Shift In pH Optimum Research Articles

Poly(methoxygalacturonide) lyase immobilized via titanium onto solid supports

AbstractPoly(methoxygalacturonide) lyase (PMGL) (E.C. 4.2.2.10) was purified from a commercial preparation and immobilized by the metal link method. The properties of DEAE‐cellulose‐Ti‐PMGL and of porous glass‐Ti‐PMGL were compared with those of the native enzyme; despite the presence of the metal and the heterogeneity of the substrate, pectin, typical substrate–enzyme‐support interactions were demonstrated by shifts in pH optima and KM values. The possible industrial application of DEAE‐cellulose‐Ti‐PMGL is discussed.

Effects of divalent cations and sodium taurocholate on pancreatic lipase activity with gum arabic-emulsified tributyrylglycerol substrates

The effects of Ca 2+ and/or sodium taurocholate on lipase activity with gum arabic-emulsified tributyrylglycerol substrates were investigated. Calcium was found to slightly increase lipase activity while bile salts showed marked inhibition except at very low concentrations. Calcium eliminated inhibition seen with low concentrations of bile salts and reduced the inhibition seen at higher bile salt concentrations. Calcium was also effective in eliminating the optimal pH shift of the enzyme from the alkaline region in the absence of bile salt to the slightly acidic region in the presence of bile salt. Calcium was shown to eliminate the time lag periods between enzyme addition and maximum rate of hydrolysis seen at low substrate concentrations and the time lag noted when bile salts were included with normal (substrate concentration not limiting) assay concentrations of substrate. Zeta potential measurements indicated that Ca 2+ reduced the negative charge on the gum arabic-emulsified particle while bile salts did not increase the negative charge. Commercial preparations of gum arabic were found to have significant concentrations of Ca 2+ and Mg 2+.

Further studies on the heterogeneity ofarylsulphatase from Proteus rettgeri and some properties of one chromatographic form of the enzyme

1. 1. Arylsulphatase in Proteus rettgeri was separated into four fractions by DEAE-cellulose chromatography. 2. 2. Fractions 2, 3 and 4 were shown by polyacrylamide-gel electrophoresis and/or sucrose density gradient centrifugation to contain more than one form of the enzyme. Some forms differed in substrate specificity. 3. 3. Arylsulphatase present in fraction 1 was previously shown to be homogeneous by paper electrophoresis and some properties of this enzyme have been determined. These include a substratedependent activation by PO 4 3− and a shift in optimum pH in the presence of CN −.

The hepatic adenylate cyclase system. II. Substrate binding and utilization and the effects of magnesium ion and pH

The kinetic characteristics of substrate utilization by hepatic adenylate cyclase were investigated under a variety of incubation conditions, including veriations in pH, [substrate], [Mg2+], and in the absence or presence of glucagon. Activities were compared with ATP and 5'-adenylylimidodiphosphate (App(NH)p) as substrates. The Km for both substrates was about 50 muM; Vmax given with App(NH)p was about 40% lower than obtained with ATP as substrate. In the presence of a saturating concentration of substrate (1 mM), basal activity was increased 4-fold by increasing [Mg2+] from 5 to 50 mM. The stimulatory effect of Mg2+ was not due to an allosteric action since basal activity was only marginally enhanced (40%) when the substrate concentration was reduced to 10 muM. As suggested by deHaen ((1974 J. Biol. Chem. 249, 2756), it is likely that Mg2+ increases enzyme activity by decreasing the concentration of an inhibitory, unchelated form of substrate that competes with the productive magnesium-substrate complex at the active site. Activity-pH profiles differed with ATP and App(NH)p as substrates; a shift in pH optimum was observed which correlated with the different pKa of the terminal phosphate groups of ATP and App(nh)p, and which reflect the concentration of protonated substrate (ATPH-3 minus) present in the incubation medium. Accordingly, protonated substrate is the predominant inhibitory species of unchelated substrate and probably has a considerably higher affinity for the active site than does the magnesium-substrate complex. Glucagon-stimulated activity was less susceptible to inhibition by protonated substrate than is the basal state as evidenced by lower stimulatory effect when the [Mg2+] was increased from 5 to 20 mM. However, increasing the [Mg2+] from 20 to 50 mM resulted in marked inhibition of glucagon-stimulated activity, particularly in the presence of 10 muM substrate. Conversely, at a fixed [Mg2+], concentrations of substrate at least 20-fold higher than the Km were required to achieve maximal hormone-stimulated activity. These findings suggest that the unchelated, fully ionized form of substrate serves as an activating ligand, as has been observed with guanine nucleotides at considerably lower concentrations. Thus, Mg2+ affects adenylate cyclase activity by forming the productive substrate complex and by titrating the inhibitory protonated and activating free forms of substrate. As a result of these effects of unchelated substrate, it proved difficult to evaluate the kinetic parameters involved in substrate binding and utilization and the effects of hormone thereon when substrate was added as the only source of activating ligand. However, linear Michaelis kinetic data were obtained by adding the activating ligand 5'-guanylylimidodiphosphate with glucagon and by making appropriate adjustments of pH and [Mg2+]. Vmax was increased 4-fold without changes in Km by the actions of 5'-guanylylimidodiphosphate and glucagon.

