Characterization of Mg2+-ATPase from sheep kidney medulla: purification.

Magnesium-dependent adenosine triphosphatase has been purified from sheep kidney medulla plasma membranes. The purification, which is based on treatment of a kidney plasma membrane fraction with 0.5% digitonin in 3 mm MgCl₂, effectively separates the Mg²⁺-ATPase from (Na⁺ + K⁺)-ATPase present in the same tissue and yields the Mg²⁺-ATPase in soluble form. The purified enzyme is activated by a variety of divalent cations and trivalent cations, including Mg²⁺, Mn²⁺, Ca²⁺, Co²⁺, Fe²⁺, Zn²⁺, Eu³⁺, Gd³⁺, and VO²⁺. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme shows two bands with R_f values corresponding to molecular weights of 150,000 and 77,000. The larger peptide is phosphorylated by [γ-³²P]ATP, suggesting that this peptide may contain the active site of the Mg²⁺-ATPase. The Mg²⁺-ATPase activity is unaffected by the specific (Na⁺ + K⁺)-ATPase inhibitor ouabain.     

Chick brain glutamine synthetase and Mn2+−Mg2+ interactions

Glutamine synthetase (GS) from the chick brain was purified to apparent homogeneity by ammonium sulfate fractionation followed by affinity chromatography, electrofocusing and Sephadex G-150 chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate analysis in polyacrylamide gel. By sedimentation equilibrium analysis and gel electrophoresis analysis, it was shown that the enzyme has a subunit molecular weight of 45,000 and a native molecular weight of 364,000, which is consistent with an octameric structure. Sedimentation analysis in the presence of Mg²⁺ revealed three different forms of macromolecules corresponding respectively to a monomer, a tetramer and an octamer. Among eight cations tested (Ca²⁺, Co²⁺, Fe²⁺, Li⁺, Mg²⁺, Mn²⁺, Ni²⁺, Zn²⁺) only Co²⁺, Mg²⁺ and Mn²⁺ supported GS activity; the order of activatory ability was Mg²⁺>Co²⁺>Mn²⁺. The maximum activating effect of Mn²⁺ occurs only within a very narrow range of concentration: with an excess of cation causing strong inhibition of GS activity. For each cation, maximal GS activity occurs at a defined cation/ATP ratio. A regulatory system in which Mn²⁺, modulates the Mg²⁺ dependent GS activity, is proposed; such cation interactions may be of significance in the intracellular control of glutamine synthesis.     

INFLUENCE OF DIVALENT CATIONS ON REGULATION OF CYCLIC GMP AND CYCLIC AMP LEVELS IN BRAIN TISSUE

—Depolarizing concentrations of K⁺ elevate levels of both adenosine 3′,5′monophosphate (cyclic AMP) and guanosine 3′,5′monophosphate (cyclic GMP) in incubated slices of mouse cerebellum. Calcium is an essential requirement for the K⁺ -induced accumulation of cyclic GMP. Barium and Sr²⁺, but not Mn²⁺ or Co²⁺, can substitute for Ca²⁺ in this process. Relatively high concentrations of Mg²⁺ inhibit the effect of Ca²⁺ on K⁺-induced accumulation of cyclic GMP. In contrast, depolarizing concentrations of K⁺ are capable of elevating cyclic AMP levels in brain slices suspended in media containing Mg²⁺ and no other divalent cations. High concentrations of Ca²⁺ (1 mm or greater) augment this Mg²⁺ -dependent, K⁺-induced accumulation of cyclic AMP, however. Strontium and Mn²⁺, but not Ba²⁺ or Co²⁺, can substitute for Ca²⁺ in this process, and high concentrations of Mg²⁺ are not inhibitory. The divalent cation ionophore, A-23187 (10 μm ), in the presence of extracellular Ca²⁺ elevates the level of cyclic GMP, but not cyclic AMP, in incubated mouse cerebellum slices. The results of this study indicate that intracellular Ca²⁺ concentration is a major factor regulating cyclic GMP levels in brain. In addition the present results suggest that, in brain tissue, depolarization-induced accumulation of cyclic GMP, but not cyclic AMP, is closely linked to some Ca²⁺-dependent mechanism(s) mediating release of intracellular substances.     

