Induction of hepatic and renal ornithine decarboxylase by cobalt and other metal ions in rats.

We previously showed that Cd2+ is able to induce hepatic and renal ornithine decarboxylase (ODC). In addition to Cd2+, the administration of Co2+ and other metal ions such as Se2+, Zn2+ and Cr2+ produced a significant increase of hepatic and/or renal ODC activity. Of the metal ions used in this study, Co2+ produced the greatest increase of ODC activity. The maximum increases in hepatic and renal ODC activity, to respectively 70 and 14 times the control values in male rats, were observed 6 h after the administration of Co2+. A similar response was seen in the liver, but not in the kidney, of female rats. Thereafter, ODC activity gradually returned to control values in the liver, but it was profoundly decreased to 7% of the control value at 24 h in the kidney. The pretreatment of animals with either actinomycin D or cycloheximide almost completely blocked the Co2+-mediated increase of ODC activity. Co2+ complexed with either cysteine or glutathione (GSH) failed to induce ODC. Depletion of hepatic GSH content by treatment of rats with diethyl maleate greatly enhanced the inducing effect of Co2+ on ODC. The inhibitors of ODC, 1,3-diaminopropane and alpha-difluoromethylornithine, were able to inhibit the induction of the enzyme, without affecting the induction of haem oxygenase by Co2+. Methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, significantly inhibited the Co2+-mediated induction of both ODC and haem oxygenase. It is suggested that the inducing effects of Co2+ on ODC and haem oxygenase are brought about in a similar manner.     

Partial purification of alkaline phosphatase from bovine polymorphonuclear neutrophils and some properties

Alkaline phosphatase was purified from bovine polymorphonuclear neutrophils by butanol extraction and a combination of ion exchange, gel filtration and affinity chromatography. The enzyme was partially purified 2300-fold with a 4.7% yield and a sp. act. of 206 units/mg of protein. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a single activity band with the mol. wt of 165,000. The pH optima for the enzyme were 10.0 with p-nitrophenylphosphate and phenylphosphate and were 9.0 when beta-glycerophosphate, AMP and ADP were used. The enzyme was activated by Mg2+, Mn2+, Co2+ and Ni2+ but was inhibited by Zn2+. The enzyme was inhibited by EDTA and the EDTA-inactivated enzyme was reactivated by Mg2+, Mn2+ and Co2+ but not Zn2+.     

Stimulation of mouse liver corticosteroid side chain isomerase by cobaltous and nickelous ions: evidence for an endogenous inhibitor of isomerase activity

Corticosteroid side chain isomerase of mouse liver cytosol was stimulated by Co2+ and Ni2+. The magnitude of stimulation increased with incubation time. For Co2+ and Ni2+, respective enhancements were 2.8- and 4.0-fold at 15 min and 3.9- and 5.0-fold at 60 min. The relationship between steroid substrate concentration (11-deoxy-[21-3H]corticosterone) and initial velocity was consistent with a model in which the cations reacted with a cytosol inhibitor of isomerase activity. Enzyme, partially purified by ammonium sulfate fractionation and gel filtration, had a 6.8-fold increased specific activity. Co2+ and Ni2+ enhanced the activity of partially purified enzyme 1.6- and 1.9-fold. Unlike the cytosol, stimulation was achieved without lag and was not altered by prolonged incubation. Metal ion chelating agents did not have a consistent effect on the activity of the partially purified enzyme. Cyanide and alpha,alpha-dipyridyl increased, and dithizone and 8-hydroxyquinoline decreased activity. The data are not consistent with the hypothesis that side chain isomerase is a metalloenzyme. It is concluded that Co2+ and Ni2+ stimulate the enzyme by removing an endogenous inhibitor.     

2,2-dipyridyl binding to metal substituted horse liver alcohol dehydrogenase

The binding of 2,2-dipyridyl to metal substituted horse liver alcohol dehydrogenase was measured by spectrophotometric titrations. Large changes in the visible absorption spectra were seen for the Co2+, Cu2+ and Ni2+ hybrids upon coordination of 2,2-dipyridyl, due to a change in coordination number. The formation constants for binding to the Co2+ and Cd2+ hybrids are of the order 10(6) M-1, which means that these hybrids have a 500-fold higher affinity for 2,2-dipyridyl than the native Zn2+ enzyme. 2,2-dipyridyl has a 100-fold higher affinity for enzyme bound Cd2+ than for aqueous Cd2+ ions, while for Cu2+ and Zn2+ the opposite is the case. None of the substituted metal ions were removed from the active site during titration with the chelator 2,2-dipyridyl.     

