A Comparison of Inhibition Constants for the Inhibition of α-Lytic Protease and Porcine Elastase by Acetylated Alanine Peptides

1973 ◽  
Vol 51 (1) ◽  
pp. 112-114 ◽  
Author(s):  
M. C. Shaw ◽  
D. R. Whitaker
Keyword(s):  
Methyl Ester ◽  
Binding Sites ◽  
Degree Of Polymerization ◽  
Inhibition Constant ◽  
Inhibition Constants

A comparison was made of the values of the inhibition constant Ki for the inhibition of α-lytic protease and porcine elastase by various N-acetylated L- and D-alanine peptides with N-benzoyl-L-alanine methyl ester as substrate for both enzymes. The Ki's for the inhibition of elastase by N-acetyl-L-alanine peptides decreased sharply as the degree of polymerization of the inhibitor increased, the respective values for monomer, dimer, and trimer being 1200 mM, 26 mM, and 2.7 mM. The corresponding estimates of Ki for α-lytic protease, viz. 310, 270, and 70 mM, showed comparatively little dependence on the degree of polymerization of the inhibitor and suggest a substantial difference in the binding sites of the two enzymes. The Ki's for inhibition of elastase by N-acetylated L-Ala-D-Ala, D-Ala-L-Ala, and D-Ala-D-Ala were, respectively, 55, 180, and > 300 mM; those for α-lytic protease were all > 300 mM.

The copurification of β-glucosidase, β-xylosidase, and 1,3-β-glucanase in two separate enzyme complexes isolated from Trichoderma harzianum E58

1987 ◽  
Vol 65 (9) ◽  
pp. 822-832 ◽  
Author(s):  
Larry U. L. Tan ◽  
Paul Mayers ◽  
Michelle Illing ◽  
John N. Saddler
Keyword(s):  
Isoelectric Point ◽  
Mercuric Chloride ◽  
Trichoderma Harzianum ◽  
Inhibition Constant ◽  
Activity Ratios ◽  
Molecular Masses ◽  
Inhibition Constants ◽  
Inhibition Studies ◽  
Enzyme Complexes

Two enzyme complexes, each with β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21), β-xylosidase (β-D-xylan xylohydrolase, EC 3.2.1.37), and 1,3-β-glucanase (laminarinase, EC 3.2.1.39) activity, were purified to near homogeneity from the cellulolytic fungus Trichoderma harzianum E58. The two complexes had the same isoelectric point of pH 8.3 and identical subunit molecular masses of 75 400 daltons. The two complexes were also similar in that all activities were sensitive to inhibition by mercuric chloride (2 mM) and D-glucono-1,5-lactone (0.2% w/v). The activity ratios of the major and minor complexes were 1:1.7:4.3 and 1:1.6:3.1 for the β-xylosidase, β-glucosidase, and 1,3-β-glucanase, respectively. Both complexes had approximately the same Km values for p-nitrophenyl β-D-glucopyranoside and salicin. The pH optima of corresponding activities of the two complexes were also similar. The major and minor complexes differed in that the Km of the former for laminarin was almost threefold lower than that of the latter. Whereas all three activities of the minor complexes were inhibited by D-glucono-1,5-lactone with the same inhibition constant, the β-glucosidase and 1,3-β-glucanase of the major complex had inhibition constants which differed by more than 80 000 times. In addition, the inhibition on the 1,3-β-glucanase in the major and minor complexes using D-glucono-1,5-lactone were noncompetitive and competitive, respectively. From the inhibition studies, the β-glucosidase, β-xylosidase, and 1,3-β-glucanase activities in the minor complex were deduced to be more interdependent than the same activities in the major complex.

scholarly journals The molecular-weight-dependence of the anti-coagulant activity of heparin

1978 ◽  
Vol 175 (2) ◽  
pp. 691-701 ◽  
Author(s):  
T C Laurent ◽  
A Tengblad ◽  
L Thunberg ◽  
M Höök ◽  
U Lindahl
Keyword(s):  
Molecular Weight ◽  
Binding Sites ◽  
Random Distribution ◽  
Degree Of Polymerization ◽  
Molecular Weight Range ◽  
High Affinity ◽  
Predicted Probability ◽  
Coagulant Activity ◽  
The Relationship ◽  
Molecular Weight Fractions

