Endosomes are acidified by association with discrete proton-pumping vacuoles in Dictyostelium.

The endocytic compartment in the amoeba Dictyostelium discoideum was labeled by feeding fluorescein 5-isothiocyanate-dextran. In homogenates containing 2 mM Mg2+, the compartments so labeled copurified with all of the vacuolar H(+)-ATPase activity in a dense peak. The fluorescence properties of the probe showed that these dense vacuoles were inherently acidic. Furthermore, after purging their residual acidity, they could be re-acidified by the addition of ATP. These data suggest that the H(+)-ATPase was structurally and functionally coupled to the endocytic space. The association of the H(+)-ATPase and endocytic compartment was reversed by the removal of either Mg2+ or traces of the cytosol. Endocytic vacuoles prepared in this way were deficient in vacuolar H(+)-ATPase activity and were not acidified upon addition of MgATP. The missing proton pumps were recovered in large buoyant vacuoles that lacked ingested fluorescein 5-isothiocyanate-dextran, acid hydrolases, and residual acidity. These vacuoles were also less susceptible than endosomes to disruption by digitonin, suggesting that their bilayers were low in sterols. These results indicate that the endocytic circuit in Dictyostelium is acidified by a discrete and separable proton-pumping organelle.     

Evidence of GTP-binding protein regulation of phospholipase A2 activity in isolated human platelet membranes

G protein regulation of human platelet membrane phospholipase A2 activity was investigated at pH 8.0 and 9.0 by studying the effects of the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), and of F-/Al3+ ions on arachidonic acid (AA) release. The membrane acted as the source of the enzyme, the substrate, and the G protein. At pH 8.0, 10 and 100 microM GTP gamma S stimulated AA mobilization at least 6-fold. Optimum AA release conditions required 1 mM Ca2+ and 5 mM Mg2+. Nonspecific nucleotide effect was excluded since similar stimulatory effects on AA release were not observed by ATP, GTP, ADP, and NADP. Although at pH 9.0 the GTP gamma S-stimulated AA release was greater than at pH 8.0, it constituted only 26% of the total. At both pH values the effect of F- (10 mM) in the presence of Al3+ (2 microM) was similar to that of GTP gamma S. The G protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), inhibited the GTP gamma S-stimulated AA release by about 80% at pH 8.0 and by 100% at pH 9.0. To determine a possible contribution to AA mobilization by the phospholipase C and diacylglycerol lipase pathway, the effects of neomycin, a phospholipase C inhibitor, were investigated. 100 microM neomycin did not inhibit the GTP gamma S-stimulated AA release at pH 8.0 and only slightly so (17%) at pH 9.0. At pH 8.0 in the presence of Ca2+ the released fatty acids consisted mainly of arachidonic and docosahexaenoic acids (80 and 8%, respectively). GTP gamma S had no effect on the fatty acid profile but only on their quantity. These results provide evidence of G protein regulation of phospholipase A2 activity in isolated platelet membranes.     

Ca2+ inhibits guanine nucleotide-activated phospholipase D in neural-derived NG108-15 cells.

We have investigated the regulation of phospholipase D (PLD) activity by guanine nucleotides and Ca2+ in cells of the NG108-15 neuroblastoma X glioma line that were permeabilized with digitonin. The nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) caused a nearly sixfold increase (EC50 = 3 microM) in production of [3H]phosphatidylethanol (specific product of the PLD transphosphatidylation reaction). Other GTP analogues were less effective than GTP gamma S, and guanosine-5'-O-(2-thiodiphosphate) inhibited PLD activation by GTP gamma S. Both basal and GTP gamma S-stimulated PLD activities were potentiated by MgATP and Mg2+. Adenosine-5'-O-(3-thiotriphosphate) and ADP also potentiated the effect of GTP gamma S, but non-phosphorylating analogues of ATP had no such effect. The activation of PLD by GTP gamma S did not require Ca2+ and was independent of free Ca2+ ions up to a concentration of 100 nM (resting intracellular concentration). Higher Ca2+ concentrations (greater than or equal to 1 microM) completely inhibited PLD activation by GTP gamma S. It is concluded that elevated intracellular Ca2+ concentrations may negatively modulate PLD activation by a guanine nucleotide-binding protein, thus affecting receptor-PLD coupling in neural-derived cells.     

