Adhesion of Pseudomonas fluorescens (ATCC 17552) to nonpolarized and polarized thin films of gold.

The adhesion of Pseudomonas fluorescens (ATCC 17552) to nonpolarized and negatively polarized thin films of gold was studied in situ by contrast microscopy using a thin-film electrochemical flow cell. The influence of the electrochemical potential was evaluated at two different ionic strengths (0.01 and 0.1 M NaCl; pH 7) under controlled flow. Adhesion to nonpolarized gold surfaces readily increased with the time of exposition at both ionic-strength values. At negative potentials (-0.2 and -0.5 V [Ag/AgCl-KCl saturated [sat.]]), on the other hand, bacterial adhesion was strongly inhibited. At 0.01 M NaCl, the inhibition was almost total at both negative potentials, whereas at 0.1 M NaCl the inhibition was proportional to the magnitude of the potential, being almost total at -0.5 V. The existence of reversible adhesion was investigated by carrying out experiments under stagnant conditions. Reversible adhesion was observed only at potential values very close to the potential of zero charge of the gold surface (0.0 V [Ag/AgCl-KCl sat.]) at a high ionic strength (0.1 M NaCl). Theoretical calculations of the Derjaguin-Landau-Verwey-Overbeek (DLVO) interaction energy for the bacteria-gold interaction were in good agreement with experimental results at low ionic strength (0.01 M). At high ionic strength (0.1 M), deviations from DLVO behavior related to the participation of specific interactions were observed, when surfaces were polarized to negative potentials.     

Effect of ionic strength on the regulatory properties of 2-oxoglutarate dehydrogenase complex.

The effects of changing ionic strength on the activity of the 2-oxoglutarate dehydrogenase complex from pig kidney cortex were explored. This enzyme complex is found to be influenced in many ways by the ionic strength of the reaction medium. The enzyme shows an optimum activity at 0.1 M ionic strength. Increase in ionic strength from 0.1 M to 0.2 M resulted in a decrease of S0.5 for 2-oxoglutarate, and in an increase of S0.5 for NAD. Changes in ionic strength over the range of 0.05-0.2 M have little, if any, effect on S0.5 for CoA. The Hill coefficient for 2-oxoglutarate and NAD at 0.2 M ionic strength was 1.0, whereas at 0.05 M ionic strength it was 0.85 and 1.2 for 2-oxoglutarate and NAD, respectively. At 0.05 M ionic strength the pH optimum of the enzyme ranges between 7.4-7.6, but at 0.15 M ionic strength the pH optimum shifts to 7.8. The magnitude of inhibition of enzyme activity by ATP is not influenced by changes in ionic strength in the absence of calcium. However, in the presence of Ca2+, increases in ionic strength lower the inhibitory effects of ATP. The Si0.5 for ATP in both presence and absence of Ca2+ was not affected by changes in ionic strength in the range of 0.1-0.2 M. In contrast, the Sa0.5 for ADP in the absence of Ca2+ decreases as ionic strength increases. In the presence of calcium and 0.2 M ionic strength ADP has no effect on 2-oxoglutarate dehydrogenase complex activity.     

Engineering of human lysozyme as a polyelectrolyte by the alteration of molecular surface charge

The surface positive charges of human lysozyme were either increased or decreased to alter the electrostatic interaction between enzyme and substrate in the lytic action of human lysozyme using site-directed mutagenesis. The amino acid substitutions accompanying either the addition or the removal of two units of positive charge have shifted the optimal ionic strength (NaCl concentration in 10 mM Mes buffer, pH 6.2) for the lysis of Micrococcus lysodeikticus cell from 0.04 M to 0.1 M and from 0.04 M to 0.02 M respectively. In addition to the change in ionic strength-activity profile, the pH-activity profile and the effect of a polycationic electrolyte, poly-L-Lys-HCl, on the lytic activity were significantly changed. Owing to the shifts in both ionic strength profiles and pH profiles the Arg74/Arg126 mutant has become a better catalyst than wild-type enzyme under the conditions of high ionic strength and high pH, and the Gln41/Ser101 mutant has become a better catalyst under the conditions of low ionic strength and low pH.     

Calf-spleen nicotinamide--adenine dinucleotide glycohydrolase. Solubilization purification and properties of the enzyme.