Reconstitution of spinach ribulose-1,5-diphosphate carboxylase from separated subunits

Summary The addition of the smaller subunit (B) to the catalytic octamer of the larger subunit, A 8 , of spinach ribulose-1,5-diphosphate carboxylase caused an appreciable stimulation of the enzymic activity, accompanied with the reconstitution of the original enzyme molecule. The reformation of the native enzyme was demonstrated by its immunochemical response to the rabbit antisera raised against larger (A) and smaller (B) subunits, whereas the catalytic oligomer only cross-reacted with the anti-[A] serum. The reconstituted enzyme exhibited the Mg 2+ -dependent optimal pH-shift to a neutral side, supporting the notion concerning the regulatory role of the smaller subunit in the Mg 2+ -effect in the enzyme catalysis.

Stimulation of hepatic drug-metabolizing enzymes by DDT, polycyclic hydrocarbons or phenobarbital in adrenalectomized or castrated mice

Benzpyrene treatment in rats increases the hepatic microsomal aniline hydroxylase pH optimum from 7 to 8. In mice treated with benzpyrene, no such shift in pH optimum of aniline hydroxylase was observed. Further studies on hormonal regulation of ethylmorphine- N-demethylase, aniline and benzpyrene hydroxylase in mice indicated that adrenalectomy, castration or adrenalectomy plus castration do not lower the basal (“uninduced”) activities of these enzymes (in rat with similar operations, basal activities are lowered by 50–90% depending on the enzyme). Also the magnitude of stimulation of hepatic microsomal drug metabolizing enzymes (HMDME) by pretreatment of mice with DDT, benzpyrene, 3-MC or phenobarbital in mice without adrenals, testes or both was not less than in sham-operated control mice (as occurs in rats), but instead was somewhat higher. These findings suggest that rats and mice differ in hormonal regulations of basal activities of HMDME and in the response of HMDME in animals pretreated with inducers of HMDME.

Properties of inosinic acid dehydrogenase from Bacillus subtilis. II. Kinetic properties.

Some of the kinetic properties of inosine 5′-phosphate (IMP) dehydrogenase purified from Bacillus subtilis have been described. K+ can be replaced as an activator by other monovalent cations, whose activities are related to their ionic radii. IMP dehydrogenase is stimulated by appropriate concentrations of adenosine 5′-phosphate, and is strongly inhibited by the antibiotic mycophenolic acid. The enzyme is inhibited by guanosine 5′-phosphate (GMP), a suspected negative feedback inhibitor, in a complex manner. Desensitization of this inhibition suggests that GMP binds at a site distinct from the catalytic site. Several properties of the enzyme change after desensitization. For example, the pH optimum shifts from pH 7.55 to pH 8.4, and whereas the inhibition by GMP is greatly reduced there is an increase in catalytic activity. These studies and the oligomeric structure of IMP dehydrogenase strongly indicate a regulatory role for the enzyme in B. subtilis.

Pancreatic lipase and co-lipase. Interactions and effects of bile salts and other detergents.

