Stimulation of Tubulin-Dependent ATPase Activity in Microtubule Proteins from Porcine Brain by Vinblastin

Vinblastine, a plant alkaloid which inhibits tubulin polymerization, stimulated an ATPase activity in microtubules. When microtubule proteins were separated into microtubule-associated proteins (MAPs) and tubulin by phosphocellulose column chromatography, vinblastine did not stimulate an ATPase activity recovered in the MAPs fraction unless tubulin was present. Therefore, vinblastine is considered to act through its binding to the tubulin molecule on MAPs ATPase. Divalent cations that activate tubulin-dependent MAPs ATPase activity were also required for the stimulation by vinblastine. In the presence of Ca2+ and vinblastine the ATPase activity was most active and the extent of stimulation reached about 200% of the original level in the absence of vinblastine. Half-maximal stimulation was attained when the molar ratio of vinblastine to tubulin was 0.5. The concentration of tubulin for half-maximal stimulation was increased in the presence of vinblastine, while divalent cation requirements were decreased. Several factors such as KCl (100 mM), alkaline pH (pH 7.5), and low temperature (10 degrees C) were not responsible for the disappearance of the stimulation. Vincristine stimulated tubulin-dependent MAPs ATPases activity as vinblastine did, whereas the activity was scarcely affected by colchicine, podophyllotoxin, strychnine, and chlorpromazine. Actin had no effect on MAPs ATPase activity in the absence and presence of vinblastine when it was used in place of tubulin.     

The presence of an adenosine-5'-triphosphatase dependent on 6S tubulin and calcium ions in rat brain microtubules.

An ATPase activity was found in rat brain microtubules prepared by successive cycles of polymerization and depolymerization. On phosphocellulose column chromatography, the ATPase activity was recovered in the fraction eluted with 0.6 M KCl and containing the microtubule associated proteins. The ATPase activity was markedly stimulated by the addition of purified brain 6S tubulin, and the stimulation was dependent on the presence of Ca2+ ions. Approximately 50 pmol of purified 6S tubulin was required for the maximal stimulation in the presence of 8 microgram of microtubule associated proteins. The specific activity was 8 to 13 nmol of ATP hydrolyzed per min per mg of protein at 37 degrees C, and the Km value for ATP was 3 X 10(-5) M in the presence of added tubulin.     

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

An ATPase activity associated with brain microtubules.

A persistent ATPase/GTPase activity has been found to be associated with highly recycled bovine brain microtubules. A GTP regeneration system was introduced to minimize the inhibitory effects of this hydrolase on microtubule polymerization. The characteristics of the ATPase indicate that it is not involved in GTP-induced mictrotubule polymerization, but is directly involved in ATP-induced polymerization. ATP-induced polymerization was also shown to require stoichiometric amounts of GDP, but higher levels of GDP inhibited both microtubule formation and the ATPase activity. An ammonium sulfate fractionation procedure was devised to separate microtubule protein into an ATPase-rich fraction and a pure tubulin fraction. The pure tubulin fraction polymerized in the presence of GTP, but not in the presence of ATP and GDP. In contrast, the ATPase-rich fraction polymerized with either ATP or GTP. It is still not known whether the microtubule associated ATPase plays a significant role in cellular microtubule function.     

Separation of active tubulin and microtubule-associated proteins by ultracentrifugation and isolation of a component causing the formation of microtubule bundles