Characterization of ATP receptor which mediates norepinephrine release in PC12 cells.

PC12 cells, a rat pheochromocytoma cell line, has been reported to release norepinephrine in response to extracellular ATP in the presence of extracellular Ca2+. The potency order of ATP analogues was adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than adenosine 5'-O-(1-thiotriphosphate) = 2-methylthioadenosine 5'-triphosphate (MeSATP) greater than 2'- and 3'-O-(4-benzoyl-benzoyl)ATP (BzATP) greater than ADP greater than 5-adenylylimidodiphosphate. Adenosine 5'-O-(2-thiodiphosphate), beta, gamma-methyleneadenosine 5'-triphosphate, AMP and adenosine were inactive. The ATP action in the absence of extracellular Ca2+, suggests a small but appreciable contribution of intracellular Ca2+ mobilization, for norepinephrine release. However, for some ATP derivatives, like BzATP, almost no contribution of the phospholipase C-Ca2+ pathway is suggested, based on their low activity in inositol phosphates production. To identify the ATP-receptor protein, PC12 cell membranes were photoaffinity-labeled with [32P]BzATP. SDS-PAGE analysis showed that a 53-kDa protein labeling was inhibited by ATP and its derivatives, as well as by P2-antagonists, suramin and reactive blue 2, which inhibit the nucleotide-induced norepinephrine release. The inhibitory activity of the nucleotides was, in parallel with their potency, to induce norepinephrine release. Despite their inability to release norepinephrine, GTP and GTP gamma S inhibited the BzATP labeling, suggesting the participation of a putative G protein in the ATP-receptor-mediated actions. We suggest that the 53-kDa protein on the PC12 cell surface is an ATP receptor, which mediates the norepinephrine release, depending, mainly, on extracellular Ca2+ gating.     

Characterization of the mechanism of endocytic vesicle fusion in vitro

A cell-free assay to monitor receptor-mediated endocytic processes has been developed that uses biotinylated transferrin and avidin-linked beta-galactosidase as receptor-associated and fluid-phase probes, respectively (Wessling-Resnick, M., and Braell, W. A. (1990) J. Biol. Chem. 265, 690-699). The fusion of vesicles from heterologous sources can be detected in this assay: endocytic vesicles from K562 cells (a human cell line) will fuse with vesicles from Chinese hamster ovary cells. Fusion between endocytic vesicles is inhibited upon treatment with N-ethylmaleimide but can be restored by the addition of untreated cytosol from either cell type. The in vitro fusion reaction is also inhibited by the nonhydrolyzable nucleotide analogs guanosine 5'-(3-thiotriphosphate) (GTP gamma S) and adenosine 5'-(3-thiotriphosphate) (ATP gamma S). Other nonhydrolyzable guanine nucleotides are found to inhibit the in vitro reaction in the following order of potency: GTP gamma S greater than 5'-guanylyl imidodiphosphate (GTP-PNP) greater than alpha,beta-methylene GTP (GTP-PCP). The inhibitory effects of the nonhydrolyzable analogs of GTP and ATP are not additive. Moreover, excess GTP relieves the inhibition by GTP gamma S more than it relieves the inhibition by ATP gamma S, while excess ATP preferentially alleviates ATP gamma S (not GTP gamma S) inhibition. These properties suggest that the two nucleotides exert their effects at distinct points in the fusion process. Although micromolar levels of excess Ca2+ also inhibit vesicle fusion, the inhibition exerted by GTP gamma S appears to proceed via a pathway independent of the divalent cation. The GTP gamma S-sensitive step in endocytic vesicle fusion is found to occur at a mechanistic stage prior to and distinct from the N-ethylmaleimide-sensitive step of the reaction. This situation permits the accumulation of a membrane vesicle intermediate in the presence of GTP gamma S; subsequent incubation of these vesicles with cytosol and GTP restores their fusion competence. Characteristics of in vitro endocytic vesicle fusion suggest that similarities exist with steps of the fusion mechanism involved with membrane traffic events of the secretory pathway.     

