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%.     

A model for the regulation of brain adenylate cyclase by ionic equilibria

Multiple-equilibrium equations were solved to investigate the individual and separate effects of Mg²⁺, Mn²⁺, Ca²⁺, ATP^4–, and their complexes on the kinetics of brain adenylate cyclase. The effects of divalent metals and/or ATP^4– (in excess of their participation in complex formation) were determined and, from the corresponding apparent affinity values, the following kinetic constants were obtained:K _m(MgATP)=1.0 mM,K _i(ATP^4–)=0.27 mM,K _m(MnATP)=0.07 mM, andK _i(CaATP)=0.015 mM. MgATP, MnATP, ATP^4–, and CaATP were shown to compete for the active site of the enzyme. Hence, it is proposed that endogenous metabolites with a strong ligand activity for divalent metals, such as citrate and some amino acids, become integrated into a metabolite feedback control of the enzyme through the release of ATP^4– from MgATP. Ca²⁺ fluxes may participate in the endogenous regulation of adenylate cyclase by modifying the level of CaATP. The free divalent metals show an order of affinityK _0.5(Ca²⁺)=0.02 mM,K _0.5(Mn²⁺)=3.8 mM,K _0.5(Mg²⁺)=4.7 mM, and an order of activity Mn²⁺>Mg²⁺>Ca²⁺. The data indicate that Mn²⁺ and Mg²⁺ ions may compete for a regulatory site distinct from the active site and increaseV _m without changingK _m(MgATP),K _m(MnATP), orK _i(ATP^4–). The interactions of ATP^4– and CaATP, which act as competitive inhibitors of the reaction of the enzyme with the substrates MgATP and MnATP, and Mg²⁺ and Mn²⁺, which act as activators of the enzyme in the absence of hormones, are shown to follow the random rapid equilibrium BiBi group-transfer mechanism of Cleland with the stipulation that neither Mg²⁺ nor Mn²⁺, in excess of their respective participation in substrate formation, are obligatorily required for basal activity. ATP^4– and CaATP are involved in dead-end inhibition. For MgCl₂ saturation curves at constant total ATP concentration, the computer-generated curves based on the RARE BiBi model predict a change in the Hill cooperativityh from a basal value of 2.6, when Mg²⁺ is not obligatorily required, to 4.0 when the addition of hormones or neurotransmitters induces an obligatory requirement for Mg²⁺.Abbreviations used: Me, divalent metal; Me_T (Mg_T or Mn_T), total Me (Me²⁺ and its complexes); ATP_T, total ATP (ATP^4– and its complexes).     

Kinetic and Structural Properties of NADP-Malic Enzyme from Sugarcane Leaves

Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP⁺, and Mg²⁺ were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (K_m = 58 micromolar), but also inhibition by high substrate (K_i = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (K_m = 120 micromolar). At both pH conditions, activity response to NADP⁺ exhibited Michaelis-Menten behavior with K_m values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg²⁺ suggested the presence of at least two Mg²⁺ - binding sites with different affinity. The K_a values for Mg²⁺ obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg²⁺ levels could be important for the physiological regulation of NADP-malic enzyme.     

Ferrochelatase of Rhodopseudomonas spheroides

Extracts of Rhodopseudomonas spheroides contain two ferrochelatases: one is soluble and forms metalloporphyrins from deuteroporphyrin and haematoporphyrin; the other is particulate and forms metalloporphyrins from protoporphyrin, mesoporphyrin, deuteroporphyrin and haematoporphyrin. Neither enzyme incorporates Mg²⁺ into porphyrins or Fe²⁺ into porphyrin cytochrome c. By using the particulate enzyme, plots of 1/v versus 1/s when one substrate was varied and the other kept constant showed that neither substrate affected the K_m of the other. The suggested sequential mechanism for the reaction is supported by derivative plots of slopes and intercepts. The K_m for deuteroporphyrin was 21.3μm and that for Co²⁺ was 6.13μm. The enzyme incorporated Co²⁺, Fe²⁺, Zn²⁺, Ni²⁺ and Mn²⁺; Cd²⁺ was not incorporated and was an inhibitor, competitive with respect to Co²⁺, non-competitive with respect to deuteroporphyrin. The K_i for Cd²⁺ was 0.73μm. Ferrochelatase was inhibited by protohaem, non-competitively with respect to Co²⁺ or with respect to deuteroporphyrin. Inhibition by magnesium protoporphyrin was non-competitive with respect to deuteroporphyrin, uncompetitive with respect to Co²⁺. The inhibitory concentrations of the metalloporphyrins are lower than those required for the inhibition of δ-aminolaevulate synthetase by protohaem. Fe²⁺ is not incorporated aerobically into porphyrins unless an electron donor, succinate or NADH, is supplied; the low aerobic rate of metalloporphyrin synthesis obtained is insensitive to rotenone and antimycin. The rate of Fe³⁺ incorporation increases as anaerobic conditions are achieved.     

