Imidazole Buffer Research Articles

Rate acceleration by stereopopulation control: models for enzyme action.

As a result of alkyl substitution in both aromatic ring and side chain, the rate constant for acid-catalyzed lactonization of hydrocoumaric acid is increased by factors as high as 10(11) and, in comparison with the bimolecular esterification of phenol and acetic acid, by almost 10(16). In the most favorable case studied, the half-life of the phenolic acid (imidazole buffer, pH 7, 30 degrees C) is 6 sec, with 90% of the total rate being due to catalysis by the buffer species. The effect is attributed to a unique interlocking of methyl groups, which produces a severe conformational restriction of the side chain and increases greatly the population of the most productive conformer. This phenomenon is presented as a model for the conformational restraint imposed by an enzyme on its substrate. The magnitude of the effect suggests that the contribution of substrate "freezing" to total rate enhancement by an enzyme can be considerably greater than previously supposed and may in fact be sufficient to complete the justification for enzyme catalysis.

Effects of fixation and substrate protection on the isoenzymes of aspartate aminotransferase studied in a quantitative cytochemical model system.

The cytochemical technique of Lee and Torack for the demonstration of aspartate aminotransferase activity was tested on a model system consisting of either total liver homogenate or the mitochondrial or soluble cytoplasmic fraction, incorporated in polyacrylamide film. After incubation of portions of film in a medium of alpha-ketoglutarate, L-aspartate, and lead nitrate, the lead oxaloacetate formed was converted to lead sulfide. The absorbance determined at 520 nm in a film spectrophotometer and expressed in terms of unit weight of film provided a measure of the contained enzymatic activity, and was directly proportional to the concentration of chemically determined oxaloacetate in the film. Both mitochondrial and "soluble" isozymes of aspartate aminotransferase reacted with the cytochemical media to a quantitatively similar degree, but were considerably inactivated after 15 min of treatment with 1% glutaraldehyde or 3.7% formaldehyde in imidazole buffer, the rate of inactivation being greater for the soluble isozyme. Application of the principle of substrate protection delayed inactivation. Thus, for both isozymes the rate of inactivation decreased if ketoglutarate was added to the fixative. Similarly, it was shown that the optimal incubation medium for the demonstration of the soluble isozyme must contain 4 mM of alpha-ketoglutarate and 20 mM of L-aspartate. Under these conditions the turnover-number for the cytochemical system is 70% of the value obtained from biochemical estimations. Cytochemical K(m) values differed for each isozyme and were in accord with values determined by biochemical techniques, indicating that the model system can be used as a link between biochemical and cytochemical data in enzymatic studies.

THE MECHANISM OF CHLORIDE TRANSPORT IN CHARACEAN CELLS

SummaryActive chloride uptake by cells of Chara corallina decreases as the pH of the bathing solution is increased, using a range of buffers. Superimposed on this effect, large stimulations of Cl− uptake can be obtained by the addition of imidazole and tris buffers or ammonium sulphate. ‘Stimulated’ or ‘normal’ Cl− influxes respond similarly to the presence of metabolic inhibitors.It is suggested that these effects are not due to changes in photosynthesis or respiration, but reflect the dependence of Cl− transport on the pH gradient across the plasmalemma. Possible ways in which Cl− transport might be linked to H+ or OH− fluxes are discussed. It is proposed that the pH gradient across the plasmalemma is controlled by charge‐separating reactions which produce an active H+ efflux, and that Cl− uptake is linked to OH− efflux by ion‐exchange reactions.The metabolic implications of this scheme are discussed in detail, with reference to previous work on Characean and other plant cells.

