Comparison of the cerebroside sulphatase and the arylsulphatase activity of human sulphatase A in the absence of activators.

A cerebroside sulphatase (cerebroside-3-sulphate 3 sulphohydrolase, EC 3.1.6.8) assay based on radio thin-layer chromatography is described. The substrate was labelled by the catalytic addition of tritium to cerebroside sulphate. Using this assay the cerebroside sulphatase activity of sulphatase A (Aryl-sulphate sulphohydrolase, EC 3.1.6.1) from human liver and kidney in the absence of activators was investigated. The pH optimum of this reaction depends on the buffer concentration, being pH 4.5 at 50 mM and 5.3 at 10 mM sodium formate. With the latter concentration the apparent Km for cerebroside sulphate is 0.06 mM; SO2-4 and nitrocatechol sulphate inhibit noncompetitively with a Ki of 4.51 mM for Na2SO4 and 0.43 mM for nitrocatechol sulphate. The cerebroside sulphatase activity of sulphatase A is highly dependent on the ionic strength. The optimum sodium formate concentration is 10 mM, and the cerebroside suophatase activity decreases rapidly with increasing buffer concentration. The same concentration dependence is observed in the inhibitory effect of cerebroside sulphate on the arylsulphatase reaction. The inhibition decreases at increasing buffer concentrations, becoming an activation at 70 mM sodium formate. The progress curve of the cerebroside sulphatase reaction shows a deviation from linearity similar to that of the arylsulphatase reaction. Investigation of the effect of preincubation with cerebroside sulphate on the arylsulphatase activity of the enzyme shows that cerebroside sluphatase activity and inactivation of the enzyme by cerebroside sulphate occur simultaneously. These observations are interpreted as supporting the assumption that cerebroside suophate and arylsulphates are degraded at an identical active site on the same enzyme. Differences in the properties of the cerebroside sulphatase and the arylsulphatase reaction of the enzyme may be attributed to the differences in the physiocochemical state of the two substrates.     

Assay of covalent intermediate of 5'-nucleotide phosphodiesterase

We report a new procedure for isolating a covalent phosphoryl enzyme (diester) intermediate of bovine intestinal 5'-nucleotide phosphodiesterase. The convenience of the procedure makes it possible to determine effects of reaction conditions on the yield of covalent intermediate. Under optimum conditions, using [methyl-3H]deoxythymidine 5'-triphosphate as substrate, more than 50% of the enzyme is recovered as thymidylyl enzyme, a 10-fold increase in yield over the previous procedure (M. Landt and L. G. Butler, 1978, Biochemistry 17, 4130-4135). Yields of thymidylyl enzyme were maximal at pH 4, whereas optimum catalytic activity is observed at pH greater than 9.     

Maximising a function of the selection differential

Summary It is shown that some problems of optimising selection response can be solved without assuming a specific form of distribution for the trait of interest. To maximise the selection limit using selection among a fixed number every generation, all above the mean should retained. If a fraction of a population is set aside as a sire breeding nucleus, and selection is at one stage, maximum response per generation occurs when the nucleus as a fraction of the whole population is the square root of the sires: dams ratio. When a trait has an optimum, but declines in value at different rates A above and B below the optimum, the population mean should be chosen so that a fraction B/(A + B) are above the optimum.     

Selection index updating

Summary When traits become evident at different ages or there are large differences in the costs of measuring various traits, selection by independent culling levels may give a higher aggregate economic return than index selection because not all traits need to be measured on all individuals. The problems with optimum independent culling selection is that general solutions are not possible and numerical integration is needed for specific cases. Recently, Xu and Muir (1991) developed a new independent culling level procedure by use of orthogonal transformation of the original characters. With their procedure, explicit solutions for optimum truncation points are possible without numerical integration. As such, the procedure is proficient for any number of stages, and generalized theoretical comparisons of alternative breeding strategies are possible. However, their procedure was limited to the case where selection is for one character at each stage. In this paper, our previous results are extended to the general case of multi-stage index selection, called selection index updating. This procedure is called selection index updating because as traits become available in latter stages, each subsequent index contains all of the traits available up to that stage.The procedure is to develop sequential indices for each stage such that correlations among indices at different stages are zero. Optimum culling points are obtained for the updating procedure by using Xu and Muir's (1991) iterative equations. Due to the property of orthogonality of the updated indices, aggregate gain can be partitioned into gains due to various stages of selection. Partitioning of aggregate economic gain is useful to breeders who desire to adjust individual trait selection intensity based on facilities available at that stage. Methods are discussed to modify the procedure to obtain maximum aggregate economic return per unit of cost associated with obtaining measures on each trait. An application of multi-stage selection is demonstrated using a set of data for Rhode Island Red layer type chickens. A second example demonstrates the use of multi-stage selection optimized with respect to aggregate economic gain and costs associated with obtaining measurements.Journal Paper No. 12813 of the Purdue University Agricultural Experiment Station     

