Function Of Enzyme Concentration Research Articles

RNase III cleaves vesicular stomatitis virus genome-length RNAs but fails to cleave viral mRNA's

The procaryotic RNA processing enzyme RNase III (endoribonuclease III [EC 3.1.4.24]) was used to probe vesicular stomatitis virus (VSV) RNAs for specific sites that could be recognized and cleaved. The effect of the enzyme on the RNAs was monitored by measuring their subsequent migration in denaturing agarose-urea gels. VSV virion RNA (negative strand; Mr, 4 X 10(6)) was cleaved by the enzyme to yield a set of discrete fragments which ranged on size from 3.5 X 10(6) to 0.2 X 10(6) daltons. The cleavage was a function of enzyme concentration, salt concentration, and time. A maximum of 20 to 22 fragments was generated under conditions of low enzyme concentration or short times of incubation. VSV genome-length intracellular RNA of both + and - polarity was also cleaved by RNase III. In contrast to the findings with virion-length RNA, however, the migration rates of VSV mRNA's purified by chromatography on polyuridylic acid-Sepharose were unaffected by treatment with RNase III. These results show that specific sites in the virion RNA and its full-length complement can be recognized by RNase III. Sites of this type are not present in the polyadenylic acid-containing mRNA, however.

Determination of binding site concentration and average binding constant by the method of equilibrium sorption: Evidence for lysine as a principal receptor site for collagen-enzyme binding

The contribution of lysyl ϵ-amino groups to the noncovalent binding on enzymes to collagen has been determined by the method of equilibrium sorption. The sorption or binding of enzyme by chemically modified, and by the corresponding unmodified membraneous collagen, was determined as a function of enzyme concentration. Chemical modification of lysyl ϵ-amino groups was accomplished by carbamylation of the collagen membrane with potassium cyanate. The collagen membranes were contacted with a purified β-galactosidase (E. coli K 12). The enzyme was purified by affinity chromato- graphy. Analysis of the resultant sorption isotherms allowed determination of the heterogeneity index (a) and the concentration of active binding sites (A o). Binding constants (K o) and the free energy of binding (δG) were also computed from the sorption data. The results of these studies have shown that lysyl ϵ-amino groups of collagen are primary receptor sites for enzyme binding (β-galactosidase, E. coli K 12) and that carbamylation of the collagen membrane resulted in a decrease in active binding sites with little or no change in the binding constant of the remaining sites.

A nitrilotriacetic acid monooxygenase with conditional NADH-oxidase activity

A tertiary amine monoxygenase from a Pseudomonas sp. was partially purified (35-fold) and characterized. In the presence of nitrilotriacetate (NTA), O 2, NADH, and Mn 2+, the enzyme yielded two sets of products: iminodiacetate, glyoxylate, NAD + and H 2O; or H 2O 2 and NAD +. Which set of products predominated was a function of enzyme concentration, ionic strength of solution, pH, and cation supplied. NTA functioned both as a modifiable substrate and as a stimulator of NADH oxidase activity. A requirement for preincubation with Mn 2+ and NTA to eliminate enzyme hysteresis and the similar Km values for NTA and Mn 2+ suggested that the substrate and metal were bound as a unit by the enzyme.

Cyclic nucleotide phosphodiesterase in heart and aorta of spontaneously hypertensive rats

Cyclic AMP and cyclic GMP phosphodiesterase (3′:5′-cyclic-AMP 5′-nucleotidohydrolase, EC 3.1.4.17) activities were compared in soluble ( 105 000 × g supernatant ) and particulate ( 105 000 × g pellet of 700 × g supernatant ) fractions of heart and aorta of hypertensive and normotensive rats at 5 μM and 100 μM substrate concentrations. Cyclic AMP phosphodiesterase activity was significantly higher ( P < 0.05) in particular fractions of hypertensive rat aorta at 100 μM, while no significant changes were observed in heart. Cyclic GMP phosphodiesterase activity was significantly increased ( P < 0.05) in particulate and soluble fractions of hypertensive rat heart compared to normotensive rat at both substrate concentrations, but there were no differences in aorta. When phosphodiesterase activities in the soluble fractions of aorta and heart were examined as a function of enzyme concentration, rectilinear rates were observed with micromolar, but not with millimolar concentrations of cyclic AMP or cyclic GMP. Double reciprocal plots for cyclic AMP phosphodiesterase activity were non-linear in heart and aorta supernatants. Similarly, cyclic GMP phosphodiesterase yielded non-linear kinetics in heart supernatant, however, the plots were linear for aorta supernatant. Particulate fractions of heart showed linear kinetics for cyclic AMP and cyclic GMP phosphodiesterase and the K m values were in the range of high K m for soluble enzymes. V in hypertensive rats was approximately doubled compared to normotensive rats for cyclic AMP and cyclic GMP phosphodiesterase. These data suggest that altered phosphodiesterase activity could result in a change in cyclic nucleotide index in the cardiovascular tissues of hypertensive rat.