A new method for separating microtubule-associated proteins (MAPs) and tubulin, appropriate for relatively large-scale preparations, was developed. Most of the active tubulin was separated from the MAPs by centrifugation after selective polymerization of the tubulin was induced with 1.6 M 2-(N-morpholino)ethanesulfonate (Mes) and GTP. The MAPs-enriched supernatant was concentrated and subsequently clarified by prolonged centrifugation. The supernatant (total soluble MAPs) contained almost no tubulin, most of the nucleosidediphosphate kinase activity of the microtubule protein, good activity in promoting microtubule assembly in 0.1 M Mes, and proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The pellet, inactive in supporting microtubule assembly, contained denatured tubulin, most of the ATPase activity of the microtubule protein, and significant amounts of protein with the electrophoretic mobility of MAP-2. Insoluble material at this and all previous stages, including the preparation of the microtubule protein, could be heat extracted to yield soluble protein active in promoting microtubule assembly and containing MAP-2 as a major constituent. The total soluble MAPs were further purified by DEAE-cellulose chromatography into bound and unbound components, both of which induced microtubule assembly. The bound component (DEAE-MAPs) contained proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The polymerization reaction induced by the unbound component (flow-through MAPs) produced very high turbidity readings. This was caused by the formation of bundles of microtubules. Although the flow-through MAPs contained significantly more ATPase, tubulin-independent GTPase, and, especially, nucleosidediphosphate kinase activity than the DEAE-MAPs, preparation of a MAPs fraction without these enzymes required heat treatment.     

Reversible inhibition of (Na+, K+) ATPase by Mg2+, adenosine triphosphate, and K+.

Adenosine triphosphate (ATP) hydrolysis catalyzed by the plasma membrane (Na+,K+)ATPase isolated from several sources was inhibited by Mg+, provided that K+ and ATP were also present. Phosphorylation of the adenosine triphosphatase (ATPase) by ATP and by inorganic phosphate was also inhibited, as was p-nitrophenyl phosphatase activity. (Ethylenedinitrilo)tetraacetic acid (EDTA) and catecholamines protected from and reversed the inhibition of ATP hydrolysis by Mg2+, K+ and ATP. EDTA was protected by chelation of Mg2+ but catecholamines acted by some other mechanism. The specificities of various nucleotides as inhibitors (in conjunction with Mg2+ and K+) and as substrates for the (Na+, K+) ATPase were strikingly different. ATP, ADP, beta,gamma-CH2-ATP and alpha,beta-CH2-ADP were active as inhibitors, whereas inosine, cytidine, uridine, and guanosine triphosphates (ITP, CTP, UTP, and GTP) and adenosine monophosphate (AMP) were not. On the other hand, ATP and CTP were substrates and beta,gamma-NH-ATP was a competitive inhibitor of ATP hydrolysis, but not an inhibitor in conjunction with Mg2+ and K+. The Ca2+-ATPase from sarcoplasmic reticulum and F1, the Mg2+-ATPase from the inner mitochondrial membrane, were also inhibited by Mg2+. Catecholamines reversed inhibition of the Ca2+-ATPase, but not that of F1.     

Biochemical and electron microscopy analysis of the endotoxin binding to microtubulesin vitro

The mechanisms involved in cellular activation and damage by bacterial endotoxins are not completely defined. In particular, there is little information about possible intracellular targets of endotoxins. Recently, the participation of a microtubule associated protein in endotoxin actions on macrophages has been suggested. In the present work, we have studied the effect ofE. coli lipopolysaccharide on the polymerization of microtubular proteinin vitro. Electrophoretic analysis of the polymerization mixtures showed that the endotoxin inhibited the polymerization when present at high concentrations. At lower concentrations, LPS selectively displaced the microtubule associated protein MAP-2 from the polymerized microtubules. Electron microscopy showed that LPS binds to microtubules of tubulin+MAPs and to microtubules of purified tubulin (without MAPs) polymerized with taxol. Gel filtration experiments confirmed the binding of LPS to tubulin, and by ligand blot assays an interaction LPS — MAP-2 was detected. The ability of LPS to interact with microtubular proteins suggests a possible participation of microtubules on the cellular effects of endotoxins.     