Biosynthesis of Urea: XIII. DISSOCIATION-ASSOCIATION KINETICS AND EQUILIBRIA OF ARGININOSUCCINASE

Abstract Further study of the cold inactivation of argininosuccinase shows that a new, more slowly sedimenting component is formed which has an S value of about 5.6 as compared to 9.3 for the active enzyme. The two components can be resolved by high speed sedimentation in phosphate but not in Tris or imidazole buffers. The proportion of each component corresponded to the extent of inactivation. On resolution in a sucrose density gradient, the 5.6 S component was found to be enzymatically inactive. Activity was regained by thermal reactivation of the fractions in this peak. Thus, cold inactivation and thermal reactivation are accompanied by reversible dissociation of the enzyme into two, probably identical, subunits which are one-half the size of the active enzyme (molecular weight 202,000). The rate of dissociation followed first order kinetics and the velocity constants increased with increasing pH. Association followed first order or second order kinetics depending on the buffer. A mechanism for the reversible dissociation has been proposed in the light of the kinetic analysis. The kinetic patterns in the direction of association reveal temperature-dependent equilibria between the dimeric and monomeric forms of the enzyme. The equilibrium constant and the negative change in free energy decreased with a decrease in temperature. The enthalpy and entropy changes were found to be 46 kcal and 189 e.u., respectively, per mole of active enzyme. The negative temperature dependence for association and the large positive value for the entropy change suggests that the active form of the enzyme is stabilized by hydrophobic bonds.

Carbonic Anhydrase from Parsley Leaves

Abstract Parsley carbonic anhydrase (EC 4.2.1.1) is a powerful catalyst for the reversible hydration of carbon dioxide, with a maximum catalytic constant of approximately 105 per sec at 25°. It comprises approximately 1% of the total protein in extracts of parsley leaves. The enzyme is purified by a procedure involving fractionation with ammonium sulfate, chromatography on diethylaminoethyl cellulose, concentration by ultrafiltration, and chromatography on agarose. Its sedimentation coefficient, s20,w0, is 8.8 S. Sedimentation equilibrium and gel filtration studies in aqueous buffer indicate a molecular weight near 180,000. In 6 m guanidine hydrochloride, or on electrophoresis on polyacrylamide gel in the presence of dodecyl sulfate, the molecular weight is approximately 29,000. This suggests that 6, possibly identical, subunits are present. Zinc analysis indicates the presence of 1 zinc atom per 29,000 molecular weight subunit. Attempts to remove the zinc without denaturing the protein were unsuccessful. The amino acid composition is somewhat similar to that of the mammalian carbonic anhydrases, although the parsley enzyme contains much more cysteine, more phenylalanine and methionine, and less tryptophan and histidine, than the mammalian enzymes. Optical rotatory dispersion and circular dichroism data suggest that the parsley enzyme may contain about 30% helix. The catalytic constant of the parsley carbonic anhydrase for carbon dioxide hydration, like that of the mammalian carbonic anhydrases, increases with increasing pH in the range from pH 6.5 to 7.5. The parsley enzyme differs from the mammalian enzymes, however, in that the Michaelis constant increases with increasing pH. It also differs from them in failing to catalyze the hydrolysis of p-and o-nitrophenyl acetates and in being reversibly inhibited by p-chloromercuribenzoate. Imidazole buffers also act as inhibitors.

The stimulating effect of angiotensin and vasopressin on adenosine triphosphatase activity in vitro.

AbstractLevin, K. The stimulating effect of angiotensin and vasopressin on adenosine triphosphatase actiuity in vitro. Acta physiol. scand. 1970. 79. 37–49.The effect of angiotensin on the levels of ATPase activity in homogenates of rat kidney cortex and other tissues has been studied. With the addition of 1.7 μg of angiotensin per ml of incubation medium containing in addition 4 mM ATP, Mg++, Na+, K+, EDTA and imidazole buffer pH 7.2, a stimulation of 8.6 ± 0.5 % of the ATPase activtiy was observed. The presence of Mg++ and Na+ was found to be necessary for the demonstration of angiotensin stimulation. The optimal pH for the effect of angiotensin was found to be 7.2 and this was confined to very narrow limits. The degree of stimulation was greatest in fresh homogenates and this rapidly decreased on storage. Centrifugation of the homogenates indicated that the ATPase activtiy which could be stimulated by angiotensin was particle bound. A similar effect to the one produced by angiontensin was obtained with vasopressin though not with oxytocin or a number of other vasoactive substances known to effect the renal handling of sodium ions. Stimdation by angiotensin could be demonstrated in kidney cortex from mouse, guinea pig, rabbit, rat and from man, and in homogenates from a number of other tissues. It was concluded that the stimulation of ATPase by angiotensin was brought about through the same mechanisms as the stimulation produced by ouabain, and that both these phenomena are closely related to Na+‐K+ ATPase. The possible physiological significance of these findings is discussed.

Partial purification and characterization of dihydrodipicolinic acid synthetase from sporulating Bacillus megaterium.