The effects of enzyme concentration on the kinetic behavior of adrenal 21-hydroxylase and 11β-hydroxylase in the pregnant rat

Experiments were designed to study the kinetic behavior of 21-hydroxylase and 11β-hydroxylase as a function of enzyme concentration (E t) during proestrus, days 5 (D5), 12 (D12), and 22 (D22) of pregnancy, and within 24 h post-partum. The enzymes were prepared from rat adrenal microsomes and mitochondria, respectively. The experiments consisted of measuring the initial velocity of each reaction for a series of substrate concentrations at three fixed E t. Double reciprocal plots were constructed and the slope (Km/Vmax) of each line estimated. Variation in the value of the slope as a function of enzyme dilution would predict the presence of an endogenous effector. The kinetic behavior of 21-hydroxylase was not altered throughout the range of E t (10–100 μg protein) at any of the reproductive stages. In contrast, kinetic behavior of 11β-hydroxylase was clearly dependent upon E t. Dilution of the enzyme preparation (25–200 μg of protein) increased the slope of the double reciprocal plot at all reproductive stages, thus suggesting that an activator substance may be present within the mitochondrial preparation. A secondary plot of the slope (Km/Vmax) versus E t described a power function (Km/Vmax = a[E t] b) with the greatest rate of change in Km/Vmax occurring at low values of E t. The rate of change in Km/Vmax per mg rise in mitochondrial protein at all dilutions of enzyme was greatest for proestrus and post-partum, followed by D22>D12>D5. In addition, repeated washing of the enzyme preparation at 4°C increased Km/Vmax to a greater extent at all E t than did the control preparation. These findings suggest the presence of a diffusible endogenous activator of 11β-hydroxylase whose influence decreases markedly at D5 and D12. On the other hand, there is no evidence to suggest the presence of a diffusible endogenous effector for 21-hydroxylase.

Spin-label assay for lysozyme

A spin-label assay for lysozyme, which is based on the enzymatic hydrolysis of spin-labeled peptidoglycan, is described. Hydrolysis of this polymer by lysozyme results in sharpening of the esr spectrum. The rate of spectral sharpening is a function of enzyme concentration. When the activities of hen egg-white and human lysozymes are compared by this method, human lysozyme is 3.5 times as active as the hen enzyme. The pH optima for both enzymes are pH 5.0. At this pH, the maximal activity for the hen egg-white lysozyme is observed at an ionic strength of 0.09. This assay is suitable for measuring lysozyme levels in biological fluids. It is a sensitive, continuous assay that measures muramidase activity on a defined substrate.

Malate dehydrogenases. The lack of evidence for dissociation of the dimeric enzyme in kinetic analyses.

The kinetic parameters of beef heart cytoplasmic and pig heart mitochondrial malate dehydrogenases have been examined over a wide range of enzyme concentration. No significant changes are observed in these properties. In conjunction with active enzyme sedimentation and sedimentation equilibrium experiments, it is concluded that there is no evidence for dissociation of the dimeric enzyme at any enyzme level in the kinetic analyses. Thus, if dissociation occurs, it must be too slow to be of significance in determining the kinetic properties of the enzyme. It is shown that unless a subunit and its dimeric form have identical kinetic and substrate binding characteristics, the kinetic parameters should change as a function of enzyme concentration.

Acid phosphatase characterization: An undergraduate biochemistry laboratory experiment

By systematically varying the general assaying procedure, students can observe reaction velocity as a function of enzyme concentration, reaction velocity as a function of pH, enzyme activity as a function of temperature, and reaction velocity as a function of substrate concentration.

DNA-relaxing enzyme from Micrococcus luteus

A DNA-relaxing enzyme which catalyzes the conversion of superhelical DNA to a non-superhelical covalently closed form has been purified from Micrococcus luteus to near homogeneity by two chromatographic steps. The enzyme is a single polypeptide chain. As determined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and gel filtration on Sephadex G 150, the molecular weight is 115,000. The DNA-relaxing activity determined as a function of enzyme concentration follows a sigmoidal curve. The enzyme requires Mg++ for activity. In the presence of 4.5 mM Mg++ addition of 50-250 mM KCl yields incompletely relaxed DNA molecules (intermediates); intermediates are also observed in the absence of KCl, when the reaction is carried out at 0 degree C or at Mg++ concentrations exceeding 10 mM.