Interaction of adenosine-5'-O-(3-thiotriphosphate) with Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum

Interaction of adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) with Ca2+,Mg2+-ATPase of sarcoplasmic reticulum was studied. The nucleotide was slowly hydrolyzed by the ATPase at 30 degrees C at a rate of about 0.5% that of ATP hydrolysis. Whereas at 0 degrees C, ATP gamma S showed only a limited reactivity toward the ATPase in that a thiophosphorylated intermediate was formed and ADP was released, but hydrolysis of the intermediate to complete the catalytic cycle did not occur. A fairly stable analog of the E-P intermediate could thus be obtained. Presence of the thiophosphorylated intermediate was indicated by the [3H]ADP in equilibrium ATP gamma S exchange reaction and also by using [35S]ATP gamma S. When the ATPase was reacted with ATP gamma S at 0 degrees C in the presence of ferricyanide, EP-forming activity was rapidly lost. Free Ca2+ ions were required for this inactivation. Disulfide bond formation between a cysteinyl residue located near the substrate binding site and the enzyme-bound ATP gamma S or the thiophosphorylated intermediate was suggested by the fact that 2-mercaptoethanol reversed the inactivation. The reaction may prove to be a useful tool for affinity labeling of the active site of the ATPase.     

ATP stimulates Ca2+ oscillations and contraction in airway smooth muscle cells of mouse lung slices

In airway smooth muscle cells (SMCs) from mouse lung slices, > or =10 microM ATP induced Ca2+ oscillations that were accompanied by airway contraction. After approximately 1 min, the Ca2+ oscillations subsided and the airway relaxed. By contrast, > or =0.5 microM adenosine 5'-O-(3-thiotriphosphate) (nonhydrolyzable) induced Ca2+ oscillations in the SMCs and an associated airway contraction that persisted for >2 min. Adenosine 5'-O-(3-thiotriphosphate)-induced Ca2+ oscillations occurred in the absence of external Ca2+ but were abolished by the phospholipase C inhibitor U-73122 and the inositol 1,4,5-trisphosphate receptor inhibitor xestospongin. Adenosine, AMP, and alpha,beta-methylene ATP had no effect on airway caliber, and the magnitude of the contractile response induced by a variety of nucleotides could be ranked in the following order: ATP = UTP > ADP. These results suggest that the SMC response to ATP is impaired by ATP hydrolysis and mediated via P2Y(2) or P2Y(4) receptors, activating phospholipase C to release Ca2+ via the inositol 1,4,5-trisphosphate receptor. We conclude that ATP can serve as a spasmogen of airway SMCs and that Ca2+ oscillations in SMCs are required to sustain airway contraction.     

Differential activation by Ca2+, ATP and caffeine of cardiac and skeletal muscle ryanodine receptors after block by Mg2+

The block of rabbit skeletal ryanodine receptors (RyR1) and dog heart RyR2 by cytosolic [Mg2+], and its reversal by agonists Ca2+, ATP and caffeine was studied in planar bilayers. Mg2+ effects were tested at submaximal activating [Ca2+] (5 microM). Approximately one third of the RyR1s had low open probability ("LA channels") in the absence of Mg2+. All other RyR1s displayed higher activity ("HA channels"). Cytosolic Mg2+ (1 mM) blocked individual RyR1 channels to varying degrees (32 to 100%). LA channels had residual P(o) <0.005 in 1 mM Mg2+ and reactivated poorly with [Ca2+] (100 microM), caffeine (5 mM), or ATP (4 mM; all at constant 1 mM Mg2+). HA channels had variable activity in Mg2+ and variable degree of recovery from Mg2+ block with Ca2+, caffeine or ATP application. Nearly all cardiac RyR2s displayed high activity in 5 microM [Ca2+]. They also had variable sensitivity to Mg2+. However, the RyR2s consistently recovered from Mg2+ block with 100 microM [Ca2+] or caffeine application, but not when ATP was added. Thus, at physiological [Mg2+], RyR2s behaved as relatively homogeneous Ca2+/caffeine-gated HA channels. In contrast, RyR1s displayed functional heterogeneity that arises from differential modulatory actions of Ca2+ and ATP. These differences between RyR1 and RyR2 function may reflect their respective roles in muscle physiology and excitation-contraction coupling.     

