Diagnostic Enzymology of a-L-Iduronidase with Special Reference to a Sulphated Disaccharide Derived from Heparin

1982 ◽  
Vol 62 (2) ◽  
pp. 193-201 ◽  
Author(s):  
J. J. Hopwood ◽  
Vivienne Muller
Keyword(s):  
Enzyme Activity ◽  
Competitive Inhibitor ◽  
Ph Profile ◽  
Ph Values ◽  
Substrate Structure ◽  
Cultured Skin ◽  
Bivalent Cations ◽  
Competitive Inhibitors ◽  
Specific Ion Effect ◽  
Hydrolysis Of

1. Iduronosyl anhydro[1-3H]mannitol 6-sulphate (IMs), iduronosyl anhydro[1-3H]mannitol, phenyl iduronide (PhI) and 4-methylumbelliferyl iduronide have been compared as substrates for the diagnostic estimation of α-l-iduronidase activity present in human leucocyte and cultured skin fibroblast homogenates. The pH profile of leucocyte and fibroblast iduronidase activity was dependent on substrate structure and concentration, the ionic strength and the nature of the buffer ion used in the assay mixture. 2. NaCl, KBr and Na2SO4 were shown to be parabolic competitive inhibitors of IMs activity, the K1 with fibroblast homogenates being 34, 13.4 and 0.22 mmol/l respectively. NaCl and KBr were shown to have a primary salt effect on the interaction between enzyme and substrate but Na2SO4 appeared to have a specific ion effect at a cationic binding site. 3. NaCl inhibited the hydrolysis of IMs at all pH values studied, whereas NaCl concentrations of 0.2 mol/l inhibited the hydrolysis of PhI at pH values below 3.8 but activated the enzyme at higher incubation pH values. 4. Cu2+ was shown to be a potent non-competitive inhibitor of IMs enzyme activity with an apparent Kl, of approximately 0.02 mmol/l. The enzyme activity was inhibited by Fe2+ (Kl 4 mmol/l), Hg2+ and Ag+, but has not significantly been affected by other univalent or bivalent cations. 5. The presence of solvent and salt effects on apparent Km but not the Vmax. suggest that the binding of IMs to the enzyme involved charge neutralization, and it is inferred that two cationic binding sites are present at the active site. It is postulated that one site specifically binds to the iduronic acid carboxyl group, the other to the 6-sulphate of the anhydromannitol moiety.

scholarly journals Mechanism of d-fructose isomerization by Arthrobacterd-xylose isomerase

1992 ◽  
Vol 283 (1) ◽  
pp. 223-233 ◽  
Author(s):  
M Rangarajan ◽  
B S Hartley
Keyword(s):  
Ring Opening ◽  
Ph Dependence ◽  
Xylose Isomerase ◽  
Competitive Inhibitor ◽  
Apparent Dissociation Constant ◽  
Ph Values ◽  
Rate Limiting Step ◽  
Competitive Inhibitors ◽  
Rate Limiting ◽  
Very High