Application of progress curve analysis to in situ enzyme kinetics using 1H NMR spectroscopy

The steady-state kinetics of enzymes in tissues, cells, and concentrated lysates can be characterized using high-resolution nuclear magnetic resonance spectroscopy; this is possible because almost invariably there are differences in the spectra of substrates and products of a reaction and these spectra are obtainable even from optically opaque samples. We used 1H spin-echo NMR spectroscopy to study the hydrolysis of alpha-L-glutamyl-L-alanine by cytosolic peptidases of lysed human erythrocytes. Nonlinear regression of the integrated Michaelis-Menten expression onto the progress-curve data yielded, directly, estimates of Vmax and Km for the hydrolase; a procedure for analyzing progress curves in this manner was adapted and compared with a commonly used procedure which employs the Newton-Raphson algorithm. We also performed a sensitivity analysis of the integrated Michaelis-Menten expression; this yielded equations that indicate under what conditions estimates of Km and Vmax are most sensitive to variations in experimental observables. Specifically, we showed that the most accurate estimates of the steady-state parameters from analysis of progress curves are obtained when the initial substrate concentration is much greater than Km. Furthermore, estimates of these parameters obtained by such an analysis are most sensitive to data obtained when the reaction is 60-80% complete, having started with the highest practicable initial substrate concentration.     

A practical approach to the choice of a suitable detergent and optimal conditions for solubilizing a membrane protein

The main objective of this laboratory practical class was to teach students how a detergent and the best experimental conditions are chosen to solubilize a given membrane protein. Kidney Na,K-ATPase was chosen as the protein of interest and anionic, neutral and zwitterionic detergents were tested. Simple laboratory experiments were designed to study the effect of the detergent on the activity of the enzyme, the effect of detergent concentration on solubilization, the effect of protein concentration on enzyme solubilization, and the effect of time and temperature of incubation during enzyme solubilization. This resulted in the selection of an appropriate detergent for the solubilization of the protein taking into account smaller inactivation factors, more effective solubilization (more effective solubilization with a better detergent-protein relationship), lower inactivation temperature and time of incubation of the membrane protein with the detergent. The results obtained showed that instantaneous incubation of Na,K-ATPase with C(12)E(8) (1:1 w/w) at 4 degrees C resulted in a more efficient solubilization and had a smaller denaturing effect on the solubilized enzyme.     

Modification and inactivation of rhodanese by 2,4,6-trinitrobenzenesulphonic acid

Bovine liver rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) is modified by 2,4,6-trinitrobenzenesulphonic acid, by the use of modifying agent concentrations in large excess over enzyme protein concentration. The end-point of the reaction, viz., the number, n, per enzyme protein molecule, of modifiable amino groups was determined graphically by the Kézdy-Swinbourne procedure. It was found that the value for n depends on the pH of the reaction medium, and ranges from 2, at pH 7.00, to 10.66, at pH 9.00. Again, the value for n increases with an increase in the concentration of 2,4,6-trinitrobenzenesulphonic acid used, with values ranging from 3.52, at 0.10 mM modifying agent, to 8.96, at 2 mM modifying agent. Rhodanese primary amino groups modification by 2,4,6-trinitrobenzenesulphonic acid is described by a summation of exponential functions of reaction time at pH values of 8.00 or higher, while at lower pH values it is described by a single exponential function of reaction time. However, the log of the first derivative, at initial reaction conditions, of the equation describing protein modification, is found to be linearly dependent on the pH of the reaction. An identical linear dependence is also found when the log of the first derivative, at the start of the reaction, of the equation describing modification-induced enzyme inactivation is plotted against the pH values of the medium used. In consequence, the fractional concentration of rhodanese modifiable amino groups essential for enzyme catalytic function is equal to unity at all reaction pH values tested. It is accordingly concluded that, when concentrations of 2,4,6-trinitrobenzenesulphonic acid in excess of protein concentration are used, all rhodanese modifiable amino groups are essential for enzyme activity. A number of approaches were used in order to establish a mechanism for the modification-induced enzyme inactivation observed. These approaches, all of which proved to be negative, include the possible modification of enzyme sulfhydryl groups, disulphide bond formation, enzyme inactivation due to sulphite released during modification, modification-induced enzyme protein polymerization, syncatalytic enzyme modification and hydrogen peroxide-mediated enzyme inactivation.     