An ESR Method for Determination of Serum Lipase Activity

Abstract An ESR method for determination of serum lipase is described in principle and demonstrated in practice. A lipid soluble nitroxide is dissolved in an olive oil emulsion. When the intensity of the ESR signal of the nitroxide in the aqueous phase is monitored as a function of time after an aliquot of lipase is added a linear plot of peak height as a function of enzyme concentration is obtained.

Cyclid 3':5'-nucleotide phosphodiesterase. Interconvertible multiple forms and their effects on enzyme activity and kinetics.

An extract of rat liver or human platelet displayed three cyclic 3':5'-nucleotide phosphodiesterase activity peaks (I, II, and III) in a continuous sucrose density gradient when assayed with millimolar adenosine 3':5'-monophosphate (cAMP) or guanosine 3':5'-monophosphate (cGMP). The three fractions obtained from each nucleotide were not superimposable. The molecular weights corresponding to the three activity peaks of cAMP phosphodiesterase in rat liver were approximately: I, 22,000; II, 75,000; and III, 140,000. In both tissues, fraction I was barely detectable when assayed with micromolar concentrations of either nucleotide, presumably because fraction I has low affinity for cAMP and cGMP. Any one of the three forms upon recentrifugation on the gradient generated the others, indicating that they were interconvertible. The multiple forms appear to represent different aggregated states of the enzyme. The ratio of the three forms of cAMP phosphodiesterase in the platelet was shifted by dibutyryl cAMP (B2cAMP) and by the enzyme concentration. B2cAMP enhanced the formation of fraction I. Low enzyme concentration favored the equilibrium towards fraction I, while high enzyme concentration favored fraction III. When phosphodiesterase activities in the extract of rat liver, human platelets, or bovine brain were examined as a function of enzyme concentration, rectilinear rates were observed with micromolar, but not with millimolar cAMP or cGMP. The specific activity with millimolar cAMP was higher with low than with high protein concentrations, suggesting that the dissociated form catalyzed the hydrolysis of cAMP faster than that of the associated form. In contrast, the specific activity with millimolar cGMP was lower with low than with high protein concentrations. Supplementing the reaction mixture with bovine serum albumin to a final constant protein concentration did not affect the activity, suggesting that the concentration of the enzyme rather than that of extraneous proteins affected the enzyme activity. A change in enzyme concentration affected the kinetic properties of phosphodiesterase. A low enzyme concentration of cAMP phosphodiesterase yielded a linear Lineweaver-Burk plot, and a Km of 1.2 X 10(-4) M (bovine), 3 X 10(-5) M (platelet), or 5 X 10(-4) M (liver), while a high enzyme concentration yielded a nonlinear plot, and apparent Km values of 1.4 X 10(-4) M and 2 X 10(-5) M (brain), 4 X 10(-5) M and 3 X 10(-6) M (platelet), or 4 X 10(-5) M and 3 X 10(-6) (liver). Since a low enzyme concentration favored fraction I, the dissociated form, whereas a high enzyme concentration favored fraction III, the associated form, these kinetic constants suggest that the dissociated form exhibits a high Km and the associated form exhibits a low Km. In contrast, a high enzyme concentration gave a linear kinetic plot for cGMP phosphodiesterase, while a low enzyme concentration gave a nonlinear plot...

Synthesis of S-alkyl-L-cysteine from pyruvate, ammonia and alkyl-mercaptan by cysteine desulfhydrase of Aerobacter aerogenes

Summary Synthesis of S-methyl-L-cysteine from pyruvate, ammonia and methylmercaptan is catalyzed by highly purified cysteine desulfhydrase from Aerobacter aerogenes . The synthetic reaction proceeds optimally at pH 10, as a function of enzyme concentration and incubation time. Methylmercaptan is replaced by ethylmercaptan and propylmercaptan to synthesize S-ethyl-L-cysteine and S-propyl-L-cysteine, respectively. The enzymatically synthesized S-methyl-L-cysteine and S-ethyl-L-cysteine were purified and identified by physicochemical meanings.