The mechanism of D-fructose isomerization by Arthrobacter D-xylose isomerase suggested from X-ray-crystallographic studies was tested by detailed kinetic analysis of the enzyme with various metal ions at different pH values and temperatures. At D-fructose concentrations used in commercial processes Mg2+ is the best activator with an apparent dissociation constant of 63 microM; Co2+ and Mn2+ bind more strongly (apparent Kd 20 microM and 10 microM respectively) but give less activity (45% and 8% respectively). Ca2+ is a strict competitive inhibitor versus Mg2+ (Ki 3 microM) or Co2+ (Ki 105 microM). The kinetics show a compulsory order of binding; Co2+ binds first to Site 2 and then to Site 1; then D-fructose binds at Site 1. At normal concentrations Mg2+ binds at Site 1, then D-fructose and then Mg2+ at Site 2. At very high Mg2+ concentrations (greater than 10 mM) the order is Mg2+ at Site 1, Mg2+ at Site 2, then D-fructose. The turnover rate (kcat.) is controlled by ionization of a residue with apparent pKa at 30 degrees C of 6.0 +/- 0.07 (Mg2+) or 5.3 +/- 0.08 (Co2+) and delta H = 23.5 kJ/mol. This appears to be His-219, which is co-ordinated to M[2]; protonation destroys isomerization by displacing M[2]; Co2+ binds more strongly at Site 2 than Mg2+, so competes more strongly against H+. The inhibition constant (Ki) for the two competitive inhibitors 5-thio-alpha-D-glucopyranose and D-sorbitol is invariant with pH, but Km(app.) in the Mg[1]-enzyme is controlled by ionization of a group with pKa 6.8 +/- 0.07 and delta H = 27 kJ/mol, which appears to be His-53. This shows that Km(app.) is a complex constant that includes the rate of the ring-opening step catalysed by His-53, which explains the pH-dependence. In the Mg[1]Mg[2]-enzyme or Co[1]Co[2]-enzyme, the pKa is lower (6.2 +/- 0.1 or 5.6 +/- 0.08) because of the extra adjacent cation. Hence the results fit the previously proposed pathway, but show that the mechanisms differ for Mg2+ and Co2+ and that the rate-limiting step is isomerization and not ring-opening as previously postulated.

Kinetics of the Inhibition of Plasmin in Acidified Human Plasma

1982 ◽  
Vol 48 (03) ◽  
pp. 257-259 ◽  
Author(s):  
H R Lijnen ◽  
M Maes ◽  
M Castel ◽  
M Samama ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasma Proteins ◽  
Competitive Inhibitor ◽  
Normal Plasma ◽  
Competitive Inhibitors ◽  
Rate Of Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of

SummaryAcid-treated human plasma is a competitive inhibitor of the hydrolysis of D-Val-Leu-Lys-Nan (S-2251) by plasmin. The rate of hydrolysis is decreased to 50% by 750 fold diluted acidified normal plasma and by 60 fold diluted acidified α2-antiplasmin depleted plasma (α2-antiplasmin concentration less than 2%). These findings suggest that α2-antiplasmin is a contributary but not the main competitive inhibitor of acidified plasma. This interpretation is supported by the finding that α2-antiplasmin depleted plasma reconstituted with purified α2-antiplasmin inhibits the hydrolysis of S-2251 by plasmin at a 125 fold dilution following acidification and by the finding that in a purified system acid inactivated α2-antiplasmin inhibits the hydrolysis of S-2251 by plasmin with a Ki of 25 nM. Thus, besides α2-antiplasmin, other plasma proteins which are at least in part eliminated by the removal of α2-antiplasmin from plasma by immunoadsorption appear to be competitive inhibitors for plasmin in acidified plasma. It is suggested that several competitive inhibitors for plasmin are present and/or generated in acidified plasma and that these inhibitors may at least in part be responsible for the variability in the results of measurements of plasminogen and/or plasmin in plasma following acidification.

scholarly journals Studies on the preparation of water-insoluble derivatives of rennin and chymotrypsin and their use in the hydrolysis of casein and the clotting of milk

1969 ◽  
Vol 115 (2) ◽  
pp. 183-190 ◽  
Author(s):  
Margaret L. Green ◽  
G. Crutchfield
Keyword(s):  
Continuous System ◽  
Competitive Inhibitor ◽  
Active Enzyme ◽  
Ph Values ◽  
Hydrolysis Of ◽  
Derivatives Of

1. Enzymically active insoluble derivatives of chymotrypsin and rennin were prepared by coupling each enzyme to agarose as described by Porath, Axén & Ernback (1967) and rennin to aminoethylcellulose by the method of Habeeb (1967). 2. Agarose–chymotrypsin was stable over the range pH2–9, but agarose–rennin released active enzyme into solution at above pH2 and aminoethylcellulose–rennin was similarly unstable at certain pH values. 3. Each derivative appeared to catalyse the clotting of milk at 30°, but this was probably entirely due to enzyme released into solution from the carrier. 4. The presence of a competitive inhibitor of chymotrypsin during its coupling to agarose had no effect on the activity or stability of the resulting derivative. 5. The characteristics of agarose and cellulose render them not entirely suitable for use in a continuous system with milk.