Effects of divalent metal ions on the calcium pump and membrane phosphorylation in human red cells

In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the divalent metal ions Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Fe²⁺. This activation is based on the formation of Me²⁺-ATP complexes which can serve as energy-donor substrates for the calcium pump, and probably, satisfy the requirement for free Me²⁺ in this transport process. Higher Me²⁺ concentrations inhibit calcium transport with various efficiencies. Mn²⁺ directly competes with Ca²⁺ at the transport site, while other divalent metal ions investigated have no such effect. The formation of the hydroxylamine-sensitive phosphorylated intermediate (EP) of the red cell membrane calcium pump from [γ-³²P]ATP is induced by Ca²⁺ while rapid dephosphorylation requires the presence of Mg²⁺. At higher concentrations Mn²⁺ and Ni²⁺ inhibit predominantly the formation of EP, while Co²⁺ and Fe²⁺ block dephosphorylation. The possible sites and nature of the divalent metal interactions with the red cell calcium pump are discussed. Hydroxylamine-insensitive membrane phosphorylation in inside-out vesicles from [γ-³²P]ATP is significantly stimulated by Mn²⁺ and Co²⁺, as compared to that produced by Mg²⁺, Fe²⁺ and Ni²⁺. Part of this labelling is found in phospholipids, especially in phosphatidylinositol. The results presented for the metal dependency of protein and lipid phosphorylation in red cell membranes may help in the characterization of ATP consumptions directly related to the calcium pump and those involved in various regulatory processes.     

Regulation of rat brainstem tryptophan 5-monooxygenase. Calcium-dependent reversible activation by ATP and magnesium.

Tryptophan 5-monooxygenase in rat brainstem cytosol was activated about twofold by incubation with 0.5 mm ATP and 5 mm MgCl₂. The activation required micromolar concentrations of Ca²⁺ but was not dependent on either cyclic AMP or cyclic GMP. Rat brain cytosol was shown to possess an endogenous protein kinase which was markedly stimulated by the addition of Ca²⁺ using endogenous protein substrates. Following activation by ATP and Mg²⁺ in the presence of Ca²⁺, tryptophan 5-monooxygenase was reversibly deactivated to the original level by incubation at 30 °C after removal of Ca²⁺ by adding ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid and was then reactivated by incubation at 30 °C after subsequent addition of Ca²⁺ and ATP. The deactivation was markedly inhibited by the omission of Mg²⁺ or by the addition of NaF.     

Characterization of calmodulin-mediated phosphorylation of cardiac muscle sarcoplasmic reticulum