Mannitol-specific enzyme II of the bacterial phosphotransferase system. II. Reconstitution of vectorial transphosphorylation in phospholipid vesicles

Purified mannitol Enzyme II from Escherichia coli was reconstituted in phospholipid vesicles employing the octylglucoside dilution procedure and was shown to catalyze vectorial mannitol 1-phosphate:mannitol transphosphorylation. Reconstitution of the enzyme into liposomes showed a marked dependency upon the octylglucoside concentration with an optimum at 1.2%. The reconstituted transphosphorylation activity exhibited an absolute dependence upon mannitol 1-phosphate as the phosphoryl donor, was sensitive to N-ethylmaleimide, and had a pH optimum near 6. The intravesicular radiolabeled mannitol phosphate could be released from the proteoliposomes by the addition of either 50 microM unlabeled mannitol or 0.5% sodium dodecyl sulfate. The rate of formation of intraliposomal mannitol phosphate, measured as a function of the mannitol Enzyme II concentration, showed a sigmoidal response, suggesting that at high enzyme concentrations the mannitol Enzyme II exists in an aggregated or oligomeric state and that this form is more active than the monomeric or dissociated form of the enzyme in catalyzing the vectorial mannitol transphosphorylation reaction.     

Interactions of inhibitors at the coenzyme binding site of 6-phosphogluconate dehydrogenase.

Nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) was purified 200-fold from chicken erythrocyte nuclei. An important feature of the purification procedure is the preliminary preparation of chromatin and extraction of the enzyme from insoluble chromatin into 0.3 m NaCl. Active enzyme in a partially purified preparation has an isoelectric point of 5.5 and a molecular weight of approximately 300,000. The most highly purified enzyme migrates as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a broad pH optimum from 6–9 and is most active at 55 °C. The activation energy for the enzyme-catalyzed reaction is 8.0 kcal/mol. The purified enzyme is able to use either nicotinate mononucleotide or nicotinamide mononucleotide as a substrate. The isolation procedure is applicable to partial purification of enzyme activity from erythrocytes of closely related birds, including pheasant, goose, and turkey. Immunochemical studies of the enzyme are reported in an accompanying article.     

Immobilized α-chymotrypsin: Pore diffusion control owing to pH gradients in the catalyst particles

The fast enzymatic hydrolysis of D ,L -phenylalanine methylester (DLE) to L -phenylalanine (LA) and D -phenylalanine methylester (DE) with immobilized α-chymotrypsin was chosen as a model reaction. Under the experimental conditions applied in the present investigations the pore diffusion is the rate-limiting step of this reaction owing to the pH gradient in the particles. The effectiveness of the catalyst is experimentally determined as a function of the substrate concentration based on measurements of the enzyme protein content of native and immobilized enzyme. The proteolytic reaction is theoretically treated by also using a pore diffusion model which takes into account the concentration gradients of substrate and product, pH- and enzyme activity profiles, as well as the change of buffer capacity of the solute in the catalyst particles. The model parameters were experimentally determined for the investigated system. It can be shown that conditions are possible for which the effectiveness of the catalyst exceeds unity.     

Deoxyribonucleic acid nucleotidyltransferase from Landschütz ascites-tumour cells. Partial purification and general properties