Guanine nucleotides decrease the free [Ca2+] required for secretion of serotonin from permeabilized blood platelets. Evidence of a role for a GTP-binding protein in platelet activation

Human platelets containing granule-bound [14C]serotonin were permeabilized, equilibrated at 0 degrees C with ATP and with various Ca2+ buffers and guanine nucleotides, and then incubated at 25 degrees C with or without a stimulatory agonist. Ca2+ alone induced the ATP-dependent secretion of [14C]serotonin (50% at a pCa of 5.1) but the sensitivity of secretion to Ca2+ was greatly enhanced by guanine nucleotides [6-fold by 100 microM GTP, 100-fold by 100 microM guanyl-5'-yl imidodiphosphate and greater than 500-fold by 100 microM guanosine 5'-O-(3-thiotriphosphate)] or by stimulatory agonists (10-fold by 2 units thrombin/ml and 4-fold by 1 microM 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine). When both GTP and a stimulatory agonist were added, they had synergistic effects on secretion. Cyclic GMP and GMP acted similarly to GTP. The effects of all these guanine nucleotides were inhibited by guanosine 5'-O-(2-thiodiphosphate), whereas those of stimulatory agonists were not. Our results demonstrate the presence in platelets of guanine nucleotide-dependent and independent mechanisms regulating the sensitivity of secretion to Ca2+.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle. Identification and characterization of an acid-stable phosphorylated intermediate of the Ca2+,Mg2+-ATPase

An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 microM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 microM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 microM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 microM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15 degrees C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16 degrees C and 14.6 kcal/mol between 16 and 30 degrees C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes.     

Properties of H+-translocating adenosine triphosphatase in vacuolar membranes of SAccharomyces cerevisiae

The properties of Mg2+-ATPase in the vacuole of Saccharomyces cerevisiae were studied, using purified intact vacuoles and right-side-out vacuolar membrane vesicles prepared by the method of Y. Ohsumi and Y. Anraku ((1981) J. Biol. Chem. 256, 2079). The enzyme requires Mg2+ ion but not Ca2+ in. Cu2+ and Zn2+ ions inhibit the activity. The optimal pH is at pH 7.0. The enzyme hydrolyzes ATP, GTP, UTP, and CTP in this order and the Km value for ATP was determined as 0.2 mM. It does not hydrolyze ADP, adenosyl-5'-yl imidodiphosphate, or p-nitrophenyl phosphate. ADP does not inhibit hydrolysis of ATP by the enzyme. The activities of intact vacuoles and of vacuolar membrane vesicles were stimulated 3- and 1.5-fold, respectively, by the protonophore uncoupler 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile and the K+/H+ antiporter ionophore nigericin. Sodium azide at a concentration exerting an uncoupler effect also stimulated the activity. The activity was sensitive to the ATPase inhibitor N,N'-dicyclohexylcarbodiimide, but not to sodium vanadate. The ATP-dependent formation of an electrochemical potential difference of protons, measured by the flow-dialysis method, was determined as 180 mV, with contribution of 1.7 pH units, interior acid, and of a membrane potential of 75 mV. It is concluded that the Mg2+-ATPase of vacuoles is a new marker enzyme for these organelles and is a N,N'-dicyclohexylcarbodiimide-sensitive, H+-translocating ATPase whose catalytic site is exposed to the cytoplasm.     

Partial characterization of the ouabain-insensitive, Na+-stimulated ATPase activity of kidney basal-lateral plasma membranes