scholarly journals The Effects of Addition of Mononucleotides on Sma nuc Endonuclease Activity

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Julia Romanova ◽  
Maria Filimonova
Keyword(s):  
Enzyme Activity ◽  
Serratia Marcescens ◽  
Competitive Inhibitor ◽  
Endonuclease Activity ◽  
Negative Bacterium ◽  
Gram Negative ◽  
Carbohydrate Residue ◽  
Partial Type ◽  
Hydrolysis Of ◽  
Hydrolysis Of Dna

Examination of the effects of mononucleotides on Sma nuc endonuclease originated from Gram negative bacteriumSerratia marcescensdisplayed that any mononucleotide produced by Sma nuc during hydrolysis of DNA or RNA may regulate the enzyme activity affecting the RNase activity without pronounced influence on the activity towards DNA. The type of carbohydrate residue in mononucleotides does not affect the regulation. In contrast, the effects depend on the type of bases in nucleotides. AMP or dAMP was classified as a competitive inhibitor of partial type. GMP, UMP, and CMP were found to be uncompetitive inhibitors that suggest a specific site(s) for the nucleotide(s) binding in Sma nuc endonuclease.

scholarly journals A BIOCHEMICAL AND CYTOCHEMICAL STUDY OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN THE PHLOEM OF NICOTIAN A TABACUM

1974 ◽  
Vol 60 (1) ◽  
pp. 221-235 ◽  
Author(s):  
Jamison Gilder ◽  
James Cronshaw
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Phloem Transport ◽  
Cytochemical Localization ◽  
Gtp Hydrolysis ◽  
Cytochemical Study ◽  
P Protein ◽  
Competitive Inhibitors ◽  
Hydrolysis Of

A biochemical and cytochemical study has been made of the distribution of ATPase in mature and differentiating phloem cells of Nicotiana tabacum and of the substrate specificity and effects of fixation on enzyme activity. Homogenates of unfixed leaf midveins and midveins fixed in formaldehyde-glutaraldehyde were assayed for enzyme activity by determining the amount of Pi, liberated per milligram of protein from various substrates in a 30 min period at pH 7.2. In fresh homogenates, hydrolysis of ATP was not significantly different from that of ITP, CTP, and UTP. Hydrolysis of GTP was slightly higher than that of ATP. ATP hydrolysis by fresh homogenates was 17% more extensive than that of ADP, 76% more extensive than that of 5'-AMP, and was inhibited by fluoride and p-chloromercuribenzoate (PCMB). There was little or no hydrolysis of the competitive inhibitors 2'- and 3'-AMP nor with the alternate substrates p-nitrophenylphosphate (PNP) or ß-glycerophosphate (ß-GP). In homogenates of material fixed in formaldehyde-glutaraldehyde for 1¼ h, ATPase activity was 13% preserved. Hydrolysis of ATP by fixed homogenates was not significantly different from that of ADP, 5'-AMP, ITP, CTP, and GTP. Hydrolysis of UTP was lower. Fluoride and PCMB inhibited fixed ATPase activity. The results of cytochemical localization experiments using a lead phosphate precipitation technique were in agreement with the biochemical results. Similar localization patterns were obtained with the nucleoside triphosphates ATP, CTP, GTP, ITP, and UTP. Activity was also localized with ADP and 5'-AMP but not with the competitive inhibitors 2'- and 3'-AMP, nor with PNP or ß-GP. Little or no reaction product was deposited in other controls incubated without substrate or with substrate plus fluoride, PCMB, or N-ethylmaleimide. ATPase activity was demonstrated chiefly at the plasma membrane of mature and differentiating phloem cells and was associated with the P-protein of mature sieve elements. It is suggested that the phloem transport system derives its energy from the demonstrated nucleoside triphosphatase activity.

