Addition Of MgATP Research Articles

Mechanisms of luteinizing-hormone exocytosis in Staphylococcus aureus-α-toxin-permeabilized sheep gonadotropes

We have used primary gonadotropes permeabilized with the pore-forming protein Staphylococcus aureus alpha-toxin to investigate luteinizing hormone (lutropin, LH) exocytosis. The diameter of the alpha-toxin pores (2-3 nm) allows the exchange of small molecules, whereas larger cytosolic proteins are retained. Because of the slow exchange of small molecules through the pores, we have developed a protocol which combines prolonged pre-equilibration of the permeabilized cells at 0 degrees C before stimulation with strong Ca2+ buffering. Under these conditions, increasing the free Ca2+ concentration from 0.1 microM to 10 microM [EC50 (concentration effecting half-maximal response) 2-3 microM] resulted in a 15-20-fold increase in LH exocytosis. LH exocytosis was maximal in the first 3 min and completed by 12 min. When permeabilized cells were equilibrated for prolonged periods in the absence of MgATP, Ca2(+)-stimulated LH secretion gradually declined (greater than 90% decrease by 60 min). Addition of MgATP (5 mM) rapidly restored full Ca2(+)-stimulated LH secretion. MgATP supported Ca2(+)-stimulated LH secretion at a half-maximal concentration of 1.5 mM. UTP and adenosine 5'-[gamma-thio]triphosphate were 40 and 31% as effective as MgATP, whereas other nucleotide triphosphates were ineffective. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA; 50 nM) stimulated LH exocytosis at free Ca2+ concentrations as low as 1 nM and was additive with Ca2+ at higher free Ca2+ concentrations. PMA-stimulated exocytosis required MgATP at concentrations similar to those required for Ca2(+)-stimulated LH exocytosis. These results demonstrate that LH exocytosis can be triggered both by micromolar Ca2+ concentrations or, in the virtual absence of Ca2+, by PKC activation. Both mechanisms of stimulated exocytosis have an absolute requirement for millimolar ATP. Because they retain cytosolic proteins, alpha-toxin-permeabilized cells may have advantages over alternative permeabilization methods provided that conditions are used that compensate for slow diffusion through alpha-toxin pores.

Monoclonal antibodies detect and stabilize conformational states of smooth muscle myosin.

Antibodies with epitopes near the heavy meromyosin/light meromyosin junction distinguish the folded from the extended conformational states of smooth muscle myosin. Antibody 10S.1 has 100-fold higher avidity for folded than for extended myosin, while antibody S2.2 binds preferentially to the extended state. The properties of these antibodies provide direct evidence that the conformation of the rod is different in the folded than the extended monomeric state, and suggest that this perturbation may extend into the subfragment 2 region of the rod. Two antihead antibodies with epitopes on the heavy chain map at or near the head/rod junction. Magnesium greatly enhances the binding of these antibodies to myosin, showing that the conformation of the heavy chain in the neck region changes upon divalent cation binding to the regulatory light chain. Myosin assembly is also altered by antibody binding. Antibodies that bind to the central region of the rod block disassembly of filaments upon MgATP addition. Antibodies with epitopes near the COOH terminus of the rod, in contrast, promote filament depolymerization, suggesting that this region of the tail is important for assembly. The monoclonal antibodies described here are therefore useful both for detecting and altering conformational states of smooth muscle myosin.

A new method for cell permeabilization reveals a cytosolic protein requirement for Ca2+-activated secretion in GH3 pituitary cells

Ca2+ is a major regulator of exocytosis in secretory cells, however, the biochemical mechanisms underlying regulation remain to be identified. To render the secretory apparatus accessible for biochemical studies, we have developed a cell permeabilization method (cell cracking) which utilizes mechanical shear. GH3 pituitary cells subjected to cracking were permeable to macromolecules but retained a normal cytoplasmic ultrastructure including secretory granules. Incubation of the permeable cells at 30-37 degrees C with 0.1-1.0 microM Ca2+ and millimolar MgATP resulted in the release of the secretory proteins, prolactin (PRL) and a proteoglycan, but not lysosomal enzymes. Extensively washed permeable cells were incapable of releasing PRL in response to Ca2+ and MgATP addition. However, addition of cytosol was found to restore Ca2+-activated, MgATP-dependent PRL release. The cytosolic factor responsible for activity was thermolabile and protease sensitive. The protein was partially purified, and its molecular mass was estimated to be equivalent to that of a globular protein of 200-350 kDa by molecular sieve chromatography. Inhibitors of calmodulin or protein kinase C (trifluroperazine, calmidazolium, H-7) failed to inhibit Ca2+-activated PRL release, and the required cytosolic protein could not be replaced by purified calmodulin, calmodulin-dependent protein kinase II, protein kinase C, or calpactin I. Further purification and characterization of the cytosolic protein should reveal the nature of biochemical events involved in regulated secretory exocytosis.