Sarcoplasmic reticulum, isolated from canine cardiac muscle, was phosphorylated in the presence of exogenous cAMP-dependent protein kinase or calmodulin. This phosphorylation has been shown previously to activate sarcoplasmic reticulum calcium uptake (LePeuch et al. (1979) Biochemistry18, 5150–5157). Calmodulin appeared to activate an endogenous protein kinase present in sarcoplasmic reticulum membranes. The incorporation of phosphate increased with time. However, once all the ATP was consumed, the level of phosphorylated protein started to decrease due to the action of an endogenous protein phosphatase. Dephosphorylation occurred even when the level of phosphorylated sarcoplasmic reticulum remained constant at high ATP concentrations. The phosphorylation of sarcoplasmic reticulum in the presence of calmodulin, increased as the pH was increased from pH 5.5 to 8.5. This phosphorylation was only inhibited by KCl concentrations greater than 100 mm. The apparent K_m of cAMP-dependent protein kinase for ATP was 5.2 ± 0.2 × 10^?5m, and of the calmodulin-dependent protein kinase for ATP was 3.67 ± 0.29 × 10^?5m. Phosphorylation was maximally activated by 5–10 mm MgCl₂; higher MgCl₂ concentrations inhibited this phosphorylation. Thus the calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum could be maximally activated at sarcoplasmic concentrations of K⁺, Mg²⁺, and ATP. The calmodulindependent phosphorylation was half-maximally activated at Ca²⁺ concentrations that were significantly greater than those required to promote the formation of the sarcoplasmic reticulum Ca-activated ATPase phosphoprotein intermediate. Thus at sarcoplasmic Ca²⁺ concentrations that might be expected during systole, the sarcoplasmic reticulum calcium pump would be fully activated before any significant calmodul-independent sarcoplasmic reticulum phosphorylation occurred. However, under certain pathological conditions when the sarcoplasmic Ca²⁺ becomes elevated (e.g., in ischemia) the kinase could be activated so that the sarcoplasmic reticulum would be phosphorylated and calcium uptake augmented. Thus, the calmodulin-dependent protein kinase may only function when the heart needs to rescue itself from a possibly fatal calcium overload.     

Studies on the interaction of metal ions with puruvate kinase from Ehrlich ascites-tumour cells and from rabbit muscle

1. Pyruvate kinase (ATP–pyruvate phosphotransferase, EC 2.7.1.40) from Ehrlich ascites-tumour cells was purified approximately fivefold by chromatography on DEAE-cellulose. The enzyme was shown to have an absolute requirement for one univalent and for one bivalent metal ion. 2. The univalent metal ion requirements were satisfied by K⁺, Rb⁺ or NH₄⁺; Na⁺ and Cs⁺ were weak activators but Li⁺ was inactive. 3. Ca²⁺ exhibited `non-competitive' and `apparent competitive' effects in relation to the K⁺ activation. 4. The bivalent metal ion requirements were satisfied by Mg²⁺, Mn²⁺ or Co²⁺; Ba²⁺, Sr²⁺, Ca²⁺, Ni²⁺, Be²⁺ and Cu²⁺ were inactive. Mn²⁺ and Co²⁺ were better activators than Mg²⁺. 5. The bivalent metal ion requirements of purified pyruvate kinase from rabbit muscle were satisfied by Mg²⁺, Mn²⁺, Co²⁺ and to a smaller extent by Ni²⁺. Mn²⁺ and Co²⁺ were better activators than Mg²⁺. 6. Ca²⁺ competitively inhibited the activation by Mg²⁺, Mn²⁺ and Co²⁺ for both the tumour and rabbit enzymes. 7. It is concluded that there are no significant differences in metal ion specificity between the tumour and rabbit enzymes. 8. The possible role of metal ions in regulating enzymic and metabolic activities is considered further.     

Glutamine synthetase of pea leaves: divalent cation effects, substrate specificity, and other properties

Purified glutamine synthetase from pea seedlings was most active with Mg²⁺ as the metal activator, but Mn²⁺ and Co²⁺ were 45 to 60% and 30 to 45% as effective, respectively, when assayed at the optimal pH for each cation. The Mg²⁺ saturation curve was quite sigmoid, and evidence indicates that MgATP is the active ATP substance. Co²⁺ also gave a sigmoidal saturation curve, but when Mn²⁺ was varied only slightly sigmoidal kinetics were seen. Addition of Mn²⁺, Ca²⁺, or Zn²⁺ at low concentrations sharply inhibited the Mg²⁺ -dependent activity, partially by shifting the pH optimum. Addition of Co²⁺ did not inhibit Mg²⁺-dependent activity. The nucleotide triphosphate specificity changed markedly when Co²⁺ or Mn²⁺ replaced Mg²⁺. Using the Mg²⁺-dependent assay, the Michaelis constant (Km) for NH₄⁺ was about 1.9 × 10⁻³ M. The Km for l-glutamate was directly proportional to ATP concentration and ranged from 3.5 to 12.4 mm with the ATP levels tested. The Km for MgATP also varied with the l-glutamate concentration, ranging from 0.14 mm to 0.65 mm. Ethylenediaminetetracetic acid activated the enzyme by up to 54%, while sulfhydryl reagents gave slight activation, occasionally up to 34%.     