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.     

The Activity of Ribulose Diphosphate Carboxylase in Extracts of Gonyaulax polyedra in the Day and the Night Phases of the Circadian Rhythm of Photosynthesis

The ribulose 1,5-diphosphate carboxylase from Gonyaulax polyedra Stein. has a half-life of about four hours in buffer, but can be stabilized by the addition of 50% glycerol. The optimum pH is 7.8 to 8.0 and the optimum Mg²⁺ concentration is 3 mm. Heavy metal ions (Cu²⁺, Hg²⁺, Ni²⁺, Zn²⁺), EDTA, pyrophosphate, and adenosine triphosphate were strongly inhibitory. Ribulose 1,5-diphosphate carboxylase from Gonyaulax was not cold-sensitive or activated by light activation factor from tomato or Gonyaulax. No difference in the activity of this enzyme was detected when extracts prepared at the maximum and the minimum of the circadian rhythm of photosynthesis were compared. The Km of HCO₃⁻ was also the same (16 to 19 mm).     

Modified nucleoside-dependent transition metal binding to DNA analogs of the tRNA anticodon stem/loop domain

Biologically active DNA analogs of tRNA^Phe (tDNA^Phe) were used to investigate metal ion interaction with tRNA-like structures lacking the 2OH. Binding of Mg²⁺ to the 76 oligonucleotide tDNA^Phe, monitored by circular dichroism spectroscopy, increased base stacking and thus the conformational stability of the molecule. Mg²⁺ binding was dependent on a d(m⁵C) in the anticodon region. In contrast to Mg²⁺, Cd²⁺ decreased base stacking interactions, thereby destabilizing the molecule. Since alterations in the anticodon region contributed to most of the spectral changes observed, detailed studies were conducted with anticodon hairpin heptadecamers (tDNA_AC ^Phe). The conformation of tDNA_AC ^Phe-d(m⁵C) in the presence of 1 mm Cd²⁺, Co²⁺, Cr²⁺, Cu²⁺, Ni²⁺, Pb²⁺, VO²⁺ or Zn²⁺ differed significantly from that of the biologically active structure resulting from interaction with Mg²⁺, Mn²⁺ or Ca²⁺. Nanomolar concentrations of the transition metals were sufficient to denature the tDNA_AC ^Phe-d(m⁵C) structure without catalyzing cleavage of the oligonucleotide. In the absence of Mg²⁺ and at [Cd²⁺] to [tDNA_Ac ^Phe-d(m⁵C)] ratios of approximately 0.2–1.0, tDNA_AC ^Phe-d(m⁵C₄₀) formed a stable conformation with one Cd²⁺ bound with a K _d = 3.7 × 10^-7. In contrast to Mg²⁺, Cd²⁺ altered the DNA analogs without discriminating between modified and unmodified tDNA_AC ^Phe. This ability of transition metals to disrupt higher order DNA structures, and possibly RNA, at M concentrations, in vitro, demonstrates that these structures are potential targets in chronic metal exposure, in vivo.     

