Apparent Affinity For ATP Research Articles

Structure-Function Relationships in Membrane Segment 5 of the Yeast Pma1 H+-ATPase

Membrane segment 5 (M5) is thought to play a direct role in cation transport by the sarcoplasmic reticulum Ca2+-ATPase and the Na+, K+-ATPase of animal cells. In this study, we have examined M5 of the yeast plasma membrane H+-ATPase by alanine-scanning mutagenesis. Mutant enzymes were expressed behind an inducible heat-shock promoter in yeast secretory vesicles as described previously (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940-7949). Three substitutions (R695A, H701A, and L706A) led to misfolding of the H+-ATPase as evidenced by extreme sensitivity to trypsin; the altered proteins were arrested in biogenesis, and the mutations behaved genetically as dominant lethals. The remaining mutants reached the secretory vesicles in sufficient amounts to be characterized in detail. One of them (Y691A) had no detectable ATPase activity and appeared, based on trypsinolysis in the presence and absence of ligands, to be blocked in the E1-to-E2 step of the reaction cycle. Alanine substitution at an adjacent position (V692A) had substantial ATPase activity (54%), but was likewise affected in the E1-to-E2 step, as evidenced by shifts in its apparent affinity for ATP, H+, and orthovanadate. Among the mutants that were sufficiently active to be assayed for ATP-dependent H+ transport by acridine orange fluorescence quenching, none showed an appreciable defect in the coupling of transport to ATP hydrolysis. The only residue for which the data pointed to a possible role in cation liganding was Ser-699, where removal of the hydroxyl group (S699A and S699C) led to a modest acid shift in the pH dependence of the ATPase. This change was substantially smaller than the 13-30-fold decrease in K+ affinity seen in corresponding mutants of the Na+, K+-ATPase (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). Taken together, the results do not give firm evidence for a transport site in M5 of the yeast H+-ATPase, but indicate a critical role for this membrane segment in protein folding and in the conformational changes that accompany the reaction cycle. It is therefore worth noting that the mutationally sensitive residues lie along one face of a putative alpha-helix.

Kinetic analyses of mutations in the glycine-rich loop of cAMP-dependent protein kinase.

The conserved glycines in the glycine-rich loop (Leu-Gly50-Thr-Gly52-Ser-Phe-Gly55-Arg-Val) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and pre-steady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s-1 in contrast to 500 s-1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the gamma-phosphate of ATP. Energy-minimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.

Erythrosin isothiocyanate selectively labels lysine464 within an ATP-protectable binding site on the Ca-ATPase in skeletal sarcoplasmic reticulum membranes.

Conditions that permit the selective modification of an ATP-protectable site on the Ca-ATPase in skeletal sarcoplasmic reticulum (SR) membranes using erythrosin isothiocyanate (Er-ITC) have been identified. The major labeling site for Er-ITC has been identified using reversed-phase HPLC and positive FAB mass spectrometry after exhaustive tryptic digestion of the Er-ITC-modified Ca-ATPase. An ATP-protectable peptide corresponding to M452NVFNTEVRNLSK464VER467 is modified by Er-ITC, the average mass of which is 2830.1 +/- 0.3 Da. The exclusive modification of lysine residues indicates Lys464 as the site of Er-ITC modification. Derivatization with Er-ITC diminishes the secondary activation of steady-state ATPase activity and the rate of dephosphorylation by millimolar concentrations of ATP. In contrast, in the presence of micromolar ATP concentrations Er-ITC modification of the Ca-ATPase does not affect (i) the apparent affinity of ATP, (ii) the maximal extent of phosphoenzyme formation by ATP, (iii) the rate of steady-state ATP hydrolysis, or (iv) the rate of dephosphorylation of the Ca-ATPase. Furthermore, ATP utilization by the Ca-ATPase is unaffected by detergent solubilization, irrespective of Er-ITC modification, indicating that the secondary activation of ATP hydrolysis involves a single Ca-ATPase polypeptide chain. Therefore, Er-ITC does not interfere with the normal structural transitions associated with phosphoenzyme decay. Rather, these results indicate that Er-ITC bound to Lys464 interferes with either ATP binding to a low-affinity site or the associated structural transitions that modulate the rate of enzyme dephosphorylation.

