共查询到20条相似文献,搜索用时 31 毫秒
1.
Juan P. Infante George E. Houghton John E. Kinsella 《Journal of theoretical biology》1980,86(1):177-201
A novel kinetic mechanism that explains the non-hyperbolic kinetics of many metal- or effector-activated enzymes is proposed as an alternative to the allosteric, hysteresis and mnemonical models. In this mechanism, the non-Michaelian behavior is generated by a reversible binding of an essential metal cation or other effector to a single site, but to at least two different enzyme forms in steady state. The model is described by a higher degree rate equation since the metal binding to more than one enzyme form generates at least two steady-state catalytic pathways of different efficiencies in addition to the recycling of the metal-enzyme species in the kinetic sequence. The proposed mechanism also explains the transition from non-hyperbolic kinetics to a Michaelian rate law, as well as the dual activation and inhibition of enzymes by the metal cation or effector, according to its concentration. This kinetic behavior is generated by the participation of the metal or effector in fast and slow competing catalytic sequences or by the competitions produced by binding as a common reactant for both the forward and reverse reactions. The model can also explain some peculiar inhibition patterns observed for some transferases. This kinetic mechanism can be tested by an experimental protocol that includes the metal cation or effector as a controlled variable reactant. The model and its complete rate equation explains the non-Michaelian behavior of choline kinase. At low ligand concentrations, an effectively ordered terreactant sequential mechanism operates (Infant. & Kinsella, 1976). The steady-state addition of choline to free enzyme is followed by the rapid-equilibrium binding of MgATp2? and the steady-state addition of Mg2+ last in the sequence. Initial velocity and product inhibition studies in the non-hyperbolic kinetic region, were consistent with a partially ordered release of reactants in which phosphocholine was the first product to dissociate from the central complex. The release sequence of the other two reactants was dependent on the prevailing Mg2+ concentration. At low Mg2+ levels, i.e. below 2·0 mM the metal cation is predominantly released after phosphocholine whereas MgADP? is the last product to dissociate under rapid-equilibrium conditions. At higher levels of the metal cation, MgADP? is predominantly released after phosphocholine leaving the Mg-enzyme complex from which Mg2+ may dissociate. However, a substantial fraction of the Mg-enzyme form is recycled in an alternate catalytic sequence in which the rapid-equilibrium binding MgATP2? to the Mg-enzyme complex is followed by the steady-state addition of choline. This pathway can also be initiated by the binding of Mg2+ to free enzyme. A third and unique sequence, which operates at low Mg2+ concentrations, includes the participation of MgADP? as an activator via a partial reversal of one of the product release sequences. In this pathway, the rapidequilibrium binding of MgADP? to free enzyme is followed by the addition of Mg2+ to the resulting transitory complex. Subsequent dissociation of MgADP? leaves the Mg-enzyme form, which is then channeled to product formation by the consecutive additions of MgATP2? and choline. 相似文献
2.
Two types of Na+-independent Mg2+ efflux exist in erythrocytes: (1) Mg2+ efflux in sucrose medium and (2) Mg2+ efflux in high Cl− media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na+-independent Mg2+ efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K+,Cl−- and Na+,K+,Cl−-symport, Na+/H+-, Na+/Mg2+-, Na+/Ca2+- and K+(Na+)/H+ antiport, Ca2+-activated K+ channel and Mg2+ leak flux. We suggest that, in choline Cl medium, Na+-independent Mg2+ efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg2+ efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg2+ to the same degree. The Kd value for inhibition of [14C]choline efflux and for inhibition of Mg2+ efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg2+ efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg2+ efflux was reduced to the same degree by these inhibitors as was the [14C]choline efflux. 相似文献
3.
Calmodulin-depleted isotonic erythrocyte ghosts contain 200 ng residual calmodulin/mg protein which is not removed by extensive washings at pCa2+ > 7. Specific activity and Ca2+-affinity of the (Ca2+ + Mg2+)ATPase increase at increasing calmodulin, with K0.5 Ca of 0.38 μM at calmodulin concentrations corresponding to that in erythrocytes. High Ca2+ concentrations inhibit the enzyme. Specific activity and Ca2+-affinity of the enzyme decrease at increasing Mg2+ concentrations. The Ca2+ ? Mg2+ antagonism is likewise observed at inhibitory Ca2+ concentrations. 相似文献
4.