Metal ion activation of S-adenosylmethionine decarboxylase reflects cation charge density

S-Adenosylmethionine decarboxylase from Escherichia coli is a pyruvoyl cofactor-containing enzyme that requires a metal cation for activity. We have found that the enzyme is activated by cations of varying charge and ionic radius, such as Li+, A13+, Tb3+, and Eu3+, as well as the divalent cations Mg2+, Mn2+, and Ca2+. All of the activating cations provide kcat values within 30-fold of one another, showing that the charge of the cation does not greatly influence the rate-limiting step for decarboxylase turnover. Cation concentrations for half-maximal activation decrease by >100-fold with each increment of increase in the cation charge, ranging from approximately 300 mM with Li+ to approximately 2 microM with trivalent lanthanide ions. The cation affinity is related to the charge/radius ratio of the ion for those ions with exchangeable first coordination sphere ligands. The exchange-inert cation Co(NH3)63+ activates in the presence of excess EDTA (and NH4+ does not activate), indicating that direct metal coordination to the protein or substrate is not required for activation. The binding of metal ions (monitored by changes in the protein tryptophan fluorescence) and enzyme activation are both cooperative with Hill coefficients as large as 4, the active site stoichiometry of this (alphabeta)4 enzyme. The Hill coefficients for Mg2+ binding and activation increase from 1 to approximately 4 as the KCl concentration increases, which is also observed with NaCl or KNO3; neither Na+ nor K+ activates the enzyme. The single tryptophan in the protein is located 16 residues from the carboxyl terminus of the pyruvoyl-containing alpha chain, in a 70-residue segment that is not present in metal ion independent AdoMet decarboxylases from other organisms. The results are consistent with allosteric metal ion activation of the enzyme, congruent with the role of the putrescine activator of the mammalian AdoMet decarboxylase.     

Substitution studies of the second divalent metal cation requirement of protein tyrosine kinase CSK

In addition to a magnesium ion needed to form the ATP-Mg complex, we have previously determined that at least one more free Mg2+ ion is essential for the activation of the protein tyrosine kinase, Csk [Sun, G., and Budde, R. J. A. (1997) Biochemistry 36, 2139-2146]. In this paper, we report that several divalent metal cations, such as Mn2+, Co2+, Ni2+, and Zn2+ bind to the second Mg2+-binding site of Csk with up to 13200-fold higher affinity than Mg2+. This finding enabled us to substitute the free Mg2+ at this site with Mn2+, Co2+, Ni2+, or Zn2+ while keeping ATP saturated with Mg2+ to study the role of the free metal cation in Csk catalysis. Substitution by these divalent metal cations resulted in varied levels of Csk activity, with Mn2+ even more effective than Mg2+. Co2+ and Ni2+ supports reduced levels of Csk activity compared to Mg2+. Zn2+ has the highest affinity for the second Mg2+-binding site of Csk at 0.65 microM, but supports no kinase activity, acting as a dead-end inhibitor. The inhibition by Zn2+ is reversible and competitive against free Mg2+, noncompetitive against ATP-Mg, and mixed against the phosphate accepting substrate, polyE4Y, significantly increasing the affinity for this substrate. Substitution of the free Mg2+ with Mn2+, Co2+, or Ni2+ also results in lower Km values for the peptide substrate. These results suggest that the divalent metal activator is an important element in determining the affinity between Csk and the phosphate-accepting substrate.     

The effect of Mn2+ and Co2+ on the activities of a zinc metallodipeptidase from a mouse ascites tumor.