Guanosine 5'-O-(3-thiotriphosphate), a potent nucleotide inhibitor of microtubule assembly

Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and the two diastereoisomers of guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) were prepared enzymatically, and their interactions with tubulin and microtubule-associated proteins (MAPs) in 0.1 M 2-(N-morpholino)ethanesulfonate, 0.5 mM MgCl2 were examined. GTP gamma S did not support microtubule assembly but instead inhibited the reaction. This analog was 1.5-2 times more potent than GDP in inhibiting both tubulin polymerization and GTP hydrolysis under conditions in which these reactions were dependent on MAPs. In contrast to the analog's inhibitory effects on polymerization and hydrolysis, however, radiolabeled GTP gamma S was only feebly bound by purified tubulin at 0 degrees C relative to the binding of GDP and GTP. There was a marked increase in the amount of GTP gamma S bound when the reaction temperature was raised to 37 degrees C or when MAPs were included in the reaction mixture. Only when both MAPs were present and the higher reaction temperature was used did the binding of GTP gamma S exceed that of GDP. Since substitution of sulfur for oxygen in a molecule should decrease its hydrophilic properties, these findings suggest that the exchangeable nucleotide binding site of tubulin becomes more hydrophobic at higher temperatures and in the presence of MAPs. The two isomers of GTP beta S were able to support MAP-dependent polymerization, although a 50-100-fold higher concentration of the analogs as compared to GTP was required. Neither isomer of GTP beta S had a significant inhibitory effect on GTP hydrolysis dependent on tubulin + MAPs.     

Effect of ovarian steroids on membrane ATPase activities in microsomes (microsomal fractions) from rat myometrium. Inhibition of a component of the Mg2+-activated ATPase by Ca2+-calmodulin and by oxytocin.

The activities of Mg2+-ATPase (Mg2+-activated ATPase), (Ca2+ + Mg2+)-activated ATPase and (Na+ + K+)-activated ATPase have been determined in microsomes (microsomal fractions) obtained from rat myometrium under different hormonal conditions. Animals were either ovariectomized and treated for a prolonged period of time with 17 beta-oestradiol or progesterone, or myometria were obtained at day 21 of pregnancy. In each case the endometrium was carefully removed. The Mg2+-ATPase consists of two components: an inactivating labile component and a second constant component. The rate of ATP hydrolysis by the labile component of the Mg2+-ATPase declines exponentially as a function of time after adding the membranes to the assay medium; this inactivation is caused by the presence of ATP in the medium. This ATPase activity inhibited by ATP is catalysed by a labile enzyme and hence it gradually diminishes within a few hours, even when the microsomes are kept on ice. This labile component has the highest activity in microsomes from pregnant rats, a lower activity in progesterone-treated rats, and the lowest in 17 beta-oestradiol-treated rats. This component of the Mg2+-ATPase is not affected by 90 nM-oxytocin. The constant component of the Mg2+-ATPase must be ascribed to a different enzyme, which, in contrast with the labile component, is very stable and not affected by the hormonal status of the animal. This constant component of the Mg2+-ATPase is inhibited both by Ca2+-calmodulin, and by oxytocin in microsomes from pregnant and from progesterone-treated animals, whereas such inhibition does not occur in microsomes from 17 beta-oestradiol-treated animals. The activity of the (Na+ + K+)-activated ATPase is not dependent on the hormonal status of the animal. Myometrial microsomes present an ATP-dependent Ca2+ transport, irrespective of the hormonal condition, but only in microsomes obtained from rats treated with 17 beta-oestradiol, can a (Ca2+ + Mg2+)-activated ATPase activity be demonstrated. This activity can be stimulated by calmodulin.     

Inhibition of microtubule assembly by poly(L-glutamic acid) and the site of its action

Poly(L-glutamic acid) (PGA) suppresses the polymerization of porcine brain microtubule proteins and induces the depolymerization in vitro in a concentration-dependent manner. The extent of inhibition increases with increasing molecular weight of the PGA tested. A 50% inhibition of the protein polymerization was observed at a PGA (molecular weight = 60,000) to microtubule protein ratio of 0.04 (w/w), and complete inhibition was obtained at a ratio of 0.07. Such an inhibition on the polymerization by PGA is greatly decreased when Mg2+ is present at a higher concentration. The addition of PGA raises the critical concentration of microtubule proteins necessary for assembly. During incubation with PGA, microtubule proteins retain the ability to assemble, i.e., substoichiometric amounts of taxol considerably relieve the inhibition of assembly by PGA. PGA interacts with microtubule-associated proteins (MAPs) preferentially, because the amount of MAPs binding to PGA-Sepharose 4B is much larger than that of tubulin. Tau proteins were observed only in adsorbed fractions, while MAP-2 was present in both unbound and adsorbed fractions.     