HORMONE AND ENZYME ASSAYS IN PREGNANCY

1975 ◽  
Vol 78 (2) ◽  
pp. 364-372
Author(s):  
Arne Christensen ◽  
P. E. Hagelid
Keyword(s):  
Enzyme Activity ◽  
Linear Part ◽  
Competitive Inhibitor ◽  
Plasma Samples ◽  
Simple Method ◽  
Normal Enzyme ◽  
Lag Period ◽  
Enzyme Activity Pattern ◽  
Hydrolysis Of

ABSTRACT A rapid and simple method for determination of the placental cystineaminopeptidase (P-CAP) activity in plasma is presented. The enzymecatalysed hydrolysis of the substrate, l-cystine-bis-p-nitroanilide, is followed in a spectrophotometer by reading the absorbance of the product p-nitroaniline at 380 nm. The absorbance increases linearily with time after a lag-period of 30 seconds to 5 min. The reaction is followed for about 10 min. and the increase in absorbtion per min is calculated from the linear part of the absorbtion curve. The test could also be performed by taking 2 readings at about 5 min intervals. In the studies of the enzyme kinetics a competitive inhibitor of the reaction was found in plasma. The amount of the inhibitory substances seemed to be nearly constant in the plasma samples studied from normal pregnant women as well as in plasma samples with high enzyme activity (twin-pregnancy) and low enzyme activity (severe pre-eclampsia). The normal enzyme activity pattern showed an increase from about 210 days of pregnancy and towards the term. A coefficient of correlation of + 0.83 with the more time consuming method using l-cystine-di-βnaphtylamide as substrate was found. It was concluded that the present method could replace the method described by Babuna & Yenen (1966) at least from about 210 days of pregnancy and where a rapid answer is necessary.

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.

scholarly journals Partial purification of a diacylglycerol lipase from bovine aorta

1994 ◽  
Vol 298 (1) ◽  
pp. 213-219 ◽  
Author(s):  
M W Lee ◽  
D L Severson
Keyword(s):  
Lipase Activity ◽  
Specific Activity ◽  
Competitive Inhibitor ◽  
Short Chain ◽  
Partial Purification ◽  
Diacylglycerol Lipase ◽  
Bivalent Cations ◽  
Triton X ◽  
Hydrolysis Of ◽  
Long Chain

A diacylglycerol (DG) lipase has been purified from a soluble subcellular fraction of bovine aorta by (NH4)2SO4 precipitation in the presence of 5.0% (w/v) Triton X-100, followed by chromatography on DEAE-Sephacel, heparin-Sepharose and octyl-Sepharose in the presence of either CHAPS or Triton X-100 detergents. Under basal conditions, the hydrolysis of a short-chain [3H]dioctanoylglycerol ([3H]diC8) substrate was much greater than that of a long-chain 1-[1-14C]palmitoyl-2-oleoyl-sn-glycerol (1-[14C]POG) substrate. Lipase activity measured with 1-[14C]POG was markedly enhanced by Triton X-100. In the presence of 0.1% Triton X-100, specific enzyme activities in the octyl-Sepharose fraction determined with 1-[14C]POG or 1-stearoyl-2-[1-14C]-arachidonoyl-sn-glycerol as substrates were the same as that measured with [3H]diC8. MgCl2 (5mM) or CaCl2 (2 mM) also selectively stimulated lipase activity (up to 10-13-fold) measured with the long-chain (1-[14C]POG) substrate only. The increase in relative specific activity in the octyl-Sepharose fraction was 60-fold and 155-fold, based on hydrolysis of [3H]diC8 and 1-[14C]POG (+ Triton X-100), respectively. Unlabelled diC8 was a competitive inhibitor of 1-[14C]POG hydrolysis, suggesting that a single lipase hydrolyses both the short-chain and long-chain DG substrates; selective stimulatory effects of non-ionic detergents and bivalent cations on the hydrolysis of 1-[14C]POG may be due to effects on the physical properties of the substrate preparation. Monoacylglycerol lipase, DG kinase and cholesterol esterase activities could not be detected in the partially purified lipase preparation.

scholarly journals Kinetics of concomitant transfer and hydrolysis reactions catalysed by the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R61

