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1.
Abstract— Mn2+ caused an 8-to 16-fold stimulation of adenylate cyclase activity in homogenates as well as synaptosomcs. isolated synaptic membranes, and slices prepared from rat brain. The stimulation occurred at low concentrations of Mn2+. with a doubling of activity at 50-60μM. and was unaffected by a 60-fold excess of Mg2+. Whether or not Mg2+ was added, inclusion of a low concentration of Mn2+ reduced, but did not prevent the stimulation of adenylate cyclase caused by dopaminc in homogenates of corpus striatum. In contrast, Ca2+. at a concentration that had little effect on basal cyclase activity, completely prevented the stimulation by dopamine. The increase of cyclase activity produced by Mn2+ in brain homogenates was potentiated by F?. Other ions, notably Hg2+. Pb2+. Cu2+ and Zn2+. in order of decreasing potency, inhibited both basal and Mn2--stimulated cyclase activity. It is proposed that the effect of Mn2+ on adenylate cyclase activity may involve only the catalytic subunit of the enzyme, and that the mechanism is different from that by which either dopamine or F? stimulates the enzyme. These results suggest that the effects of low concentrations of Mn2+ and certain other divalent metal ions on adenylate cyclase activity may be involved in their neuropsychiatrie or other toxic effects, and that such ions may also participate in normal physiological mechanisms involving cyclic nucleotides. 相似文献
2.
《Comparative biochemistry and physiology. B, Comparative biochemistry》1994,107(2-3):359-369
GTP has been shown to inhibit AlF4−-stimulated, and to activate forskolin-stimulated adenylyl cyclase activity in the presence of Mg2+ in cell membranes from human embryonic kidney 293 cells. The maximal inhibitory response of AlF4−-stimulated adenylyl cyclase activity by GTP was not dependent on the concentration of Mg2+, but was so in the case of forskolin-activated activity at all forskolin concentrations assayed. Mn2+ ions stimulated AlF4−- or forskolin-activated adenylyl cyclase activity to a greater extent than Mg2+. The inhibition of AlF4−-stimulated cyclase by GTP was still observed with Mn2+, but the activation of forskolin-stimulated cyclase by GTP was not. When assayed together, Mn2+ and Mg2+ showed non-additive behaviours with respect to the amount of cyclic AMP formed after AlF4−-stimulation of adenylyl cyclase. The temperature dependence of the activation of adenylyl cyclase by forskolin, AlF4− or under basal conditions was observed to be somehow different in the presence of Mn2+ than in the presence of Mg2+ ions. Cholera toxin treatment produced a markedly increased cyclase activity, specially when assayed with AlF4−. In the case of forskolin-activated adenylyl cyclase, UTP and CTP were unable to reproduce the cyclase activation detected with GTP. However, in the case of AlF4−-stimulated adenylyl cyclase, UTP was as good as GTP at inhibiting cyclase activity, and CTP virtually eliminated the activation of the cyclase with AlF4−. 相似文献
3.