Magnesium requirements for guanosine 5'-O-(3-thiotriphosphate) induced assembly of microtubule protein and tubulin

Two conflicting interpretations on the role of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in microtubule protein and tubulin assembly have been previously reported. One study finds that GTP gamma S promotes assembly while another study reports that GTP gamma S is a potent inhibitor of microtubule assembly. We have examined the potential role of Mg2+ to learn if the conflicting interpretations are due to a metal effect. Turbidity, electron microscopy, and nucleotide binding and hydrolysis were used to analyze the effect of the Mg2+ concentration on GTP gamma S-induced assembly of microtubule protein (tubulin + microtubule-associated proteins) in the presence of buffer +/- 30% glycerol and in buffer with GTP added before or after GTP gamma S. GTP gamma S substantially lowers the Mg2+ concentration required to induce cross-linked or clustered rings of tubulin. These cross-linked rings do not assemble well into microtubules, and GTP only partially restores microtubule assembly. However, taxol will promote GTP gamma S-induced cross-linked rings of microtubule protein to assemble into microtubules. The effect of GTP gamma S on microtubule protein assembly in the presence of Zn2+ with and without added Mg2+ suggests that GTP gamma S also effects the formation of Zn2+-induced sheet aggregates. Purified tubulin was used in assembly experiments with Mg2+, Zn2+, and taxol to better understand GTP gamma S interactions with tubulin. The optimal Mg2+ concentration for assembly of tubulin is lower with GTP gamma S than with GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of Mg2+ on hepatic microsomal Ca2+ and Sr2+ transport

The ATP-dependent uptake of Ca2+ by rat liver microsomal fraction is dependent upon Mg2+. Studies of the Mg2+ requirement of the underlying microsomal Ca2+-ATPase have been hampered by the presence of a large basal Mg2+-ATPase activity. We have examined the effect of various Mg2+ concentrations on Mg2+-ATPase activity, Ca2+ uptake, Ca2+-ATPase activity and microsomal phosphoprotein formation. Both Mg2+-ATPase activity and Ca2+ uptake were markedly stimulated by increasing Mg2+ concentration. However, the Ca2+-ATPase activity, measured concomitantly with Ca2+ uptake, was apparently unaffected by changes in the Mg2+ concentration. In order to examine the apparent paradox of Mg2+ stimulation of Ca2+ uptake but not of Ca2+-ATPase activity, we examined the formation of the Ca2+-ATPase phosphoenzyme intermediate and formation of a Mg2+-dependent phosphoprotein, which we have proposed to be an attribute of the Mg2+-ATPase activity. We found that Ca2+ apparently inhibited formation of the Mg2+-dependent phosphoprotein both in the absence and presence of exogenous Mg2+. This suggests that Ca2+ may inhibit (at least partially) the Mg2+-ATPase activity. However, inclusion of the Ca2+ inhibition of Mg2+-ATPase activity in the calculation of Ca2+-ATPase activity reveals that this effect is insufficient to totally account for the stimulation of Ca2+ uptake by Mg2+. This suggests that Mg2+, in addition to stimulation of Ca2+-ATPase activity, may have a direct stimulatory effect on Ca2+ uptake in an as yet undefined fashion. In an effort to further examine the effect of Mg2+ on the microsomal Ca2+ transport system of rat liver, the interaction of this system with Sr2+ was examined. Sr2+ was sequestered into an A23187-releasable space in an ATP-dependent manner by rat liver microsomal fraction. The uptake of Sr2+ was similar to that of Ca2+ in terms of both rate and extent. A Sr2+-dependent ATPase activity was associated with the Sr2+ uptake. Sr2+ promoted formation of a phosphoprotein which was hydroxylamine-labile and base-labile. This phosphoprotein was indistinguishable from the Ca2+-dependent ATPase phosphoenzyme intermediate. Sr2+ uptake was markedly stimulated by exogenous Mg2+, but the Sr2+-dependent ATPase activity was unaffected by increasing Mg2+ concentrations. Sr2+ uptake and Sr2+-dependent ATPase activity were concomitantly inhibited by sodium vanadate. In contrast to Ca2+, Sr2+ had no effect on Mg2+-dependent phosphoprotein formation. Taken together, these data indicate that Mg2+ stimulated Ca2+ and Sr2+ transport by increasing the Ca2+ (Sr2+)/ATP ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