Smooth muscle gelsolin and a Ca2+‐sensitive contractile cell model

Smooth muscle gelsolin, termed smooth muscle 90-kDa protein in our previous paper (Kanno et al. FEBS Lett. 1985; 184:202-206), was purified from bovine aorta. Antibody prepared against smooth muscle gelsolin was used to detect the presence of gelsolin in human lung fibroblast MRC-5 cells permeabilized with Triton X-100 (MRC-5 cell models). These cells contracted in the presence of MgATP and Ca2+ in doses over 1 microM. Immunofluorescence microscopy using phalloidin and antigelsolin antibody showed that gelsolin was distributed along the stress fibers, except for a marginal bundle of cells, when MRC-5 cells were growth-arrested in serum-depleted medium. Making use of immunoblotting and indirect immunofluorescence techniques, we demonstrated that gelsolin is not retained in the MRC-5 cell models. We used purified smooth muscle gelsolin as a specific agent to sever the actin filaments. Preincubation of MRC-5 cell models with gelsolin led to a destruction of stress fibers, in a dose- and Ca2+ -dependent manner. The contractility was also lost, in the same manner described above, thereby indicating that a continuous distribution of actin filaments within the stress fibers is required for cell contraction. Treatment of MRC-5 cells with the Ca2+ ionophore A23187 induced an extracellular Ca2+ -dependent contraction but not a massive destruction of stress fibers, thereby indicating that most of the endogenous gelsolin was inactive under these conditions. Our interpretation of these results is that increases in cytoplasmic Ca2+ concentrations are sufficient for the contraction but may be too transient to activate endogenous gelsolin and thereby disrupt the stress fibers. Indeed, the inhibition of contraction of the MRC-5 cell, as induced by smooth muscle gelsolin, required preincubation in the presence of Ca2+, before the addition of MgATP. These results suggest that destruction of the stress fibers by endogenous gelsolin, which leads to inhibition of cell contraction, may occur if the cytoplasmic Ca2+ is maintained at high concentrations for a few minutes.

Glutamate uptake into synaptic vesicles of bovine cerebral cortex and electrochemical potential difference of proton across the membrane

Measurements have been made of the ATP-dependent membrane potential (delta psi) and pH gradient (delta pH) across the membranes of the synaptic vesicles purified from bovine cerebral cortex, using the voltage-sensitive dye bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxanol and the delta pH-sensitive fluorescent dye 9-aminoacridine respectively. A pre-existing small delta pH (inside acidic) was detected in the synaptic vesicles, but no additional significant contribution by MgATP to delta pH was observed. In contrast, delta psi (inside positive) increased substantially upon addition of MgATP. This ATP-dependent delta psi was reduced by thiocyanate anion (SCN-), a delta psi dissipator, or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonmotive-force dissipator. Correspondingly, a substantially larger glutamate uptake occurred in the presence of MgATP, which was inhibited by SCN- and FCCP. A nonhydrolysable analogue of ATP, adenosine 5'-[beta gamma-methylene]triphosphate, did not substitute for ATP in either delta psi generation or glutamate uptake. The results support the hypothesis that a H+-pumping ATPase generates a protonmotive force in the synaptic vesicles at the expense of ATP hydrolysis, and the protonmotive force thus formed provides a driving force for the vesicular glutamate uptake. The delta psi generation by ATP hydrolysis was not affected by orthovanadate, ouabain or oligomycin, but was inhibited by N-ethylmaleimide, quercetin, trimethyltin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid. These results indicate that the H+-pumping ATPase in the synaptic vesicle is similar to that in the chromaffin granule, platelet granule and lysosome.