The present paper characterizes the Na+-stimulated ATPase activity present in basal-lateral plasma membranes from guinea-pig kidney proximal tubular cells. These characteristics are compared with those of the (Na+ + K+)-stimulated ATPase activity, and they are: (A) Na+-ATPase activity: (1) requires Mg2+; (2) may be activated by mu molar quantities of Ca2+; (3) optimal ratio Mg:ATP = 5:1-2 and Ka for Mg:ATP = 3:0.60 mM; (4) Ka for Na+:8 mM; (5) does not require K+; (6) is only stimulated by Na+ and Li+ (in a lower extent); (7) is similarly stimulated by the Na+ salt of different anions; (8) hydrolyzes only ATP; (9) optimal temperature: 47 degrees C; (10) optimal pH: 6.9; (11) is ouabain insensitive; (12) is totally inhibited by 1.5 mM ethacrynic acid, 2 mM furosemide and 0.75 mM triflocin. (B) (Na+ + K+)-ATPase activity: (1) also requires Mg2+; (2) is inhibited by Ca2+; (3) optimal ratio Mg:ATP = 1.25:1 and Ka for Mg:ATP = 0.50: 0.40 mM; (4) Ka for Na+: 14 mM (data not shown); (5) needs K+ together with Na+; (6) K+ may be substituted by: Rb+ greater than NH+4 greater than Cs+; (7) is anion insensitive; (8) hydrolyzes mostly ATP and to a lesser extent GTP, ITP, UTP, ADP, CTP; (9) optimal temperature: 52 degrees C; (10) optimal pH: 7.2; (11) 100% inhibited by 1 mM ouabain; (12) 63% inhibited by 1.5 mM ethacrynic acid, 10% inhibited by 2 mM furosemide and insensitive to 0.75 mM triflocin.     

Polarized expression and function of P2Y ATP receptors in rat bile duct epithelia

Extracellular nucleotides may be important regulators of bile ductular secretion, because cholangiocytes express P2Y ATP receptors and nucleotides are found in bile. However, the expression, distribution, and function of specific P2Y receptor subtypes in cholangiocytes are unknown. Thus our aim was to determine the subtypes, distribution, and role in secretion of P2Y receptors expressed by cholangiocytes. The molecular subtypes of P2Y receptors were determined by RT-PCR. Functional studies measuring cytosolic Ca2+ (Ca) signals and bile ductular pH were performed in isolated, microperfused intrahepatic bile duct units (IBDUs). PCR products corresponding to P2Y1, P2Y2, P2Y4, P2Y6, and P2X4 receptor subtypes were identified. Luminal perfusion of ATP into IBDUs induced increases in Ca that were inhibited by apyrase and suramin. Luminal ATP, ADP, 2-methylthioadenosine 5'-triphosphate, UTP, and UDP each increased Ca. Basolateral addition of adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), but not ATP, to the perifusing bath increased Ca. IBDU perfusion with ATP-gamma-S induced net bile ductular alkalization. Cholangiocytes express multiple P2Y receptor subtypes that are expressed at the apical plasma membrane domain. P2Y receptors are also expressed on the basolateral domain, but their activation is attenuated by nucleotide hydrolysis. Activation of ductular P2Y receptors induces net ductular alkalization, suggesting that nucleotide signaling may be an important regulator of bile secretion by the liver.     

Structures of the mono- and divalent metal nucleotide complexes in the myosin ATPase

The structure of both the mono- and the divalent metal nucleotide complexes active in the myosin subfragment 1 ATPase has been determined using the phosphorothioate analogs of ATP in the presence of various cations. Both the Sp and the Rp diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) were substrates in the presence of Mg2+, Ca2+, Mn2+, Co2+, Zn2+, and Cd2+ as well as with NH4+ and T1+. The Sp/Rp activity ratios obtained were largely independent of the cation. The simplest explanation of these results is that both mono- and divalent cations do not coordinate to the alpha-phosphate group. With adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), essentially only the Sp diastereomer was active with Mg2+ with Sp/Rp ratio of greater 3000. As the divalent metal ion was varied in the series given above, this ratio was progressively lowered to the value of 0.2 found with Cd2+. Similar changes in stereoselectivity were seen with monovalent cations. Thus, with NH4+, an Sp/Rp ratio of 8 was observed, whereas with T1+, this figure was reduced to 0.04. These data indicate that both mono- and divalent cations coordinate to the beta-phosphate group of the nucleoside triphosphate substrate. These results obtained with ATP alpha S and ATP beta S suggest that myosin uses the mono- or divalent cation delta, beta, gamma-bidentate nucleotide chelate as substrate.     