Evidence That Ser775 in the α Subunit of the Na,K-ATPase Is a Residue in the Cation Binding Pocket

Substitution of alanine for Ser775 in a ouabain-resistant alpha1 sheep isoform causes a 30-fold decrease in apparent affinity for K+ as an activator of the Na,K-ATPase, as well as an increase in apparent affinity for ATP (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). This study was carried out to determine whether Ser775 is a direct cation-ligating residue or whether the change in apparent affinity for K+ is secondary to a conformational alteration as evidenced in the change in ATP affinity, with the following results. Kinetics of K+(Rb+) influx into intact cells show that the change is due to a change in K+ interaction at the extracellular surface. The K+ dependence of formation of K+-occluded enzyme (E2(K)) and of the rate of formation of deoccluded enzyme from E2(K) indicate that the Ser775 --> Ala mutation results in a marked increase (>/=30-fold) in rate of release of K+ from E2(K). The high affinity Na+-like competitive antagonist 1,3-dibromo2,4,6-tris-(methylisothiouronium)benzene (Br2TITU), which interacts with the E1 conformation and blocks cytoplasmic cation binding (Hoving, S., Bar-Shimon, M., Tijmes, J. J. , Tal, D. M., and Karlish, S. J. D. (1995) J. Biol. Chem. 270, 29788-29793), inhibits Na+-ATPase of the mutant less than the control enzyme. With intact cells, Br2TITU acts as a competitive inhibitor of extracellular K+ activation of both the mutant and control enzymes. In this case, the mutant was more sensitive to inhibition. With vanadate as a probe of conformation, a difference in conformational equilibrium between the mutant and control enzymes could not be detected under turnover conditions (Na+- ATPase) in the absence of K+. These results indicate that the increase in apparent affinity for ATP effected by the Ser775 --> Ala mutation is secondary to a change in intrinsic cation affinity/selectivity. The large change in affinity for extracellular K+ compared with cytoplasmic Na+ and to Br2TITU binding supports the conclusion that the serine hydroxyl is either part of the K+-gate structure or a direct cation-ligating residue that is shared by at least one Na+ ion, albeit with less consequence on rate constants for Na+ binding or release compared with K+.

Kinase Activity of EnvZ, an Osmoregulatory Signal Transducing Protein ofEscherichia coli

EnvZ is an inner membrane protein present inEscherichia colithat is important for osmosensing and required for porin gene regulation. EnvZ is phosphorylated by intracellular ATP, and EnvZ-P phosphorylates OmpR, which then binds to the porin promoters to regulate their expression. An overexpressed, truncated form of the enzyme, EnvZ115, was used to characterize the kinase reactionin vitro.Using a filter binding assay, we report the first direct measurements of the kinase activity, including the apparent affinity for ATP of 200 μm. The phosphorylation reaction is dependent on MgCl2, and the phosphoenzyme has the expected stability of a phosphohistidine; i.e., it is stable in base and less stable in acid at room temperature. The addition of OmpR and ATP to solutions containing EnvZ resulted in an OmpR-stimulated, EnvZ-dependent ATPase activity that was not vanadate-sensitive. Thein vivokinase activity of EnvZ and two mutants that were deficient in porin expression were studied using an immune complex kinase reaction. Interestingly, a mutation located in the periplasmic domain of EnvZ exhibited kinase activity that was identical to that of the wild-type enzyme, while a mutation located close to the phosphorylation site showed a significant decrease in both kinase and phosphotransferase activities. These data provide support for models of EnvZ consisting of separate sensing and kinase domains.

Glucose-independent inhibition of yeast plasma-membrane H+-ATPase by calmodulin antagonists.

Glucose metabolism causes activation of the yeast plasma-membrane H+-ATPase. The molecular mechanism of this regulation is not known, but it is probably mediated by phosphorylation of the enzyme. The involvement in this process of several kinases has been suggested but their actual role has not been proved. The physiological role of a calmodulin-dependent protein kinase in glucose-induced activation was investigated by studying the effect of specific calmodulin antagonists on the glucose-induced ATPase kinetic changes in wild-type and two mutant strains affected in the glucose regulation of the enzyme. Preincubation of the cells with calmidazolium or compound 48/80 impeded the increase in ATPase activity by reducing the Vmax of the enzyme without modifying the apparent affinity for ATP in the three strains. In one mutant, pma1-T912A, the putative calmodulin-dependent protein kinase-phosphorylatable Thr-912 was eliminated, and in the other, pma1-P536L, H+-ATPase was constitutively activated, suggesting that the antagonistic effect was not mediated by a calmodulin-dependent protein kinase and not related to glucose regulation. This was corroborated when the in vitro effect of the calmodulin antagonists on H+-ATPase activity was tested. Purified plasma membranes from glucose-starved or glucose-fermenting cells from both pma1-P890X, another constitutively activated ATPase mutant, and wild-type strains were preincubated with calmidazolium or melittin. In all cases, ATP hydrolysis was inhibited with an IC50 of approximately 1 microM. This inhibition was reversed by calmodulin. Analysis of the calmodulin-binding protein pattern in the plasma-membrane fraction eliminates ATPase as the calmodulin target protein. We conclude that H+-ATPase inhibition by calmodulin antagonists is mediated by an as yet unidentified calmodulin-dependent membrane protein.