1. A purification procedure for DNA nucleotidyltransferase from Landschütz ascites-tumour cells is described. The enzyme can be separated from endogenous nucleic acid and from triphosphatase and deoxyribonuclease activities measurable at pH7.5. 2. The basic properties of the nucleotidyltransferase reaction are as follows. The enzyme has optimum activity at pH7.2-7.4. It displays an absolute requirement for DNA-primer, thermally-denatured DNA serving three to ten times as efficiently in this respect as native DNA. Maximum synthesis of polydeoxyribonucleotide occurs in the presence of all four deoxyribonucleoside 5'-triphosphates, but a limited incorporation of mononucleotide into polynucleotide is observed when the system is provided with only one triphosphate, or with various combinations of mono-, di- and tri-phosphates. The reaction requires the presence of a bivalent cation, and of those tested, Mg(2+) ions were by far the most effective. Manganous ions promoted synthesis but to a much smaller extent. Calcium ions did not support synthesis at all. At the appropriate concentrations, the univalent cations (sodium and potassium) stimulated the reaction by 25% and 125% respectively. The presence of EDTA in the reaction mixture stimulates the system five- to ten-fold. 3. The storage characteristics of the enzyme (as well as the activities of the various fractions) improve markedly if EDTA and 2-mercaptoethanol are included in the enzyme solution and in all preparative buffer solutions. 4. The enzyme loses more than 95% of its activity after heating for 1min. at 45 degrees . If the heating is conducted in the presence of DNA, the enzyme becomes relatively heat-resistant (presumably as a consequence of complex-formation with the DNA) and may actually display an activation effect. This is discussed in relation to a possible molecular conformation of the enzyme. 5. The product of the nucleotidyltransferase reaction is precipitable by acid or ethanol, and is susceptible to the actions of deoxyribonucleases I and II, snake-venom and spleen phosphodiesterases, and micrococcal nuclease. It forms a band in a density gradient of caesium chloride at a density similar to that of the DNA-primer. 6. By the criteria of nearest-neighbour frequency analyses, the product of the nucleotidyltransferase reaction has the characteristics to be expected of a polynucleotide synthesized in accordance with the template directions of the primer.     

Determination of inhibition constants, I50 values and the type of inhibition for enzyme-catalyzed reactions

A procedure is proposed for determining whether an inhibitor of an enzyme-catalyzed reaction is competitive, noncompetitive, or uncompetitive with respect to the substrate. The method is based on fitting the equation for noncompetitive inhibition to data obtained by measuring the rate of the reaction over a range of substrate and inhibitor concentrations. The results of this fit may suggest that the inhibition may be either competitive or uncompetitive, whereupon the data are reanalyzed using the appropriate equation. Comparison of this second fit with the first using an F test permits a statistical decision to be made on the type of inhibition. The chosen fit yields values and standard errors for the Michaelis-Menten parameters (maximum velocity and Michaelis constant), as well as the inhibition constant(s). From these values it is then possible to predict the I50, and its standard error, at any chosen substrate concentration, thereby facilitating comparison with results obtained with similar inhibitors, for homologous enzymes, or in other laboratories.     

Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity

These studies report the development of a simple, specific, and highly sensitive fluorometric assay for rat liver peroxisomal fatty acyl-CoA oxidase activity. In this in vitro procedure fatty acyl-CoA-dependent H2O2 production was coupled in a peroxidase-catalyzed reaction to the oxidation of scopoletin (6-methoxy-7-hydroxycoumarin), a highly fluorescent compound, to a nonfluorescent product. Enzyme-catalyzed reaction rates as low as 5 pmol of H2O2 produced per minute could readily be detected. The reaction was studied in liver homogenates from normal rats with respect to absolute activity, time course, protein concentration dependence, substrate concentration dependence, pH optimum, substrate specificity, and cofactor requirements. The properties of the enzyme activity as assessed by the fluorometric assay agree well with those determined by other investigators using other assay methods. After subcellular fractionation of liver homogenates by differential centrifugation, the fatty acyl-CoA oxidase activity distributed like known peroxisomal marker enzymes. These results demonstrate that the fluorometric assay of fatty acyl-CoA oxidase should be useful in studying the distribution, properties, and subcellular localization of the enzyme, particularly in enzyme sources of low activity or in situations when only small amounts of material are available.     

Screening of Commercial Enzymes for Transfructosylation of Tyrosol: Effect of Process Conditions and Reaction Network

Enzymatic fructosylation of organic acceptors other than saccharides brings new possibilities to synthesize molecules that do not exist in nature. The introduction of fructosyl moiety may lead to glycosides possessing enhanced physicochemical and bioactive properties which could be useful in the pharmaceutical and cosmetic industry. In this work, the regioselective synthesis of tyrosol β‐d ‐fructofuranoside (TF) catalyzed by β‐fructofuranosidase is investigated. In the first step, 32 commercial enzyme preparations are screened for fructoside‐hydrolyzing activity. The most active preparations are subsequently examined for fructofuranosyl transfer from sucrose to tyrosol. The best candidate, Novozym 188, is chosen to study the effect of reaction conditions on the product formation in a batch reactor. The effects of substrate concentration, temperature, pH, time, and enzyme dosage on the concentration of TF produced are studied using the design of experiments methodology. The maximal product concentration of 3.8 g L^?1 is achieved for the sucrose concentration of 1.5 m , tyrosol concentration of 29 g L^?1, temperature of 41 °C, and pH 5.1. Besides the main transfructosylation reaction between sucrose and tyrosol, several side reactions take place. A reaction network includes also the formation of fructooligosaccharides and the hydrolysis of sucrose and all reaction products.     