Sporulation of Bacillus megaterium Km (ATCC 13632) was synchronized by a technique employing three 10% transfers. The culture was harvested when 60% of the cells contained spore forms. Dihydrodipicolinic acid synthetase was purified 150-fold by ammonium sulfate fractionation at pH 6.5, heating for 15 min at 45 C at pH 6.0, ammonium sulfate fractionation at pH 6.0, and subsequent chromatography on diethylaminoethyl cellulose. During the final stage of the purification procedure, the enzyme exhibited sensitivity to refrigeration temperatures. The enzyme had a pH optimum of 7.65 in imidazole buffer. The apparent K(m) values were 4.6 x 10(-4) and 5.0 x 10(-4)m for beta-aspartyl semialdehyde and pyruvate, respectively. All attempts to demonstrate cofactor requirements were unsuccessful. Sulfhydryl inhibiting reagents and lysine did not inhibit the enzymatic reaction. The enzyme exhibited maximal thermal resistance at pH 10.5. The thermal stability of the enzyme at 75 C was increased more than 1,800-fold by the addition of 0.3 m pyruvate. The E(a) was 67,300 cal/mole for the thermal denaturation of the enzyme. At 60 C, the DeltaF, DeltaH, and DeltaS values for the thermal denaturation of the enzyme were 22,250, 66,700, and 133 cal per mole per degree, respectively.

Partial purification of a NADPH-dependent 21-hydroxysteroid dehydrogenase from human placenta

A NADPH-dependent 21-hydroxysteroid dehydrogenase was purified 70- to 117-fold from term human placenta. Properties of the enzyme were studied using 21-dehydrocortisol synthesized by an improved procedure. Final specific activity ranged from 0.026 to 0.048 μmoles NADPH oxidized per min per mg of protein. Optimum pH was at 6.7 in sodium phosphate and 6.3 in imidazole buffers. K m values for several 21-dehydrosteroids were approx. 1·10 −3 M. In addition, methylglyoxal and phenylglyoxal, but not glyoxal, were reduced. K m value for NADPH was 1.2·10 −5 M. Excess NADPH inhibited 21-dehydrocortisol reduction. The inhibition constant, K iB , was 1.3·10 −4 M. NAD + and NADP + also inhibited the enzyme. Androgens, estrogens and progestagens were inactive as substrates, and did not inhibit reduction of 21-dehydrocortisol. The 21-hydroxysteroids were neither reduced nor oxidized by the enzyme system, and were weak competitive inhibitors of the reduction of 21-dehydrocortisol. Sulfhydryl groups on the enzyme appeared to be necessary for activity. Molecular weight was estimated at 33 000 to 35 000 by gel filtration. Oxidation of 21-hydroxysteroids by the dehydrogenase could not be demonstrated. It is suggested that 21-hydroxysteroid dehydrogenase functions to control the level of 21-dehydrosteroids in the placenta formed by other pathways.

Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase: IV. Properties of a lubrol-solubilized beef brain microsomal enzyme

Lubrol extracts containing (Na + K)-activated adenosine triphosphatase of high specific activity could be prepared from NaI-treated beef-brain microsomes. In the presence of 20 m m imidazole buffer (pH 6.0), 2.5 m m Na 2ATP, 40 m m NaCl, and 6% Lubrol the enzyme retained about 80% of its initial activity after 5 days' storage at 5 °. Gel chromatography of Lubrol extracts on 6% agarose columns not containing Lubrol gave a single peak of (Na + K)-activated adenosine triphosphatase with an apparent molecular weight of 670,000, which is identical to that previously found for the enzyme in Lubrol extracts of guinea pig microsomes. The fact that the same apparent molecular weight was obtained when the enzyme was prepared from brains from different species and by different methods lends support to the view that the 670,000 molecular weight component is a true molecular species. Sucrose density gradient centrifugation partially separated the (Na + K) -activated adenosine triphosphatase from the ouabain-insensitive adenosine triphosphatase, the unbound Lubrol, and other protein. At equilibrium the (Na + K)-activated adenosine triphosphatase peak gave a density of 1.05, which is intermediate between that of the α- and β-lipoproteins of serum. The Na-K ATPase could be separated from half to two-thirds of the non-Na-K ATPase protein by chromatography on DEAE-cellulose. It is concluded that the (Na + K)-activated adenosine triphosphatase is a lipoprotein which can be solubilized and separated from other protein in the membrane and from unbound solubilizing detergent.