1973 ◽  
Vol 135 (3) ◽  
pp. 483-492 ◽  
Author(s):  
J.-M. Frère ◽  
J.-M. Ghuysen ◽  
H. R. Perkins ◽  
M. Nieto
Keyword(s):  
Enzyme Activity ◽  
Binding Site ◽  
Water Content ◽  
Diaminopimelic Acid ◽  
Acceptor Molecule ◽  
Competitive Inhibitors ◽  
Hydrolysis Pathway ◽  
High Concentrations ◽  
Kinetics Of ◽  
Hydrolysis Of

When Ac2-l-Lys-d-Ala-d-Ala and either meso-diaminopimelic acid or Gly-l-Ala are exposed to the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R61, transpeptidation reactions yielding Ac2-l-Lys-d-Ala-(d)-meso- diaminopimelic acid and Ac2-l-Lys-d-Ala-Gly-l-Ala occur concomitantly with the hydrolysis of the tripeptide into Ac2-l-Lys-d-Ala. The proportion of the enzyme activity which can be channelled in the transpeptidation and the hydrolysis pathways depends upon the pH and the polarity of the environment. Transpeptidation is favoured both by increasing the pH and by decreasing the water content of the reaction mixtures. Kinetics suggest that the reactions proceed through an ordered mechanism in which the acceptor molecule (meso-diaminopimelic acid or Gly-l-Ala) binds first to the enzyme. Both acceptors behave as non-competitive inhibitors of the hydrolysis pathway. Transpeptidation is inhibited by high concentrations of Gly-l-Ala but not by high concentrations of meso-diaminopimelic acid. The occurrence on the enzyme of an additional inhibitory binding site for Gly-l-Ala is suggested.

scholarly journals Таблеткові суміші, що містять іммобілізований лізоцим і кверцетин: отримання, властивості

2017 ◽  
Author(s):  
I. I. Romanovska ◽  
S. S. Dekina ◽  
O. V. Sevastyanov ◽  
Ye. A. Rogozha
Keyword(s):  
Enzyme Activity ◽  
Chemical Properties ◽  
Lysozyme Activity ◽  
Biologically Active ◽  
Alkaline Ph ◽  
Acidic Media ◽  
Ph Profile ◽  
Ph Values ◽  
Physico Chemical ◽  
Different Origin

Introduction. Lysozyme (EC 3.2.1.17), which has antibacterial, anti-inflammatory, immunomodulatory actions is increasingly used in medicine in a different dosage forms: tablets, wound coatings, capsules, gels, etc. Tablet is a convenient dosage form for usage of lysozyme in combination with other biologically active substances. The targeted stabilization of enzyme by immobilization into polymeric matrices determines the relevance of tablet form development of complex compositions of lysozyme and quercetin.The aim of the study – development of tablet blends of complex preparation of immobilized lysozyme and quercetin, their quantitative determination and the study of the properties of active substances.Methods of the research. Lysozyme activity was determined by bacteriolytic method. The protein content was controlled according to Lowry-Hartree, quercetin – using zirconium chloride.Results and Discussion. Using the earlier obtained data about the interaction of lysozyme with polymeric supports of different origin and structure, for the development of tableting mixtures with lysozyme and quercetin, as polymeric binders the poly-N-vinylpyrrolidone, sodium salt of carboxymethyl cellulose and gelatin were chosen. The comparative analysis of physico-chemical properties of immobilized in polymers lysozyme had shown prospects of gelatin usage as a matrice, which promotes widening of pH-profile of enzyme activity both in areas of acidic and alkaline pH values, stability in acidic media. For making the lysozyme and quercetin – containing tableting mixtures, the fillers, disintegrants, flavours and wet granulation method were chosen. The total preservation of lysozyme activity and quercetin content in obtained granules was shown.conclusions. Tablet mixtures with lysozyme and quercetin with usage of polymeric carriers of different origin and structure were developed. The analysis of biochemical and physico-chemical properties of immobilized lysozyme had shown prospects of gelatin usage as a matrice. Immobilization promotes widening of pH-profile of enzyme activity both in areas of acidic and alkaline pH values, stability in acidic media. 

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