On the Inhibition Mechanism of Sarcoplasmic or Endoplasmic Reticulum Ca2+-ATPases by Cyclopiazonic Acid

Ca2+-ATPase inhibition by stoichiometric and substoichiometric concentrations of cyclopiazonic acid was studied in sarcoplasmic reticulum preparations from rabbit fast-twitch muscle. The apparent affinity of the nonphosphorylated enzyme for ATP showed a Kd of approximately 3 microM in the absence of cyclopiazonic acid and approximately 28 microM in the presence of the drug. Fractional saturation of the enzyme by cyclopiazonic acid was accompanied by the appearance of two ATP-binding populations (enzyme with and without drug) and a progressive increase in the half-maximal concentration for saturating the ATP-binding sites. Enzyme turnover in the presence of stoichiometric concentrations of cyclopiazonic acid displayed lower apparent affinity for ATP and lower maximal hydrolytic activity than in the absence of the drug. When cyclopiazonic acid is in the substoichiometric range, the observed kinetic parameters will correspond to the simultaneous contribution of two different reaction cycles sustained by the enzyme with and without drug. The inhibition could be elicited by adding ATP to allow the enzyme turnover when cyclopiazonic acid was preincubated with the enzyme in the presence of Ca2+. The onset of inhibition during enzyme cycling was observed over a period of seconds, revealing the existence of a low inhibition rate constant. It is concluded that cyclopiazonic acid decreases enzyme affinity for ATP in non-turnover conditions by approximately one order of magnitude. This allows enzyme cycling after drug binding, provided that a high ATP concentration is used. Cyclopiazonic acid and ATP do not compete for the same binding site.

Substitutions of Glutamate 781 in the Na,K-ATPase α Subunit Demonstrate Reduced Cation Selectivity and an Increased Affinity for ATP

The intramembrane Glu781 residue of the Na,K-ATPase alpha subunit has been postulated to have a role in the binding and/or occlusion of cations. To ascertain the role of Glu781, the residue was substituted with an aspartate, alanine, or lysine residue and the mutant Na,K-ATPases were coexpressed with the native beta 1 subunit in Sf9 insect cells using the baculovirus expression system. All alpha mutants are able to efficiently assemble with the beta 1 subunit and produce catalytically competent Na,K-ATPase molecules with hydrolytic activities comparable to that of the wild-type enzyme. Analysis of the kinetic properties of the mutated enzymes showed a decrease in apparent affinity for K+ compared to wild-type Na,K-ATPase, with the lysine and alanine substitutions displaying the greatest reduction. All Na,K-ATPase mutants demonstrated a significant increase in apparent affinity for ATP compared to wild-type Na,K-ATPase, while the sensitivity to the cardiotonic inhibitor, ouabain, was unchanged. The dependence on Na+, however, differs among the mutant enzymes with both the Glu781-->Asp and Glu781-->Ala mutants displaying a decrease in the apparent affinity for the cation, while the Glu781-->Lys mutant exhibits a modest increase. Furthermore, in the absence of K+, the Glu781-->Ala mutant displays a Na(+)-ATPase activity and a cellular Na+ influx suggesting that Na+ is substituting for K+ at the extracellular binding sites. The observation that trypsin digestion of the Glu781-->Ala mutant in Na+ medium produces a K(+)-stabilized tryptic fragment also intimates a decreased capacity of the mutant to discriminate between Na+ and K+ at the extracellular loading sites. All together, these data implicate Glu781 of the Na,K-ATPase alpha subunit as an important coordinate of cation selectivity and activation, although the modest effect of Glu781-->Lys substitution seemingly precludes direct involvement of the residue in the cation binding process. In addition, the fifth membrane segment is proposed to represent an important communicative link between the extramembraneous ATP binding domain and the cation transport regions of the Na,K-ATPase.