Ubiquitin adenylate: structure and role in ubiquitin activation

The acid precipitate of the ubiquitin activating enzyme after reaction with ATP and ubiquitin contains one enzyme equivalent of ubiquitin adenylate in which the carboxyl-terminal glycine of ubiquitin and AMP are in an acyl-phosphate linkage. The recovered ubiquitin adenylate has the catalytic properties proposed for it as a reaction intermediate. Thus, upon reaction with fresh enzyme in the absence of Mg2+ or ATP, the product complex, E-ubiquitin . AMP-ubiquitin, is formed. This complex is capable of generating ubiquitin-protein isopeptide derivatives when added to a reticulocyte fraction that catalyzes protein conjugation. This reproduces the effect previously shown to require ubiquitin, ATP, and Mg2+. In the presence of activating enzyme, ubiquitin adenylate is converted to ATP and free ubiquitin in a step requiring PPi and Mg2+. On the basis of studies of [32P]PPi/nucleoside triphosphate exchange, the activating enzyme could be used to generate 2'-deoxy-AMP-, 2'-deoxy-IMP-, and 2'-deoxy-GMP-ubiquitin but not pyrimidine nucleotide-ubiquitin derivatives. The enzyme shows a modest preference for the pro-S diastereomers of adenosine 5'-O-(1-thiotriphosphate) and adenosine 5'-O-(2-thiotriphosphate). Inorganic phosphate, arsenate, methyl phosphate, and tripolyphosphate, but not nucleoside triphosphates, can serve as alternate substrates in place of PPi in the reverse of ubiquitin adenylate formation. Therefore, the enzyme catalyzes the unusual reaction ATP + Pi in equilibrium ADP + PPi in the presence of ubiquitin.     

Stereochemical probes of the argininosuccinate synthetase reaction

The stereochemical course of the argininosuccinate synthetase reaction has been determined. The SP isomer of [alpha-17O,alpha-18O,alpha beta-18O]ATP is cleaved to (SP)-[16O,17O,18O]AMP by the action of argininosuccinate synthetase in the presence of citrulline and aspartate. The overall stereochemical transformation is therefore net inversion, and thus the enzyme does not catalyze the formation of an adenylylated enzyme intermediate prior to the synthesis of citrulline adenylate. The RP isomer of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) is a substrate in the presence of Mg2+, but the SP isomer is a substrate when Cd2+ is used as the activating divalent cation. Therefore, the lambda screw sense configuration of the beta,gamma-bidentate metal--ATP complex is preferred by the enzyme as the actual substrate. No significant discrimination could be detected between the RP and SP isomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) when Mg2+ or Mn2+ are used as the divalent cation. Argininosuccinate synthetase has been shown to require a free divalent cation for full activity in addition to the metal ion needed to complex the ATP used in the reaction.     

Dissociation of Tetrahymena 30 S dynein into 14 S subunit by sonication.

The sonication of 30 S dynein obtained from Tetrahymena cilia induced dissociation into 14-S subunits, some of the enzyme still remaining as intact 30 S dynein and partially dissociated dynein (21 S) in a minor amount. It was demonstrated that the enzymatic properties of the 14 S subunit are quite similar to those of 30 S dynein except for the Ca2+:Mg2+ ratio. ATPase (EC 3.6.1.3) (ATP phosphohydrolase activity of the 14 S subunit was steadily enhanced by increasing concentrations of Mg2+. It was also activated by Ca2+ with an optimum at 6 mM but inhibited by a further increase in concentration. The Ca2+:Mg2+ ratio at 1 mM was about 0.62. 0.6 M KCl stimulated ATPase activity of the 14 S subunit two-fold. The Mg2+-ATPase had an optimum at pH 6.2 and revealed a high activity over pH 10. The Ca2+-ATPase showed two optima at pH 6.2 and 9.5. The Km for ATP was 10 muM. Only 10% of the 14 S subunit recombined with the outer fibers in the presence of Mg2+. The 14 S subunit was shown to have the same mobility as that of 30 S dynein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

ATP-dependent interaction of propranolol and local anaesthetic with sarcoplasmic reticulum. Stimulation of Ca2+ efflux.