Kinetic studies of the effect of addition of Co2+ or Mn2+ to a highly purified dipeptidase from Ehrlich-Lettré mouse ascites tumor cells show that these metals specifically activate the hydrolyses of certain classes of dipeptides. This enzyme was previously (S. Hayman and E. K. Patterson, 1971, J. Biol. Chem. 246, 660) reported to be a Zn-metalloenzyme. It is now shown that Zn is the only metal that can partially restore the activity of the EDTA-inhibited dipeptidase in cleaving Ala-Gly. Addition of Co2+ increases the Vmax of N-terminal Gly-dipeptides with increase in Km while addition of Mn2+ primarily activates the hydrolysis of Pro-Gly, again with increases in both Vmax and Km. Prior incubation (5 min, 30 degrees) of the dipeptidase with the appropriate metal ions causes decrease in initial lag time in the Co2+-activated hydrolysis of Gly-Gly and the Mn2+-activated hydrolysis of Pro-Gly. Long-term (6-19 hr, 0 degrees) incubation of the enzyme with Co2+ results in loss of activity toward Ala-Gly with a concomitant 13-fold increase in the rate of Gly-Gly hydrolysis and loss of 70% of the Zn2+ from the dipeptidase; these effects can be partially reversed by addition of Zn2+. In contrast, long-term incubation of the enzyme with Mn2+ results in no loss of Zn2+ and a twofold increase in activity toward Pro-Gly. One affinity constant of 1.4 muM for Co2+ and two constants of 0.23 and 27 muM for Mn2+ were determined by kinetic experiments. Comparison of the properties of this tumor enzyme with a dipeptidase purified in our laboratory from Escherichia coli B, and with mammalian dipeptidases highly purified by others, shows remarkable similarities in molecular weights and molecular activities toward the preferred substrates but in the case of bacterial dipeptidase, differences in substrate specificities and in the effect of metal ions.     

Induction of mouse epidermal ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate: dependence on calcium availability

The role of calcium in epidermal ornithine decarboxylase (ODC) induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) was determined in adult mouse skin pieces incubated in serum-free minimal essential medium (MEM). Addition of TPA to skin pieces incubated in serum-free MEM, which contains 1.82 mM Ca2+ and 0.83 mM Mg2+, resulted in about a 200-fold increase in epidermal ODC activity at about 8 h after TPA treatment. TPA failed to induce epidermal ODC in skin pieces incubated in calcium-free medium. Similarly, chelation of extracellular calcium by ethyleneglycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented ODC induction by TPA, which could be resumed upon calcium restoration in the medium. Furthermore, calcium ionophore A23187, which facilitates efflux of Ca2+ across cellular membranes, induced ODC activity in incubated skin pieces. Epidermal ODC activity increased by TPA appears to be the result of an increase in both the amount of ODC protein and the level of hybridizable ODC messenger. Inhibition of the induction of ODC activity by EGTA was the result of the inhibition of the amount of active ODC protein and the level of ODC mRNA.     

Estrogen- and antiestrogen-induced ornithine decarboxylase activity and uterine growth in the rat

The estrogen antagonists tamoxifen and monohydroxytamoxifen are also classified as partial estrogen agonists. In infantile rats, estradiol induced a single peak of uterine ODC activity at 6h following injection regardless of the extent of induction by various estradiol doses. By contrast, the timing of the ODC activity peak induced by tamoxifen and monohydroxytamoxifen was highly dependent upon the dosing conditions and was delayed to 18 h at lower tamoxifen doses. In immature rats, tamoxifen and monohydroxytamoxifen induced two peaks of uterine ODC activity resembling those induced by estradiol. Both ODC activity peaks were delayed by 9 h, without decreases in peak heights, by a 50-fold tamoxifen dose reduction. In all experiments the initial appearance of antiestrogen- and estradiol-induced ODC activity corresponded to initial uterine wet weight gain regardless of dosing condition. Thus, when dose-related temporal shifts are taken into account, tamoxifen and monohydroxytamoxifen are complete agonists with respect to induction of uterine weight gain and ODC activity.     

Early alterations of polyamine metabolism induced after acute administration of clenbuterol in mouse heart.

An acute treatment of mice with clenbuterol, a beta-adrenergic agonist, produced a marked increase of polyamines levels in heart, particularly during the early phase of administration of the drug. A single dose of 1.5 mg/kg caused as much as a 10 fold induction in activity of ornithine decarboxylase (ODC) and 3 to 4 fold increase in levels of putrescine, spermidine and spermine in mouse heart. Maximum changes were observed 3 to 4 hours post-administration of clenbuterol. This treatment did not produce any change in S-adenosylmethionine decarboxylase activity. The induction of cardiac ODC by clenbuterol was also dose dependent with a peak at about 5 micromol/kg. Co-administration of difluoromethylornithine, an irreversible inhibitor of ODC, or propranolol, a nonspecific beta-antagonist, with clenbuterol completely prevented the induction of ODC activity as well as the increase in polyamine levels in heart. However, pretreatment with alprenolol or metoprolol, the specific beta1 and beta2-antagonists, respectively, produced only partial prevention. The cardiac ODC from controls as well as clenbuterol treated mice exhibited similar affinity (Km) for its substrate, ornithine, while maximum enzyme activity (Vmax) was about 14 fold higher in clenbuterol treated mouse heart than in the control. Clenbuterol produced no change in the level of specific ODC mRNA or the protein, but the enzyme from the drug-treated mouse heart was considerably more stable than the control. Pretreatment of mice with either cycloheximide or actinomycin D followed by administration of clenbuterol could not prevent the induction in ODC activity suggesting that de novo biosynthesis of the enzyme protein or ODC mRNA was not responsible for induction of ODC activity. Post-translational changes in ODC may be responsible for an early increase of ODC activity due to clenbuterol treatment.     