The effect of conjugated bile salts on the activity of pancreatic lipase depends on their concentration in relation to their critical micellar concentration.Below the critical micellar concentration, conjugated bile salts slightly stimulate the initial rate of hydrolysis of tributyrine, an effect that may be caused by a protection of the enzyme from inactivation at the substrate‐water interface.Above the critical micellar concentration, conjugated bile salts almost completely inhibit lipase. The inhibition is more marked in alkaline reactions resulting in a pH optimum shift with increasing bile salt concentration. Bile salt inhibition of lipase is related both to the concentration of bile salt and to the substrate concentration or rather to substrate surface area, and most probably is complete when the interface is saturated with detergent.Co‐lipase, in the absence of bile salts, stimulates the activity of lipase, 1.3 to 1.4‐fold in the whole pH range of its activity. Co‐lipase overcomes the inhibition of lipase caused by bile salts with a shift in the pH optimum to 6–7 compared to 8–9 for lipase alone.The different conjugated bile salts have similar effects, consideration being taken to differences in their critical micellar concentration; free bile salts have a less inhibitory effect on lipase and the stimulation by co‐lipase shows no pH optimum shift.Detergents of the acyltaurine type such as decanoyltaurine and dodecanoylsarcosyltaurine inhibit lipase in a similar manner to the conjugated bile salts and this inhibition is also released by co‐lipase. Detergents such as dodecylsulphate above the micellar concentration, irreversibly inhibit lipase. The simultaneous presence of bile salts protects the enzyme from being irreversibly inactivated.Lipase and co‐lipase interact in a stoichiometrical relationship and it appears justified to classify co‐lipase as a co‐enzyme for lipase.

Stimulation of adenylate cyclase activity in lymph nodes of rats with graft-versus-host disease

Adenylate cyclase activity in the lymph node particles from rats undergoing graft-versus-host reaction was found to increase markedly. Such a change in the enzyme activity does not appear to be due to a shift in pH optima. Sodium fluoride failed to stimulate the adenylate cyclase activity in preparations from animals with graft-versus-host disease. These results suggest that the cellular immune process is associated with alteration in the adenylate cyclase activity.

Two different effects of mutation on the binding of folate antagonists by a microbial dihydrofolate reductase

Two types of mutant dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP + oxidoreductase, EC 1.5.1.3) with altered inhibitor binding have been identified with antifolate-resistant strains of Diplococcus pneumoniae. One class (mutant Type A) exhibits a small decrease in affinity for 4-aminofolate analogs (about 3-fold higher average K i ). The estimated K i for binding of aminopterin and amethopterin by the wild-type enzyme is somewhat less than 10 −10. There is also a small decrease in affinity for dihydrofolate (1.5–2 fold higher K m ) and an increase in affinity for NADPH (2–4-fold lower K m ). The mutations which determine this class of enzyme are distributed at 4 distinct sites within the dihydrofolate reductase structural gene. A second class (mutant Type B) of mutant enzyme shows a marked decrease in affinity for 4-aminofolate analogs (50–100-fold higher K i ) and a shift in pH optimum for dihydrofolate reduction from 7.3 to 6.7. The mutations, which determine these enzymic effects, are clustered at one region of the structural gene. Binding of folate to the wild-type enzyme was almost 10 6-fold less than that of the 4-aminofolates, aminopterin and amethopterin. Binding of folate by mutant and wild-type enzymes was not dependent on pH within the range of 5.9 to 9.5. Binding of aminopterin by the wild-type enzyme was independent of pH within the range of 5.9 to 7.4, but decreased at pH levels above and below this range. The binding by a B mutant enzyme shows a somewhat greater dependence on ph within the same range. A discussion of these results in terms of the possible mode of binding of the pyrimidine moiety of these pteridine analogs is presented. Two related 2,4-diaminopyrimidines (NSC-110 180 and NSC-110 191) bearing both aminobenzoyl and aspartyl moieties were bound to the mutant and wild-type enzymes to nearly the same extent as the 4-aminofolates. The 5-aryl diaminopyridine, trimethoprim ( K i = 3.7·10 −9) was bound better than the related pyrimethamine ( K i = 2.46·10 −8) to A mutant and wildtype enzymes. Binding to a B mutant enzyme was reduced only 5-fold for pyrimethamine, but 100-fold for trimethoprim.

Characterization of arylsulfatase isoenzymes from Pseudomonas aeruginosa.