Incorporation of ethanolamine-1,2-14C into plant microsomal phospholipids.

The incorporation of ethanolamine-1,2-14C into phospholipid and the decarboxylation of phosphatidyl-serine-1,2-14C by etiolated pea seedling "microsomal suspensions" have been investigated. Optimum incorporation occurred with phosphate buffer, pH 8.5, in the presence of 2.9 × 10−3 M CaCl2. Toluene, phospholipase D, and reduced glutathione stimulated ethanolamine-1,2-14C incorporation. Higher concentrations of CaCl2, imidazole buffer, and Tris buffer were inhibitory. Other divalent cations, ATP, CTP, and CMP were without effect. L-Serine and choline were also incorporated into phospholipids by pea "microsomal suspensions" and competitively inhibited the incorporation of ethanolamine-1,2-14C. Inhibition was also observed with D-serine and glycerol, but not with methanol, ethanol, propane-1-ol, and propane-2-ol. The formation of 14CO2 by the decarboxylation of serine-U-14C by "microsomal suspensions" was also stimulated by Ca2+ and inhibited by ethanolamine. The experimental results suggest that the incorporation of ethanolamine and the decarboxylation of serine in plants proceeds by essentially the same Ca2+-requiring mechanisms reported for animals.

IMPROVED METHOD FOR HISTOCHEMICAL DEMONSTRATION OF PHOSPHOGLUCOMUTASE

A method for the histochemical demonstration of phosphoglucomutase was improved in cryostat sections of liver and muscle of rabbit under light microscope.Several biochemical activators and a specific inhibitor for the enzyme were applied. Magnesium ions (1 mM) and L-histidine (5 mM) had an advantage of histochemical demonstration of the enzyme activity. The specific inhibitor, beryllium ions (1 mM) were effective to prove histochemically the specificity of the enzyme. Imidazole buffer (4×10-2M pH 7.4) was qualified for the incubation of demonstrating the enzyme reaction.Suitable conditions for histochemical reaction for the enzyme activity were determined as follows: 80 mg glucose-1-phosphate, 5 mg adenosine triphosphate, 2.5 mg nicotinamide adenine dinucleotide phosphate (NADP), 1 mM magnesium chloride, 5 mM L-histidine, 5 ml nitro blue tetrazolium (Nitro BT, 1 mg/ml), 10 ml imidazole buffer (pH 7.4), 5 ml 3% gelatin, 0.04 ml glucose-6-phosphate dehydrogenase solution (1 mg/ml) and distilled water in a total volume of 25 ml. Two control procedures were prepared, using the incubation mixture without glucose-1-phosphate and the substrate mixture with 1 mMberyllium sulphate.The reaction products, dark blue granules of diformazan were intensely demonstrated in cytoplasms of rabbit liver cells in all areas of lobuli, without nuclei. They were moderately revealed in intermyofibrillar sarcoplasms of the large fibers and weakly in the small fibers.

Regulation of glutamine synthetase. VII. Adenylyl glutamine synthetase: a new form of the enzyme with altered regulatory and kinetic properties.

synthetase, it became obvious that conditions of growth of the organism played a decisive role in the catalytic properties of the enzyme purified from it. It was found that enzyme purified from E. coli grown on NH4Cl and glucose (preparation. I) was in general less sensitive to inhibition by feedback effectors than was enzyme purified from E. coli grown on glutamate and glycerol (preparation I). M\/Ioreover, in the biosynthetic reaction. catalyzed by the enzyme, the two preparations show markedlydifferent responses to divalent cations. Examination for structural differences in the two forms of the enzyme has shown that the dramatie kinetic alterations are not due to differences in, primary structure, bu-t rather to adenylylati.on of glutanrine synthetase I which converts it to glutamine synthetase I. rThis report presents experirnents which demonstrate that the two forms of the enzyme have identical amino acid composition but differ in the amount of adenosine 5'-phosphate (5'A1\P) bound in phosphodiester linkage to the protein. Materials and Methods.-E. coli strain W was grown aerobically in a 300-liter fermenlter as previously described., 2 Preparation I was the enzyme purified from cells grownl onl 4 mnM NH4CI and 11 mM glucose. Preparation II was the enzyme purified from cells grownl on a medium in which 19 mM glutamate replaced the NJI4Cl and 21 mM glycerol replaced the glucose. Cells were harvested anid frozen in liquid nitrogen until ready for the purification procedure, which was as previously described,' anid the resultanit enzyme preparations were homogeneous on disk gel electrophoresis anid in the ultracentrifuge. Samples of enzyme were dialyzed exhaustively against 0.01 M imidazole buffer, pH 7.0, before chemical analyses were performed. All chemicals were of the highest quality commercially available. Cellulose and polyethylenimine-cellulose thinlayer chromatography plates were purchased from Brinkmann Instruments, Inc. Snake venom phosphodiesterase (Crotalus ademanteus) and E. coli alkaline phosphatase were purchased from Worthington Biochemical Co. 5'-Adenylic acid deaminase (muscle) was obtained from Sigma