Characterization of Mg2+-ATPase from sheep kidney medulla: magnetic resonance and kinetic studies.

Magnesium-dependent adenosine triphosphatase, purified from sheep kidney medulla using digitonin, has been characterized in a series of kinetic and magnetic resonance studies. Kinetic studies of divalent metal activation using either Mg²⁺ or Mn²⁺ indicate a biphasic response to divalent cations. Apparent K_m values of 23 μm for free Mg²⁺ and 3.3 μm for free Mn²⁺ are obtained at low levels of added metal, while K_m values of 0.50 mm for free Mg²⁺ and 0.43 mm for free Mn²⁺ are obtained at much higher levels of divalent cations. In all cases the kinetic data indicate that the binding of divalent metals is independent of the substrate, ATP. Kinetic studies of the substrate requirements of the Mg²⁺-ATPase also yield biphasic Lineweaver-Burk plots. At low ATP concentrations, kinetic studies yield apparent K_m values for free ATP of 6.0 and 1.4 μm with Mg²⁺ and Mn²⁺, respectively, as the activating divalent metals. At much higher levels of ATP the response of the enzyme to ATP changes so that K_m values for free ATP of 8.0 and 2.0 mm are obtained for Mg²⁺ and Mn²⁺, respectively. In both cases, however, the binding of ATP is independent of added metal. ADP inhibits the Mg²⁺-ATPase and the kinetic data indicate that ADP competes with ATP at both the high and low affinity sites. Dixon plots of the data are consistent with competitive inhibition at both ATP sites, with K_i values of 10.5 μm and 4.5 mm. Electron paramagnetic resonance and water proton relaxation rate studies show that the enzyme binds 1 g ion of Mn²⁺ per 469,000 g of protein. The Mn²⁺ binding studies yield a K_D for Mn²⁺ at the single high affinity site of 2 μm, in good agreement with the kinetically determined activator constant for Mn²⁺ at low Mn²⁺ levels. Moreover, the EPR binding studies also indicate the existence of 34 weak sites for Mn²⁺ per single high affinity Mn²⁺ site. The K_D for Mn²⁺ at these sites is 0.55 mm, in good agreement with the kinetic activator constant for Mn²⁺ of 0.43 mm, consistent with additional activation of the enzyme by the large number of weaker metal binding sites. The enhancement of water proton relaxation by Mn²⁺ in the presence of the enzyme is also consistent with the tight binding of a single Mn²⁺ ion per 469,000 M_r protein and the weaker binding of a large number of divalent metal ions. Analysis of the data yields a value for the enhancement for bound Mn²⁺ at the single tight site, ?_b, of 5 and an enhancement at the 34 weak sites of 11. The frequency dependence of water proton relaxation by Mn²⁺ at the single tight site yields a dipolar correlation time (constant from 8–60 MHz) of 3.18 × 10^?9 s. The kinetics and metal binding studies, together with the effect of temperature on ATPase activity at high and low levels of ATP, are consistent with the existence in this preparation of a single Mg²⁺-ATPase, with high and low affinity sites for divalent metals and for ATP. Observations of both high and low affinities for ATP have been made with two other purified ATPases. The similarities of these systems to the Mg²⁺-ATPase described here are discussed.     

Control of flagellar motility in Euglena and Chlamydomonas. Microinjection of EDTA, EGTA, Mn(2)+, and Zn(2)+.