A method is described for the separation and isolation of two electrophoretically distinct arylsulfatases (arylsulfatase EC.3.1.6.1) from Pseudomonas aeruginosa.Characterization of the enzymes revealed that they were type I arylsulfatases, with similar kinetic properties. They differed, however, in respect to charge, pH optima for substrate hydrolysis, and activation by anions. In addition, the enzymes displayed dual pH optima curves with both substrates used and the curious property of a shift in pH optima with varied p-nitrophenyl sulfate concentrations.

Studies on the shift in the pH optimum of hepatic microsomal aniline p-hydroxylation following treatment of rats with 3,4-benzpyrene

Benzpyrene injection in rats is associated with changes in the pH optimum of the hepatic microsomal metabolism in vitro of aniline to p-aminophenol. The shift in pH optimum (upwards from pH 7.0 in controls to as high as pH 8.0 in microsomes from benzpyrene-treated rats) was dependent on substrate concentration; low substrate concentrations totally obscured this change in pH optimum. Phospholipase C treatment in vitro of hepatic microsomes from control rats resulted in a shift in pH optimum of aniline p-hydroxylation similar to that produced by benzpyrene injections ( in vivo). Adding hexobarbital to microsomes from control rats also displaced upward the pH optimum of microsomal aniline p-hydroxylation in vitro. Taken together with some proposals and results of others, the present studies lead to the conclusion that the microsomal type I substrate-binding site is involved in determining the pH optimum of a type II substrate (aniline) of the microsomal mixed function oxidase system.

Linoleate oxidation induced by lipoxygenase and heme proteins: A direct spectrophotometric assay

Conditions are described for the preparation of a Tween-solubilized linoleic acid substrate which remained clear over a broad range of pH and permitted the direct spectrophotometric recording of initial rates of conjugated diene formation in the reaction mixture. The catalytic effects of lipoxygenase and heme proteins on linoleate oxidation were studied, using such a substrate. Catalase, peroxidase, and cytochrome c were relatively weak catalysts for diene formation compared to soybean lipoxygenase. Optimal pH values for heme proteins ranged from 4 to 5, whereas lipoxygenase displayed maximal activity around pH 9, a shift in optimal pH toward 7 occurring at high linolcate and Tween coneentrations. Tween 20 acted as a competitive inhibitor for lipoxygenase, but it did not inhibit heme proteins.

A rapid micro assay method for amylo-1,6-glucosidase

A micro-scale method for the determination of amylo-1,6-glucosidase activity has been developed. The method utilizes the ability of the glucosidase to incorporate radioactive glucose moieties into glycogen which is then precipitated on filter paper squares by immersion in aqueous alcohol. The papers are counted directly for radioactivity by placing them in a vial containing scintillation fluid. The method was found to be entirely specific for amylo-1,6-glucosidase in tissue extracts. The pH optimum for the glucosidase was the same as that measured when assaying for the enzyme in the degradative direction using glycogen phosphorylase limit dextrin as a substrate, and a similar shift in pH optimum and inhibition by Tris were noted. The method was found to be applicable to muscle and liver extracts and to erythrocyte hemolyzates as well as to purified enzyme. The method has the advantage that it is rapid, that the substrates are readily available, and that only micro amounts of enzyme are required.

Effects of cetyltrimethylammonium bromide on catalytic properties of kidney microsomal glucose-6-phosphatase, inorganic pyrophosphate-glucose phosphotransferase and inorganic pyrophosphatase activities