Unusual kinetic behavior of Endothia parasitica protease in hydrolysis of small peptides

Endothia parasitica protease hydrolyzes l-leucyl- l-leucine amide and l-leucyl- l-phenylalanine amide at the peptide bond. l-Phenylalanyl- l-leticine amide, N-carbobenzoxy- l-leucyl- l-phenylalanine amide, N-carbobenzoxy- l-leucyl- l-pheml-alanine, N-carbobenzoxy- l-phenylalanyl- l-valine amide, and l-leucyl-β-naphthyl-amide are not hydrolyzed. In contrast to the kinetics of hydrolysis of casein and oxidized B-chain of insulin and activation of trypsinogen by Endothia parasitica protease which are normal, reaction progress curves for hydrolysis of l-leucyl- l-leucine amide and l-leucyl- l-phenylalanine amide are sigrnoidal. Initially, the reaction rates were of the order of 0.5–2.5% of the maximum rates eventually attained. With increasing time of incubation the reaction rates became faster and faster until maximum rates were achieved. This abnormal behavior was not eliminated by recrystallization of substrate or by incubation of enzyme alone or with products of the reaction prior to addition of substrate. Addition of a new aliquot of substrate, viz l-leucyl- l-leucine amide, to the reaction prior to complete hydrolysis of all of a previous aliquot of the same substrate, or reactions containing a mixture of oxidized B chain of insulin and l-leucyl- l-leucine amide, gave normal reaction progress curves. The duration of abnormal behavior before a maximum rate was attained was a function of enzyme concentration and temperature but not of substrate concentration even though substrate was in less than saturating amounts. The reaction data follow second-order autocatalytic kinetics with respect to enzyme concentration. It is proposed that most of the enzyme is in an inactive form in absence of substrate but is rapidly converted to the active form on combination with a good substrate such as trypsinogen, casein, or oxidized B chain of insulin. However, with a poor substrate such as l-leucyl- l-leucine amide, conversion to active enzyme is mediated through formation of an active enzyme-inactive enzyme complex followed by combination with substrate and hydrolysis.

Validation of a “clearing” assay for milk lipoprotein lipase in agarose gel

We have developed a simplified method for the quantitative assay of lipoprotein lipase in cow's milk based on the hydrolysis of a glyceride emulsion in semisolid agarose gel. The area of clearing produced thereby is a function of enzyme concentration. Absolute molar rates for unknown samples may be calculated if standards of known activity are used concurrently. The precision of the simplified assay compared favorably with a method based on titrimetric determination of the rate of free fatty acid release. A modified assay has been used to assess the potency of lipoproteins in lipoprotein lipase activation.

Lysophospholipases of Rat Brain

Abstract 1. The properties of rat brain lysophospholipase were investigated. Three subcellular fractions were employed: a particulate preparation which sedimented at 26,000 x g, a microsomal preparation which sedimented at 100,000 x g, and the supernatant of the above. The soluble enzyme represented only a small fraction of the total activity; it was further purified by treatment with protamine and ammonium sulfate and by gel filtration through Sephadex G-50. Attempts to solubilize the particulate or microsomal enzymes yielded only 1% or less of their activity as a soluble enzyme. 2. When reaction rates were plotted as a function of enzyme concentration, straight lines were obtained with the soluble enzyme. With the particulate or microsomal enzymes, these curves were straight lines only at lysolecithin concentrations below 0.05 to 0.1 mm. Above these concentrations, the curves were parabolic. 3. When reaction rates were plotted as a function of substrate concentration, the curves were biphasic. Assymmetrical bell-shaped curves were obtained with the particulate or microsomal enzymes. When using the soluble enzyme, the curve ascended as a hyperbola, but reached a maximal value above which the reaction rates were constant. With the particulate or microsomal enzymes the concentration of substrate at which the maximal activity was obtained increased with increasing protein concentrations. 4. Albumin increased the reaction rates catalyzed by the particulate or microsomal enzymes at all substrate concentrations. It changed the parabolic V/E curves into straight lines and the biphasic V/S curves into rectangular hyperbolas. When using the soluble enzyme, low concentrations of albumin had no effect, while higher concentrations decreased the reaction rates. 5. Lysolecithin was adsorbed onto the particulate or microsomal enzymes; this was counteracted by serum albumin. 6. These data are discussed in light of the hypothesis that rat brain lysophospholipase utilizes monomers but probably not micelles of the substrate.