Inhibition of Neutral Lipase from Castor Bean Lipid Bodies by Coenzyme A (CoA) and Oleoyl-CoA

The neutral lipase (EC 3.1.1.3) in lipid body membranes isolated from the endosperm of 4 day old castor (Ricinus communis L.) seedlings catalyzes the hydrolysis of [(14)C]trioleoylglycerol, releasing [(14)C]oleic acid for up to 4 hours. However, the addition of Mg-ATP and coenzyme A (CoA), which are present in the cytoplasm of plant cells, caused a progressive inhibition of the neutral lipase such that after 15 minutes, release of [(14)C]oleic acid was almost undetectable. A fatty acyl CoA synthetase was found in the lipid body membrane which converts [(14)C]oleic acid produced from the lipase reaction to [(14)C]oleoyl-CoA under these conditions. The concentration of free oleoyl-CoA in the reaction mixture when the lipase was inhibited by 50% was calculated to be about 21 micromolar. It was found that a mixture of exogenously added oleoyl-CoA and CoA was most effective in causing lipase inhibition. Little inhibition of lipase was detected in the presence of CoA alone. It is possible that this effect is important In vivo in coordinating lipase activity with fatty acid oxidation.

Nitrite, a new substrate for nitrogenase.

We have examined the reactivity of the purified component proteins of Azotobacter vinelandii nitrogenase (Av1 and Av2) toward nitrate and nitrite. Nitrate has no effect on H2 evolution or C2H2 reduction by nitrogenase and thus is neither a substrate nor an inhibitor. Nitrite dramatically inhibits H2 evolution. This inhibition has two components, one irreversible and one reversible upon addition of CO. The irreversible inhibition is due to nitrite inactivation of the Fe protein. The rate of this inactivation is greatly enhanced by addition of MgATP, suggesting the [4Fe-4S] cluster is the site of nitrite attack. The reversible inhibition does not represent an inhibition of electron flow but rather a diversion of electrons away from H2 evolution and into the six-electron reduction of nitrite to ammonia. Thus, nitrogenase functions as a nitrite reductase.

Isolation and properties of an asparaginase from leaves of Pisum sativum

Asparaginase (EC 3.5.1.1), which displays a diurnal variation in activity in developing leaves of Pisum sativum, has been isolated from half-expanded leaves obtained at the end of the dark (dark-harvested) or during the light (light-harvested) periods and its properties compared to determine if variation in activity is a result of changes to the protein. Both dark- and light-harvested asparaginase preparations required K + and Ca 2+ for maximum activity and had similar M r (58 000), pH optima (7.8–9.8), and K m for asparagine (3.2–3.7 mM). Asparaginase activity from light- or dark-harvested leaves co-eluted during size-exclusion, ion-exchange and hydroxylapatite chromatography. Addition of MgATP to crude preparations inhibited activity non-competitively ( K ies 0.2 mM, K ies , 0.8 mM), but no inhibition of activity was noted by ATP in purified preparations. The activity of purified asparaginase was not affected by a range of monovalent or divalent ions (except for Zn 2+), or by any of the protein amino acids. These results indicate that changes in asparaginase activity during the photoperiod are not a result of post-translational modification of existing protein, and support our previous suggestion that increased activity is a result of de novo synthesis.

Involvement of the 60 kDa phosphoprotein in the regulation of Ca 2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles

Junctional sarcoplasmic reticulum (SR) vesicles isolated from back muscles of normal and malignant hyperthermia susceptible (MHS) pigs were phosphorylated by addition of MgATP in the presence of 5 mM Ca 2+ and 1 μM calmodulin (CaM). The major site of phosphorylation was a 60 kDa protein both in normal and MHS SR. The maximal amount of phosphorylation in MHS SR (5 pmol P/mg SR) was significantly lower than that in the normal SR (12 pmol P/mg SR). The phosphorylated 60 kDa protein was spontaneously dephosphorylated both in normal and MHS SR. Ca 2+ release from the passively loaded SR was induced by a Ca 2+-jump, and monitored by stopped-flow fluorometry using chlorotetracycline. In the absence of preincubation with MgATP, no significant difference was found in any of the kinetic parameters of Ca 2+ release between normal and MHS SR. Upon addition of 20 μM MgATP to the passively loaded SR to phosphorylate the 60 kDa protein, the initial rate of Ca 2+ release in normal SR significantly decreased from 659 ± 102to361 ± 105 nmol Ca 2+/mg SR per s, whereas in MHS SR the rate decreased from 749 ± 124 to 652 ± 179nmol Ca 2+/mg SR per s. Addition of 20 μM adenosine 5′-[β,γ-imido]triphosphate (p[NH]ppA) did not significantly alter the initial rate of Ca 2+ release both in normal and MHS SR. These results suggest that the previously reported higher Ca 2+ release rate in MHS SR (Kim et al. (1984) Biochim. Biophys. Acta 775, 320–327) is at least partly due to the reduced extent of the Ca 2+/CaM-dependent phosphorylation of the 60 kDa protein. Two-dimensional gel electrophoresis study showed that amount of a protein with M r = 55 000 was significantly lower in MHS SR than in normal SR suggesting that the abnormally lower amount of 55 kDa protein would cause the lower amount of phosphorylation of the 60 kDa protein in MHS SR.