Low concentrations of Mn2+ supported the basal adenylate cyclase activity in crude and purified sarcolemmal membranes from cardiac muscle more effectively than did relatively high concentrations of Mg2+; at saturating concentrations the cyclase activities obtained with Mg2+ or Mn2+ were similar. In contrast, Mg2+ supported the basal cyclase activities of crude membrane fractions and purified sarcolemmal membranes from skeletal muscle far more effectively than did Mn2+; at saturating concentrations of either metal ion the Mg2+-supported cyclase activities were 5- to 10-fold greater than Mn2+-supported activities. Further, compared to Mg2+, Mn2+ supported the cyclase activities very poorly in all the primary subcellular fractions of skeletal muscle, whereas this cation was at least as effective as Mg2+ in all fractions of cardiac muscle. The apparent affinities of the cyclase for Mn2+ in heart as well as skeletal muscle appeared to be greater compared to those for Mg2+. The skeletal muscle cyclase displayed greater apparent affinity for MnATP2? (app. Km 0.10 mm) compared to MgATP2? (app. Km 0.32 mm) whereas the heart enzyme displayed greater apparent affinity for MgATP2? (app. Km 0.07 mm) compared to MnATP2? (app. Km 0.19 mm). Following preactivation with guanyl-5′-yl imidodiphosphate and isoproterenol, Mn2+ (0.15 to 2 mm) supported the cyclase activity of skeletal muscle even more effectively than did optimally effective concentrations of Mg2+. With the heart enzyme the relatively greater potency of Mn2+ persisted following preactivation. Significant enhancement in the Mn2+-sensitivity of skeletal muscle cyclase was also observed when assayed in the presence of GTP and isoproterenol or in the presence of NaF. Preactivation of both heart and skeletal muscle cyclases caused selective enhancement in the enzyme's apparent affinity for free Me2+ (Mg2+ or Mn2+) without influencing the apparent Km for MeATP2? (MgATP2? or MnATP2?). Evidences were obtained to show that the poor effectiveness of Mn2+ in supporting the basal activity of skeletal muscle cyclase is not related to (a) potentiation by Mn2+ of adenosine-mediated inhibition of the cyclase, (b) Mn2+-induced lability of the cyclase, (c) indirect effects of Mn2+ on ATP-regenerating system, or (d) the presence of different cation-specific molecular forms of the cyclase. It is also shown that the onset of enhanced Mn2+ sensitivity of the skeletal muscle enzyme following preactivation is not accompanied by a general loss of cation specificity of the cyclase. These results suggest that cations support the catalytic activity of adenylate cyclase by interacting with an enzymeregulatory free metal binding site and that the differential cation sensitivity of nonactivated (basal) cyclases from heart and skeletal muscle is likely due to differences in the properties of such an allosteric metal site. Furthermore, the metal site appears to undergo a conformational change following interaction of the cyclase system with the guanyl nucleotide and isoproterenol since the cation sensitivity of the cyclase and the relative potency of cations depend on the conformational status of the enzyme. 相似文献
4.
Cytosolic fructose-1,6-bisphosphatase from spinach (Spinacia oleracea L.) leaves was purified over 1700-fold. The final preparation was specific for fructose-1,6-bisphosphate in the presence of either Mg2+ or Mn2+, and was free of interfering enzyme activities. Ca2+ was an effector of fructose-1,6-bisphosphatase activity, and showed different kinetics, depending on whether Mg2+ or Mn2+ was used as cofactor. In the presence of 5 millimolar Mg2+, Ca2+ appeared as activator or as inhibitor of the enzyme at low or high levels of substrate, respectively. In both cases, a rise in affinity for fructose-1,6-bisphosphate was observed. A model is proposed to describe the complex interaction of fructose-1,6-bisphosphatase with its substrate and Ca2+. However, with Mn2+ (60 micromolar) as cofactor, Ca2+ exhibited the Michaelis-Menten kinetics of a noncompetitive inhibitor. When assayed at constant substrate concentration, Ca2+ behaves as a competitive or noncompetitive inhibitor, depending on the use of Mg2+ or Mn2+ as cofactor, respectively, with a positive cooperativity in both cases. Fructose-2,6-bisphosphate showed a classic competitive allosteric inhibition in the presence of Mg2+ as cofactor, but this effect was low with Mn2+. From these results we suggest that Ca2+ plays a role in the in vivo regulation of cytosolic fructose-1,6-bisphosphatase. 相似文献
5.
