Manganese-dependent inhibition of human liver arginase by borate

Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn2+ for 10 min at 60 degrees C, resulted in increased Vmax and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (Ki = 2 +/- 0.5 mM), L-lysine (Ki = 2.5 +/- 0.4 mM), and guanidinium chloride (Ki = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)4- being the inhibitor, rather than trigonal B(OH)3. We suggest that arginase activity is associated with a tightly bound Mn2+ whose catalytic action may be stimulated by addition of a more loosely bound Mn2+, to generate a fully activated enzyme form. The Mn2+ dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn2+ and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor.     

Spectroscopic study on the structure and stability of beef liver arginase

The secondary and tertiary structure of the oligomeric arginase (EC 3.5.3.1) from beef liver was investigated by circular dichroism (CD) and fluorescence measurements. The far-ultraviolet CD spectrum of the enzyme at neutral pH is indicative of high helical content. The intrinsic fluorescence emission of the protein is due to tryptophan, the contribution of tyrosine being small. Upon excitation at 295 nm, the maximum of emission occurs at 330 nm, implying that the tryptophan residues are rather buried in a hydrophobic interior of the protein. Ethylenediaminetetraacetic acid (EDTA), which inactivates the enzyme by removing the functional Mn2+-ion from the enzyme, does not dissociate the enzyme into subunits, nor affect noticeably its secondary and tertiary structure. Inactivation occurs in the acid pH range, being complete at pH below 4. However, acidification up to pH 1.5 produced only limited changes in the far-ultra-violet CD spectrum and intrinsic fluorescence emission properties. The enzyme shows noteworthy thermal stability, as shown by measuring the residual activity after heating and by evaluating the temperature dependence of the CD signal at 220 nm and the intensity of emission fluorescence. A temperature of half inactivation (Tm) of 77 degrees was determined upon heating the enzyme at pH 7.5 in the presence of Mn2+-ions for 10 min; in the presence of EDTA, Tm is shifted to 55 degrees. Taken together, these observations indicate that the structural stability of beef liver arginase arises from a clustering of hydrophobic amino acids and from Mn2+-ion binding.     

Roles of metal ions in the maintenance of the tertiary and quaternary structure of arginase from Saccharomyces cerevisiae

Arginase from Saccharomyces cerevisiae has long been known to be a metal ion-requiring enzyme as it requires heating at 45 degrees C in the presence of 10 mM Mn2+ for catalytic activation. Metals are also thought to play a structural role in the enzyme, but the identity of the structural metal and its precise structural role have not been defined. Analysis of the metal ions that bind to yeast arginase by atomic absorption spectroscopy reveals that there is a weakly associated Mn2+ that binds to the trimeric enzyme with a stoichiometry of 1.04 +/- 0.05 mol of Mn2+ bound per subunit and an apparent K'D value of 26 microM at pH 7.0 and 4 degrees C. A more tightly associated Zn2+ ion can only be removed by dialysis against chelating agents. In occasional preparations, this site contained some Mn2+; however, Zn2+ and Mn2+ together bind to high affinity sites with a stoichiometry of 1.14 +/- 0.25/mol of subunit. Both the loosely associated catalytic Mn2+ ion and the more tightly associated structural Zn2+ ion confer stability to the enzyme. Removal of the weakly bound Mn2+ ion results in a 3 degree C decrease in the midpoint of the thermal transition (T 1/2) (from 57 by 54 degrees C) as monitored by UV difference absorption spectroscopy. Removal of the tightly bound Zn2+ ion produces a 19 degrees C decrease in T 1/2 (to 38 degrees C). Similar results are obtained by circular dichroism measurements. When the Zn2+ ion is removed, the steady-state fluorescence intensity increases 100% as compared to the holoenzyme, with a shift in the emission maximum from 337 to 352 nm. This suggests that in the folded trimeric metalloenzyme, the tryptophan fluorescence is quenched and that upon removal of the structural metal, the quenching is relieved as tryptophan residues become exposed to more polar environments. Equilibrium sedimentation experiments performed after dialysis of the enzyme against EDTA demonstrate that arginase exists in a reversible monomer-trimer equilibrium, in the absence of metal ions, with a KD value of 5.05 x 10(-11) M2. In contrast, the native enzyme exists as a trimer with no evidence of dissociation when Mn2+ and Zn2+ are present (Eisenstein, E., Duong, L.T., Ornberg, R. L., Osborne, J.C., Jr., and Hensley, P. (1986) J. Biol. Chem. 261, 12814-12819). In summary, the study presented here demonstrates that binding of a weakly bound Mn2+ ion confers catalytic activity. In contrast, binding of a more tightly associated Zn2+ ion confers substantial stability to the tertiary and quaternary structure of the enzyme.     