Calcium uptake and release characteristics of the dense tubules of digitonin-permeabilized human platelets

The kinetics of ATP-driven Ca 2+ uptake by the dense tubules were studied in digitonin-permeablized human blood platelets. Digitonin at 3 μg/ml was shown capable of permeablizing the plasma membrane to lactate dehydrogenase and the cytoplasmic Ca 2+ indicator Quin2 without increasing the passive permeability of the dense tubular membrane for Ca 2+. Experimentation was carried out with platelets treated with 3 μg/ml digitonin reisolated and resuspended in detergent-free medium (‘digitonin-permeablized’ platelets). Active Ca 2+ accumulation, which occurs over a period of minutes, was monitored by the increase in the fluorescence of chlorotetracycline after the addition of Mg-ATP (37°C). The active uptake is inhibited by 15 μM trifluoperazine. The process is saturable with respect to external [Ca 2+], with a K m of 180 ± 5 nM and a Hill coefficient (n) of 1.40 ± 0.05. Analysis of the maximal uptake in steady state gave similar results ( K m = 160 ± 5 nM, n = 1.50 ± 0.05). The rate of uptake at [Ca 2+] ≈ K m is increased when the digitonin-permeablized platelets are preincubated with 100 nM phorbol 12-myristate 13-acetate. Actively accumulated Ca 2+ is rapidly released (less than 1 min) by addition of d -myo- inositol triphosphate (IP 3). The maximal extent of release is 50%; the EC 50 for IP 3 is approx. 12 μM. The data are compared with findings for fractionated dense tubular membrane vesicles and for the intact platelet.

NaCl Induces a Na+/H+ Antiport in Tonoplast Vesicles from Barley Roots

Evidence was found for a Na(+)/H(+) antiport in tonoplast vesicles isolated from barley (Hordeum vulgare L. cv California Mariout 72) roots. The activity of the antiport was observed only in membranes from roots that were grown in NaCl. Measurements of acridine orange fluorescence were used to estimate relative proton influx and efflux from the vesicles. Addition of MgATP to vesicles from a tonoplast-enriched fraction caused the formation of a pH gradient, interior acid, across the vesicle membranes. EDTA was added to inhibit the ATPase, by chelating Mg(2+), and the pH gradient gradually dissipated. When 50 millimolar K(+) or Na(+) was added along with the EDTA to vesicles from control roots, the salts caused a slight increase in the rate of dissipation of the pH gradient, as did the addition of 50 millimolar K(+) to vesicles from salt-grown roots. However, when 50 millimolar Na(+) was added to vesicles from salt-grown roots it caused a 7-fold increase in the proton efflux. Inclusion of 20 millimolar K(+) and 1 micromolar valinomycin in the assay buffer did not affect this rapid Na(+)/H(+) exchange. The Na(+)/H(+) exchange rate for vesicles from salt-grown roots showed saturation kinetics with respect to Na(+) concentration, with an apparent K(m) for Na(+) of 9 millimolar. The rate of Na(+)/H(+) exchange with 10 millimolar Na(+) was inhibited 97% by 0.1 millimolar dodecyltriethylammonium.

Myosin from pancreatic acinar carcinoma cells. Isolation, characterization and demonstration of heavy- and light-chain phosphorylation.