《Biochimica et Biophysica Acta (BBA) - Enzymology》1979,566(2):371-384
The interactions of divalent cations, Mn2+, Mg2+, and Ca2+, with the cytosolic guanylate cyclase (GTP pyrophosphate-lyase (cyclizing, EC 4.6.1.2) from different tissues were studied. Guanylate cyclase activities of the kidney, liver, and lung were strongly dependent on Mn2+. In contrast, the enzyme in smooth muscle of the colon, aorta, and vas diferens was active with Mg2+ as well as with Mn2+. Ca2+ was ineffective in all tissues. Preincubation, at 30°C, of colon extracts, but not those of kidney and liver, increased guanylate cyclase activity. The Mg2+-dependent activity was preferentially enhanced by this treatment. These results suggest that when the enzyme was autoactivated by endogenus factors it became more Mg2+ dependent. Dithiothreitol strongly inhibited the Mg2+-dependent colon enzyme, whereas activities in kidney and liver were not affected and the response of the enzyme in lung was intermediate. This suggests that autoactivation involved an oxidative-reductive alteration of the enzyme. Ca2+ markedly inhibited the Mg2+-dependent activity in smooth muscles but Mg2+-dependent activities in lung, liver, and kidney were not influenced appreciably. Exogenous activators, dehydroascorbate and NaN3, increased guanylate cyclase, assayed with either Mg2+ or Mn2+. However, the relative stimulation of the enzyme assayed with Mg2+ was greater than with Mn2+. When activated by these exogenous agents, guanylate cyclase in all tissues became inhibitable by Ca2+. These findings suggest that guanylate cyclase in smooth muscle, as prepared, was in a partially activated form.The endogenous activating factors in colon smooth muscle were heat-stable, largely extractable with chloroform/methanol, and cochromatographed with authentic fatty acids. Arachidonic acid stimulated colon guanylate cyclase and enhancement of the Mg2+-dependent activity was blocked by Ca2+. This strongly infers that a significant part of the endogenous activating factors in the colon was fatty acids or their derivatives. The colon activators had only minimal affects on the enzyme in the kidney. Similarly prepared activator extracts from the kidney increased colon guanylate cyclase but did not stimulate the renal enzyme. Thus, the ability of the enzyme to be stimulated by endogenous activators was dependent on the tissue from which the enzyme was derived.The possibility that the interactions of Mg2+ and Ca2+ on guanylate cyclase in smooth muscles are of regulatory significance to the contractile response of the muscle was discussed. It was proposed as a working hypothesis that cyclic GMP and Ca2+ might participate in reciprocal negative feedback mechanisms. 相似文献
6.
Certain biochemical characteristics of an adenylate cyclase that is activated by low concentrations of histamine (Ka, 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 Km (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. 相似文献
7.
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
8.
9.
《BBA》1986,848(2):224-229
Evidence is presented for the presence of divalent cation binding sites in purified F1-ATPase from Micrococcus lysodeikticus (Micrococcus luteus). Electron paramagnetic resonance studies of native F1-ATPase indicate that the enzyme binds Mn2+ and Cu2+. Scatchard-type plot for Mn2+ binding to the enzyme indicates the presence of 3–4 independent and identical sites with a dissociation constant of 18.3 · 10−6 M. Cu2+ binds to the enzyme at only one kind of site(s). This Cu2+ binding site(s) is characterized by a moderately ionic ligand field provided by the protein and by a tetragonal symmetry of nitrogen and/or oxigen ligands. Competition studies indicate that Mg2+ binds at these Mn2+ and Cu2+ binding sites. 相似文献
10.
S.K. Mukerji 《Archives of biochemistry and biophysics》1977,182(1):352-359
Differences in the kinetic properties of corn leaf phosphoenolpyruvate (PEP) carboxylase isoenzymes were found, depending on whether Mg2+ or Mn2+ was used as the metal cofactor of the reaction. Also, differences in kinetic constants with respect to Mg2+ and Mn2+ were noticed between the two isoenzymes which further differentiates the two proteins. The catalytic activity of the enzyme in the Mg2+-activated system was dependent on a PEP-Mg2+ complex and not on the concentration of free Mg2+ or free PEP. Kinetics in the presence of total Mg2+ and those of PEP-Mg2+ suggest a negative cooperative effect with respect to ligand binding with concurrent progressive substrate activation. Magnesium ions, thus, have a special regulatory role in the corn leaf PEP carboxylase reaction. 相似文献
11.
Louis J. Ignarro Jonathan N. Degnan William H. Baricos Philip J. Kadowitz Michael S. Wolin 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,718(1):49-59
Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containg forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 μM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent Km for GTP in the presence of excess Mn2+ or Mg2+ was 10 μM and 85–120 μM, respectively, for unactivated guanylate cyclase. The apparent Km for GTP in the presence of Mn2+ was not altered but the Km in the presence of Mg2+ was lowered to 58 μM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg2+ or Mn2+. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO nitroso compounds. 相似文献
12.