Influence of pH on the Mn2+ activation of and binding to yeast enolase: a functional study.

The influence of pH on the activation of yeast enolase by Mn2+ was measured by steady-state kinetics. The pH influence on the binding of Mn2+ to apoenolase and the enolase-substrate complex was measured by EPR spectroscopy. At pH values above 6.6, activation by Mn2+ is fit by Michaelis-Menten kinetics, but at higher concentrations of Mn2+, inhibition is observed. Under conditions analogous to the kinetic studies, the enzyme binds two Mn2+ per dimer with a Kd in the micromolar range. In the presence of the substrate 2-phosphoglycerate, three thermodynamically distinct cation binding sites per monomer are detected and the binding constants are determined by a fit to the data. As the pH decreases, the reaction velocity decreases and the cation inhibition becomes minimal. Under these conditions, only two Mn2+ binding sites per monomer are observed; the third site must be the inhibitory site. The velocity and kinetic constants are minimally affected by buffer except at pH 5.8 with PIPES. Under these conditions, the velocity is only about 40% that observed with other buffers and only a single binding site for Mn2+ per monomer is detected in the presence or absence of substrate. A direct role in the catalytic mechanism by the second cation is called to question. The binding constant for Mn2+ at site I is independent of pH over the range from 7.5 to 5.2, and the binding at site II increases only slightly over this same pH range. These results indicate that the cation sites at positions I and II contain ligands that are pH independent over this range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical studies in marine species--I. NADP(+)-dependent isocitrate dehydrogenase from turbot liver (Scophthalmus maximus L.)

1. An NADP+-dependent isocitrate dehydrogenase was extracted from turbot liver. The enzyme required divalent cations (Mg2+ or Mn2+) for its activity but was inhibited by high salt concentrations. 2. The enzyme had an optimum activity in the pH range between 7.5 and 9.0. The enzymic activity increased with temperature (in the range of 5 to 68 degrees C) with an Ea of 23.5 kJ/mol and a Q10 of 1.34. 3. The apparent Km values for the substrates were 6.5 microM for NADP+, 56 microM for Mg2+, 87 microM for Mn2+ and 4.2 and 73.5 microM for the complexes Mg-isocitrate and Mn-isocitrate, respectively. The physiological significance of these results is discussed. 4. The enzyme was inhibited by citrate and adenine nucleotides. The degree of inhibition depended on the relation between the concentrations and that of magnesium. A possible regulating mechanism is proposed.     

Characterisation of arginase from the extreme thermophile 'Bacillus caldovelox'