Myosin has been identified in a variety of non-muscle cells, and is believed to play a role in maintenance of cell shape, locomotion, cytokinesis, exocytosis and other cellular functions. In this paper we describe the purification of myosin from a pancreatic acinar-cell carcinoma of the rat which forms solid tumours, but retains many differentiated functions. The purified myosin was composed of a 200,000 Da heavy chain and two or three classes of light chains. Electron-microscopic examination of rotary-shadowed preparations revealed that individual molecules had two globular heads and a long tail measuring approx. 149 nm. The myosin was soluble in high-salt buffers and became sedimentable as the ionic strength was lowered. Examination of negative-stained preparations showed that this sedimentable myosin consisted of short, bipolar, thick filaments which had a strong tendency to aggregate in a head-to-head manner. The ATPase activity of the purified myosin was stimulated by EDTA or Ca2+, but not by Mg2+. In low ionic strength the Mg2+-dependent ATPase activity was activated by muscle f-actin. The pancreatic myosin bound to actin and could be dissociated by the addition of MgATP. Myosin purified from cells cultured in media containing [32P]Pi was phosphorylated on one of the light chains as well as the heavy chain. Thus pancreatic acinar cells contain a typical non-muscle myosin, and the subunits of this molecule are subject to post-translational modification by phosphorylation.

Study of sidedness and tightness to H + of corn root plasmalemma vesicles: preparation of a fraction enriched in inside-out vesicles

The latency of the Mg 2+-ATPase of corn root plasmalemma was measured using Triton X-100, Zwittergent 3–14, sodium dodecyl sulfate and lysophosphatidylcholine. Lysophosphatidylcholine was the only permeabilizing agent which did not inhibit the enzyme. It gave a higher latency than the other surfactants. The sidedness of plasmalemma vesicles obtained after sucrose gradient purification was estimated from: (i) the Mg 2+-ATPase latency; (ii) the fraction of Mg 2+-ATPase resistant to trypsin attack; (iii) the effect of the specific binding of concanavalin A on the microelectrophoretic mobility of the vesicles; and (iv) the separation of the vesicles by chromatography on a Sepharose-Con A column. These four kinds of independent data were consistent. They led to the conclusion that this membrane preparation contained only sealed vesicles (impermeable to MgATP), the majority of which (approx. 65%) were oriented right-side out. Addition of 0.25 M sucrose in the same way inhibited the MgATP hydrolysis measured with or without lysophosphatidylcholine, so that the latency of the activity remained unchanged. The inhibition was higher in the presence of 0.25 M inositol and trehalose, again without modifying the latency. Finally, this preparation contained no significant proportion of tightly sealed active vesicles, as indicated by the absence of quinacrine quenching after addition of MgATP. Tightly sealed vesicles, displaying quinacrine quenching, were obtained according to De Michelis and Spanswick ((1986) Plant Physiol. 81, 542–547). The sidedness in this vesicle preparation was the same as the previous one (approx. 65% right-side out). Washing with Triton X-100 described by Clement et al. ((1986) Physiol. Vég. 24, 25–35) was applied. The simultaneous measurements of quinacrine quenching and Mg 2+-ATPase latency suggested a general inversion of the vesicle sidedness (approx. 65% of inside-out vesicles) after 0.1% treatment with (w / v) Triton X-100. Furthermore, the total Mg 2+-ATPase activity was recovered in spite of protein loss of approx. 60%, and the specific activity increased by approx. 150% in the presence of lysophosphatidylcholine. In the absence of lysophosphatidylcholine, coupling of Mg 2+-ATPase activity with H + transport was indicated by a 30% increase of MgATP hydrolysis upon addition of gramicidin or NH 4Cl.

Further Characterization of Ribosome Binding to Thylakoid Membranes

Previous work indicated more polysomes bound to pea (Pisum sativum cv Progress No. 9) thylakoids in light than in the dark, in vivo (LE Fish, AT Jagendorf 1982 Plant Physiol 69: 814-825). With isolated intact chloroplasts incubated in darkness, addition of MgATP had no effect but 24 to 74% more RNA was thylakoid-bound at pH 8.3 than at pH 7. Thus, the major effect of light on ribosome-binding in vivo may be due to higher stroma pH. In isolated pea chloroplasts, initiation inhibitors (pactamycin and kanamycin) decreased the extent of RNA binding, and elongation inhibitors (lincomycin and streptomycin) increased it. Thus, cycling of ribosomes is controlled by translation, initiation, and termination. Bound RNA accounted for 19 to 24% of the total chloroplast RNA and the incorporation of [(3)H]leucine into thylakoids was proportional to the amount of this bound RNA. These data support the concept that stroma ribosomes are recruited into thylakoid polysomes, which are active in synthesizing thylakoid proteins.