Subcellular localization,metal ion requirement and kinetic properties of arginase from the gill tissue of the bivalve Semele solida

Arginase activity (3.1 ± 0.5 units/g (wet wt) of tissue) was found associated to the cytosolic fraction of the gill cells of the bivalve Semele solida. The enzyme, with a molecular weight of 120,000 ± 3000, was partially purified, and some of the enzymic properties were were examined. The activation of the enzyme by Mn²⁺ followed hyperbolic kinetics with a K_Mn value of 0.10 ± 0.02 μM. In addition to Mn²⁺, the metal ion requirement of the enzyme was satisfied by Ni²⁺, Cd²⁺ and Co²⁺; Zn²⁺ was inhibitory to ail the Values of K_m for arginine and K_i for lysine inhibition, were the same, regardless of the metal ion used to activate the enzyme; K_m values were 20 mM at pH 7.5 and 12 mM at the optimum pH of 9.5. Competitive inhibition was caused by ornithine, lysine and proline, whereas branched chain amino acids were non competitive inhibitors of the enzyme.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase

Both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase were recovered mainly from the supernatant fractions of guinea-pig pancreas, but a higher proportion of the activity of the former was associated with the pellet fractions. The activities in the supernatant were not separated by gel filtration, but were clearly separated by subsequent chromatography on an anion-exchange resin. The activities of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase had high-affinity (K_m 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (K_m 0.56±0.05mm and 0.32±0.03mm respectively) components. The activity of neither enzyme was affected by the pancreatic secretogens, cholecystokinin-pancreozymin, secretin and carbachol. Removal of ions by gel filtration resulted in a marked reduction in cyclic nucleotide phosphodiesterase activity, which could be restored by addition of Mg²⁺. Mn²⁺ (3mm) was as effective as Mg²⁺ (3mm) in the case of cyclic AMP phosphodiesterase, but was less than half as effective in the case of cyclic GMP phosphodiesterase. The metal-ion chelators, EDTA and EGTA, also decreased activity. Ca²⁺ (1mm) did not affect the activity of cyclic nucleotide phosphodiesterase when the concentration of Mg²⁺ was 3mm. At concentrations of Mg²⁺ between 0.1 and 1mm, 1mm-Ca²⁺ was activatory, and at concentrations of Mg²⁺ below 0.1mm, 1mm-Ca²⁺ was inhibitory. These results are discussed in terms of the possible significance of cyclic nucleotide phosphodiesterase in the physiological control of cyclic nucleotide concentrations during stimulus–secretion coupling.     

Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster

dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40μm, gave a hyperbolic curve for activity versus dCMP concentration and a K_m value for dCMP of 0.91mm. dCTP activation required the presence of Mg²⁺ or Mn²⁺ ions. dTTP inhibited the deaminase maximally at 15μm; the inhibition required the presence of Mg²⁺ or Mn²⁺ ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v).     

Purification and properties of adenosine diphosphoglucose pyrophosphorylase from sweet corn

A 40-fold purification of adenosine diphosphoglucose pyrophosphorylase from sweet corn (Zea mays var. Golden Beauty) revealed the enzyme to be specific for adenosine triphosphate. The enzyme has an absolute requirement for Mg²⁺ and is activated by 3-phosphoglycerate and to a lesser extent by ribose-5-phosphate and fructose-6-phosphate. The apparent Km values of the enzyme for glucose-1-phosphate, adenosine triphosphate, pyrophosphate, and adenosine diphosphoglucose are 1.9 × 10⁻⁴, 3.2 × 10⁻⁵, 3.3 × 10⁻⁵, and 6.2 × 10⁻⁴m, respectively. Pyrophosphate inhibits adenosine diphosphoglucose synthesis competitively (Ki = 3.8 × 10⁻⁷m), while orthophosphate and sulfate appear to inhibit the reacion noncompetitively. These results show that the production of this sugar nucleotide can be controlled by the concentration of pyrophosphate.     