A thermostable arginase (L-arginine amidinohydrolase, EC 3.5.3.1) was purified from the extreme thermophile 'Bacillus caldovelox' (DSM 411) by a procedure including DEAE-Sepharose chromatography, and gel filtration, anion exchange and hydrophobic-interaction fast-protein liquid chromatography, with substantial retention of the metal ion cofactor. The purified enzyme is a hexamer with a subunit Mr of 31,000 +/- 2000 and contains greater than or equal to 1 Mn atom per subunit. Maximum activation on incubation with Mn2+ is 29%. Activity is optimal at pH 9 and at 60 degrees C the Km for arginine is 3.4 mM and Ki(ornithine) is 0.55 mM. Incubation in 0.1 M Mops/NaOH buffer (pH 7) causes rapid inactivation at 60 degrees C (t1/2 (half life) = 4.5 min) and individually 0.1 mM Mn2+ or 1 mg/ml BSA (bovine serum albumin) increase the t1/2 of arginase activity 4-fold, but combined they produce greater than 1000-fold increase and a t1/2 = 105 min at 95 degrees C. Aspartic acid and other species that bind Mn2+ can replace BSA, and it is suggested that arginase can be inactivated by free Mn2+. A strong chelating agent causes inactivation without subunit dissociation, but arginase dissociates rapidly at pH 2.5. Reassociation occurs at pH 9 and is unusual in that it does not require Mn2+.     

Purification and characterization of NADP+-linked isocitrate dehydrogenase from an alkalophilic Bacillus

We have succeeded in purifying to homogeneity a very labile NADP+-linked isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) from a strain of alkalophilic Bacillus, by a simple method, with an overall yield over 76% of the original activity. The molecular weight on Sephadex G-200 was around 90,000; and that by electrophoresis on SDS-polyacrylamide gels was about 44,000. The sedimentation coefficient (s020,w) and isoelectric point of the enzyme were determined to be 3.22 S and pH 4.7, respectively. The enzyme required Mn2+ for the reaction and for stability. The optimum pH for the reaction was in the range 7.8-8.4 at 30 degrees C; the optimum temperature at pH 8.0 was 75 degrees C; the activation energy of the reaction was 6.2 kcal/mol. The Km values for threo-Ds-isocitrate, DL-isocitrate, and NADP+ were 5.4 microM, 9.9 microM, and 7.3 microM, respectively. This enzyme was inhibited by NADPH, glyceraldehyde 3-phosphate, 3-phosphoglycerate, phosphoenol pyruvate, cis-aconitate, alpha-ketoglutarate, and oxaloacetate. In addition, it was subject to a concerted inhibition by a combination of glyoxylate and oxaloacetate, and also to a cumulative inhibition by nucleoside triphosphates.     

Factors affecting the manganese and iron activation of the phosphoenolpyruvate carboxykinase isozymes from rabbit.

Timed assays in which GTP and GDP were separated and quantitated by HPLC were developed and used to study the metal activation of the mitochondrial and cytosolic isozymes of phosphoenolpyruvate carboxykinase purified from rabbit liver. These assays allowed both directions of catalysis to be studied under similar conditions and in the absence of coupling enzymes. The mitochondrial enzyme is rapidly inactivated by preincubation with Fe2+, as had been shown previously for the cytosolic isozyme. The greatest activation by Fe2+ was obtained by adding micromolar Fe2+ immediately after enzyme to form the complete assay mixture that also contained millimolar Mg2+. In the direction of synthesis of OAA from Pep, the K0.5 values for Mn2+ and Fe2+ were in the 3-7 microM range when a nonchelating buffer, Hepes, was used. The buffer used strongly affected activation by Fe2+ at pH 7.4; activation was eliminated in the case of phosphate and K0.5 increased several-fold over that obtained with Hepes when imidazole was used. In non-chelating buffer, the pH optimum was near 7.4 for both isozymes and for both directions of catalysis. However, the near optimal pH range extended below 7.4 for the direction of oxaloacetate synthesis while the range was above 7.4 for Pep synthesis. In the direction of oxaloacetate synthesis: (1) Both isozymes required the presence of micromolar Mn2+ or Fe2+ in addition to millimolar Mg2+ in order to shown significant activity. (2) Fe2+ was as effective an activator as Mn2+ at pH 7 and below. In the direction of Pep synthesis: (1) Micromolar Mn2+ was a much better activator than Fe2+ at the higher pH values needed for optimal activity in this direction. (2) With increasing pH, decreasing activation was obtained with Fe2+ while the activity supported by Mg2+ alone increased. The results demonstrate the potential for regulation of either isozyme of Pep carboxykinase by the availability of iron or manganese.     

Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of the steady-state kinetic and proton relaxation rate properties of the Mn(II)-containing enzyme

The essential Zn(II) in bovine liver dihydropyrimidine amidohydrolase (DHPase) was removed by incubation with 2,6-dipicolinic acid and replaced with Mn(II). Electron paramagnetic resonance studies of Mn(II) binding show that there are four binding sites per tetramer, and the dissociation constant at pH 7.5 is 13.5 microM. The substitution of Mn(II) for Zn(II) increases the specific activity of the enzyme approximately sixfold but has only a small effect (twofold increase) on the Km for 5-bromo-5,6-dihydrouracil (BrH2Ura). The pH dependence of the catalytic properties of Mn(II)-DHPase is the same as for the Zn(II) enzyme (Lee, M., Cowling, R., Sander, E., and Pettigrew, D. (1986) Arch. Biochem. Biophys. 248, 368-378). The pH dependence is well described in terms of the ionization of a single group with a pK of about 6 in the free enzyme. The ionization of this group is required for catalytic activity. The substitution of Mn(II) for Zn(II) does not affect the pH dependence of DHPase catalysis and therefore strongly suggests that the ionizable group is an amino acid residue at or near the active site, rather than a metal-bound water molecule. The pH dependence of the enhancement of the paramagnetic effect of the DHPase-Mn complex on the relaxation rate of the solvent water protons also is well described in terms of the ionization of a single group with a pK of about 6. Ionization of the group which is involved in catalysis also perturbs the environment of the bound Mn(II). The ionization of the active site group does not affect the number of exchangeable water molecules but does affect the symmetry of the environment of the bound Mn(II) and its electron relaxation.     

Effect of protons on the amidase activity of human alpha-thrombin. Analysis in terms of a general linkage scheme

The amidase activity of human alpha-thrombin has been studied in the pH range 5.5 to 10, and at four different chloride concentrations from 5 mM to 1 M. The Michaelis-Menten constant, Km, shows a bell-shaped dependence over the pH range studied, with a minimum around pH 8. The pH dependence of the catalytic constant, kcat, shows multiple inflection points especially at low (less than 0.1 M) chloride concentrations, thereby implicating the existence of multiple catalytic forms of the enzyme. A general linkage scheme is proposed for the analysis of the effect of protons on thrombin amidase activity, and experimental data have globally been analysed over the entire pH range in terms of such a scheme. Four proton-linked ionizable groups seem to be involved in the control of thrombin amidase activity. Two of these groups change their pK value upon substrate binding to the enzyme and account for the pH dependence of Km. All four groups control the catalytic activity of the enzyme which decreases with increasing protonation. Chloride has little effect on Km, while kcat changes significantly at pH less than 8. This effect is due to an increased enzymatic activity of the highly protonated intermediates at high chloride concentrations, as well as to the pK shift of two proton-linked ionizable groups.     

Purification of intact and nicked forms of a zinc-containing, Mg2+-dependent, low Km cyclic AMP phosphodiesterase from bakers' yeast