Carbamoyl-phosphate synthetase II of the mammalian CAD protein: kinetic mechanism and elucidation of reaction intermediates by positional isotope exchange.

The kinetic mechanism of carbamoyl-phosphate synthetase II from Syrian hamster kidney cells has been determined at pH 7.2 and 37 degrees C. Initial velocity, product inhibition, and dead-end inhibition studies of both the biosynthetic and bicarbonate-dependent adenosinetriphosphatase (ATPase) reactions are consistent with a partially random sequential mechanism in which the ordered addition of MgATP, HCO3-, and glutamine is followed by the ordered release of glutamate and Pi. Subsequently, the binding of a second MgATP is followed by the release of MgADP, which precedes the random release of carbamoyl phosphate and a second MgADP. Carbamoyl-phosphate synthetase II catalyzes beta gamma-bridge:beta-nonbridge positional oxygen exchange of [gamma-18O]ATP in both the ATPase and biosynthetic reactions. Negligible exchange is observed in the strict absence of HCO3- (and glutamine or NH4+). The ratio of moles of MgATP exchanged to moles of MgATP hydrolyzed (nu ex/nu cat) is 0.62 for the ATPase reaction, and it is 0.39 and 0.16 for the biosynthetic reaction in the presence of high levels of glutamine and NH4+, respectively. The observed positional isotope exchange is suppressed but not eliminated at nearly saturating concentrations of either glutamine or NH4+, suggesting that this residual exchange results from either the facile reversal of an E-MgADP-carboxyphosphate-Gln(NH4+) complex or exchange within an E-MgADP-carbamoyl phosphate-MgADP complex, or both. In the 31P NMR spectra of the exchanged [gamma-18O]ATP, the distribution patterns of 16O in the gamma-phosphorus resonances in all samples reflect an exchange mechanism in which a rotationally unhindered molecule of [18O3, 16O]Pi does not readily participate. These results suggest that the formation of carbamate from MgATP, HCO3-, and glutamine proceeds via a stepwise, not concerted mechanism, involving at least one kinetically competent covalent intermediate, such as carboxyphosphate.

The Ca2+-Transport ATPase of Plant Plasma Membrane Catalyzes a nH+/Ca2+ Exchange

Microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings accumulate Ca(2+) upon addition of MgATP. MgATP-dependent Ca(2+) uptake co-migrates with the plasma membrane H(+)-ATPase on a sucrose gradient. Ca(2+) uptake is insensitive to oligomycin, inhibited by vanadate (IC(50) 40 micromolar) and erythrosin B (IC(50) 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca(2+) uptake is insensitive to protonophores. These results indicate that Ca(2+) transport in these microsomal vesicles is catalyzed by a Mg(2+)-dependent ATPase localized on the plasma membrane. Ca(2+) strongly reduces DeltapH generation by the plasma membrane H(+)-ATPase and increases MgATP-dependent membrane potential difference (Deltapsi) generation. These effects of Ca(2+) on DeltapH and Deltapsi generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca(2+) uptake into plasma membrane vesicles. The Ca(2+)-induced increase of Deltapsi is collapsed by permeant anions, which do not affect Ca(2+)-induced decrease of DeltapH generation by the plasma membrane H(+)-ATPase. The rate of decay of MgATP-dependent DeltapH, upon inhibition of the plasma membrane H(+)-ATPase, is accelerated by MgATP-dependent Ca(2+) uptake, indicating that the decrease of DeltapH generation induced by Ca(2+) reflects the efflux of H(+) coupled to Ca(2+) uptake into plasma membrane vesicles. It is therefore proposed that Ca(2+) transport at the plasma membrane is mediated by a Mg(2+)-dependent ATPase which catalyzes a nH(+)/Ca(2+) exchange.

Catalytic and allosteric mechanism of AMP nucleosidase from primary, beta-secondary, and multiple heavy atom kinetic isotope effects.