When a Euglena, in a medium containing ATP, is microinjected with 7 × 10^?14 l of 0.02 M EDTA, which binds Ca²⁺ and Mg²⁺, flagellar motility stops. Flagellar arrest in Chlamydomonas occurs with the injection of 2 × 10^?14 l of 0.02 M EDTA. The injection of similar amounts (7 × 10^?14 l in Euglena and 3 × 10^?14 l in Chlamydomonas) of 0.02 M EGTA, which preferentially binds Ca²⁺, did not significantly alter flagellar motility. This suggests that a decrease in the internal Ca²⁺ concentration in Euglena or Chlamydomonas did not stimulate flagellar beating. Further, flagellar motility decreased when internal Mg²⁺ was chelated. The microinjection of Zn²⁺ into these cells caused a decrease in flagellar frequency analogous to the decrease in frequency caused by the injection of Ca²⁺ and EDTA. The microinjection of 7 × 10^?14 l of 0.2 M Mn²⁺ caused an approx. 1.5-fold increase in Euglena flagellar motility. Chlamydomonas flagella, which cease to beat upon impalement in an Mg²⁺-free medium, resume a flagellar frequency of 18 Hz when injected with 3 × 10^?14 l of 0.2 M Mn²⁺. In the experiments reported here, Mn²⁺ acts as an analog of Mg²⁺.     

Characterization of cAMP-dependent protein kinase from the pupal wing epidermis of Manduca sexta

Cyclic AMP-dependent protein kinase (cAMP-PK) activity in the wing epidermis of day zero pupae of Manduca sexta was characterized. The preferred exogenous substrates were histones, subfractions H1 and H2b, casein and protamine sulfate; histone H2a was only phosphorylated moderately, while free base protamine and bovine serum albumin were poor substrates for cAMP-PK. cAMP-PK activity required Mg²⁺ and was optimal in the presence of 1 mM Mg²⁺. Co²⁺ and Mn²⁺ did not substitute for Mg²⁺, and Ca²⁺ inhibited cAMP-PK activity. The effective concentration of cAMP for activation of the cAMP-PK was substantially lower than that of cGMP (EC₅₀ 1.3 × 10⁻⁸ and 1.2 × 10⁻⁶ M, respectively). The type II isozyme of cAMP-PK comprised approx. 75% of the total cytosolic wing cAMP-PK as determined by DEAE anion exchange chromatography. Photoaffinity labeling of the whole cell homogenate with 8-azido cAMP revealed the presence of only type II isozyme. The distribution of the cAMP-PK isozymes was also determined for whole cell homogenates of brain, prothoracic glands, hemolymph, trachea, nerve cord, fat body, muscle and midgut.     

Purification and some properties of Δ2-isopentenylpyrophosphate:5′AMP Δ2-isopentenyltransferase from the cellular slime mold Dictyostelium discoideum

Δ²-Isopentenylpyrophosphate:5′AMP Δ²-isopentenyltransferase, which catalyzes the formation of isopentenyl-AMP from Δ²-isopentenylpyrophosphate and 5′AMP, was purified 6800-fold from the fruiting body of the cellular slime mold Dictyostelium discoideum using several separation procedures including 5′AMP^ox-redAH-Sepharose 4B affinity column chromatography. The final preparation was very unstable and lost its activity in a day. Various properties of the 1000-fold-purified enzyme preparation were examined. The molecular mass was 40,000 ± 2000 Da, as determined by Sephadex G-100 superfine gel filtration. The divalent metal ions Mn²⁺, Zn²⁺, and Mg²⁺ profoundly affected the enzymatic activity depending on their concentration, and also altered the optimum pH and temperature. Of the compounds tested, 5′AMP was the best acceptor of the isopentenyl group and, interestingly, ADP also served as a substrate, being 60–80% as effective as 5′AMP. Adenine, adenosine, and ATP were not substrates for this enzyme. Under the optimum assay conditions (pH 7.0, 1 mm Zn²⁺, and 25 °C) the K_m values for 5′AMP and Δ²-isopentenylpyrophosphate were 1.0 × 10^?7m and 2.2 × 10^?6m, respectively.     