Preincubation of sarcoplasmic reticulum (SR) with propranolol or tetracaine inhibits Ca2+ accumulation and stimulates ATPase activity by more than 2-fold. This effect is obtained only when the preincubation is carried out in the presence of ATP or other nucleoside triphosphates. The (ATP + drug)-induced inhibition of Ca2+ accumulation is pH-dependent, increasing as the pH rises above 7.5. The presence of micromolar concentrations of Ca2+ or Mg2+ during the preincubation prevents the inhibitory effect of ATP plus drug on Ca2+ accumulation or ATPase activity. The (ATP + drug) modification of SR vesicles resulted in stimulation of a rapid Ca2+ efflux from passively loaded vesicles. The ATP-dependent inhibition of Ca2+ accumulation by the drug is obtained with other local anaesthetics. The drug concentration required for 50% inhibition was 0.15 mM for dibucaine and 0.4 mM for both propranolol and tetracaine, whereas it was 5 mM, 8 mM and greater than 10 mM for lidocaine, benzocaine and procaine respectively. The heavy SR vesicles were only slightly affected by the incubation with propranolol or tetracaine in the presence of ATP, but their sensitivity increased markedly after storage at 0 degrees C for 24-48 h. These results suggest that propranolol and some local anaesthetics, in the presence of ATP, stimulate Ca2+ efflux by modifying a protein factor(s) rather than the phospholipid bilayer.     

The interaction of phosphorothioate analogues of ATP with phosphomevalonate kinase. Kinetic and 31P NMR studies

The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) could act as substrates for phosphomevalonate kinase in the presence of Mg2+ and Cd2+ as activating divalent metal cations. The Sp diastereomer of ATP alpha S was the preferred substrate regardless of the metal ion used, consistent with the metal ion not binding to the alpha-phosphate. With ATP beta S, the Sp diastereomer was the preferred substrate with Mg2+, and the Rp diastereomer was the preferred substrate with Cd2+. The reversal of specificity establishes that the metal is chelated through the beta-phosphate in the active site of the phosphomevalonate kinase reaction. A comparison of the Vmax values as a function of substitution of oxygen by sulfur showed the order for Mg2+ to be: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Sp) greater than ATP gamma S greater than ATP beta S(Rp). With Cd2+ as the activating metal ion, the order was: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Rp) greater than ATP gamma S greater than ATP beta S(Sp). It is concluded that the chelate structure of metal ATP substrate in the phosphomevalonate kinase reaction is the delta, beta, gamma-bidentate complex. 31P NMR measurements and radioassay with [2-14C] phosphomevalonate were used to measure the equilibrium of the reaction catalyzed by phosphomevalonate kinase with ATP and phosphorothioate analogues of ATP as the phosphoryl group donor. The order as a phosphate donor as determined by both methods in the phosphomevalonate kinase reaction is ATP beta S greater than ATP alpha S greater than ATP greater than ATP gamma S. Except for ATP gamma S, the equilibrium is shifted in the direction of formation of ADP alpha S and ADP beta S relative to ADP formation. Thus, ATP beta S rather than ATP would be effective for the synthesis of diphosphomevalonate. The phosphomevalonate kinase reaction could also be used to synthesize mevalonate 5-(2-thiodiphosphate) using ATP gamma S as the phosphoryl group donor.     

Human neutrophils have a novel purinergic P2-type receptor linked to calcium mobilization

Stimulation of suspensions of fura-2-loaded human neutrophils with ATP resulted in an elevation in cytosolic free calcium concentration ([Ca2+]i) from a basal value of 0.1 microM to a transient peak of 1 microM. The response is due to Ca2+ release from intracellular stores and influx of extracellular Ca2+. Release from intracellular stores is shown by the response in the absence of extracellular Ca2+. The greater and more maintained response in the presence of extracellular Ca2+ is indicative of stimulated Ca2+ entry and a stimulated influx pathway was confirmed by using Mn2+ as a surrogate for Ca2+. A variety of purinergic agonists were used to characterize the pharmacology of this [Ca2+]i response. Their rank order of potency was ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) greater than ADP much greater than 2-methylthioadenosine 5'-triphosphate (2Me-SATP), where ATP had an EC50 value of 3 microM and 2MeSATP had an EC50 value of 1000 microM. Adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), adenylyl (alpha,beta-methylene)- diphosphonate (AMPCPP) and adenosine were inactive at 1 mM. These results suggest that neutrophils have a novel type of purinergic P2 receptor that is neither P2x nor P2y.