Taxol effect on tubulin polymerization and associated guanosine 5'-triphosphate hydrolysis

Taxol has been used as a tool to investigate the relationship between microtubule assembly and guanosine 5'-triphosphate (GTP) hydrolysis. The data support the model previously proposed [Carlier, M.-F., & Pantaloni, D. (1981) Biochemistry 20, 1918] that GTP hydrolysis is not tightly coupled to the polymerization process but takes place as a monomolecular process following polymerization. The results further indicate that the energy liberated by GTP hydrolysis is not responsible for the subsequent blockage of GDP on polymerized tubulin. When tubulin is polymerized in the presence of 10-100 microM taxol, the rapid formation of a large number of very short microtubules (l less than 1 micron) is accompanied by the development of turbidity to a lesser extent than what is observed when the same weight amount of longer microtubules (l = 5 microns) is formed. A slower subsequent turbidity increase corresponds to the length redistribution of these short microtubules into 3-5-fold longer ones without any change in the weight amount of polymer. The evolution of the rate of length redistribution with the concentration of taxol suggests a model within which taxol would bind to dimeric tubulin and to tubulin present at the ends of microtubules with a somewhat 10-fold lower affinity than to polymerized tubulin embedded in the bulk of microtubules. In agreement with this model, binding of taxol to the tubulin-colchicine complex in the dimeric form could be measured from the increase in the GTPase activity of the tubulin-colchicine complex accompanying taxol binding.     

Ca2+- or Mg2+-Dependent Enzymic ATP Hydrolysis Associated with the Microsomal Fraction of Frog Sciatic Nerves

The microsomal fraction of frog sciatic nerves was found to contain Ca2+- or Mg2+-dependent hydrolytic activity toward different nucleoside di- and triphosphates. In the presence of Ca2+ substrate specificity was in the order CTP > UTP > GTP > ATP. When Mg2+ was used, the triphosphates were approximately equally good substrates. ATP hydrolytic activity was very similar with Ca2+ or Mg2+ as the cofactor, whereas Ca2+ was the more potent activator of hydrolysis of the other triphosphates tested. The preparation showed some activity toward the nucleoside diphosphates but none toward the monophosphates or p-nitrophenylphosphate. The enzymic properties of ATP hydrolysis were more closely studied. The hydrolysis was optimal at 18--24 degrees C in the presence of 1 mM-Ca2+ or 1 mM-Mg2+. Ca2+- and Mg2+-ATP hydrolysis displayed pH maxima around 8.0--8.5 and 7.4--8.0, respectively. Vmax values for Ca2+- and Mg2+-ATP hydrolysis similar: approx. 12 mumol Pi per h per mg protein with a Km value of approx. 0.05 mM. The ATP hydrolysis activity was inhibited by NaF but unaffected by ouabain, vanadate, cytochalasin B, and various drugs known to influence ATPase activity of mitochondria. Zn2+ stimulated the ATP hydrolysis activity at low concentrations (10(-6)-10(-5) M) and inhibited it at higher concentrations. The possibility that these observations account for stimulation and inhibition of axonal transport in frog sciatic nerves exposed to similar concentrations of Zn2+ is discussed.     