Uptake of Lead and Cadmium by Maize Seedlings and the Effect of Heavy Metals on the Activity of Phosphoenolpyruvate Carboxylase Isolated from Maize

Maize seeds and five-day-old maize seedlings were incubated in media containing Pb²⁺ at concentrations of 50, 100, and 200 mg 1^-1 and Cd²⁺ at concentrations of 1, 5, 10 and 50 mg 1^-1. After five days of incubation, both heavy metals were determined by means of AAS following wet mineralisation of roots and shoots. The results obtained indicate that Pb²⁺ were transported to shoots from roots at a lower rate than Cd²⁺. Phosphoenolpyruvate carboxylase (PEPC) isolated from germinating maize seeds was inhibited to a comparable degree by solutions containing 0.001 mmol 1^-1 Pb²⁺, 0.01 mmol 1^-1 Cd²⁺, and 0.005 mmol 1^-1 Cu²⁺. The enzyme was protected against this inhibition by the addition of mercaptoethanol, the substrate (PEP), or the cofactor (Mg²⁺). The inhibition increased during a 20 min incubation of the enzyme with salts of the metals. Mn²⁺, Ni²⁺, and Co²⁺ ions could partially substitute for the metal cofactor Mg²⁺. K_m values for these metal ions were as follows: for Mg²⁺ 0.07 mmol 1^-1 in the range from 0 to 0.30 mmol 1^-1 Mg²⁺; 0.71 mmol 1^-1 for 0.30 to 2.50 mmol 1^-1 Mg²⁺; for Mn²⁺ 0.36 mmol 1^-1; for Ni²⁺ 0.34 mmol 1{-1}; and for Co²⁺ 0.20 mmol 1^-1. The activity of the enzyme reached with Mn²⁺ 85 %, with Ni²⁺ 65 %, and with Co²⁺ 55 % of the activity recorded with Mg²⁺.     

Cd2+ activation of l-threonine dehydrogenase from Escherichia coli K-12

Homogeneous preparations of l-threonine dehydrogenase (l-threonine: NAD⁺ oxidoreductase, EC 1.1.1.103) from Escherichia coli K-12, after having been dialyzed against buffers containing Chelex-100 resin, have a basal level of activity of 10–20 units/mg. Added Cd²⁺ stimulates dehydrogenase activity approx. 10-fold; this activation is concentration-dependent and is saturable with an activation K_d = 0.9 μM. Full activation by Cd²⁺ is obtained in the absence of added thiols. The pH-activity profile of the Cd²⁺-activated enzyme conforms to a theoretical curve for one-proton ionization with a pK_a = 7.85. Mn²⁺, the only other activating metal ion, competes with Cd²⁺ for the same binding site. K_m values forl-threonine and NAD⁺ as well as the V_max for ‘demetallized’, Cd²⁺-activated, and Mn²⁺-activated threonine dehydrogenase were determined and compared.     

Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum

Cytosolic NADP-specific isocitrate dehydrogenase was isolated from leaves of Pisum sativum. The purified enzyme was obtained by ammonium sulfate fractionation, ion exchange, affinity, and gel filtration chromatography. The purification procedure yields greater than 50% of the total enzyme activity originally present in the crude extract. The enzyme has a native molecular weight of 90 kilodaltons and is resolved into two catalytically active bands by isoelectric focusing. Purified NADP-isocitrate dehydrogenase exhibited K_m values of 23 micromolar for dl-isocitrate and 10 micromolar for NADP, and displayed optimum activity at pH 8.5 with both Mg²⁺ and Mn²⁺.     

5-Oxoprolinase (l-Pyroglutamate Hydrolase) in Higher Plants: Partial Purification and Characterization of the Wheat Germ Enzyme

5-Oxoprolinase has been found to be widely distributed in higher plants. This enzyme catalyzes the ATP-dependent hydrolysis of 5-oxo-l-proline (l-pyrollidone carboxylate, l-pyroglutamate) to glutamate. The enzyme has been purified almost 60 fold from wheat germ (Triticum aestivum L). This enzyme requires a divalent cation, either Mn²⁺ or Mg²⁺, and a combination of both appears to be the most effective. There is also an absolute requirement for a monovalent cation best fulfilled by either NH₄⁺ or K⁺. The K_m for ATP is 0.4 mm and for 5-oxo-l-proline is 14 μm. A small amount of activity is observed when other purine nucleotides such as ITP and GTP replace ATP. The substitution of the pyrimidine nucleotides CTP and UTP for ATP yield almost completely inactive preparations. The enzyme appears to have an active sulfhydryl group since there is an increase in activity in the presence of dithioerythritol. Preincubation with reagents such as N-ethylmaleimide or iodoacetamide lead to complete inactivation. The presence of this enzyme leads to the speculation of the possible presence of a γ-glutamyl cycle in higher plants.     