A low Km cyclic AMP phosphodiesterase was purified to homogeneity from microsomes of bakers' yeast. "Intact" enzyme, purified from microsomes prepared in the presence of the protease inhibitor phenylmethylsulfonyl fluoride, had a specific activity of 0.6 mumol/min/mg of protein (30 degrees C, pH 8.0, 1 microM cyclic AMP), a pI of 6.65 +/- 0.15, and a molecular weight of 61,000 determined by gel electrophoresis in the presence of sodium dodecyl sulfate. Gel filtration of native enzyme suggested it is a monomer. When phenylmethylsulfonyl fluoride was omitted, a product ("nicked" enzyme) was obtained with a specific activity of 1.2 mumol/min/mg of protein, the same pI, and a similar amino acid composition; but gel electrophoresis now showed two bands, with molecular weights of 45,000 and about 17,000, together with a small amount of the 61,000 band. Apart from the higher specific activity of the nicked enzyme, no difference was found between the catalytic properties of the two enzyme forms. Between 40 nM and 1 microM cyclic AMP, an apparent Km of 170 nM was observed at pH 8.0, but at higher cyclic AMP concentrations (2-30 microM), Hofstee plots curved upwards. Cyclic deoxy-AMP was a substrate, but cyclic GMP was not and did not affect the activity towards cyclic AMP. Both enzyme forms contained tightly bound zinc. The metal chelators, 8-hydroxyquinoline and orthophenanthroline , caused progressive partial inactivation of the enzyme and a decrease in its affinity for cyclic AMP. Dialysis against Zn2+, Cu2+, Co2+, or Mn2+ (but not Mg2+ or Ni2+) reversed these changes.     

Comparison of biochemical properties of liver arginase from streptozocin-induced diabetic and control mice

Arginase activity is elevated in livers of diabetic animals compared to controls and there is evidence that this is due in part to increased specific activity (activity/mg arginase protein). To investigate the molecular basis of this increased activity, the physicochemical and kinetic properties of hepatic arginase from diabetic and control mice were compared. Two types of arginase subunits with molecular weights of 35,000 and 38,000 were found in both the diabetic and control animals and the subunits in these animals had similar, multiple ionic forms. Kinetic parameters of purified preparations of arginase for arginine (apparent Km and Vmax values) and the thermal stability of these preparations from diabetics and controls were also similar. Furthermore, no difference was found in the distribution of arginase activity among different subcellular liver fractions. Separation of basic and acidic oligomeric forms of arginase by fast-protein liquid chromatography resulted in a slightly different distribution of activity among the forms in the normal and diabetic group. The apparent Km values for Mn2+ of the basic form of the enzyme were 25 and 33 microM for the enzyme from normal and diabetic animals, respectively; for acidic forms, for which two apparent Km values were measured, the values were 8 and 197 microM for arginase from controls and 35 and 537 microM from diabetics. These results indicate that in diabetes, while no marked changes in the physicochemical characteristics of arginase are obvious, some changes are found in the interaction of arginase with its cofactor Mn.     

Interaction of bacteriophage lambda protein phosphatase with Mn(II): evidence for the formation of a [Mn(II)]2 cluster.

The interaction of bacteriophage lambda protein phosphatase with Mn2+ was studied using biochemical techniques and electron paramagnetic resonance spectrometry. Reconstitution of bacteriophage lambda protein phosphatase in the presence of excess MnCl2 followed by rapid desalting over a gel filtration column resulted in the retention of approximately 1 equiv of Mn2+ ion bound to the protein. This was determined by metal analyses and low-temperature EPR spectrometry, the latter of which provided evidence of a mononuclear high-spin Mn2+ ion in a ligand environment of oxygen and nitrogen atoms. The Mn2+-reconstituted enzyme exhibited negligible phosphatase activity in the absence of added MnCl2. The EPR spectrum of the mononuclear species disappeared upon the addition of a second equivalent of Mn2+ and was replaced by a spectrum attributed to an exchange-coupled (Mn2+)2 cluster. EPR spectra of the dinuclear (Mn2+)2 cluster were characterized by the presence of multiline features with a hyperfine splitting of 39 G. Temperature-dependent studies indicated that these features arose from an excited state. Titrations of the apoprotein with MnCl2 provided evidence of one Mn2+ binding site with a micromolar affinity and at least one additional Mn2+ site with a 100-fold lower affinity. The dependence of the phosphatase activity on Mn2+ concentration indicates that full enzyme activity probably requires occupation of both Mn2+ sites. These results are discussed in the context of divalent metal ion activation of this enzyme and possible roles for Mn2+ activation of other serine/threonine protein phosphatases.     