NAD glycohydrolase of calf spleen was solubilized with pancreatic lipase and purified approximatively 800-fold to a specific activity of 7 units/mg of protein by successive DEAE-cellulose and carboxymethyl-cellulose chromatography. The purified enzyme has a molecular weight of 24,000 and is characterized by a double band on disc gel electrophoresis. Some kinetic properties of the NAD-glycohydrolase-catalyzed hydrolsis of NAD have been examined using a titrimetric assay for enzyme activity. The reaction is subject to inhibition be excess of substrate, which disappears at high ionic strength and low pH. At a pH below 5 the kinetic displays an apparent activation by substrate. The effects of pH (4.5-9.0) on the kinetic parameters do not reveal an essential ionizable group in the catalytic process.     

Hepatic microsomal glucose-6-phosphatase of normal and alloxan-diabetic rats. Thermotropic effects on kinetics and interaction with deoxycholate and 1-anilino-8-naphthalene sulfonate

Thermotropic effects on the kinetics of glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) activity of hepatic microsomes from normal and alloxan-diabetic rat liver were investigated by determining V, Km and Ki (substrate inhibition) values. Influence of deoxycholate (0.1%) and 1-anilino-8-naphthalene sulfonate (2.5 mM) on the kinetics was also evaluated. 1. Substrate inhibition occurred at 0.06 M for the enzyme from normal rats and at 0.0-0.025 M for the enzyme from diabetic rats. 2. The enzyme from diabetic rats showed a transition that extended between 22.7 and 27 degrees C in the Arrhenius plot (log V vs. T-1) instead of at 19.5 degrees C. 3. Deoxycholate increased the V value of both enzymes without affecting substrate inhibition at all the temperatures but did not completely abolish the transition in the Arrhenius plot of the enzyme from diabetic rats. 4. 1-Anilino-8-naphthalene sulfonate eliminated substrate inhibition and activated the enzyme of normal rats above 27.5 degrees C by increasing both V and Km values. Below this temperature, the enzyme showed biphasic or allosteric kinetics. At low substrate concentrations it was activated as both V and Km values were increased. The enzyme from diabetic rats, on the other hand, was activated at all the temperatures and exhibited linear kinetics. 5. Binding of 1-anilino-8-naphthalene sulfonate to the microsomal fraction increased with decreasing temperature as revealed by the increase of relative fluorescence. The microsomal fraction of diabetic rats showed a more anomalous fluorescence response between 13-18 degrees C. 6. Enthalpy changes for glucose 6-phosphate binding to the inhibition site were slightly larger than binding to the active site. Calculated entropies of activation for transition state complex of glucose-6-phosphatase reaction were fairly large and negative. The free energy of activation (28-30 kcal/mol) was independent of temperature and experimental conditions. 7. In the microsomal fraction (total as well as rough), phospholipid content and fatty acid unsaturation index of phospholipids were decreased after diabetes. The level of free cholesterol remained unchanged but the molar ratio of cholesterol to phospholipid increased. The different thermal response and 1-anilino-8-naphthalene sulfonate interaction to the enzyme from diabetic rat and liver could be ascribed to the altered lipid environment of the enzyme on the endoplasmic reticulum membrane.     

Adhesion of Pseudomonas fluorescens (ATCC 17552) to Nonpolarized and Polarized Thin Films of Gold

The adhesion of Pseudomonas fluorescens (ATCC 17552) to nonpolarized and negatively polarized thin films of gold was studied in situ by contrast microscopy using a thin-film electrochemical flow cell. The influence of the electrochemical potential was evaluated at two different ionic strengths (0.01 and 0.1 M NaCl; pH 7) under controlled flow. Adhesion to nonpolarized gold surfaces readily increased with the time of exposition at both ionic-strength values. At negative potentials (−0.2 and −0.5 V [Ag/AgCl-KCl saturated {sat.}]), on the other hand, bacterial adhesion was strongly inhibited. At 0.01 M NaCl, the inhibition was almost total at both negative potentials, whereas at 0.1 M NaCl the inhibition was proportional to the magnitude of the potential, being almost total at −0.5 V. The existence of reversible adhesion was investigated by carrying out experiments under stagnant conditions. Reversible adhesion was observed only at potential values very close to the potential of zero charge of the gold surface (0.0 V [Ag/AgCl-KCl sat.]) at a high ionic strength (0.1 M NaCl). Theoretical calculations of the Derjaguin-Landau-Verwey-Overbeek (DLVO) interaction energy for the bacteria-gold interaction were in good agreement with experimental results at low ionic strength (0.01 M). At high ionic strength (0.1 M), deviations from DLVO behavior related to the participation of specific interactions were observed, when surfaces were polarized to negative potentials.     