It is proposed that the anti-coagulant activity of heparin is related to the probability of finding, in a random distribution of different disaccharides, a dodecasaccharide with the sequence required for binding to antithrombin. It is shown that this probability is a function of the degree of polymerization of heparin. The hypothesis has been been tested with a series of narrow-molecular-weight-range fractions ranging from 5,600 to 36,000. The fractions having mol.wts. below 18,000 (comprising 85% of the original preparation) followed the predicted probability relationship as expressed by the proportion of molecules capable of binding to antithrombin. The probability that any randomly chosen dodecasaccharide sequence in heparin should bind to antithrombin was calculated to 0.022. The fraction with mol.wt. 36,000 contained proteoglycan link-region fragments, which may explain the deviation of the high-molecular-weight fractions from the hypothetical relationship. The relationship between anti-coagulant activity and molecular weight cannot be explained solely on the basis of availability of binding sites for antithrombin. The activity of high-affinity heparin (i.e. molecules containing high-affinity binding sites for antithrombin), determined either by a whole-blood clotting procedure or by thrombin inactivation in the presence of antithrombin, thus remained dependent on molecular weight. Possible explanations of this finding are discussed. One explanation could be a requirement for binding of thrombin to the heparin chain adjacent to antithrombin.

scholarly journals The interaction of α-N-(p-toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester with thrombin and trypsin

1978 ◽  
Vol 169 (1) ◽  
pp. 157-167 ◽  
Author(s):  
Y S Klausner ◽  
M Rigbi ◽  
T Ticho ◽  
P J De Jong ◽  
E J Neginsky ◽  
...  
Keyword(s):  
Methyl Ester ◽  
Mixed Type ◽  
Peptide Inhibitors ◽  
Inhibition Constant ◽  
Inhibitor Concentration ◽  
Bovine Trypsin ◽  
Human Thrombin ◽  
Phenylalanine Methyl Ester ◽  
Esterase Activities

The syntheses are described of p-guanidino-L-phenylalanine and some of its derivatives. alpha-N-(p-Toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester is an excellent substrate of bovine trypsin (EC 3.4.21.4) (Km 57 micron; kcat. 320s-1 at pH 7.4-8.0) and a very poor substrate of human thrombin (EC 3.4.21.5) (Km 190 micron, kcat. 0.2s-1) and bovine chymotrypsin (EC 3.4.21.1). The ester inhibits thrombin clotting activity. It also inhibits the amidase and esterase activities of human thrombin, this inhibition being of the mixed type. The inhibition constant, K1, of the order of 1 micron, increases with increasing inhibitor concentration. This suggests that the enzyme binds the inhibitor at multiple sites. The importance of the residue at the P1 position [notation of Berger & Schechter (1970) Philos. Trans. R. Soc. London Ser. B 257, 249-264] in determining the selectivity of a substrate or quasi-substrate among trypsin-like enzymes is borne out. p-Guanidino-L-phenylalanine may have a use in the synthesis of selective peptide inhibitors of thrombin.

scholarly journals A kinetic study of pig liver pyruvate kinase activated by fructose diphosphate

1974 ◽  
Vol 139 (3) ◽  
pp. 499-508 ◽  
Author(s):  
Neil Macfarlane ◽  
Stanley Ainsworth
Keyword(s):  
Kinetic Study ◽  
Pyruvate Kinase ◽  
Binding Sites ◽  
Experimental Results ◽  
Enzyme Complex ◽  
Pig Liver ◽  
Michaelis Constants ◽  
Inhibition Constants ◽  
Complex Forms

The paper reports a study of the reaction between phosphoenolpyruvate, ADP and Mg2+ catalysed by pig liver pyruvate kinase when activated by fructose diphosphate and K+. The experimental results are consistent with two non-sequential mechanisms in which the substrates and products of the reaction are phosphoenolpyruvate, ADP, Mg2+, pyruvate and MgATP. Pyruvate release occurs before ADP binding. Two Mg2+ ions are involved, though the two Mg2+-binding sites cannot be occupied simultaneously. An isomerized enzyme complex forms before release of MgATP. Values were determined for the Michaelis constants of the reaction. Apparent MgATP inhibition constants are also given.

scholarly journals Characterization and localization of Ac-SDKP receptor binding sites using125I-labeled Hpp-Aca-SDKP in rat cardiac fibroblasts

2007 ◽  
Vol 292 (2) ◽  
pp. H984-H993 ◽  
Author(s):  
Jia L. Zhuo ◽  
Oscar A. Carretero ◽  
Hongmei Peng ◽  
Xiao C. Li ◽  
Domenico Regoli ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Collagen Synthesis ◽  
Cardiac Fibroblasts ◽  
Biologically Active ◽  
Inhibition Constant ◽  
Maximal Effect ◽  
Ang Ii ◽  
Protein Dissociation