Regulation of the L-lactase dehydrogenase from Lactobacillus casei by fructose-1,6-diphosphate and metal ions.

The lactate dehydrogenase of Lactobacillus casei, like that of streptococci, requires fructose-1,6-diphosphate (FDP) for activity. The L. casei enzyme has a much more acidic pH optimum (pH 5.5) than the streptococcal lactate dehydrogenases. This is apparently due to a marked decrease in the affinity of the enzyme for the activator with increasing pH above 5.5; the concentration of FDP required for half-maximal velocity increase nearly 1,000-fold from 0.002 mM at pH 5.5 to 1.65 mM at 6.6. Manganous ions increase the pH range of activity particularly on the alkaline side of the optimum by increasing the affinity for FDP. This pH dependent metal ion activation is not specific for Mn2+. Other divalent metals, Co2+, Cu2+, Cd2+, Ni2+, Fe2+, Fe2+, and Zn2+ but not Mg2+, will effectively substitute for Mn2+, but the pH dependence of the activation differs with the metal ion used. The enzyme is inhibited by a number of commonly used buffering ions, particularly phosphate, citrate, and tris (hydroxymethyl) aminomethane-maleate buffers, even at low buffer concentrations (0.02 M). These buffers inhibit by affecting the binding of FDP.     

Comparative kinetics of D-xylose and D-glucose isomerase activities of the D-xylose isomerase from Thermus aquaticus HB8

The D-xylose isomerase from T. aquaticus accepts, besides D-xylose, also D-glucose, and, with lower efficiency, D-ribose, and D-arabinose as alternative substrates. The activity of the enzyme is strictly dependent on divalent cations. Mn2+ is most effective in the D-xylose isomerase reaction and Co2+ in the D-glucose isomerization. Mg2+ is active in both reactions, Zn2+ only in the further one. The enzyme is strongly inhibited by Cu2+, and weakly by Ni2+, Fe2+, and Ca2+. A hyperbolic dependence of the reaction velocity of the D-xylose isomerase on the concentration of D-xylose xylose and of D-glucose was found, while biphasic saturation curves were obtained by variation of the metal ion concentrations. The D-glucose isomerization reaction shows normal behaviour with respect to the metal ions. A kinetic model was derived on the basis of the assumption of two binding sites for divalent cations, one cofactor site with higher affinity and a second, low affinity site, which modulates the activity of the enzyme.     

Calcium ion activation of rabbit liver alpha 1,2-mannosidase

Rabbit liver alpha 1,2-mannosidase is a calcium ion requiring enzyme involved in processing the asparagine-linked oligosaccharides of glycoproteins. Ca2+ activation occurs with an apparent Ka of 1.1 microM. The major effect of the metal ion activator is on Km rather than Vmax. The kinetic mechanism of the enzyme is that of an ordered equilibrium in which Ca2+ must bind before substrate and the metal ion cannot release once the substrate has added to the enzyme. Several other divalent cations including Co2+, Mn2+, and Zn2+ were competitive with Ca2+ and inhibited the enzyme. Significantly, Mg2+ had no effect on enzyme activity. 1-Deoxymannojirimycin and Tris, which inhibit glycoprotein processing in vivo, are inhibitors of the mannosidase competitive with substrate. The effect of Ca2+ on the affinity of the enzyme for substrate may be a determinant in regulation of enzyme activity in vivo.     