Quantitative Histochemistry of the Primate Skin: VIII Enolase

Quantitative Histochemistry of the Primate Skin: VIII Enolase

Amperometric argentometric titration of thiol groups in imidazole buffer

Amperometric argentometric titration of thiol groups in imidazole buffer

The activity of bovine carbonic anhydrase in imidazole buffers

Abstract The variation of activity with substrate concentration has been measured for bovine carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) at several hydrogen-ion concentrations between pH 6.2 and pH 7.7 in imidazole buffers containing a constant concentration of 1 anion. A simple kinetic picture emerges. In the hydration reaction K m does not vary between pH 6.7 and pH 7.7. ν max varies as if dependent on the ionisation of one group with a p K near 6.9, only the basic form being active. In the dehydration reaction in the range pH 6.2–7.45 the rate is proportional to the bicarbonate concentration: there is thus no apparent substrate binding. The rate varies with pH as though it depends on the ionisation of one group with p K near 6.9 only the acid form being active. The kinetic parameters for hydration and dehydration reactions obey the requirement for a reversible enzyme reaction throughout the range of hydrogen-ion concentration in which measurements on both reactions were made. It is suggested that the apparent binding of bicarbonate as substrate reported by other workers was due to an anionic inhibition of the enzyme by this ion in the dilute phosphate buffers they employed.

The sulfite-activated oxidation of reduced pyridine nucleotides by peroxidase

1. 1. The aerobic oxidation of sulfite ions is initiated by peroxidase in the presence of Mn ++ or certain phenolic compounds such as thyroxine or estradiol. An inhibition of sulfite oxidation by the peroxidase-thyroxine-O 2 system was produced by epinephrine, glutathione, sodium thiosulfate, ascorbic acid, catalase and reduced diphosphopyridine nucleotide. 2. 2. The oxidation of reduced diphosphopyridine nucleotide (or reduced triphosphopyridine nucleotide) by horseradish peroxidase, Mn ++ and oxygen is stimulated by sulfite ions. Hemoglobin, myoglobin, myeloperoxidase, a uterine homogenate and relatively large concentrations of inorganic iron can replace horseradish peroxidase in this reaction. Sulfite was most effective when cacodylate, phosphate, and to a lesser extent, arsenate buffers were employed, and was less effective in the presence of Tris, glycylglycine, or imidazole buffers at pH 7.0. The sulfite-activated oxidation of reduced diphosphopyridine nucleotide was inhibited by copper, hydrogen peroxide, sodium thiosulfate, glutathione, epinephrine, and ascorbic acid. 3. 3. Sulfite and thyroxine, or sulfite and estradiol have a synergistic effect on the oxidation of reduced diphosphopyridine nucleotide by the Mn ++-peroxidase-O 2 system. Sulfite ions have been employed in this way to magnify the response of the uterine enzyme to thyroxine and estradiol.

Paroxysmal Nocturnal Hemoglobinuria : The Role of Antibodies in the Diagnostic Thrombin Test

Abstract 1. The thrombin test for paroxysmal nocturnal hemoglobinuria (PNH) consists in the demonstration that PNH hemolysis in vitro is increased by the addition of a small amount of thrombin. This ability of thrombin is not substantially changed by preliminary removal of the heterophil antibodies which are present in commercial preparations of bovine thrombin, provided the test is performed in the way it was originally described. 2. Dilution of the serum with saline and the addition of imidazole buffer abolish the phenomenon of thrombin enhancement of PNH hemolysis. 3. Certain commercial thrombin preparations contain additives which inactivate the PNH hemolytic system. These materials are unsuitable for use in the thrombin test.