Activation and labelling of the purified skeletal muscle ryanodine receptor by an oxidized ATP analogue.

We have tested the periodate-oxidized ATP analogue 2',3'-dialdehyde adenosine triphosphate (oATP) as a ligand for the skeletal muscle ryanodine receptor/Ca(2+)-release channel. Ca2+ efflux from passively loaded heavy sarcoplasmic reticulum vesicles of skeletal muscle is biphasic. oATP stimulates the initial phase of Ca2+ release in a concentration-dependent manner (EC50 160 microM), and the efflux proceeds with a half-time in the range 100-200 ms. This oATP-modulated initial rapid Ca2+ release was specifically inhibited by millimolar concentrations of Mg2+ and micromolar concentrations of Ruthenium Red, indicating that the effect of oATP was mediated via the ryanodine receptor. The purified Ca(2+)-release channel was incorporated into planar lipid bilayers, and single-channel recordings were carried out to verify a direct interaction of oATP with the ryanodine receptor. Addition of oATP to the cytoplasmic side activated the channel with an EC50 of 76 microM, which is roughly 30-fold higher than the apparent affinity of ATP. The oATP-induced increase in the open probability of the ryanodine receptor displays a steep concentration-response curve with a Hill coefficient of approximately 2, which suggests a co-operativity of the ATP binding sites in the tetrameric protein. oATP binds to the ryanodine receptor in a quasi-irreversible manner via Schiff base formation between the aldehyde groups of oATP and amino groups in the nucleotide binding pocket. This allows for the covalent specific incorporation of [alpha-32P]oATP by borhydride reduction. A typical adenine nucleotide binding site cannot be identified in the primary sequence of the ryanodine receptor. Our results demonstrate that oATP can be used to probe the structure and function of the nucleotide binding pocket of the ryanodine receptor and presumably of other ATP-regulated ion channels.

Functional consequences of amino-terminal diversity of the catalytic subunit of the Na,K-ATPase.

One region of marked sequence diversity among the highly homologous alpha isoforms of the Na,K-ATPase is the lysine-rich NH2 terminus. Expression of a mutant cDNA encoding an alpha 1 protein, minus the 32 NH2-terminal residues, results in a modified enzyme (alpha 1M32), which behaves similarly to alpha 1 in overall Na/K exchange activity (Vmax) and apparent affinities for intracellular Na+ and extracellular K+. However, with membranes isolated from HeLa cells expressing the rat alpha 1M32 mutant, as well as membranes from cells expressing the rat alpha 1 and the ouabain-resistant mutated forms of rat alpha 2 (alpha 2*) and alpha 3 (alpha 3*) developed by Jewell and Lingrel (Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925-16930), distinct Na,K-ATPase kinetics are observed. Thus, at 1 microM ATP, the effects of K+ on the Na-ATPase activity of alpha 2* and alpha 1M32 are similar; both are activated, whereas alpha 1 and alpha 3 are inhibited by the addition of K+ at low (0.1 mM) concentration. These effects are attributed to different rates of a step involved in K+ deocclusion (E2(K)<-->E1K<-->E1 + K+) and are consistent with our earlier evidence (Wierzbicki, W., and Blostein, R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 70-74) for a role of the NH2 terminus in the K+ deocclusion pathway of the Na,K-ATPase reaction. These differences are not directly related to differences in apparent affinities for ATP, since alpha 3* has alpha 1-like high affinity K+ inhibition but resembles alpha 2* and alpha 1M32 with respect to a lower K'ATP. Na-ATPase activities of alpha 2*, alpha 3*, and alpha 1M32, but not alpha 1, are activated by Li+ but not Rb+, consistent with a relatively faster rate of Li+ deocclusion (Post, R. L., Hegyvary, C., and Kume, S. (1972) J. Biol. Chem. 247, 6530-6540), as well as higher affinity of alpha 3 for extracellular K+ (Li+) activation of dephosphorylation (E2P + K+<-->E2(K) + Pi). Inhibition of Na-ATPase by higher concentrations (> or = 1 mM) K+ is observed with all isoforms and is attributed to K+ acting at inhibitory cytoplasmic sites.(ABSTRACT TRUNCATED AT 400 WORDS)

The Two Km's for ATP of Corn-Root H+-ATPase and the Use of Glucose-6-Phosphate and Hexokinase as an ATP-Regenerating System.