RNase E is a major intracellular endoribonuclease in many bacteria and participates in most aspects of RNA processing and degradation. RNase E requires a divalent metal ion for its activity. We show that only Mg2+ and Mn2+ will support significant rates of activity in vitro against natural RNAs, with Mn2+ being preferred. Both Mg2+ and Mn2+ also support cleavage of an oligonucleotide substrate with similar kinetic parameters for both ions. Salts of Ni2+ and Zn2+ permitted low levels of activity, while Ca2+, Co3+, Cu2+, and Fe2+ did not. A mutation to one of the residues known to chelate Mg2+, D346C, led to almost complete loss of activity dependent on Mg2+; however, the activity of the mutant enzyme was fully restored by the presence of Mn2+ with kinetic parameters fully equivalent to those of wild-type enzyme. A similar mutation to the other chelating residue, D303C, resulted in nearly full loss of activity regardless of metal ion. The properties of RNase E D346C enabled a test of the ionic requirements of RNase E in vivo. Plasmid shuffling experiments showed that both rneD303C (i.e., the rne gene encoding a D-to-C change at position 303) and rneD346C were inviable whether or not the selection medium was supplied with MnSO4, implying that RNase E relies on Mg2+ exclusively in vivo. 相似文献
13.
《Enzyme and microbial technology》1986,8(11):696-700
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 Km-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. 相似文献
14.
15.
Guanylate cyclase activity was determined in a 1000g particulate fraction derived from rabbit heart homogenates using Mg2+ or Mn2+ as sole cation in the presence and absence of Triton X-100. With Mg2+, very little guanylate cyclase activity could be detected in the original particulate fraction assayed with or without Triton, or in the particulate fraction treated with varying concentrations of Triton (detergent-treated mixture) prior to enzyme assay. However, the detergent-solubilized supernatants as well as the detergent-insoluble residues (pellets) derived from detergent-treated mixtures possessed appreciable Mg2+-supported enzyme activity. With Mn2+, significant enzyme activity was detectable in the original particulate fraction assayed without Triton. Much higher activity was seen in particulate fraction assayed with Triton and in detergent-treated mixtures; the supernatants but not the pellets derived from detergent-treated mixtures possessed even greater activity. The sum of enzyme activity in pellet and supernatant fractions greatly exceeded that of the mixture. When the pellets and supernatants derived from detergenttreated mixtures were recombined, measured enzyme activities were similar to those of the original mixture. With Mg2+ or Mn2+, the specific activity of guanylate cyclase in pellet and supernatant fractions varied considerably depending on the concentration of Triton used for treatment of the particulate fraction; treatment with low concentrations of Triton (0.2–0.7 μmol/mg protein) gave supernatants showing high activity whereas treatment with relatively greater concentrations of the detergent (>0.7 μmol/mg protein) gave pellets showing high activity. The relative distribution of guanylate cyclase in pellet and supernatant fractions expressed as a function of Triton concentration during treatment (of the particulate fraction) showed that 50 to 80% of the recovered enzyme activity remained in supernatants at low detergent concentrations whereas 50 to 80% of the recovered activity resided in the pellets at higher detergent concentrations. Inclusion of excess Triton in the enzyme assay medium did not alter the specific activity profiles and the relative distribution patterns of the cyclase in pellet versus supernatant fractions. The results demonstrate the inherent potential of cardiac particulate guanylate cyclase to utilize Mg2+ in catalyzing the synthesis of cyclic GMP. However, it appears that some factor(s) endogenous to the cardiac particulate fraction severely impairs the expression of Mg2+-dependent activity; Mn2+-dependent activity is also affected by such factor(s) but apparently less severely. Further, the results suggest that previously reported activities of cardiac particulate guanylate cyclase, despite being assayed with Mn2+ and in the presence of Triton X-100, represent underestimation of what otherwise appears to be a highly active enzyme system capable of utilizing physiologically relevant divalent cation such as Mg2+. 相似文献
16.
Isocitrate dehydrogenase kinase/phosphatase (AceK) is a bifunctional enzyme with both kinase and phosphatase activities that are activated by Mg2+. We have studied the interactions of Mn2+and Mg2+ with AceK using isothermal titration calorimetry (ITC) combined with molecular docking simulations and show for the first time that Mn2+ also activates the enzyme activities. However, Mn2+ and Mg2+ exert their effects by different mechanisms. Although they have similar binding constants (of 1.11?×?105 and 0.98?×?105 M?1, respectively) for AceK and induce conformational changes of the enzyme, they do not compete for the same binding site. Instead Mn2+ appears to bind to the regulatory domain of AceK, and its effect is transmitted to the active site of the enzyme by the conformational change that it induces. The information in this study should be very useful for understanding the molecular mechanism underlying the interaction between AceK and metal ions, especially Mn2+ and Mg2+. 相似文献
17.
