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1.
Glutamine synthetase of pea leaves: divalent cation effects, substrate specificity, and other properties 总被引:1,自引:0,他引:1
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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%. 相似文献
2.
Heripsime Ohanian Kamran Borhanian Salette de Farias Alfred Bennun 《Journal of bioenergetics and biomembranes》1981,13(5-6):317-355
Multiple-equilibrium equations were solved to investigate the individual and separate effects of Mg2+, Mn2+, Ca2+, ATP4–, and their complexes on the kinetics of brain adenylate cyclase. The effects of divalent metals and/or ATP4– (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(ATP4–)=0.27 mM,K
m(MnATP)=0.07 mM, andK
i(CaATP)=0.015 mM. MgATP, MnATP, ATP4–, 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 ATP4– 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(ATP4–). The interactions of ATP4– 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. ATP4– 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; MeT (MgT or MnT), total Me (Me2+ and its complexes); ATPT, total ATP (ATP4– and its complexes). 相似文献
3.
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 (Km = 58 micromolar), but also inhibition by high substrate (Ki = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (Km = 120 micromolar). At both pH conditions, activity response to NADP+ exhibited Michaelis-Menten behavior with Km 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 Ka 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. 相似文献
4.
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 Km of the other. The suggested sequential mechanism for the reaction is supported by derivative plots of slopes and intercepts. The Km 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 Ki 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. 相似文献
5.
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 Km values of 23 μm for free Mg2+ and 3.3 μm for free Mn2+ are obtained at low levels of added metal, while Km 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 Km 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 Km 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 Ki 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 KD 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 KD 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 Mr 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. 相似文献
6.
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). 相似文献
7.
Biologically active DNA analogs of tRNAPhe (tDNAPhe) were used to investigate metal ion interaction with tRNA-like structures lacking the 2OH. Binding of Mg2+ to the 76 oligonucleotide tDNAPhe, 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 (tDNAAC
Phe). The conformation of tDNAAC
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 tDNAAC
Phe-d(m5C) structure without catalyzing cleavage of the oligonucleotide. In the absence of Mg2+ and at [Cd2+] to [tDNAAc
Phe-d(m5C)] ratios of approximately 0.2–1.0, tDNAAC
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 tDNAAC
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. 相似文献
8.
《Comparative biochemistry and physiology. B, Comparative biochemistry》1994,107(4):683-689
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 KMn 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 Km for arginine and Ki for lysine inhibition, were the same, regardless of the metal ion used to activate the enzyme; Km 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. 相似文献
9.
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 (Km 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (Km 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. 相似文献
10.
Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster 总被引:4,自引:3,他引:1
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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 Km 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). 相似文献
11.
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. 相似文献
12.
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+. Km 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+. 相似文献
13.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1988,952(2):222-229
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 Kd = 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 pKa = 7.85. Mn2+, the only other activating metal ion, competes with Cd2+ for the same binding site. Km values forl-threonine and NAD+ as well as the Vmax for ‘demetallized’, Cd2+-activated, and Mn2+-activated threonine dehydrogenase were determined and compared. 相似文献
14.
Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum 总被引:2,自引:2,他引:0
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 Km 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+. 相似文献
15.
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 Km 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. 相似文献
16.
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. 相似文献
17.
《Biochemical medicine and metabolic biology》1986,35(2):115-119
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 Km (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 Vmax of the Mg2+-dependent enzyme is lower than that of the latter. However, for each enzyme, the Km remains almost constant and Vmax varies during ontogenic development. Vmax 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%. 相似文献
18.
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 (Kd) 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 Kds also near 10 μm. However, the binding of metals by HRG exhibits positive cooperativity, so that the apparent Kds 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. 相似文献
19.
H. P. M. Vijverberg T. Leinders-Zufall R. G. D. M. van Kleef 《Cellular and molecular neurobiology》1994,14(6):841-857
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. 相似文献
20.
Free ribulose bisphosphate (RuBP4?) rather than its magnesium complex (RuBP-Mg2?) was the apparent substrate for spinach ribulose bisphosphate carboxylase/oxygenase. The apparent Km 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 Km for RuBP-Mg2? 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 Km values for uncomplexed RuBP4? were independent of the (saturating) concentration of Mg2+ (Km=2.2 μM). The Vmax did not vary with the changing concentrations of Mg2+. In contrast, the Km for total RuBP remained constant with varying Mg2+ concentrations (Km=59.5 μM) for the enzyme from R. rubrum. The apparent Km for the RuBP-Mg2? 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. 相似文献