Partial Purification and Properties of d-Glucosamine 6-Phosphate N-Acetyltransferase from Phaseolus aureus

d-Glucosamine-6-P N-acetyltransferase (EC 2.3.1.4) from mung bean seeds (Phaseolus aureus) was purified 313-fold by protamine sulfate and isoelectric precipitation, ammonium sulfate and acetone fractionation, and CM Sephadex column chromatography. The partially purified enzyme was highly specific for d-glucosamine-6-P. Neither d-glucosamine nor d-galactosamine could replace this substrate. The partially purified enzyme preparation was inhibited up to 50% by 2 × 10⁻²m EDTA, indicating the requirement of a divalent cation. Among divalent metal ions tested, Mg²⁺ was required for maximum activity of the enzyme. Mn²⁺ and Zn²⁺ were inhibitory, while Co²⁺ had no effect on the enzyme activity. The pH optimum of the enzyme in sodium acetate and sodium citrate buffers was found to be 5.2. The effect of Mg²⁺ on the enzyme in sodium acetate and sodium citrate buffers was particularly noticeable in the range of optimum pH. Km values of 15.1 × 10⁻⁴m and 7.1 × 10⁻⁴m were obtained for d-glucosamine-6-P and acetyl CoA, respectively. The enzyme was completely inhibited by 1 × 10⁻⁴mp-hydroxymercuribenzoate, and this inhibition was partially reversed by l-cysteine; indicating the presence of sulfhydryl groups at or near the active site of the enzyme.     

Characterization of microsomal ATPases from developing human placenta

Activities and some properties of microsomal ATPases have been studied in developing human placenta. The enzyme activities (Na⁺ + K⁺ + Mg²⁺, Mg²⁺, and Ca²⁺ dependent) in the placenta increase steadily with gestational age until the 18th to 21st week, and decrease in the second half of pregnancy. Mg²⁺-dependent and Na⁺ + K⁺ + Mg²⁺-dependent ATPases possess nearly the same K_m (apparent) for ATP, while the Ca²⁺-dependent enzyme shows a different one. Mg²⁺-dependent ATPase shows higher substrate affinity than Ca²⁺-dependent ATPase, although the V_max of the Mg²⁺-dependent enzyme is lower than that of the latter. However, for each enzyme, the K_m remains almost constant and V_max varies during ontogenic development. V_max of the enzymes decline at term. The enzymes are heat-labile, unaffected by amino acids, namely, l-phenylalanine, l-leucine, and l-tryptophan, and deoxycholate inhibits the enzyme activities by about 50%.     

The interaction of zinc, nickel and cadmium with serum albumin and histidine-rich glycoprotein assessed by equilibrium dialysis and immunoadsorbent chromatography

Human serum albumin (HSA) has been shown to bind 2–3 mol of Zn²⁺, Ni²⁺, or Cd²⁺ per mole of protein with apparent dissociation constants (K_d) in the range of 10 μm. Rabbit histidine-rich glycoprotein (HRG) binds 13, 9, and 6 mol of Zn²⁺, Ni²⁺, and Cd²⁺ per mole of protein, respectively, with apparent K_ds also near 10 μm. However, the binding of metals by HRG exhibits positive cooperativity, so that the apparent K_ds may underestimate HRGs true affinity for metal ions. The relative affinities of HSA and HRG for metal ions were found to be Zn²⁺ > Ni²⁺ > Cd²⁺. In addition, histidine (a serum metal chelator) affected the binding of Ni²⁺ by both proteins but not that of Zn²⁺ or Cd²⁺. At physiological concentrations of HSA (250 μm), HRG (2.5 μm), and histidine (100 μm), HRG bound 36% of the Zn²⁺, 9% of the Ni²⁺, and 13% of the Cd²⁺ at a total metal concentration of 25 μm. Under the same conditions HSA held 37% of the Zn²⁺, 14% of the Ni²⁺, and 56% of the Cd²⁺. Thus, HSA appears to have a lower intrinsic affinity for the three metals than HRG but would be expected to bind a higher proportion of these metals in serum. A specific immunoadsorbent column was prepared and used to study the metal binding by HRG in serum directly. Both ⁶⁵Zn²⁺ and ⁶³Ni²⁺ were associated with HRG in aliquots of rabbit serum after incubation with the corresponding metal ion. This evidence indicates that HRG must be considered as a metal binding component of serum.     