We have shown that the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibited endothelin-1 (ET-1)-induced cell proliferation and collagen synthesis in cultured rat cardiac fibroblasts (CFs) and reduced left ventricle collagen deposition in rats with aldosterone (salt)- and ANG II-induced hypertension. However, it is not known whether these effects are mediated by receptor binding sites specific for Ac-SDKP. We hypothesized that Ac-SDKP exerts antifibrotic effects by binding to specific receptor sites in cultured rat CFs, which mediate the inhibitory effects of Ac-SDKP on ET-1-stimulated collagen synthesis. Ac-SDKP binding sites in rat CFs and hearts were characterized by a specific radioligand,125I-labeled 3-( p-hydroxyphenyl)-propionic acid (or desaminotyrosine) (Hpp)-Aca-SDKP, a biologically active analog of Ac-SDKP.125I-labeled Hpp-Aca-SDKP bound to rat CFs and fractionated membranes with similar affinities and specificity in a concentration- and time-dependent fashion. Scatchard plot analyses revealed a single class of high-affinity Hpp-Aca-SDKP binding sites (maximal binding: 1,704 ± 198 fmol/mg protein; dissociation constant: 3.3 ± 0.6 nM).125I-labeled Hpp-Aca-SDKP binding in CFs was displaced by unlabeled native peptide Ac-SDKP (inhibition constant: 0.69 ± 0.15 nM) and the analog Hpp-Aca-SDKP (inhibition constant: 10.4 ± 0.2 nM) but not the unrelated peptide ANG II or ET-1 (10 μM). In vitro, both Ac-SDKP and Hpp-Aca-SDKP inhibited ET-1-stimulated collagen synthesis in CFs in a dose-dependent fashion, reaching a maximal effect at 1 nM (control: 7.5 ± 0.4, ET-1: 19.9 ± 1.2, ET-1+SDKP: 7.7 ± 0.4, ET-1+Hpp-Aca-SDKP: 9.7 ± 0.1 μg/mg protein; P < 0.001). Ac-SDKP also significantly attenuated ET-1-induced increases in intracellular calcium and MAPK ERK1/2 phosphorylation in CFs. In the rat heart, in vitro autoradiography revealed specific125I-labeled Hpp-Aca-SDKP binding throughout the myocardium, primarily interstitially. We believe that these results demonstrate for the first time that Hpp-Aca-SDKP is a functional ligand specific for Ac-SDKP receptor binding sites and that both Ac-SDKP and Hpp-Aca-SDKP exert antifibrotic effects by binding to Ac-SDKP receptors in rat CFs.

scholarly journals Thiol-dependent changes in the properties of rat liver sulphotransferases

1971 ◽  
Vol 123 (3) ◽  
pp. 427-434 ◽  
Author(s):  
D. J. Barford ◽  
J. G. Jones
Keyword(s):  
Ionic Strength ◽  
Methyl Ester ◽  
Molecular Size ◽  
Degree Of Polymerization ◽  
Prolonged Storage ◽  
Kinetic Properties ◽  
Female Rat ◽  
Km Value ◽  
Low Ionic Strength ◽  
Rat Livers

1. Two enzymes (A and B) which catalyse the sulphation of p-nitrophenol and l-tyrosine methyl ester have been isolated from female rat livers. One of these enzymes (A) also catalyses the sulphation of dehydroepiandrosterone. 2. The Km values for the sulphation of p-nitrophenol and l-tyrosine methyl ester by enzyme B at pH7.5 are 1.5μm and 2.9mm respectively. 3. Enzyme B is oxidized on keeping at 0°C when the Km and Vmax. values for the sulphation of p-nitrophenol are increased approx. 200-fold and fourfold respectively. This oxidized preparation of enzyme B fails to catalyse the sulphation of l-tyrosine methyl ester. 4. When the oxidized form of enzyme B is kept at 0°C and low ionic strength then further forms of p-nitrophenol sulphotransferase are produced having even lower affinities for the sulphate acceptor. 5. The Km value for adenosine 3′-phosphate 5′[35S]-sulphatophosphate is not affected during storage of the enzyme under these conditions. 6. Prolonged storage of enzyme B at low ionic strength leads to a considerable degree of polymerization of p-nitrophenol sulphotransferase and l-tyrosine methyl ester sulphotransferase. 7. The changes in the kinetic properties and molecular size of enzyme B during storage are reversed by dithiothreitol.