Resistance of Dextran-Modified Hyaluronidase to Inhibition by Heparin

Properties of native and aldehyde dextran-modified hyaluronidase (with surface amino group modification about 98%) were investigated. Optimal endoglycosidase activity of the native enzyme was observed at 0.15 M NaCl and pH 5.5 and electrostatic interactions influenced the enzyme activity. The inhibitory effect of heparin on hyaluronidase activity slightly differed at pH 5.5 (1.5-fold inhibition) and 7.5 (1.2-fold inhibition). Ionic strength of the reaction medium only slightly influenced the effect of heparin. Modification of hyaluronidase with dextran increased hydrophobic interactions and steric hindrance. Conjugation with dextran increased the resistance of hyaluronidase activity to denaturing agents (urea, guanidinium hydrobromide) and extended the optimal conditions for maximal endoglycosidase activity (pH 4.5-6.5, the range of NaCl concentration from 0.1 to 0.3 M). The conjugation also reduced electrostatic effects on the active site of hyaluronidase and efficacy of heparin inhibition. At pH 7.5 the enzyme was almost insensitive to heparin. The resistance of dextran-modified hyaluronidase to heparin points to approaches for subsequent studies of the heparin binding site of this enzyme and biomedical trial of the stabilized enzyme for the treatment of acute cardiovascular lesions.     

Enzymatic characterization of the chondrocytic alkaline phosphatase isolated from bovine fetal epiphyseal cartilage.

Purified chondrocytic alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and inorganic pyrophosphate. Optimal activities for p-nitrophenyl phosphate, ATP and inorganic pyrophosphate are found at pH 10.5, 10.0 and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations. p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasng substrate concentration. Heat inactivation studies indicate that both phosphorolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg2+ (0.1-10.0 mM) and Mn2+ (0.01-0.1 mM) show a small stimulation of p-nitrophenyl phosphate-splitting activity at pH 10.5. Levamisole, Pi, CN-, Zn2+ and L-phenylalanine are all reversible inhibitors of the phosphomonoesterase activity. Pi is a competitive inhibitor with a Ki of 10.0 mM. Levamisole and Zn2+ are potent non-competitive inhibitors with inhibition constants of 0.05 and 0.04 mM, respectively. The chondrocytic alkaline phosphatase is inhibited irreversibly by Be2+, EDTA, EGTA, ethane-1-hydroxydiphosphonate, dichloromethane diphosphonate, L-cysteine, phenyl-methylsulfonyl fluoride, N-ethylmaleimide and iodoacetamide. NaCL, KCL and Na2SO4 at 0.5-1.0 M inhibit the enzyme. At pH 8.5, the cleavage of inorganic pyrophosphate (pyrophosphate phosphohydrolase, EC 3.6.1.1) by the chondrocytic enzyme is slightly enhanced by low levels of Mg2+ and depressed by concentrations higher than 1mM. Ca2+ show only inhibition. Similar effects of Mg2+ and Ca2+ on the associated ATPase (ATP phosphohydrolase, EC 3.1.6.3) activity were observed. Arrhenius studies using p-nitrophenyl phosphate and AMP as substrates have accounted for the ten-fold difference in V in terms of small differences in both the enthalpies and entropies of activation which are 700 cal/mol and 2.3 cal/degree per mol, respectively.     

Linkage between proton binding and amidase activity in human gamma-thrombin.

The amidase activity of human gamma-thrombin has been studied in the pH range 6-10 as a function of NaCl concentration and temperature. As recently found for human alpha-thrombin [Di Cera, E., De Cristofaro, R., Albright, D.J., & Fenton, J.W., II (1991) Biochemistry 30, 7913-7924], the Michaelis-Menten constant, Km, shows a bell-shaped dependence over this pH range with a minimum around pH 7.9 in the presence of 0.1 M NaCl at 25 degrees C. The catalytic constant, kcat, has a bell-shaped pH dependence with a maximum around pH 8.6. A thermodynamic analysis of these parameters has enabled a characterization of the linkage between proton and substrate binding, its dependence on NaCl concentration, and the relevant entropic and enthalpic contributions to binding and catalytic events. Three groups seem to be responsible for the control of gamma-thrombin amidase activity as a function of pH. One of these groups has pK values that are significantly different from those found for alpha-thrombin, and all groups show slightly perturbed enthalpies of ionization. The dependence of gamma-thrombin amidase activity on NaCl concentration is different from that of alpha-thrombin. Increasing NaCl concentration always decreases the substrate affinity for the enzyme in the case of alpha-thrombin, regardless of pH. In the case of gamma-thrombin, such an effect is observed only in the pH range 7.5-9, and a reversed linkage is observed at pH less than 7 and greater than 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoacylglycerol hydrolase in human platelets.