On the fidelity of DNA replication: manganese mutagenesis in vitro

Manganese is mutagenic in vivo and in vitro in studies with a variety of enzymes and templates. Using Escherichia coli DNA polymerase I with poly[d(A-T)] and phi X174 DNA templates, we analyzed the mechanism of manganese mutagenesis by determining the dependence of error rate on free Mn2+ concentration and comparing this to measured dissociation constants of Mn2+ from enzyme, template, and deoxynucleoside triphosphate substrates. This comparison suggests several conclusions: (1) At very low Mn2+ concentrations, the enzyme is activated at high fidelity. Thus, it is unlikely that activation with manganese per se significantly alters the conformation of the enzyme so as to affect nucleotide selection. (2) At low free Mn2+ concentrations (less than 100 microM), manganese causes errors in incorporation via its interaction with the DNA template. The concentration dependence of mutagenesis is determined by the strength of binding Mn2+ to the particular DNA template used. The data do not allow one to rule out the possibility that Mn2+-deoxynucleoside triphosphate interactions contribute to mutagenesis in selected situations. This range of free Mn2+ concentrations is the one of greatest relevance for in vivo mutagenesis. (3) At higher concentrations (between 500 microM and 1.5 mM), further mutagenesis by Mn2+ occurs. This mutagenesis probably is due either to binding of manganese to single-stranded regions within the DNA or to weak accessory sites on the enzyme.     

NADP-dependent isocitrate dehydrogenase from bass (Dicentrarchus labrax L.) liver

The isocitrate dehydrogenase from bass liver was purified to homogeneity by gel filtration, affinity and ion exchange chromatographies. The molecular weight was estimated by gel filtration chromatography to about 120,000. Analysis of the enzyme on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed it to be a dimeric protein. The enzyme showed maximum activity in the pH range between 7.0 and 8.0 while its maximum activity was at pH 7.5. DL-Isocitrate and Mn2+ stabilized the enzyme, while NADP had the opposite effect. The Km for isocitrate was 0.31 mM and the Km for NADP was 36 microM.     

Assay and kinetics of arginase

A sensitive colorimetric assay for arginase was developed. Urea produced by arginase was hydrolyzed to ammonia by urease, the ammonia was converted to indophenol, and the absorbance was measured at 570 nm. The assay is useful with low concentrations of arginase (0.5 munit or less than 1 ng rat liver arginase) and with a wide range of arginine concentrations (50 microM to 12.5 mM). Michaelis-Menten kinetics and a Km for arginine of 1.7 mM were obtained for Mn2+-activated rat liver arginase; the unactivated enzyme did not display linear behavior on double-reciprocal plots. The kinetic data for unactivated arginase indicated either negative cooperativity or two types of active sites on the arginase tetramer with different affinities for arginine. The new assay is particularly well suited for kinetic studies of activated and unactivated arginase.     

Isolation and characterization of NADP+-linked isocitrate dehydrogenase of germinating pea seeds (Pisum sativum)

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.     

The divalent cation dependence of bovine brain calmodulin-dependent phosphatase

The divalent cation dependence of a calmodulin-stimulated phosphatase from bovine brain has been characterized kinetically using phosphorylated myelin basic protein and casein as substrates. At saturating concentrations of calmodulin, dephosphorylation of both myelin basic protein and casein was catalyzed 8- to 10-fold more rapidly at saturating concentrations of Mn2+ than at saturating concentrations of Ca2+. Half-maximal rates of dephosphorylation of both substrates occurred at either 15 microM Mn2+ or 1 microM Ca2+, and the Kact for each ion was not influenced appreciably by the presence of calmodulin. Half-maximal rates of dephosphorylation were observed at concentrations of calmodulin ranging from 3 X 10(-8) to 10(-6) M at saturating concentrations of divalent cations depending on the substrate used and the particular cation chosen. Trypsin treatment of the phosphatase activated the enzyme several-fold, eliminated its calmodulin dependence, but did not alter the Mn2+ concentration dependence of the activity. Ca2+ (10 microM) increased dephosphorylation rates without altering the Mn2+ concentration dependence of the phosphatase activity regardless of the presence of calmodulin. Mg2+ at millimolar concentrations did not alter the Ca2+ or Mn2+ concentration dependence of the activity. As measured without calmodulin, Ca2+ (90 microM) or Mn2+ (200 microM) produced nearly identical alterations of the far ultraviolet circular dichroic spectrum of the phosphatase.     