Some properties of a ficin-papain inhibitor from avian egg white

A procedure has been established for the isolation, from sheep liver, of 6-phosphogluconate dehydrogenase which is homogeneous according to the criteria of the analytical ultracentrifuge, and isoelectric focusing. A systematic determination of the effects of pH, ionic strength, metal ions, and temperature, on the kinetic parameters of the isolated 6-phosphogluconate dehydrogenase has been carried out. Double-reciprocal plots of enzyme rate measurements as a function of substrate concentration indicate K_m values of 15 μm for 6-phosphogluconate, and 7 μm for NADP⁺, under optimum assay conditions, and show no effect of the concentration of one substrate on the K_m of the other substrate under the assay conditions employed. Ionic strength, monovalent and divalent metals, are apparently interchangeable in their ability to activate the enzyme, and act by decreasing the K_m values of the enzyme, not by increasing the reaction rate. Concentrations of metals above the optimum are strongly inhibitory. Plots of ?log K_m vs pH show inflection points at 8.3 for 6-phosphogluconate, and 6.5 for NADP⁺. At low substrate concentrations the pH optimum of the enzyme is at pH 7.7, but plots of V vs pH increase up to pH 9.1 (the enzyme is unstable at higher pH values). An Arrhenius plot shows a straight line between temperatures of 8.6 and 39.4 °C, and an energy of activation of 15,450 cal mole^?1.     

THE INFLUENCE OF HYDROGEN ION CONCENTRATION ON THE INACTIVATION OF PEPSIN SOLUTIONS

1. Pepsin in solution at 38°C. is most stable at a hydrogen ion concentration of about 10^–5 (pH 5.0). 2. Increasing the hydrogen ion concentration above pH 5.0 causes a slow increase in the rate of destruction of pepsin. 3. Decreasing the hydrogen ion concentration below pH 5.0 causes a very rapid increase in the rate of destruction of the enzyme. 4. Neither the purity of the enzyme solution nor the anion of the acid used has any marked effect on the rate of destruction or on the zone of hydrogen ion concentration in which the enzyme is most stable. 5. The existence of an optimum range of hydrogen ion concentration for the digestion of proteins by pepsin cannot be explained by the destruction of the enzyme by either too weak or too strong acid.     

Potentiometric and colorimetric methods for the assay of 2',3'-cyclic nucleotide 3'-phosphohydrolase

Abstract— A potentiometric titration method for the assay of 2′,3′-cyclic nucleotide 3′-phosphohydrolase is presented. Progress curves of the reaction were recorded automatically by pH-stat. 2-Mercaptoethanol was added to the reaction mixture to maintain a linear rate of reaction. The method is suitable for obtaining kinetic parameters and can be used for the rapid assay of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in nervous tissues. An improved colorimetric method for estimation of 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity at the optimum pH is described. This method employs the two-step procedure in which decyclization by 2′,3′-cyclic nucleotide 3′-phosphohydrolase and dephosphorylation by Escherichia coli alkaline phosphatase (EC 3.1.3.1) are carried out separately under the optimum conditions for each enzyme. The method is sensitive and most convenient for routine assays.     

Polyvinylferrocenium modified Pt electrode for anaerobic glucose monitoring

An amperometric enzyme electrode for the determination of glucose under anaerobic solution conditions was developed by immobilizing glucose oxidase and then by adsorbing ferrocene in polyvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of ferrocene that the reduced flavin adenine dinucleotide centers of glucose oxidase was measured at a constant potential. The response characteristics of the enzyme electrode were investigated. The effects of the thickness of the polymeric film, the amount of the enzyme immobilized, the amount of the mediator, the glucose concentration, the applied potential, operating pH and temperature on the response of the enzyme electrode were studied. The response time and the optimum pH were found to be 30-40 s and pH 7.4 at 25 degrees C, respectively. The linear response was observed up to 5.0 mM glucose concentration that the produced detectable current was 0.0075 mM glucose concentration. The activation energy (E(a)) of immobilized enzyme reaction was calculated to be 41.3 kJ mol(-1) from the Arrhenius plot. The apparent Michaelis-Menten constant (K(Mapp)) was found to be 6.05 mM glucose according to the Lineweaver-Burk graph of the Michaelis-Menten equation under the optimum conditions. The interference signal due to the most common electrochemical interfering species was also evaluated.