C A Carvalho 《Life sciences》1979,25(1):73-82
Synaptosomes isolated from sheep brain cortex accumulate Ca2+, Sr2+ and Mg2+ when incubated in isosmotic sucrose media containing 5 mM of either of these cations. The maximal levels of cations retained per mg of protein are 100 nmol of Ca2+, 85 nmol of Mg2+ and 80 nmol of Sr2+. The loss of Ca2+ or Sr2+ from the preloaded synaptosomes is increased by monovalent cations in the following order: Na+> K+ > Li+> choline, whereas for the loss of Mg2+ this order is different: K+ > Na+ > Li ~ choline. The efflux of Ca2+ or Sr2+ induced by monovalent cations decreases as the temperature is lowered and it is nearly abolished at 0°C, whereas the efflux of Mg2+ is much less influenced by temperature. The results suggest that the mechanism of exchange of Ca2+ for Na+ in synaptosomes operates similarly for Sr2+, but not for Mg2+. 相似文献
5.
Kinetic studies of the reverse reaction catalysed by adenosine triphosphate–creatine phosphotransferase. The inhibition by magnesium ions and adenosine diphosphate
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1. Kinetic investigations of the reaction catalysed by ATP–creatine phosphotransferase have been carried out. 2. No firm conclusions could be reached about the reaction of Mg2+ at the nucleotide-binding site of the enzyme. The value of the kinetic constant for this reaction depends on the value used for the apparent stability constant of the metal ion–nucleotide complex and, to a smaller extent, on the method of plotting the results. 3. At higher concentrations Mg2+ is a non-competitive inhibitor of the enzyme with respect to both MgADP− and phosphocreatine. 4. ADP3− is a competitive inhibitor of the enzyme with respect to MgADP− and a non-competitive inhibitor with respect to phosphocreatine. 5. The concentration of phosphocreatine has little, if any, effect on the kinetic constants for the nucleotide reactants. 相似文献
6.
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
7.
Free ribulose hisphosphate (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
8.
The effect of Mg2+ on the thermal inactivation and unfolding of calf intestinal alkaline phosphatase has been studied at different temperatures
and Mg2+ concentrations. Increasing the Mg2+ concentration in the denatured system significantly enhanced the inactivation and unfolding of the enzyme during thermal
inactivation. The analysis of the kinetic course of substrate reaction during thermal inactivation showed that at 47°C the
increased free Mg2+ concentration caused the inactivation rate to increase. Increasing the temperature strengthened the effect of Mg2+ on the thermal inactivation. Control experiment showed that this is not due to salt effect. The time course of fluorescence
emission spectra showed that the emission maximum for Mg2+-containing system was always higher than that of Mg2+-free system, and the higher temperature enhanced this difference. In addition, Mg2+also enhanced the unfolding rate of the enzyme at 47°C. The potential biological significance of these results are discussed. 相似文献
9.
K. J. Lendzian 《Planta》1978,141(1):105-110
Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly affected by interactions between Mg2+, proton, and substrate concentrations. Mg2+ activates the enzyme to different degrees; however, it is not essential for enzyme activity. The Mg2+-dependent activation follows a maximum curve, magnitude and position of the maximum being dependent on pH and NADPH/NADP+ ratios. At a ratio of zero and pH 7.2, maximum activity is observed at 10 mM Mg2+. Increasing the NADPH/NADP+ ratio up to 1.7 (a ratio measured in the stroma during a light period), maximum activity is shifted to much lower Mg2+ concentrations. At pH 8.2 (corresponding to the pH of the stroma in the light) and at a high NADPH/NADP+ ratio, enzyme activity is not affected by the Mg2+ ion. The results are discussed in relation to dark-light-dark regulation of the oxidative pentose phosphate cycle in spinach chloroplasts.Abbreviations DTT
dithiothreitol
- G-6-P
glucose-6-phosphate
- G-6-PDH
glucose-6-phosphate dehydrogenase (EC 1.1.1.49)
- PPC
pentose phosphate cycle 相似文献
10.
The K+-stimulated ATPase activity associated with the purified gastric microsomes from the pig gastric mucosa can be completely inactivated by treatment with 15% ethanol for 60 s at 37 °C but not at 25 °C. Sequential exposure of the microsomes to 15% ethanol at 25 and 37 °C caused the release of 2.9 and 4.3% of the total membrane phospholipids, respectively, consisting entirely of phosphatidyl choline and phosphatidyl ethanolamine. The ethanol-treated (37 °C) membrane had high basal (with Mg2+ as the only cation in the assay mixture) activity, which was further enhanced during reconstitution with phosphatidyl choline or phosphatidyl ethanolamine. The high basal activities could be reduced to the normal control level by assaying the enzyme in presence of the “activator protein,” partially purified from the soluble supernatant of the pig gastric cells. Phosphatidyl choline was somewhat more effective than phosphatidyl ethanolamine in the restoration of the activity of the ethanol-treated enzyme while phosphatidyl serine, phosphatidyl inositol, and sphingomyelin were without any effect. Synthetic phosphatidyl choline with various fatty acid substitutions were tested for their effectiveness in the restoration of the ethanol-inactivated enzyme. The distearoyl (18:0), dioleoyl (18:1), and dilinoleoyl (18:2) derivatives of phosphatidyl choline were almost equally effective while dipalmitoyl (16:0) phosphatidyl choline was somewhat less effective in the reconstitution process. Cholesterol appeared to interfere with phosphatidyl choline in the restoration of the activity of ethanol-treated enzyme. The fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine extracted by 15% ethanol at 37 °C was clearly different than those of the total microsome. Our data suggest that the phospholipids extracted by 15% ethanol at 37 °C are derived primarily from the immediate lipid environment of the enzyme and ATP together with Mg2+ and K+ help the partially delipidated enzyme to retain the appropriate conformation for the subsequent reconstitution. Furthermore, ethanol appears to either release or inactivate the membrane-associated activator protein, demonstrated to be essential for the K+-stimulated activity of the pig gastric ATPase. 相似文献
11.