The modifying effects of the cationic detergent cetyltrimethylammonium bromide (CTAB) on catalytic properties of kidney microsomal d- glucose-6-P phosphohydrolase (EC 3.1.3.9) and associated inorganic pyrophosphatase and PP 1-glucose phosphotransferase activities have been studied. Initial reaction velocities, apparent Michaelis constant values and apparent maximal reaction velocity values were determined at various H + concentrations with microsomal preparations which had been supplemented with the detergent to a final concentration (w/v), of 0.00, 0.05, 0.10, 0.20 or 0.30% 15 min prior to assay. Measurements of initial reaction velocities at various assay pH values indicated that CTAB treatment produced a shift in pH optimum of glucose-6-P phosphohydrolase activity towards acid pH values and in that of inorganic pyrophosphatase and phosphotransferase activities in the direction of neutrality. Both on the basis of initial reaction velocity values and apparent maximal reaction velocity values extrapolated to infinite substrate concentrations, lower concentrations of CTAB (0.05 and 0.10%, w/v) were found to elevate all activities, while higher concentrations (0.20 and 0.30%, w/v) were less effective in this respect or actually inhibited. In contrast with these polyphasic effects of the detergent on velocity values, apparent Michaelis constant values for all phosphate substrates, assayed at pH 4.5, 5.5, and 6.5, all decreased progressively as CTAB concentrations were increased from 0.05% to 0.30% (w/v). Plots (see J. S. Friedenwald and G. D. Meangwyn-Davies, in W. D. McElroy and B. Glass, The Mechanism of Enzyme Action, Johns Hopkins Press, Baltimore 1954, p. 180), of reciprocals of apparent Michaelis constant values for these compounds versus CTAB concentrations were linear, supporting the concept that CTAB acts as a “coupling” activator at lower concentrations and as a noncompetitive inhibitor at higher concentrations. Apparent Michaelis constants for PP i—0.3–0.4 mM with 0.30% (w/v) CTAB (pH 5.5)—are the lowest such values yet reported. The apparent Michaelis constant value for glucose in the phosphotransferase reaction, in contrast, was only modestly changed by CTAB treatment of microsomes. Several alternative mechanistic interpretations are considered briefly.

Comparison of lipid effects on K+-Mg2+ activated p-nitrophenyl phosphatase and Na+-K+-Mg2+ activated adenosine triphosphatase of membrane.

Abstract— A comparison was made between K+‐Mg2+ activated p‐nitrophenyl phosphatase and Na+‐K+‐Mg2+ activated adenosine triphosphatase with a solubilized enzyme preparation from a membrane fraction of cerebral cortex. The NPPase showed activity even in the absence of phospholipid, whereas the ATPase required the lipid for its activity. More varied types of phospholipids were effective in activating the NPPase than the ATPase, and with each phospholipid the extent and the pattern of the NPPase activation differed from that of the ATPase. By deoxycholate treatment the pH optimum of the NPPase was shifted independently from the pH optimum shift of the ATPase. The specific activity ratio of the NPPase to the ATPase was not constant during purification. These two enzymes were, however, not separable with ammonium sulphate fractionation, and their thermo‐lability was identical regardless of the presence of phospholipid. The results suggested two possibilities: (1) the NPPase is a separate enzyme entity from the ATPase; (2) although the NPPase is a part of the ATPase system, the mechanism of action of lipids on the former part differs from that on the rest of the system.

Structure and function of chloroplast proteins: VII. Ribulose-1,5-diphosphate carboxylase of Chlorella ellipsoidea

Ribulose-1, 5-diphosphate carboxylase isolated from autotrophically grown cells of the green algae, Chlorella ellipsoidea, was partially purified. The nature of the enzyme closely resembles the spinach leaf enzyme in its elution patterns on Sephadex gel filtration and DEAE-cellulose column chromatography, migration on starch gel electrophoresis, and the immunological specificity. Chlorella RuDP carboxylase exhibits essentially identical kinetic parameters of the regulatory enzyme with those of spinach enzyme: (a) homotropic effect of NaHCO 3, (b) allosteric activating effect of Mg 2+ with respect to NaHCO 3, and (c) shift of optimum pH by elevating the Mg 2+ concentrations.

Some properties of variously activated microsomal glucose-6-phosphatase, inorganic pyrophosphatase and inorganic pyrophosphate-glucose phosphotransferase. Shift in pH optimum