Characterization of Calcium-Activated and Magnesium-Activated ATPases of Brain Nerve Endings

The properties of Ca2+-activated and Mg2+-activated ATPases of nerve endings from mouse brain were investigated. Ca2+ and Mg2+ each can activate ATP hydrolysis in synaptosomes and its subfractions. Both Ca2+-ATPase and Mg2+-ATPase exhibit high and low affinity for their respective cations. At millimolar concentrations of Ca2+ or Mg2+, several nucleoside triphosphates could serve as substrate for the two enzymes and their specific activities were about three to four times higher in synaptic vesicles than in synaptosomal plasma membranes (SPM). Both in SPM and in synaptic vesicles the relative activity in the presence of Ca2+ was in the order of CTP greater than UTP greater than GTP = ATP, but with Mg2+ the activity was higher with ATP than with the other three triphosphates. Mg2+-ATPase was more active than Ca2+-ATPase in SPM, but in synaptic vesicles the two enzymes exhibited similar activity. Kinetic studies revealed that Mg2+-ATPase was inhibited by excess ATP and not by excess Mg2+. The simultaneous presence of Na+ + K+ stimulated Mg2+-ATPase and inhibited Ca2+-ATPase activity in intact synaptosomes and SPM. The stimulation of Mg2+-ATPase by Na+ + K+ was further increased by increasing Mg2+ concentration and was inhibited by Ca2+ and by ouabain. When Ca2+ and Mg2+ are present together in SPM or synaptic vesicles, the total Pi liberated by the two cations may either increase or decrease, depending on their relative concentrations. Kinetic analyses indicate that Ca2+ and Mg2+ bind independently to the enzyme alone or together at different sites. The results suggest that Ca2+-ATPase and Mg2+-ATPase in SPM or synaptic vesicles may be separate and distinct systems.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

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 an ionized Ca2+ concentration of approx. 1 microM. 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.     

Adenosine triphosphatases associated with bovine brain microtubules. II. Properties of ATPases I and II

The catalytic properties of two ATPases which had been purified from bovine brain microtubules (Tominaga, S. & Kaziro, Y. (1983) J. Biochem. 93, 1085-1092) were studied. ATPase I, which had a molecular weight of 33,000, required the presence of 1.0 microM tubulin, 0.2 mM Mg2+, and 10 mM Ca2+ for maximal activity. The activation of ATPase I by tubulin was specific to the native form of tubulin, which could not be replaced by F-actin or tubulin denatured either by heat or more mildly by dialysis in the absence of glycerol. ATPase I was not specific to ATP, and GTP, and to a lesser extent, UTP and CTP were also hydrolyzed. Km for ATP of ATPase I was about 0.04 mM. ATPase I was inhibited by 5 mM Mg2+, 0.04 M K+, 10(-3) M vanadate, 10 mM N-ethylmaleimide, or 20% (v/v) glycerol. ATPase II, which was associated with membrane vesicles, required the presence of 0.2-2.0 mM Mg2+ and 20 mM KCl for activity. Tubulin stimulated the reaction of ATPase II only partially, and the addition of Ca2+ was rather inhibitory. ATPase II was specific to ATP with a Km value of 0.14 mM. It was inhibited by 1.6 mM N-ethylmaleimide and 20% (v/v) glycerol, but was not very sensitive to vanadate. Instead, ATPase II was inhibited by trifluoperazine, chlorpromazine, and nicardipin at 10(-3) M.     

Division of divalent cations into two groups in relation to their effect on the coupling of the F0F1-ATPase of Rhodospirillum rubrum to the protonmotive force