Plasma membrane vesicles derived from corn (Zea mays L.) roots retain a membrane-bound H+-ATPase that is able to form a H+ gradient across the vesicle membranes. The activity of this ATPase is enhanced 2- to 3-fold when Triton X-100 or lysophosphatidylcholine is added to the medium at a protein:detergent ratio of 2:1 (w/w). In the absence of detergent, the ATPase exhibits only one Km for ATP (0.1-0.2 mM), which is the same as for the pumping of H+. After the addition of either Triton X-100 or lysophosphatidylcholine, two Km's for ATP are detected, one in the range of 1 to 3 [mu]M and a second in the range of 0.1 to 0.2 mM. The Vmax of the second Km for ATP increases as the temperature of the assay medium is raised from 15[deg]C to 38[deg]C. The Arrhenius plot reveals a single break at 30[deg]C, both in the absence and in the presence of detergents. In the presence of Triton X-100 the H+-ATPase catalyzes the cleavage of glucose-6-phosphate when both hexokinase and ADP are included in the assay medium. There is no measurable cleavage when the apparent affinity for ATP of the H+-ATPase is not enhanced by Triton X-100 or when 1 mM glucose is included in the assay medium. These data indicate that when the high-affinity Km for ATP is unmasked with the use of detergent, the ATPase can use glucose-6-phosphate and hexokinase as an ATP-regenerating system.

Functional comparisons between plant plasma membrane H(+)-ATPase isoforms expressed in yeast.

To examine the functional properties of the three major isoforms of plasma membrane H(+)-ATPase expressed in Arabidopsis thaliana (AHA1, AHA2, and AHA3), we employed a system for the heterologous expression of functional plant plasma membrane H(+)-ATPase in yeast (Villalba, J. M., Palmgren, M. G., Berberian, G. E., Ferguson, C., and Serrano, R. (1992) J. Biol. Chem. 267, 12341-12349). Each isoform was expressed efficiently but appeared to be retained in the endoplasmic reticulum of yeast. All isoforms displayed qualitatively similar enzymatic properties, but quantitative differences were found. When compared with AHA3, AHA1 and AHA2 had an apparent higher turnover rate for ATP hydrolysis, exhibited a 10-fold higher apparent affinity for ATP, and a 3-fold higher sensitivity toward vanadate. In addition, AHA2 had a slightly lower apparent affinity for H+ and seemed to be more susceptible to activation by lysophosphatidylcholine than did AHA1 and AHA3. This study represents the first comparison of the functional properties of isoforms of the plant plasma membrane H(+)-ATPase.

Glutamate 329 located in the fourth transmembrane segment of the alpha-subunit of the rat kidney Na+,K+-ATPase is not an essential residue for active transport of sodium and potassium ions.

An allelic variant of the ouabain-insensitive rat kidney Na+,K(+)-ATPase alpha 1-isoform was identified by chance in a cDNA library. The variant differed from the wild-type rat kidney Na+,K(+)-ATPase by a single G to C base substitution in the cDNA, which on the amino acid level gave rise to a glutamine in place of the glutamate residue Glu329, previously suggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS-1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentration of ouabain highly cytotoxic to the parental cells containing only the endogenous COS-1 cell Na+,K(+)-ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain-insensitive Glu329-->Gln variant, whose cDNA was shown by polymerase chain reaction amplification to be stably integrated into the COS-1 cell genome. Functional analysis on isolated plasma membranes demonstrated that the Glu329-->Gln variant was able to catalyze Na(+)- and K(+)-activated ATPase activity with a maximum turnover number similar if not identical to that of the wild type, but the variant exhibited a reduced affinity for both cations corresponding to a 2-fold increase in K0.5 for Na+ and a 6-fold increase in K0.5 for K+. Moreover, the apparent affinity for ATP was increased 15-fold in the variant relative to wild-type Na+,K(+)-ATPase. The Na+,K(+)-ATPase activity of the variant displayed an anomalous pH dependence with a down-shift of the pH optimum and a nearly constant rate in the range between pH 7.0 and 8.7.