scholarly journals Partially esterified oligogalacturonides are the preferred substrates for pectin methylesterase of Aspergillus niger

2003 ◽  
Vol 372 (1) ◽  
pp. 211-218 ◽  
Author(s):  
Gert-Jan W. M. van ALEBEEK ◽  
Katrien van SCHERPENZEEL ◽  
Gerrit BELDMAN ◽  
Henk A. SCHOLS ◽  
Alphons G. J. VORAGEN
Keyword(s):  
Methyl Ester ◽  
Aspergillus Niger ◽  
Methyl Esters ◽  
Pectin Methylesterase ◽  
Degree Of Polymerization ◽  
Specific Pattern ◽  
Specific Product ◽  
Ionization Time ◽  
Active Site Cleft

Investigations on the mode of action of Aspergillus niger pectin methylesterase (PME) towards differently C6- and C1-substituted oligogalacturonides (oligoGalpA) are described. De-esterification of methyl-esterified (un)saturated oligoGalpA proceeds via a specific pattern, depending on the degree of polymerization. Initially, a first methyl ester of the oligomer is hydrolysed, resulting in one free carboxyl group. Subsequently, this first product is preferred as a substrate and is de-esterified for a second time. This product is then accumulated and hereafter de-esterified further to the final product, i.e. oligoGalpA containing one methyl ester located at the non-reducing end residue for both saturated and unsaturated oligoGalpA, as found by post-source decay matrix-assisted laser-desorption/ionization–time-of-flight MS. The saturated hexamer is an exception to this: three methyl esters are removed very rapidly, instead of two methyl esters. When unsaturated oligoGalpA were used, the formation of the end product differed slightly, suggesting that the unsaturated bond at the non-reducing end influences the de-esterification process. In vivo, PME prefers methyl esters, but the enzyme appeared to be tolerant for other C6- and C1-substituents. Changing the type of ester (ethyl esterification) or addition of a methyl glycoside (C1) only reduced the activity or had no effect respectively. The specific product pattern was identical for all methyl- and ethyl-esterified oligoGalpA and methyl-glycosidated oligoGalpA, which strongly indicates that one or perhaps two non-esterified oligoGalpA are preferred in the active-site cleft.

scholarly journals A CYTOCHEMICAL STUDY OF THE L.E. BODIES OF SYSTEMIC LUPUS ERYTHEMATOSUS

1957 ◽  
Vol 106 (4) ◽  
pp. 575-592 ◽  
Author(s):  
Gabriel C. Godman ◽  
Arline D. Deitch
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Lupus Erythematosus ◽  
Binding Sites ◽  
Degree Of Polymerization ◽  
Methyl Green ◽  
Dilute Acid ◽  
Systemic Lupus ◽  
Cytochemical Study ◽  
Associated Proteins ◽  
The Mean

The composition, with respect to nucleic acids, of the L.E. bodies resulting from the action of the plasma of patients with systemic lupus erythematosus on substrate leukocyte nuclei in different kinds of preparations was compared microspectrophotometrically with that of control lymphocyte nuclei. Binding of the basic dye methyl green to DNA was uniformly found to be depressed in L.E. bodies as compared with control nuclei. Since Feulgen-revealed DNA, which served as a standard of reference, was relatively unchanged in amount, the Feulgen:methyl green ratios of L.E. bodies were higher than those of lymphocyte nuclei. Acetylation, which covers basic groups of proteins, was found to increase methyl green uptake by DNA of L.E. bodies to values approximating those of control nuclei, with consequent revision, after acetylation of the Feulgen: methyl green ratios of L.E. bodies to values similar to those of lymphocyte nuclei. Ribonuclease was found to reduce methyl green staining; extraction with dilute acid had little effect. These data have been interpreted to indicate (a) the presence in L.E. bodies of DNA-associated proteins whose basic groups compete with the cationic dye for binding sites of DNA and so inhibit methyl green staining, and (b) the DNA itself is not detectibly altered in state or degree of polymerization. Photometric comparison of the mean Feulgen-stainable DNA content per L.E. body with that of control nuclei showed that DNA is not lost in the L.E. transformation of nuclei.