In the present paper we show for the first time monoacylglycerol hydrolase in human platelets. No monoacylglycerol hydrolase activity could be demonstrated in the other blood cells. The monoacylglycerol hydrolase of platelets could not be released from the cells by heparin, thus the enzyme is distinct from the postheparin plasma lipases. The enzyme could be solubilized by a non-ionic detergent, Triton X-100. The solubilized monoacylglycerol hydrolase from platelets was optimally active at pH between 7 and 8 and at ionic strength corresponding to [NaCl] between 0.1 and 0.3 M. The optimal assay temperature was 37 degrees C. The enzyme activity was sensitive to HgCl2 but not to NaF. Accordingly, it was stabilized by 2-mercaptoethanol.     

Thiol groups of Escherichia coli citrate synthase and their influence on activity and regulation.

The modification of Escherichia coli citrate synthase (citrate oxaloacetatelyase(pro-3S-CH2.COO- leads to acetyl-CoA, EC 4.1.3.7) with 5,5'-dithiobis-(2-nitrobenzoic acid) has been investigated. (1) In low ionic strength (20 mM Tris.HCl, pH 8.0): (A) Eight thiol groups per tetramer of the native enzyme reacted with Nbs2. (b) Two of the eight accessible thiols were modified rapidly with the loss of 26% enzyme activity but with no change in the NADH inhibition. The remaining six were modified more slowly, resulting in a further 60% loss of activity and complete densensitization to NADH. (c) The 2nd-order rate constant for the modification of the rapidly reacting thiols is 2.5.10(4) M-1.min-1. At the reagent concentrations used (0.1 to 0.2 mM) the modification of the six thiols in the slow kinetic set appeared to be 1st-order; at 0.1 mM dithionitrobenzoic acid their rate of modification was approximately 30 times slower than the thiols in the fast kinetic set. (2) In high ionic strength (20 mM Tris.HCl, pH 8.0, 0.1 M KCl): (a) Four thiol groups were modified in a single kinetic set and it appeared that these thiols are four of the six slowly modified in the absence of KCl. (b) The modification resulted in 70% loss of enzyme activity and complete loss of NADH inhibition. (3) From the kinetic analysis it is proposed that the four thiol groups accessible to dithionitrobenzoic acid in the absence and presence of 0.1 M KCl are those involved in the response of NADH. Modification of any one of these four groups produced no reduction in the inhibition; instead, loss of NADH sensitivity was coincident with the appearance of tetrameric protein possessing three substituted thiols, whereas enzyme with one or two modified groups was still fully inhibited by NADH.     

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves. Kinetic properties of different oligomeric structures

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves was inhibited by increasing the ionic strength in the assay medium. The inhibitory effect was higher at pH 7.0 than 8.0, with median inhibitory concentrations (IC50) of 89 mM and 160 mM respectively, for inhibition by NaCl. Gel-filtration experiments indicated that the enzyme dissociated into dimers and monomers when exposed to high ionic strength (0.3 M NaCl). By using the enzyme-dilution approach in the absence and presence of 0.3 M NaCl, the kinetic properties of each oligomeric species of the protein was determined at pH 7.0 and 8.0. Tetrameric, dimeric and monomeric structures were shown to be active but with different V and Km values. The catalytic efficiency of the oligomers was tetramer greater than dimer greater than monomer, and each quaternary structure exhibited higher activity at pH 8.0 than 7.0. Dissociation constants for the equilibria between the different oligomeric forms of the enzyme were determined. It was established that Kd values were affected by pH and Mg2+ levels in the medium. Results suggest that the distinct catalytic properties of the different oligomeric forms of NADP-dependent malate dehydrogenase and changes in their equilibrium could be the molecular basis for an efficient physiological regulation of the decarboxylation step of C4 metabolism.     

Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.     

The effect of pH on the kinetics of beef-liver fructose bisphosphatase.