High-conductance, Ca2+-activated K+ channels from the basolateral membrane of rabbit distal colon epithelial cells were reconstituted into planar phospholipid
bilayers to examine the effect of Mg2+ on the single-channel properties. Mg2+ decreases channel current and conductance in a concentration-dependent manner from both the cytoplasmic and the extracellular
side of the channel. In contrast to other K+ channels, Mg2+ does not cause rectification of current through colonic Ca2+-activated K+ channels. In addition, cytoplasmic Mg2+ decreases the reversal potential of the channel. The Mg2+-induced decrease in channel conductance is relieved by high K+ concentrations, indicating competitive interaction between K+ and Mg2+. The monovalent organic cation choline also decreases channel conductance and reversal potential, suggesting that the effect
is unspecific. The inhibition of channel current by Mg2+ and choline most likely is a result of electrostatic screening of negative charges located superficially in the channel entrance.
But in addition to charge, other properties appear to be necessary for channel inhibition, as Na+ and Ba2+ are no (or only weak) inhibitors. Mg2+ and possibly other cations may play a role in the regulation of current through these channels.
Received: 25 August 1995/Revised: 16 November 1995 相似文献
12.
Phosphatidyl inositol and lysophosphatidyl choline have been identified as activators of a partially purified brain cyclic nucleotide phosphodiesterase previously shown to be regulated in vitro by Ca2+ and a Ca2+-binding protein. Microgram quantities of either phospholipid produced a linear, immediate and reversible activation of the enzyme in the absence of Ca2+ and the Ca2+-dependent regulator (CDR). Fatty acids were also found to activate the phosphodiesterase to varying degrees, with oleic acid being the most effective. Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and lysophosphatidyl ethanolamine were not effective as activators. Only sodium dodecyl sulfate, of a variety of nonionic, cationic, and anionic detergents tested, activated the phosphodiesterase. Sodium dodecyl sulfate produced a modest degree of activation over a narrow concentration range, followed by enzyme denaturation at higher concentrations.The interaction of the phosphodiesterase with the phospholipid activators has been compared to its interaction with the Ca2+·CDR complex. Both Ca2+·CDR and lysophosphatidyl choline decreased the thermal stability of the enzyme to a similar extent. The apparent Km of the lysophosphatidyl choline-dependent phosphodiesterase activity was approximately 30 μm with guanosine-3′,5′-monophosphate (cGMP) as substrate and 1 mm with adenosine-3′,5′-monophosphate (cAMP) as substrate. With increasing lysophosphatidyl choline concentration, the apparent Km for each nucleotide remained unchanged while the V increased. The apparent Kd for Mg2+ of the lysophosphatidyl choline-dependent phosphodiesterase activity was approximately 3 μm and was unaffected by lysophosphatidyl choline concentration. Activation of the phosphodiesterase by lysophosphatidyl choline was characterized by a high degree of positive cooperativity, exhibiting a Hill coefficient of 3.8. Fluphenazine was a competitive inhibitor of both Ca2+·CDR and lysophosphatidyl choline activation of the enzyme. 相似文献
13.
The activation of muscle pyruvate kinase by divalent cations was studied by steady-state kinetics. Under experimental conditions the enzyme exhibits activation by Mg2+, Co2+, Mn2+, Ni2+, and Zn2+ in descending order of maximal velocity. Combinations of cations were also studied. A synergistic activation was observed with a fixed concentration of Mg2+ and varying concentrations of Mn2+ or of Co2+. This synergism indicates at least two roles for the cations for enzymatic activation and a differential specificity among the cations for the separate functions. Synergistic activation was also observed with fixed Co2+ and varying Mn2+. These results are consistent with a cation specifically required to activate the enzyme and a cation which serves as a cation-nucleotide complex which is a substrate for the reaction. The response observed suggests that Mn2+ is a better activator of the enzyme than is Mg2+, however, MgADP is a better substrate than is MnADP. The lack of a synergistic effect by Ni2+ or Zn2+ with Mg2+ suggests that Ni2+ and Zn2+ are poor activators either because they serve one catalytic function poorly but bind to that site tightly or they serve both catalytic functions poorly in contrast to Mg2+. These studies yield the first simple kinetic evidence that muscle pyruvate kinase, under catalytic conditions of the overall reaction, has a dual divalent cation requirement for activity. 相似文献
14.