Activation of yeast enolase by Cd(II)

Activation of yeast enolase by Cd2+ exhibits properties similar to activation by the physiological cofactor Mg2+. The activity is weakly stimulated, then inhibited by increasing ionic strength. The activity increases, then falls with increasing Cd2+ concentration. The effect of pH on activity produced by Cd2+ is very similar to that produced by Mg2+, except that the Cd2+ profile is shifted one pH unit to more alkaline values, and the maximum activity of the Cd2+-enzyme is about 10% of that of the Mg2+-enzyme. The apparent kinetic parameters of Cd2+ activation show little effect of pH except for inhibition by high concentrations of Cd2+: the apparent Ki increases sharply with pH. This is interpreted as the result of Cd2+ being a less effective "catalytic" metal ion, and Cd2+ being more effective in stabilizing the enzyme at alkaline pH's. The similarity of effects of ionic strength, divalent cation, and pH may be due to interaction with the same six sites per mole of enzyme. We also characterized the dependence of what is believed to be the enzyme-catalyzed enolization of a substrate analog, D-tartronate semialdehyde-2-phosphate (TSP) on similar parameters of pH, ionic strength, etc. The putative enolization is dependent on catalytic metal ion, although the TSP binds to the conformational Cd2+-enzyme complex. The reaction is very slow and very pH dependent, increasing with pH with a midpoint of reaction velocity at pH 8.7. There is a strong qualitative correlation between pH dependencies of reaction velocity of substrate conversion and TSP enolization and absorbance of the enzyme-bound TSP enolate, whether with Mg2+ or Cd2+ as cofactor. The slowness of the Cd2+-TSP reaction is not limited by proton release or any reaction involving covalent bonds to hydrogen. The apparent reaction rate constant increases linearly with Cd2+ concentration. Addition of excess ethylenediaminetetraacetic acid reverses the TSP reaction, but again very slowly. The binding of Cd2+ to the catalytic sites is characterized by low association and dissociation rate constants.     

Submicromolar manganese dependence of Golgi vesicular galactosyltransferase (lactose synthetase)

1. Manganese(II) buffers were set up with inorganic triphosphate, trimethylenediaminetetraacetate and tetramethylenediaminetetraacetate to study the Mn dependence of beta 1,4-galactosyltransferase (lactose synthetase) in preparations of rat mammary gland. 2. In intact particulate preparations, treated with the calcium ionophore A23187, lactose synthesis was abolished by chelators and restored by bivalent transition metal ions in a manner characteristic of activation site I of the pure enzyme. Ni(II) also activated, as did Mg at high concentration. 3. Only Mn(II) could restore endogenous rates, giving an apparent Km of 0.1-0.2 microM, and eliciting about 70% full activity without addition of a site II activator. 4. In purified Golgi membrane vesicles, Mn gave an apparent Km of 0.4 microM. This increased sharply to about 10 microM on permeabilization with filipin, lysis with detergents, solubilization with Triton X-100, or in the pure enzyme. Preparations of chemically undamaged Golgi vesicles, known to include a proportion of the enzyme on exposed membranes, exhibited both low-Km and high-Km components. 5. The response of particulate galactosyltransferase to apparently physiological concentrations of free Mn(II) ion is interpreted as either due to a sensitizing factor within the Golgi lumen, or to the accumulation of Mn at elevated concentrations. Alternatively, the high Km of the soluble enzyme may reflect proteolytic damage.