Divalent cations are divided into two groups in relation to their ability to promote ATP synthase catalyzed reactions. In the presence of Mg2+, the following pattern rules: (i) uncoupler-stimulated ATP hydrolysis of Rhodospirillum rubrum chromatophores which shows an optimum concentration of the divalent cation; (ii) ATP-induced proton pumping in chromatophores; (iii) light-induced ATP synthesis in chromatophores; (iv) no or very low ATPase activity of purified F1-ATPase unmasked by diethylstilbestrol or n-octyl beta-D-glucopyranoside. In the presence of Ca2+, the following pattern occurs: (i) no stimulation of the ATP hydrolysis in chromatophores by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; (ii) no ATP-induced proton pumping; (iii) no light-induced ATP synthesis; (iv) a high ATPase activity of the purified F1-ATPase which is inhibited by diethylstilbestrol and n-octyl beta-D-glucopyranoside. Co2+, Mn2+, and Zn2+ are members of the "Mg2+-group", whereas Cd2+ is suggested to fall between the two groups. Intrinsic uncoupling of the membrane-bound ATP synthase has been suggested to account for the effect caused by Ca2+ in chloroplasts [Pick, U., & Weiss, M. (1988) Eur. J. Biochem. 173, 623-628]. Such an interpretation is consistent with our results on chromatophores. The uncoupling cannot occur at the level of the membrane since neither light-induced nor Mg-ATP-induced proton pumping is affected by Ca2+. A conformational change is suggested to be the reason for this intrinsic uncoupling, and it is proposed to be controlled by the diameters of the divalent cations (Ca2+ greater than Cd2+ greater than Mn2+ greater than Co2+ greater than Zn2+ greater than Mg2+).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tubulin-myosin interaction. Some properties of binding between tubulin and myosin

This report presents evidence suggesting the direct binding between tubulin and myosin: (1) coprecipitation of tubulin with myosin occurred at a low ionic strength at which no precipitation of tubulin by itself occurred; (2) the amount of tubulin coprecipitated was unchanged when the coprecipitate was washed thoroughly; (3) about 2 mol of tubulin dimer could bind per mol of myosin at the maximum under our experimental conditions. The binding of about 1 mol of tubulin dimer was influenced by the presence of F-actin, but that of the other 1 mol of tubulin dimer was uninfluenced. In the former binding, tubulin or actin which bound first to myosin was suggested to have a priority. With regard to the priority of the binding, a similar result was obtained from the experiments of tubulin interference in actin activation of myosin Mg2+-ATPase. The tubulin-myosin binding occurred moderately even at 0 degrees C and was not affected by Ca2+ (2 mM), colchicine (200 microM), or Mg-ATP (4 mM), reflecting that the ability of tubulin to bind to myosin was different from the ability of tubulin to form microtubules and that the nature of tubulin-myosin binding was different from that of F-actin-myosin binding. Besides tubulin-myosin interaction, a possible interaction between microtubule-associated proteins (MAPs) and actomyosin was suggested from the data that MAPs activated actomyosin MG2+-ATPase activity while purified tubulin inhibited the activity.     

Characterization of rat heart plasma membrane Ca2+/Mg2+ ATPase

The Ca2+/Mg2+ ATPase of rat heart plasma membrane was activated by millimolar concentrations of Ca2+ or Mg2+; other divalent cations also activated the enzyme but to a lesser extent. Sodium azide at high concentrations inhibited the enzyme by about 20%; oligomycin at high concentrations also inhibited the enzyme slightly. Trifluoperazine at high concentrations was found inhibitory whereas trypsin treatment had no significant influence on the enzyme. The rate of ATP hydrolysis by the Ca2+/Mg2+ ATPase decayed exponentially; the first-order rate constants were 0.14-0.18 min-1 for Ca2+ ATPase activity and 0.15-0.30 min-1 for Mg2+ ATPase at 37 degrees C. The inactivation of the enzyme depended upon the presence of ATP or other high energy nucleotides but was not due to the accumulation of products of ATP hydrolysis. Furthermore, the inactivation of the enzyme was independent of temperature below 37 degrees C. Con A when added into the incubation medium before ATP blocked the ATP-dependent inactivation; this effect was prevented by alpha-methylmannoside. In the presence of low concentrations of detergent, the rate of ATP hydrolysis was reduced while the ATP-dependent inactivation was accelerated markedly. Both Con A and glutaraldehyde decreased the susceptibility of Ca2+/Mg2+ ATPase to the detergent. These results suggest that the Ca2+/Mg2+ ATPase is an intrinsic membrane protein which may be regulated by ATP.