The inhibition of NaK-ATPase (EC 3.6.1.3) from human red cells by Mg2+ is markedly dependent on the relative concentrations of Na+ and K+. Inhibition increases with increasing K+ and decreases with increasing Na+. The inhibition appears to be a combined effect of Mg2+ and K+ at sites distinct from the sites at which these cations activate the enzyme. The kinetics of activation of the enzyme by Na+ with inhibitory levels of Mg2+ and K+ are biphasic, indicating both low and high affinity Na+ sites. At noninhibitory levels of Mg2+ and K+ only high affinity Na+ sites are seen. The results are inconsistent with any model in which Mg2+ and K+ compete with Na+ at a single site. A kinetic model is proposed to explain the mechanism of inhibition by Mg2+ and K+. 相似文献
15.
Glutamine synthetase (GS) was purified to electrophoretic homogeneity from the halophilic archaebacterium Halobacterium salinarium. The enzyme was purified 300-fold to homogeneity with 30% yield. By gel filtration and SDS gel electrophoresis, it was shown that the enzyme has a native molecular weight of 495,000 and a subunit molecular weight of 62,000. This indicates an octameric quaternary structure. The amino acid composition and the isoelectric point of 4.9 are similar to other GSs. The enzyme shows highest stability in 4 M NaCl or KCl and at temperatures up to 45°C. Lower salt concentrations or higher temperatures lead to rapid and irreversible denaturation. By low concentrations of Mg2+ or Mn2+, the salt dependence was decreased and the thermostability increased. Mg2+ or Mn2+ are essential cofactors. The two resulting activities show differences in pH and salt concentrations required for optimal activity, different K
m-values and different sensitivity to inhibition by amino acids. The enzyme is not adenylylated like the GS from some eubacteria but cytidylylated. The covalently bound CMP increases Mn2+-and Mg2+-dependent activities at a different extent. 相似文献
16.
The properties of membrane-associated ATPase of cucumber (Cucumis sativus cv. Seiriki No. 2) roots cultured in a complete medium (complete enzyme) and in a medium lacking Ca2+ (Ca2+-deficient enzyme) were investigated. The basal activity of membrane-associated ATPase increased during Ca2+ starvation, while Mg2+-activation of the enzyme decreased and even resulted in inhibition by high Mg2+ concentration at the late stage of the Ca2+ starvation. The complete enzyme had low basal activity and showed a Mg2+-activated hyperbolic reaction curve in relation to ATP concentration. Ca2+-deficient enzyme with high basal activity showed a biphasic reaction curve and Mg2+-activation was seen only at high ATP concentrations. Activation of membrane-associated ATPase by various cations was decreased or lost during Ca2+ starvation. The basal ATPase activity of Ca2+-deficient enzyme increased for various substrates including pyrophosphate, p-nitrophenyl phosphate, glucose-6 phosphate, β-glycerophosphate, AMP, ADP and ATP. Mg2+-activation was found only for ADP and ATP in both the complete and Ca2+-deficient enzymes, but the activation for ATP was greatly reduced by Ca2+ starvation. The heat inactivation curves for basal and Mg2+-activated ATPase did not differ much between the complete and Ca2+-deficient enzyme. The delipidation of membrane-associated enzyme by acetone affected the protein content and the basal activity slightly, but inhibited the Mg2+-activated ATPase activity clearly with somewhat different behaviour between the complete and Ca2+-deficient enzyme. 相似文献
17.
Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATpase of cardiac sarcoplasmic reticulum
Larry R. Jones 《生物化学与生物物理学报:生物膜》1979,557(1):230-242
ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold.Activation of K+-stimulated ATPase activity by Ca2+ was maximal at anionized Ca2+ concentration of approx. 1 μM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme. 相似文献
18.
Luciana R. Mazzitelli 《生物化学与生物物理学报:生物膜》2007,1768(7):1777-1783
The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg2+ and K+. Micromolar concentrations of Ca2+ inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca2+ concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca2+. The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K+ but the apparent affinity of the enzyme for Mg2+ increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg2+. Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca2+ free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca2+ and the activation improves the interaction of the enzyme with Mg2+. 相似文献
19.
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