and also monomethyl succinate and succinamic acid are consistent with a scheme which assumes the formation of an E.I2 complex. The observed specificity of dicarboxylic acid binding is used to postulate a schematic diagram for binding of these species to the enzyme, and an interpretation of this diagram is suggested on the basis of the crystallographically determined structure of the enzyme.

Reversible Inhibition of Carboxypeptidase A. IV. Inhibition of Specific Esterase Activity by Hippuric Acid and Related Species and other Amino Acid Derivatives and a Comparison with Substrate Inhibition

1975 ◽  
Vol 53 (13) ◽  
pp. 1993-2004 ◽  
Author(s):  
John W. Bunting ◽  
Chester D. Myers
Keyword(s):  
Amino Acids ◽  
Hippuric Acid ◽  
Substrate Inhibition ◽  
Enzymic Hydrolysis ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Peptide Substrates ◽  
Uncompetitive Inhibition ◽  
Hydrolysis Of ◽  
Noncompetitive Inhibitors

The anions of each of the following carboxylic acids exhibit uncompetitive inhibition of the hydrolysis of O-hippuryl-L-3-phenyllactic acid by bovine carboxypeptidase A at pH 7.5, 25°, ionic strength 0.2: hippuric acid, p-chloro- and p-nitrohippuric acids, hippurylglycine, carbobenzoxyglycine, phenaceturic acid, N'-(3-phenylpropanoyl)glycine, benzoxyacetic acid, 3-benzoylpropanoic acid, and O-hippuryl-D-mandelic acid. In each case, this uncompetitive inhibition is consistent with the ordered binding of substrate and inhibitor to the enzyme; i.e. the inhibitor binds to E.S but not to the free enzyme. Evidence is presented for the binding site for uncompetitive inhibitors being the same as for inhibitory ester substrate molecules. Comparison of the specificities of uncompetitive inhibitors and esters which display substrate inhibition provides evidence for a critical conformational change which controls the binding of uncompetitive inhibitors and inhibitory substrate molecules.D-Phenylalanine, D-leucine, D-p-nitrophenylalanine, glycyl-L-tyrosine, glycyl-L-phenylalanine, and glycyl-L-leucine are competitive inhibitors of the enzymic hydrolysis of O-hippuryl-L-3-phenyllactic acid, whereas the N-chloroacetyl derivatives of L-tyrosine, L-phenylalanine, and L-leucine are noncompetitive inhibitors. For the above D-amino acids, glycyl dipeptides, and N-chloroacetyl amino acids, the phenylalanine derivative in each case is a considerably stronger inhibitor than the corresponding leucine derivative. This preference is similar to that observed for the binding of peptide substrates but the reverse of that observed for ester substrates and simple mono- and dicarboxylate ion inhibitors.The peptide substrates carbobenzoxyglycylglycyl-L-phenylalanine and N-chloroacetyl-L-phenylalanine are noncompetitive inhibitors of the enzymic hydrolysis of O-hippuryl-L-3-phenyllactic acid. This clearly demonstrates the presence of different ester and peptide binding sites in this enzyme, which is consistent with conclusions from recent studies in other laboratories.

Reversible Inhibition of Carboxypeptidase A. III. Inhibition of Specific Esterase Activity by Substituted Benzoate and Related Anions

1975 ◽  
Vol 53 (13) ◽  
pp. 1984-1992 ◽  
Author(s):  
John W. Bunting ◽  
Chester D. Myers
Keyword(s):  
Ionic Strength ◽  
Esterase Activity ◽  
Competitive Inhibition ◽  
Chemical Properties ◽  
Enzyme Molecule ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Reversible Inhibition ◽  
Noncompetitive Inhibition ◽  
Hydrolysis Of

The reversible inhibition of the hydrolysis of O-hippuryl-L-3-phenyllactic acid by bovine carboxypeptidase A, has been studied for a series of para-substituted benzoate ions (p-XC6H4-CO2−) at pH 7.5, 25°, ionic strength 0.2. For X = H, F, CN, NH2, CH3 competitive inhibition occurs, whereas non-competitive inhibition occurs for X = CF3, NO2, Cl, Br, (CH3)2N, CH3O, (CH3)2CH, (CH3)3C. For X = C2H5 mixed inhibition is observed and this can be separated into individual competitive and noncompetitive components. Uncompetitive inhibition occurs with X = I. The distinction between competitive and noncompetitive inhibition appears to depend on the size of X rather than on its chemical properties. The p-tolylacetate and 3-(p-tolyl)propanoate ions display partially competitive inhibition consistent with the formation of E.I2 species. The inhibition by the 3-(p-iodophenyl)propanoate ion is complex and depends on the binding of at least two inhibitor ions per enzyme molecule.

CATHEPSIN B AND AMINOPEPTIDASE ACTIVITY IN THE POSTERIOR MIDGUT OF EUSCHISTUS EUSCHISTOIDES (HEMIPTERA: PENTATOMIDAE)

1984 ◽  
Vol 116 (10) ◽  
pp. 1393-1396 ◽  
Author(s):  
Jon G. Houseman ◽  
W. K. MacNaughton ◽  
A. E. R. Downe
Keyword(s):  
Cathepsin B ◽  
Blood Feeding ◽  
Aminopeptidase Activity ◽  
Posterior Midgut ◽  
Hydrolysis Of

AbstractThe posterior midgut of a seed-feeding pentatomid, Euschistus euschistoides (Vollenhoven), contains the proteinases cathepsin B and aminopeptidase. Cadiepsin B hydrolysis of benzoyl-DL-arginine-2-naphthylamide is activated by thiol chemicals and EDTA. Aminopeptidase hydrolysis of leucine-p-nitroanilide is activated by MgCl2 and inhibited by cysteine, glutathione, EDTA, and CaCl2. These results are similar to those obtained for cathepsin B and aminopeptidase from blood-feeding Hemiptera and support the hypothesis that catheptic proteinases are unique to this order.

Identification and partial characterization of digestive proteinases from two species of bedbug (Hemiptera: Cimicidae)

1982 ◽  
Vol 60 (8) ◽  
pp. 1837-1840 ◽  
Author(s):  
Jon G. Houseman ◽  
A. E. R. Downe
Keyword(s):  
Cathepsin B ◽  
Horse Serum ◽  
Cimex Lectularius ◽  
Aminopeptidase Activity ◽  
Blood Sucking ◽  
Digestive Proteinases ◽  
Cimex Hemipterus ◽  
Hydrolysis Of ◽  
Optimal Ph

The digestive midgut of Cimex hemipterus (Fabr.) and Cimex lectularius L. contains cathepsin B, aminopeptidase, and an acidic proteinase that hydrolyzes haemoglobin at an optimal pH of 3.4. Cathepsin B was demonstrated by hydrolysis of benzoyl-DL-arginine-β-napthylamine at an optimal pH of 5.4. Hydrolysis of the substrate was activated by thiol chemicals and EDTA and inhibited by iodoacetamide (IAA) and horse serum. Maximum aminopeptidasehydrolysis of leucine-p-nitroanilide occurred at pH 8.4. Aminopeptidase activity was activated by MgCl2 and inhibited by EDTA, thiol chemicals, and CaCl2. Only C. hemipterus aminopeptidase was inhibited by IAA. The digestive proteinases in bedbugs are similar to those reported for other blood-sucking hemipteran insects.

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