1. The kinetics of the reaction catalysed by fructose bisphosphatase have been studied at pH 7.2 and at pH 9.5. The activity of the enzyme was shown to respond sigmoidally to increasing concentrations of free Mg2+ or Mn2+ ions at pH 7.2, whereas the dependence was hyperbolic at pH 9.5. At both pH values the enzyme responded hyperbolically to increasing concentrations of fructose 1,6-bisphosphate, although inhibition was observed at higher concentrations of this substrate. This high substrate inhibition was shown to be partial in nature and the enzyme was found to be more sensitive at pH 7.2 than at pH 9.5. 2. The properties of the enzyme, are consistent with the enzyme obeying either a random-order equilibrium mechanism or a compulsory-order steady-state mechanism in which fructose bisphosphate binds to the enzyme before the cation. 3. Reaction of the enzyme with a four-fold molar excess of p-chloromercuribenzoate caused activation of the enzyme when its activity was assayed in the presence of MN2+ ions but inhibition when Mg2+ ions were used. Higher concentrations of p-chloromercuribenzoate caused inhibition. This activation at low p-chloromercuribenzoate concentrations, and the reaction of 5,5'-dithio-bis(2-nitrobenzoate) with the four thiol groups in the enzyme that reacted rapidly with this reagent, were prevented or slowed by the presence of inhibitory, but not non-inhibitory, concentrations of fructose bisphosphate. After reaction with a four-fold molar excess of p-chloromercuribenzoate the enzyme was no longer sensitive to high substrate inhibition by fructose bisphosphate.     

Intracellular localization of rat-liver uracil-DNA glycosylase. Purification and properties of the chromatin enzyme

Most of the uracil-DNA glycosylase of the rat liver cell is located in chromatin; there is, however, some activity in the nuclear sap and in the cytoplasm. The chromatin uracil-DNA glycosylase has been purified; the preparation is devoid of endonuclease and exonuclease activities; the enzyme does not need divalent cations, has a broad optimum pH around 8, is strongly inhibited by increasing ionic strength and free uracil. The apparent Km is independent of the strandedness of the DNA substrate containing uracil, but V is slightly higher with the single-stranded substrate. The frequency of uracil substitution in the double-stranded DNA influences the kinetic parameters: a higher frequency increases both Km and V. The inhibitory effects of NaCl and free uracil are greater when the substrate is double-stranded rather than single-stranded. It is speculated that, acting either on the DNA or on the enzyme, both oppose the opening of the double helix necessary for the formation of the enzyme-substrate complex. The increased reaction rate with a higher frequency of uracil residues in double-stranded DNA is interpreted as a tendency for the repair enzyme to work in a processive way. It is supposed that processivity also occurs with single-stranded DNA and that it is opposed by both NaCl and free uracil, explaining a greater inhibition when the single-stranded substrate has a higher uracil content.     

alpha-D-Mannosidase of Saccharomyces cerevisiae. Characterization and modulation of activity.

A unique and interesting alpha-D-mannosidase (alpha-D-mannoside mannohydrolase EC 3.2.1.24) activity has been isolated from Saccharomyces cerevisiae. The enzyme was localized in a crude particulate fraction of the cell extract and was not solubilized by treatment with detergents or high ionic strength NaCl. The enzyme had a pH optimum of 6.3, Km 50 micron with p-nitrophenyl-alpha-D-mannopyranoside, and was competitively inhibited by D-mannose (Ki 20 mM). The enzyme is not affected by ethylenediaminetetraacetic acid, a number of different cations, or sulfhydryl reagents. It was inhibited by p-chloromercuriphenyl sulfonic acid and this inhibition is prevented by the addition of substrate. The cellular concentration of alpha-D-mannosidase is inversely proportional to growth rate, suggesting that the enzyme is under catabolite repression. The level of enzyme was found to increase approx. 8-fold during sporulation. This is apparently due to de novo synthesis, since inhibition of protein synthesis by cycloheximide prevents the increase in enzyme activity.     

How much is secondary structure responsible for resistance of double-stranded RNA to pancreatic ribonuclease A?