Synthesis of L-tyrosine from pyruvate, ammonia and phenol by crystalline tyrosine phenol lyase

Synthesis of L -tyrosine from pyruvate, ammonia and phenol is catalyzed by crystalline tyrosine phenol lyase prepared from cells of Escherichia intermedia . The synthetic reaction proceeds optimally at pH 8.5–9.0, as a function of enzyme concentration and incubation time. The Km values for pyruvate and phenol are determined to be 5.1 × 10 −3 M and 4.4 × 10 −3 M, respectively, and the maximal velocity was 7.2 μmoles /min./mg of protein. Phenol is replaced by pyrocatechol and resorcinol to synthesize 3,4-dihydroxyphenyl- L -alanine and 2,4-dihydroxyphenyl- L -alanine, respectively. Addition of pyruvate, ammonia and phenol to holotyrosine phenol lyase results in the appearance of a new spectral band near 500 mμ which has been ascribed to the intermediates in many pyridoxal phosphate dependent reactions.

The Subunit Interactions of Fumarase

Abstract The subunit interactions of fumarase have been examined as a function of enzyme concentration, pH, and concentration of guanidine hydrochloride. Fumarase was found to dissociate, with loss of activity, either at low concentrations of the enzyme (l1 x 10-4 mg per ml), below pH 6, above pH 10, or in solutions of guanidine hydrochloride (g1 m). At pH 9 a species of enzyme was produced which was fully associated, but whose hydrodynamic structure was different from that of the native enzyme. Fumarase dissociated in dilute acid or alkali or by guanidine hydrochloride could be reassociated with nearly complete recovery of activity. The reassociation of the acid-dissociated fumarase was observed to follow first order kinetics with respect to enzyme concentration. l-Malate, citrate, phosphate, and ATP all reduced the rate of dissociation and increased the extent of recombination of the subunits. As judged by optical rotatory dispersion measurements, little change in polypeptide chain conformation was noted between native fumarase and the enzyme dissociated between pH 5 and 11. The thiol groups of the dissociated enzyme, however, were found to be more exposed to solvent than the same groups located in the tetrameric molecule.

The effect of 2,2-diphenyl-1-picrylhydrazyl and p-cresol on the oxidative degradation of indole-3-acetate

A stable free radical compound, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and an oxidogenic substrate of peroxidase, p-cresol, both markedly stimulate the catalytic activity of 10 −8 m horseradish peroxidase in the oxidation of indole-3-acetate. However, at higher enzyme concentrations (10 −5 m), the oxidative degradation of the plant growth regulator is inhibited or prevented by these same compounds. The new finding that the overall effect of these agents is a function of enzyme concentration is interpreted in terms of the concentration-dependent reactivity of a reduced, oxygenated enzyme intermediate, compound III, in a second reaction with the promoting agents. Under some conditions the essential intermediate is destroyed rapidly in a second reaction with p-cresol or DPPH. However, formation of the enzyme compound is apparently accelerated at both high and low enzyme concentrations. IAA reacts directly with peroxidase and oxygen in an initial rapid reaction to cause accumulation of a mixture of reduced enzyme intermediates which exhibit absorbauce maxima near 418 and 430 mμ. However, oxygen is consumed in a second reaction which is presumed to be catalyzed by compound III (oxyferroperoxidase) which contains the bound superoxide anion. At low enzyme concentrations the first reaction is rate-limiting since it appears that compound III must accumulate before the oxygen-consuming reactions may proceed. Under these conditions, DPPH and p-cresol stimulate the initial process and the overall reaction. At higher enzyme levels the essential intermediate is formed more rapidly but is removed by the promoting agents leading to overall inhibition of IAA degradation.

Lecithin-agar assay for lecithinase antibodies in serum.

A technique for assay of lecithinase antibodies in serum was developed in this laboratory by using a lecithin-agar plate diffusion procedure based on a combination of described plate assays. Egg yolk lipoprotein composed primarily of lecithin was used as a substrate for reaction with free or non-neutralized lecithinase C after incubation of known amounts of lecithinase C with various dilutions of control and test sera. It was found that the size of the reaction zone was a function of enzyme concentration and inversely proportional to the antibody concentration. Accuracy and precision of the assay were determined. In addition, lecithinase antibody levels in sera from experimentally inoculated rats and rabbits and sera from randomly selected human patients were studied.

Lecithin-Agar Assay for Lecithinase Antibodies in Serum

A technique for assay of lecithinase antibodies in serum was developed in this laboratory by using a lecithin-agar plate diffusion procedure based on a combination of described plate assays. Egg yolk lipoprotein composed primarily of lecithin was used as a substrate for reaction with free or non-neutralized lecithinase C after incubation of known amounts of lecithinase C with various dilutions of control and test sera. It was found that the size of the reaction zone was a function of enzyme concentration and inversely proportional to the antibody concentration. Accuracy and precision of the assay were determined. In addition, lecithinase antibody levels in sera from experimentally inoculated rats and rabbits and sera from randomly selected human patients were studied.