Adenosine 5'-phosphate was synthesized with specific heavy atom substitutions to permit measurement of V/K kinetic isotope effects for the N-glycohydrolase activity of the allosteric AMP nucleosidase and the acid-catalyzed solvolysis of these compounds. The effects of allosteric activation on the kinetic isotope effects together with the kinetic mechanism of AMP nucleosidase [DeWolf, W. E., Jr., Emig, F. A., & Schramm, V. L. (1986) Biochemistry 25, 4132-4140] indicate that the kinetic isotope effects are fully expressed. Comparison of individual primary and secondary kinetic isotope effects with combined isotope effects and the isotope effect of the reverse reaction indicated that kinetic isotope effects in AMP nucleosidase arise from a single step in the reaction mechanism. Under these conditions, kinetic isotope effects can be used to interpret transition-state structure for AMP nucleosidase. Changes in kinetic isotope effects occurred as a function of allosteric activator, demonstrating that allosteric activation alters transition-state structure for AMP nucleosidase. Kinetic isotope effects, expressed as [V/K(normal isotope]/[V/K(heavy isotope)], were observed with [2'-2H]AMP (1.061 +/- 0.002), [9-15N]AMP (1.030 +/- 0.003), [1'-2H]AMP (1.045 +/- 0.002), and [1'-14C]AMP (1.035 +/- 0.002) when hydrolyzed by AMP nucleosidase in the absence of MgATP. Addition of MgATP altered the [2'-2H]AMP effect (1.043 +/- 0.002) and the [1'-2H]AMP effect (1.030 +/- 0.003) and caused a smaller decrease of the 14C and 15N effects. Multiple heavy atom substitutions into AMP caused an increase in observed isotope effects to 1.084 +/- 0.004 for [1'-2H,1'-14C]AMP and to 1.058 +/- 0.002 for [9-15N,1'-14C]AMP with the enzyme in the absence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction between actin and HMM

It is shown that the interaction between actin and HMM results in a rapid precipitation of acto-HMM gel upon addition of MgATP. This is a simple demonstration of the idea that the formation of myosin filaments is not essential for mechanochemical reaction (muscle contraction) to occur and that the soluble myosin heads are competent to interact with actin to produce mechanical effect. Our findings also strongly support earlier suggestion that each head of one HMM molecule is able to bind to a different actin filament.

Reactivated melanophore motility: differential regulation and nucleotide requirements of bidirectional pigment granule transport.

To study the molecular basis for organized pigment granule transport, procedures were developed to lyse melanophores of Tilapia mossambica under conditions in which pigment granule movements could be reactivated. Gentle lysis of the melanophores resulted in a permeabilized cell model, which, in the absence of exogenous ATP, could undergo multiple rounds of pigment granule aggregation and dispersion when sequentially challenged with epinephrine and cAMP. Both directions of transport required ATP, since aggregation or dispersion in melanophores depleted of nucleotides could be reactivated only upon addition of MgATP or MgATP plus cAMP, respectively. Differences between the nucleotide sensitivities for aggregation and dispersion were demonstrated by observations that aggregation had a lower apparent Km for ATP than did dispersion and could be initiated at a lower ATP concentration. Moreover, aggregation could be initiated by ADP, but only dispersion could be reactivated by the thiophosphate ATP analog, ATP gamma S. The direction of pigment transport was determined solely by cAMP, since pigment granules undergoing dispersion reaggregated when cAMP was removed, and those undergoing aggregation dispersed when cAMP was added. These results provide evidence that pigment granule motility may be based on two distinct mechanisms that are differentially activated and regulated to produce bidirectional movements.

Isolation and characterization of a second nitrogenase Fe-protein from Azotobacter vinelandii.

Wild-type Azotobacter vinelandii strain UW was transformed with plasmid pDB12 to produce a species (LS10) unable to synthesize the structural proteins of component 1 and component 2 of native nitrogenase. A spontaneous mutant of this strain was isolated (LS15) which can grow by nitrogen fixation in the presence or absence of either Mo or W. It is proposed that LS15 fixes nitrogen solely by an alternative nitrogen-fixing system which previously has been hypothesized to exist in A. vinelandii. Under nitrogen-fixing conditions, LS15 synthesizes a protein similar to component 2 (Av2) of native nitrogenase in that it can complement native component 1 (Av1) for enzymatic activity. Isolation and characterization of this second component 2 shows it to be a 4Fe-4S protein of molecular mass about 62 kDa and is antigenically similar to Av2. This protein is also similar to Av2 in that in the reduced state it possesses a rhombic ESR spectrum in the g = 2 region, which changes to an axial spectrum upon addition of MgATP. It is suggested that this second Fe-protein is associated with the alternative nitrogen-fixing system in A. vinelandii.