1. 1. The activity of microsomal glucose-6-phosphatase (EC 3.1.3.9) and the related enzymatic functions, acid inorganic pyrophosphatase and inorganic pyrophosphate- glucose phosphotransferas is greatly increased by pretreatment of ‘microsomes’ with hydroxyl ion at pH 9.5–9.8. The thermal stability of the preparations so activated is in marked contrast to the great instability observed when a corresponding activation is accomplished with deoxycholate or Triton X-100. 2. 2. It is suggested that comparative levels of these enzyme activities be determined on NH 4OH-treated homogenates or particulate fractions rather than on untreated or detergent-activated preparations. 3. 3. After activation with NH 4OH, deoxycholic acid, or Triton X-100 the optimum pH of all three activities is slufted in the alkaline direction. 4. 4. A similar activation and pH optimum shift is observed with liver mirosomes from fed, fasted and diabetic rats, in which the absolute level of activity varies many-fold. 5. 5. Erroneous results are obtained if activities of untreated microsomes are compared with activated ones at the same pH. This is particularly true for the transferase activity in which the quantitative effect of the pH optimum shift is much greater than for the hydrolase activities. 6. 6. A difference between glucose-6-phosphatase and the related PP i-utilizing enzymatic activites has been noted and studied. Glucose 6-phosphatase hydrolysis by liver microsomes is inhibited by lower concentrations of deoxycholate or Triton X-100 than are required to cause activation. PP i hydrolysis and glucose 6-phosphatase synthesis from PP i, on the other hand, are only activated by these low concentrations of detergents. 7. 7. The results lead to the suggestion that some of the active sites, particularly those for glucose, are on the inner surfaces of the endoplasmic reticulum or are impeded by the spatial conformation of the membrane or by the attachment of ribosomes on the outer surfaces.

Genetically alterable transport of amethopterin in Diplococcus pneumoniae. II. Impairment of the system associated with various mutant genotypes.

Six mutations determining resistance to amethopterin were examined for their effects on the active transport of the drug. In strains bearing each of the mutations and exhibiting resistance levels varying from 10- to 100-fold, transport at limiting concentrations of H(3)-amethopterin was reduced from 2.5 to 10 times the rate characteristic of the wild type. Kinetic analysis of transport showed an increase in the value for K(m) of the system in all of the mutants. Values for the wild-type system were 0.9 x 10(-6)m and for the mutants varied between 2.5 x 10(-6)m and 9.0 x 10(-6)m. Values for V(max) were approximately the same for each system. The mutant transport systems also exhibited a shift in pH optimum from near 6.0 (wild-type) to below 5.0. The results were interpreted as an alteration in the binding properties of the permease in the mutant strains.

Polygalacturonase of Erwinia carotovora

Abstract The polygalacturonase, clearly differentiated from polygalacturonic acid trans-eliminase, produced in the culture fluid of Erwinia carotovora was purified 56-fold by means of acetone precipitation, acetate buffer extraction, and two successive treatments by chromatography on carboxymethyl cellulose. The purified enzyme is specific for nonmethoxylated polygalacturonic acid. It shows an optimum pH between 5.2 and 5.4; no shift of optimum pH is observed when tetragalacturonic acid is used as a substrate. The activity is stimulated by 0.1 m sodium, potassium, and ammonium ions, but not by 0.001 m calcium, magnesium, and barium ions. At 0.1 m, phosphate (pH 5.2), chloride, and sulfate ions are somewhat inhibitory as compared to acetate ions, but 0.1 m phosphate (pH 4.8) and 0.001 m polymetaphosphate ions are not inhibitory. The major end products are mono- and digalacturonic acids. Digalacturonic acid is not hydrolyzed further. The relative initial rates of the reactions on substrates of different chain lengths are: polygalacturonic acid, 100; shorter chain polygalacturonic acid (average degree of polymerization, 12), 52; hexagalacturonic acid, 15.8; pentagalacturonic acid, 12.8; tetragalacturonic acid, 2.2; trigalacturonic acid, 1.7. Hexamer is cleaved predominantly at the central bond, producing 2 molecules of trimer; slower reactions yielding dimer and tetramer were also observed. Pentamer is cleaved only into dimer and trimer. Tetramer is cleaved at Bond 1 to yield trimer and monomer; the central bond of tetramer is also cleaved at a lower but significant rate to form dimer. Both reduced and oxidized tetramer are cleaved much more slowly, and at the central bond only, resulting in an accumulation of normal and reduced or oxidized dimer. Simultaneous attack of Bonds 1 and 2 is postulated for trimer hydrolysis, since only the monomer is found as the reaction product and no dimer is detected, although Erwinia polygalacturonase cannot hydrolyze the dimer. Both reduced and oxidized trimer are cleaved at Bond 1 after a long incubation.