Differential effects of heavy metal ions on Ca2+-dependent K+ channels

Summary 1. The ability of various divalent metal ions to substitute for Ca²⁺ in activating distinct types of Ca²⁺-dependent K⁺ [K⁺(Ca²⁺] channels has been investigated in excised, inside-out membrane patches of human erthrocytes and of clonal N1E-115 mouse neuroblastoma cells using the patch clamp technique. The effects of the various metal ions have been compared and related to the effects of Ca²⁺.2. At concentrations between 1 and 100 µM Pb²⁺, Cd²⁺ and Co²⁺ activate intermediate conductance K⁺(Ca²⁺) channels in erythrocytes and large conductance K⁺(Ca²⁺) channels in neuroblastoma cells. Pb²⁺ and Co²⁺, but not Cd²⁺, activate small conductance K⁺(Ca²⁺) channels in neuroblastoma cells. Mg²⁺ and Fe²⁺ do not activate any of the K⁺(Ca²⁺) channels.3. Rank orders of the potencies for K⁺(Ca²⁺) activation are Pb²⁺, Cd²⁺>Ca²⁺, Co²⁺>>Mg²⁺, Fe²⁺ for the intermediate erythrocyte K⁺(Ca²⁺) channel, and Pb²⁺, Cd²⁺>Ca²⁺>Co²⁺>>Mg²⁺, Fe²⁺ for the small, and Pb²⁺>Ca²⁺>Co²⁺>>Cd²⁺, Mg²⁺, Fe²⁺ for the large K⁺(Ca²⁺) channel in neuroblastoma cells.4. At high concentrations Pb²⁺, Cd²⁺, and Co²⁺ block K⁺(Ca²⁺) channels in erythrocytes by reducing the opening frequency of the channels and by reducing the single channel amplitude. The potency orders of the two blocking effects are Pb²⁺>Cd²⁺, Co²⁺>>Ca²⁺, and Cd²⁺>Pb²⁺, Co²⁺>>Ca²⁺, respectively, and are distinct from the potency orders for activation.5. It is concluded that the different subtypes of K⁺(Ca²⁺) channels contain distinct regulatory sites involved in metal ion binding and channel opening. The K⁺(Ca²⁺) channel in erythrocytes appears to contain additional metal ion interaction sites involved in channel block.     

The response to ribulose bisphosphate4− (RuBP4−) and RuBP-Mg2− in catalysis by structurally divergent RuBP carboxylase/oxygenases

Free ribulose bisphosphate (RuBP^4?) rather than its magnesium complex (RuBP-Mg^2?) was the apparent substrate for spinach ribulose bisphosphate carboxylase/oxygenase. The apparent K_m for total RuBP (pH 8.0 at 30° C) increased with increasing Mg²⁺ concentrations from 11.6 μM at 13.33 mM Mg²⁺ to 32.6 μM at 40.33 mM Mg²⁺. Similarly the apparent K_m for RuBP-Mg^2? complex increased with increasing Mg²⁺ from 9.4 μM at 13.33 mM Mg²⁺ to 29.7 μM at 40.33 mM Mg²⁺. However, the K_m values for uncomplexed RuBP^4? were independent of the (saturating) concentration of Mg²⁺ (K_m=2.2 μM). The V_max did not vary with the changing concentrations of Mg²⁺. In contrast, the K_m for total RuBP remained constant with varying Mg²⁺ concentrations (K_m=59.5 μM) for the enzyme from R. rubrum. The apparent K_m for the RuBP-Mg^2? complex decreased with increasing Mg²⁺ concentrations from 16.0 μM at 7.5 mM Mg²⁺ to 5.9 μM at 27.5 mM Mg²⁺. The initial velocity for the C. vinosum enzyme was also found to be independent of the (saturating) concentration of Mg²⁺ when total RuBP was varied in the assay. Thus the response to total RuBP by these two bacterial enzymes, which markedly differ in structure, was closely similar.