A Direct Enzymatic Assay for the Esterolytic Activity of Activated Hageman Factor

1974 ◽  
Vol 31 (01) ◽  
pp. 030-039 ◽  
Author(s):  
Richard J Ulevitch ◽  
David Letchford ◽  
C. G Cochrane
Keyword(s):  
Methyl Ester ◽  
Substrate Inhibition ◽  
Competitive Inhibitor ◽  
Enzymatic Assay ◽  
Inhibition Constant ◽  
Turnover Number ◽  
Hageman Factor ◽  
Saturation Kinetics ◽  
Proteolytic Activities ◽  
Esterolytic Activity

SummaryA direct enzymatic assay for activated Hageman factor (HFa) has been developed utilizing N-α-acetylgiycine lysine methyl ester (AGLME). This assay allowed the measurement of the esterolytic activity of purified Hageman factor (HF) which had been activated by enzymatic (trypsin) and non-enzymatic (kaolin) mechanisms. No esterolytic activity was detected in preparations of purified HF prior to activation.Studies with purified HF activated on kaolin demonstrated the following: (1) Simple saturation kinetics (Km = 9 mM, Turnover number = 2.5 min–1) at AGLME concentrations less than 0.04 M and substrate inhibition at AGLME concentrations greater than 0.04 M. (2) The substrate AGLME behaved as a competitive inhibitor of the activation of prekallikrein (PK) by HFa with an inhibition constant (Ki) of about 10 mM. (3) Both the esterolytic (AGLME hydrolysis) and the proteolytic activities (PK activation) were inhibited by diisopropylfluorosphosphate (DFP) with an approximate Ki of 5 × 10–4 M.

Synthesis of 6-Aryloxy- and 6-Arylalkoxy-2-chloropurines and Their Interactions with Purine Nucleoside Phosphorylase from Escherichia coli

1999 ◽  
Vol 54 (12) ◽  
pp. 1055-1067 ◽  
Author(s):  
Agnieszka Bzowska ◽  
Lucyna Magnowska ◽  
Zygmunt Kazimierczuk
Keyword(s):  
Mixed Type ◽  
Purine Nucleoside Phosphorylase ◽  
Phase Transfer ◽  
Competitive Inhibition ◽  
Purine Nucleoside ◽  
Inhibition Constant ◽  
Purine Base ◽  
Nucleoside Phosphorylase ◽  
E Coli ◽  
Inhibition Constants

The phase transfer method was applied to perform the nucleophilic substitution of 2,6- dichloropurines by modified arylalkyl alcohol or phenols. Since under these conditions only the 6-halogen is exchanged, this method gives 2-chloro-6-aryloxy- and 2-chloro-6-arylalkoxypurines. 2-Chloro-6-benzylthiopurine was synthesized by alkylation of 2-chloro-6-thiopurine with benzyl bromide. The stereoisomers of 2-chloro-6-(1-phenyl-1-ethoxy)purine were obtained from R- and S-enantiomers of sec.-phenylethylalcohol and 2,6-dichloropurine. All derivatives were tested for inhibition with purified hexameric E. coli purine nucleoside phosphorylase (PNP). For analogues showing IC50 < 10 μm, the type of inhibition and inhibition constants were determined. In all cases the experimental data were best described by the mixed-type inhibition model and the uncompetitive inhibition constant, Kiu, was found to be several-fold lower than the competitive inhibition constant, Kic. This effect seems to be due to the 6-aryloxy- or 6-arylalkoxy substituent, because a natural PNP substrate adenine, as well as 2-chloroadenine, show mixed type inhibition with almost the same inhibition constants Kiu and KiC. The most potent inhibition was observed for 6-benzylthio-2-chloro-, 6-benzyloxy-2-chloro-, 2-chloro-6-(2-phenyl-l-ethoxy), 2-chloro-6-(3-phenyl-l-propoxy)- and 2-chloro-6-ethoxypurines (Kiu = 0.4, 0.6, 1.4, 1.4 and 2.2 μm, respectively). The R-stereoisomer of 2-chloro-6-(1pheny-1-ethoxy)purine has Kiu = 2.0 μm, whereas inhibition of its S counterpart is rather weak (IC50> 12 μm). More rigid (e.g. phenoxy-), non-planar (cyclohexyloxy-), or more bulky (2,4,6-trimethylphenoxy-) substituents at position 6 of the purine base gave less potent inhibitors (IC50 = 26, 56 and >100 μm, respectively). The derivatives are selective inhibitors of hexameric “high-molecular mass” PNPs because no inhibitory activity vs. trimeric Cellulomonas sp. PNP was detected. By establishing the ligand-dependent stabilization pattern of the E. coli PNP it was shown that the new derivatives, similarly as the natural purine bases, are able to form a dead-end ternary complex with the enzyme and orthophosphate. It was also shown that the derivatives are substrates in the reverse synthetic direction catalyzed by E. coli PNP

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