Manganese enhances the phosphorylation of membrane-associated proteins isolated from barley roots

Microsomal membranes isolated from barley roots (Hordeum vulgare L. cv. CM72) contained endogenous protein phosphorylation activities that were greatly enhanced by Mn²⁺. Mg²⁺ions also stimulated protein phosphorylation, but to a lesser extent than Mn²⁺. Ca²⁺ enhanced Mg²⁺, but not Mn²⁺-dependent phosphorylation. It is proposed that this strong enhancement by Mn²⁺ may be due to a greater affinity of Mn²⁺ than either Ca²⁺ or Mg²⁺ for both the Ca²⁺ and Mg²⁺ binding sites of certain kinases. Some Mn²⁺ stimulated kinase activity was eliminated from the membrane by washing with 0.2 mol/L KCl. The KCl extract contained histone and casein kinase activities, and 4 major phosphoproteins that were phosphorylated on serine and threonine residues. Phosphorylation of a 52 kDa polypeptide corresponded with the characteristics of the histone kinase activity and may represent the autophosphorylation of a CDPK-type kinase. Phosphorylation of a 36 kDa polypeptide was Ca²⁺ stimulated and may represent the autophosphorylation of a different type of unknown kinase. Polypeptides of 18 and 15 kDa had characteristics that suggest they were autophosphorylating subunits of a membrane bound nucleotide di-phosphokinase.     

Calcium and adenosine affect human sperm adenylate cyclase activity

The adenylate cyclase activity of human ejaculated spermatozoa in broken-cell preparations was investigated. In the presence of 5 mM metal cations and 0.1 mM ATP, the relative enzyme activity with Mn²⁺, Ca²⁺, Mg²⁺, Ba²⁺ was 1.00, 0.28, 0.22, and 0.03, respectively. Added Ca²⁺ appeared to activate the enzyme in the presence of Mn²⁺ or Mg²⁺. The human sperm adenylate cyclase was stimulated by ～ 2-fold by free Ca²⁺ (lmM) in the presence of Mg²⁺ (5 mM). If the GTP analogue, 5′-guanylyl imidophosphate (Gpp(NH)p) was added to the sperm homogenate in the presence of 200 μM ethylene-glycol-bis (β-aminoethylether) N,N′-tetraacetic acid (EGTA), the adenylate cyclase activity was increased by approximately 25%, but with the addition of 280 μM Ca²⁺ there was a decrease in enzyme activity. A similar response to low concentrations of Ca²⁺ was obtained after complementation of the sperm enzyme with the guanine nucleotide regulatory component from human erythrocytes, where the addition of 40 μM Gpp(NH)p, 200 μM EGTA, and Ca²⁺ (≤ 160 μM) stimulated the sperm enzyme ～ 3–4-fold, but the further addition of Ca²⁺ (280 μM, final) neutralized the stimulatory effect. The addition of adenosine, and the nucleotides 5′-AMP and 5′-ADP inhibited the enzyme, whereas guanine and 5′-GMP had no appreciable effect. Human follicular fluid and serum also had little direct effect on the sperm adenylate cyclase. These resuls suggest that Ca²⁺ might be an important physiological modulator of the human sperm adenylate cyclase.     

Phosphoinositol kinase from germinating mung bean seeds

Phosphoinositol kinase (adenosine triphosphate-inositolmonophosphate phospho—tranferase) has been isolated from cotyledons; about 300-fold purification has been achieved, with a recovery of 11%. The enzyme has a pH optimum at 7·4. It can mediate phosphorylation of lower inositol phosphates to their corresponding higher homologues, ATP being the phosphate donor. ATP can be replaced partially by UTP and PEP. The enzyme requires divalent cations for the reaction. Mn²⁺ has been found to be twice as effective as Mg²⁺, Ca²⁺ being inhibitory. Phosphoinositol kinase has been found to be different from inositol kinase.     