1. Double-stranded f2 sus11 or Qbeta RNAs, resistant to bovine pancreatic RNAase A in 0.15 M NaCl/0.015 M sodium citrate (SSC), are quickly and completely degraded at 10-fold lower ionic strength (0.1 X SSC) under otherwise similar conditions. At this ionic strength the secondary structure of double-stranded RNA is maintained, as judged by the following: (a) the unchanged resistance of double-stranded RNA and DNA, under similar low ionic strength conditions, to nuclease S1 from Aspergillus oryzae, in contrast with the sensitivity of the corresponding denatured nucleic acids to this enzyme, specific for single-stranded RNA and DNA; (b) the co-operative pattern of the thermal-transition profile of double-stranded RNA (with a Tm of 89 degrees C) in 0.1 X SSC. 2. Whereas in SSC bovine seminal RNAase (RNAase BS-1) and whale pancreatic RNAase show an activity on double-stranded RNA significantly higher than that of RNAase A, in 0.1 X SSC the activity of the latter enzyme on this substrate becomes distinctly higher than that of RNAase BS-1, and similar to that of whale RNAase. 3. From these results it is deduced that the secondary structure is probably not the only nor the most important variable in determining the susceptibility double-stranded RNA to ribonuclease. Other factors, such as the effect of ionic strength on the enzyme and/or the binding of enzyme to nucleic acids, may play an important role in the process of double-stranded RNA degradation by ribonucleases specific for single-stranded RNA.     

Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui

D-Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui has been partially purified by ammonium-sulfate fractionation, hydrophobic and ion exchange chromatography. Catalytic activity of the enzyme requires salt concentrations beyond 1M NaCl: optimum conditions are 4M NaCl or KCl, pH 6-8, 50 degrees C. Michaelis constants for NADH and pyruvate under optimum conditions of enzymatic activity are 0.070 and 4.5mM, respectively. As for other bacterial D-specific lactate dehydrogenases, fructose 1,6-bisphosphate and divalent cations (Mg2+, Mn2+) do not affect the catalytic activity of the enzyme. As shown by gel-filtration and ultracentrifugal analysis, the enzyme under the conditions of the enzyme assay is a dimer with a subunit molecular mass close to 36 kDa. At low salt concentrations (less than 1M), as well as high concentrations of chaotropic solvent components and low pH, the enzyme undergoes reversible deactivation, dissociation and denaturation. The temperature dependence of the enzymatic activity shows non-linear Arrhenius behavior with activation energies of the order of 90 and 25 kJ/mol at temperatures below and beyond ca. 30 degrees C. In the presence of high salt, the enzyme exhibits exceptional thermal stability; denaturation only occurs at temperatures beyond 55 degrees C. The half-time of deactivation at 70 and 75 degrees C is 300 and 15 min, respectively. Maximum stability is observed at pH 7.5-9.0.     

Linkage between proton binding and amidase activity in human alpha-thrombin: effect of ions and temperature

The amidase activity of human alpha-thrombin has been studied at steady state in the pH range 6-10, as a function of NaCl concentration from 1 mM to 1 M and temperature from 10 to 40 degrees C. The Michaelis-Menten constant, Km, shows a bell-shaped dependence over this pH range with a minimum around pH 7.5 in the presence of 0.1 M NaCl at 25 degrees C. The catalytic constant, kcat, also has a bell-shaped pH dependence with multiple inflection points that are more evident at low NaCl concentrations and a maximum around pH 8.2 in the presence of 0.1 M NaCl at 25 degrees C. A detailed analysis of the results in terms of a general linkage scheme has allowed a thorough characterization of the linkage between proton and substrate binding and its dependence on NaCl concentration, as well as the relevant entropic and enthalpic contributions to binding and catalytic events. Formulation of detailed partition functions for each enzyme intermediate involved in the catalytic cycle suggests that (at least) three groups are responsible for the control of thrombin amidase activity as a function of pH. One group is to be identified with the active site His, due to its pK values in the free enzyme and the adduct and its enthalpy of ionization. The effect of NaCl concentration on amidase activity seems to be extremely specific. Comparative steady-state measurements carried out in the presence of NaCl, NaBr, NaI, KCl, and MgCl2 show that human alpha-thrombin is capable of discriminating among different cations and anions. This suggests that small ions participate as allosteric effectors in the regulation of thrombin activity. The linkage with NaCl is strongly pH dependent and increases with decreasing pH. The present results provide information on the basic aspects of human alpha-thrombin activity and regulation and enable a rigorous thermodynamic approach to other important regulatory interactions in human alpha-thrombin and its structurally perturbed derivatives.     

The hamster adipocyte adenylate cyclase system I. Regulation of enzyme stimulation and inhibition by manganese and magnesium ions

In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E₁, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn²⁺ and Mg²⁺, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn²⁺ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E₁ (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn²⁺, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E_1^?, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn²⁺, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn²⁺ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg²⁺, although about 100-fold higher concentrations of Mg²⁺ than of Mn²⁺ were required. The data indicate that Mn²⁺at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn²⁺ on the functions of the guanine nucleotide site(s) in the system.