Elena Fabbri Maria Enrica Ferretti Marco Buzzi Maria Luisa Colamussi Prof. Carla Biondi 《Neurochemical research》1995,20(12):1511-1517
Sheep olfactory epithelium contains an adenylyl cyclase which is stimulated by many but not all odorants. Here we report that this enzyme is activated by calmodulin in a dose-dependent manner, and that calcium ions are required for this response. Odorant stimulation of adenylyl cyclase is unaffected by the complex Ca2+/calmodulin, as suggested by the results obtained both in Ca2+/calmodulin-depleted membranes and under calmodulin antagonist treatment; this confirms the prediction that the Ca2+ binding protein and odorants stimulate the olfactory adenylyl cyclase through parallel mechanisms. The persistent activation of the regulatory component of adenylyl cyclase by GppNHp does not alter the response of the enzyme to either odorant or Ca2+/calmodulin. In sheep olfactory epithelium a cAMP-phosphodiesterase activity is also present, which is highly inhibited by IBMX and aminophylline, searcely by RO 20-1724, and unaffected by Ca2+/calmodulin. The modulatory role exerted by calcium on cAMP system in sheep olfactory signal transduction is discussed. 相似文献
18.
A comparison was made between the activation of membrane-bound adenylate cyclase from rat fat cell membranes and the enzyme solubilized with digitonin. The isoprenaline stimulation of the particulate enzyme was enhanced by GTP, both in the presence of Mg2+ and Mn2+, but no effect of the metal ion nor of GTP was found on the Ka of isoprenaline. The Ka of sodium fluoride for enzyme stimulation was shifted to 3-fold higher concentrations when Mg2+ was replaced by Mn2+, whereas V decreased. GTP did not influence the Ka of sodium fluoride but reduced V, irrespective of the metal ion. After digitonin solubilization the enzyme was no longer responsive to isoprenaline or GTP; however, V of the sodium fluoride activation was higher in the presence of Mn2+ than in the presence of Mg2+, and the Ka was found at 15-fold higher concentrations. Both the solubilized and the particulate adenylate cyclase were inhibited by adenosine; this inhibition was also seen with the fluoride stimulated enzyme. We conclude that solubilization with digitonin did not result in an enzyme preparation which preferentially turns over MnATP2+, although the fat cell adenylate cyclase possesses a metal ion regulatory site with a higher affinity for Mn2+ than for Mg2+. The data suggest that the guanyl nucleotide regulatory site and the sodium fluoride-sensitive site are located on different subunits while there is an interaction between the metal ion regulatory site and the fluoride-sensitive site. 相似文献
19.
M S Coleman 《Archives of biochemistry and biophysics》1977,182(2):525-532
Methods of extraction and assay of terminal deoxynucleotidyl transferase (TdT) from human lymphoblasts and calf thymus were compared. A high salt concentration was mandatory for complete enzyme extraction, while dialysis of the crude extract resulted in a major loss of enzyme activity. In addition, TdT was partially purified from lymphoblasts of patients with acute lymphoblastic leukemia. The Km for the monomer, deoxy-guanosine 5′-triphosphate (dGTP), is high (~0.1 mm) in the presence of either Mg2+ or Mn2+, whereas the Km for the initiator, poly(deoxyadenylic acid , with an average chain length of 50 residues, is 2.5 μm in the presence of Mg2+ and 0.3 μm in the presence of Mn2+. The maximum velocity is higher for the calf thymus TdT in the presence of Mg2+ than in Mn2+. Human TdT catalyzes the polymerization of dGTP at a higher rate in the presence of Mn2+ than with Mg2+. These data illustrate that partially purified human TdT differs in catalytic properties from the purified calf thymus enzyme. Therefore, optimal conditions for assay of TdT in extracts from calf and human tissues differ. 相似文献
20.
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