Biochemical characterization of histamine-sensitive adenylate cyclase in mammalian brain.

Certain biochemical characteristics of an adenylate cyclase that is activated by low concentrations of histamine (K_a, 8 μm) and that is present in cell-free preparations from the dorsal hippocampus of guinea pig brain have been studied. Histamine increased the maximal reaction velocity of adenylate cyclase without altering the K_m (0.18 mm) for its substrate, MgATP. Increasing concentrations of free Mg²⁺ stimulated enzymatic activity; the kinetic properties of this activation by Mg²⁺ suggest the existence of a Mg²⁺ allosteric site on the enzyme. Histamine increased the affinity of this apparent site for free Mg²⁺. Free ATP was a competitive inhibitor with respect to the MgATP substrate. The apparent potency of free ATP as an inhibitor increased in the presence of histamine. In the presence of Mg²⁺, low concentrations of Ca²⁺ markedly inhibited adenylate cyclase activity; half-maximal inhibition of both basal and histamine-stimulated enzyme activity occurred at 40 μm Ca²⁺. Other divalent cations, including Zn²⁺, Cu²⁺, and Cd²⁺, were also inhibitory. Of the divalent cations tested, only Co²⁺ and Mn²⁺ could replace Mg²⁺ in supporting histamine-stimulated adenylate cyclase activity. The nucleoside triphosphates GTP and ITP increased basal adenylate cyclase activity and markedly potentiated the stimulation by histamine. Preincubation of adenylate cyclase with 5′-guanylylimidodiphosphate dramatically increased enzyme activity; in this activated state, the adenylate cyclase was relatively refractory to further stimulation by histamine or F^?. The subcellular distribution of histamine-sensitive adenylate cyclase activity was studied in subfractions from guinea pig cerebral cortex. The highest total and specific activities were observed in those fractions enriched in nerve endings, while adenylate cyclase activity was not detectable in the brain cytosol fraction. A possible physiological role for this histamine-sensitive adenylate cyclase in neuronal function is discussed.     

Divalent metal ion effects on a mutant histidinol phosphate phosphatase from Salmonella typhimurium

The effect of divalent metal ions on the activity of a mutant histidinol phosphate phosphatase has been studied. The enzyme was isolated from strain TA387, a mutant of Salmonella typhimurium with a nonsense lesion near the midpoint of the bifunctional hisB gene. Mn²⁺, Mg²⁺, Co²⁺, and Zn²⁺ shift the optimal pH of phosphatase activity to 6.5 while Be²⁺ and Ca²⁺ have no effect on the shape of the pH profile. In the absence of divalent metal ions, the pH optimum is 7.5. Four Me²⁺ ions, Mn²⁺, Co²⁺, Zn²⁺, and Fe²⁺ decreased the K_m of histidinol phosphate at pH 6.5 from 5.5 mm (without Me²⁺) to 0.14 mm. Ni²⁺ and Be²⁺ increased the K_m to 22.2 and 25.0 mm, respectively, and Ca²⁺ and Mg²⁺ had an intermediate effect. Changes in maximal velocity were substantially less, only about 2-fold changes being observed. It was shown that the maximal velocity at optimal pH was the same in the absence and presence of Mn²⁺. Kinetic analysis indicated that there was a rapid equilibrium-ordered addition of Mn²⁺ to the enzyme before the addition of the substrate, histidinol phosphate. A k_imn²⁺ of 4.3 μm was calculated for the metal ion activation at both pH 6.5 and 7.5. Addition of ethyl-enediaminetetracetate (EDTA) strongly inhibited the phosphatase; inhibition could be reversed by addition of several Me²⁺ ions, Mg²⁺ being the most efficient followed by Mn²⁺. Prolonged incubation with EDTA led to irreversible inactivation.     