Interaction of EGTA with a hydrophobic region inhibits particulate adenylate cyclase from rat cerebral cortex: a study of an EGTA-inhibitable enzyme by using alamethicin

Washed membranes isolated from rat cerebral cortex (gray matter) showed the presence of EGTA-inhibitable and EGTA-insensitive forms of adenylate cyclase activity. The former activity was stimulated by low concentrations (microM) of various divalent cations (Mn2+, Ca2+, Co2+ and Sr2+) assayed with MgATP2- and MgCl2. At higher concentrations (mM), only Mn2+ stimulated this enzyme whereas Ca2+, Co2+ and Sr2+ were inhibitory. Alamethicin markedly (up to 30-fold) increased the activity of EGTA-inhibitable form and only moderately of EGTA-insensitive form of the enzyme. The increased activity due to alamethicin does not result from solubilization of the enzyme from membranes. Our results suggest the presence of two distinct metal binding sites--one of high (Site I) and other of low (Site II) affinity. Divalent metals via interacting with these produce divergent effects on the enzyme. Site I appears to be located in the hydrophobic region of catalytic unit of the enzyme or of membrane-associated calmodulin. The likely significance of these results is briefly presented.     

Determination of the metal ion dependence and substrate specificity of a hydratase involved in the degradation pathway of biphenyl/chlorobiphenyl

BphH is a divalent metal ion-dependent hydratase that catalyzes the formation of 2-keto-4-hydroxypentanoate from 2-hydroxypent-2,4-dienoate (HPDA). This reaction lies on the catabolic pathway of numerous aromatics, including the significant environmental pollutant, polychlorinated biphenyls (PCBs). BphH from the PCB degrading bacterium, Burkholderia xenoverans LB400, was overexpressed and purified to homogeneity. Atomic absorption spectroscopy and Scatchard analysis reveal that only one divalent metal ion is bound to each enzyme subunit. The enzyme exhibits the highest activity when Mg2+ was used as cofactor. Other divalent cations activate the enzyme in the following order of effectiveness: Mg2+ > Mn2+ > Co2+ > Zn2+ > Ca2+. This differs from the metal activation profile of the homologous hydratase, MhpD. UV-visible spectroscopy of the Co2+-BphH complex indicates that the divalent metal ion is hexa-coordinated in the enzyme. The nature of the metal ion affected only the kcat and not the Km values in the BphH hydration of HPDA, suggesting that cation has a catalytic rather than just a substrate binding role. BphH is able to transform alternative substrates substituted with methyl- and chlorine groups at the 5-position of HPDA. The specificity constants (kcat/Km) for 5-methyl and 5-chloro substrates are, however, lowered by eight- and 67-fold compared with the unsubstituted substrate. Significantly, kcat for the chloro-substituted substrate is eightfold lower compared with the methyl-substituted substrate, showing that electron withdrawing substituent at the 5-position of the substrate has a negative influence on enzyme catalysis.     

Further purification and some properties of a gelatin-specific proteinase of human leucocytes

A latent gelatin-specific proteinase (gelatinase) was isolated from the crude extract of human leukocytes. The enzyme was purified about 180-fold (7040 units/mg) with an overall yield of 23%. The isolated protein migrated as a single band on sodium dodecyl sulfate polyacrylamide-gel electrophoresis. Its mobility was unaffected by reducing agent. The protein band corresponded to approx. 90-94 kDa. Gelatinase activity was strongly inhibited by chelating agents, such as EDTA and 1,10-phenanthroline. This inhibition was reversed by Zn2+ and Co2+; other metal ions were less or not at all effective in reversing the inhibition. Moreover, Co2+ stimulated gelatinase activity. These results indicate that Zn2+ and/or Co2+ are essential for the activity of this gelatinase.     

Two-step purification of mouse kidney ornithine decarboxylase

We developed a simple two-step purification procedure for ornithine decarboxylase (ODC, EC 4.1.1.17), consisting of DEAE-Cellulofine chromatography and affinity chromatography on a HO-101 monoclonal anti-rat liver ODC antibody-Affi-Gel 10 column. By this method, ODC was purified 1700-fold to homogeneity with about 80% yield from the kidney of ICR mice treated with testosterone enanthate. The final specific activity range between 1.0 x 10(6)-1.4 x 10(6) nmol/h.mg protein. On SDS-polyacrylamide gel electrophoretic analysis, the final preparations gave a major protein band of Mr 54,000 and a minor band of Mr 51,000. Although relative staining intensity of the two bands varied depending on preparations, both bands could be stained by immunoblotting and labeled by a preincubation with [14C]difluoromethylornithine (DFMO). On Oudin double diffusion immunoanalysis, a single fused precipitin line was formed between purified anti-mouse kidney ODC IgG and both the purified enzyme and crude mouse kidney extract. In contradiction with earlier reports, no significant difference was observed between mouse kidney ODC and rat liver ODC in either final specific activity or specific binding of labeled DFMO.