Catalytic properties of d-xylose isomerase from Streptomyces violaceoruber

The catalytic properties and stability of d-xylose isomerase from Streptomyces violaceoruber have been studied. The enzyme was activated by Mg²⁺, Co²⁺ and Mn²⁺ but Ni²⁺, Ca²⁺, Zn²⁺, Cu²⁺ and Hg were ineffective. Optimum catalytic conditions were obtained at 80°C in the pH range 7.5–9.5 and in the presence of 10 mm Mg²⁺. The specific activity of the enzyme increased after treatment with 10 mm EDTA (factor 2.4). A further increase of activity (factor 2.0–2.8) was observed after preincubation of the enzyme with Mg²⁺ or Co²⁺, the preincubation time depending on the incubation temperature. The thermal stability of the enzyme is very high. At 60°C the enzyme retained optimum activity following 30 days of storage in the presence of 1 mm Co²⁺ or 10 mm Mg²⁺. At 80°C, Co²⁺ is superior as a protector against thermal denaturation. At saturating concentrations of Mg²⁺ (35°C) the K_m-values of the EDTA-treated enzyme with respect to d-xylose and d-glucose were 2.8 and 149 mm and the dissociation constants of the enzyme-Mg²⁺ complex for xylitol and d-sorbitol were 0.455 and 4.47 mm, respectively.     

Some properties of a deoxyribonuclease isolated from Aspergillus niger

A deoxyribonuclease, isolated from the mycelia of Aspergillus niger, grown as surface cultures on a liquid medium, was partially purified and had an optimum pH of 9.5 and an optimum temperature of 37°C. The enzyme was strongly activated by Mg²⁺ while Mn²⁺, Ca²⁺ or Co²⁺ activated the enzyme to lesser extents. Thiol reagents, mercaptoethanol and dithiothreitol (DTT) activated the enzyme. S-Adenosylmethionine at low concentration (2.8?5.0 × 10^?2 mM) activated the enzyme but at a higher concentration (11.5 × 10^?2 mM) and above it inhibited the enzyme. The effect of EDTA on the enzyme was variable. The enzyme had both ATP-dependent and independent activities, the former usually being higher. ATP could be replaced by CTP or GTP. The nucleoside diphosphates ADP, CDP and GDP could replace ATP but they were not as effective as the triphosphates.     

Purification and characterization of human lung calmodulin-independent cyclic AMP phosphodiesterase

A high-affinity calmodulin-independent cyclic AMP phosphodiesterase was purified to homogeneity from human lung tissue. This enzyme has a molecular weight of 60,000, a sedimentation coefficient of 3.2–3.4 S, and an isoelectric pH of 4.6–4.8. Neither Ca²⁺ nor calmodulin (in the presence or absence of added Ca²⁺) stimulates the enzymatic activity. This enzyme appears to be very similar to that described previously from dog kidney (W. J. Thompson, P. M. Epstein, and S. J. Strada, (1979) Biochemistry18, 5228–5237). Hydrolysis of cyclic AMP is greatly enhanced by Mg²⁺ (25–30× at 10 mm Mg²⁺) and Mn²⁺ (20× at 10 mm Mn²⁺). Zn²⁺, Cu²⁺, and Co²⁺ are ineffective at these concentrations. Cyclic AMP is the exclusive substrate with a K_m of 0.7–0.8 μm. The I₅₀ of cyclic GMP is 1 mm using 1 μm cyclic AMP as substrate. In contrast, aminophylline, MIX, and SQ 20009 have I₅₀s of 0.28, 0.021, and 0.001 mm, respectively). The purified enzyme is susceptible to temperature inactivation and protease degradation. Significant (10%) inhibition is seen at 37 °C for 20 min. Trypsin, at 0.1 μg/ml, destroys 50% of the activity in 30 min at 25 °C. Our observations concerning its lability to temperature and proteases coupled with its lack of response to calmodulin suggest this enzyme is a basic catalytic subunit of other cyclic AMP phosphodiesterases present within human lung tissue.