Ischemic Brain Injury Selectively Alters ATP Binding of Calcium and Calmodulin-Dependent Protein Kinase II

Calmodulin and ATP affinity and total binding capacity were characterized for CaM kinase II isolated from control and ischemic animals. Ischemic CaM kinase II exhibited equivalent apparent affinity and total binding for calmodulin when compared to control enzyme. However, ischemic CaM kinase II exhibited a significant decrease in apparent affinity for ATP in saturation experiments. ATP binding was characterized using the ATP photoaffinity analog [α-32P] Azido-ATP. A significant decrease in total binding and binding affinity for ATP was observed for the alpha (50 kDa) and beta (60 kDa) subunits. The observation that ischemia induced an alteration of ATP binding without affecting calmodulin binding is consistent with the hypothesis that ischemia directly affects the ATP binding of CaM kinase II which results in subsequent inhibition of the enzyme.

Functional consequences of alterations to Pro 328 and Leu 332 located in the 4th transmembrane segment of the α-subunit of the rat kidney Na +,K +-ATPase

Site-specific mutagenesis was used to analyse the functional roles of the residues Pro 328 and Leu 332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the α 1-subunit of the ouabain resistant rat kidney Na +,K +-ATPase. cDNAs encoding either of the Na +,K +-ATPase mutants Pro 328→Ala and Leu 332→Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 μM ouabain were isolated, and the Na +, K +, ATP and pH dependencies of the Na +,K +-ATPase activity measured in the presence of 10 μM ouabain were analysed. Under these conditions the exogenous expressed Na +,K +-ATPase contributed more than 95% of the Na +,K +-ATPase activity. The Pro 328→Ala mutant displayed a reduced apparent affinity for Na + ( K 0.5 (Na +) 13.04 mM), relative to the wild type ( K 0.5 (Na +) 7.13 mM). By contrast, the apparent affinity for Na + displayed by the Leu 332→Ala mutant was increased ( K 0.5 (Na +) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K + relative to the wild type ( K 0.5 (K +) 2.46 mM for Pro 328→Ala and 1.97 mM for Leu 332→Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type ( K 0.5 (ATP) 0.086 mM for Pro 328→Ala and 0.042 mM for Leu 332→Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)→E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro 328 and Leu 332 in the stabilization of the E2 form and of Pro 328 in Na + binding. The possible role of the mutated residues in K + binding is discussed.

Subunit interactions of Rubisco activase: Polyethylene glycol promotes self-association, stimulates ATPase and activation activities, and enhances interactions with Rubisco

The effect of polyethylene glycol (PEG) on the enzymatic and physical properties of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase was examined. In the presence of PEG, Rubisco activase exhibited higher ATPase and Rubisco activating activities, concomitant with increased apparent affinity for ATP and Rubisco. Specific ATPase activity, which was dependent on Rubisco activase concentration, was also higher in the presence of Ficoll, polyvinylpyrrolidone, and bovine serum albumin. The ability of Rubisco activase to facilitate dissociation of the tight-binding inhibitor 2-carboxyarabinitol 1-phosphate from carbamylated Rubisco was also enhanced in the presence of PEG. Mixing experiments with Rubisco activase from two different sources showed that tobacco Rubisco activase, which exhibited little activation of spinach Rubisco by itself, was inhibitory when included with spinach Rubisco activase. Polyethylene glycol improved the ability of tobacco and a mixture of tobacco plus spinach Rubisco activase to activate spinach Rubisco. Estimates based on rate zonal sedimentation and gel-filtration chromatography indicated that the apparent molecular mass of Rubisco activase was two- to fourfold higher in the presence of PEG. The increase in apparent molecular mass was consistent with the propensity of solvent-excluding reagents like PEG to promote self-association of proteins. Likewise, the change in enzymatic properties of Rubisco activase in the presence of PEG and the dependence of specific activity on protein concentration resembled changes that often accompany self-association. For Rubisco activase, high concentrations of protein in the chloroplast stroma would provide an environment conducive to self-association and cause expression of properties that would enhance its ability to function efficiently in vivo.

A residue substitution in phosphoribulokinase of Synechocystis PCC 6803 renders the mutant light-sensitive.

We have isolated a light-sensitive mutant (BRLS) of the photosynthetic cyanobacterium Synechocystis 6803 (S. 6803) that does not survive exposure to bright light: 70% of BRLS cells die upon exposure to light of greater than 3,000 lux for 2 h. A complementing DNA fragment from wild-type cells and the corresponding DNA from the BRLS cells have been cloned and sequenced. An open reading frame is found to encode phosphoribulokinase, a key enzyme in the enzyme system for photosynthetic carbon reduction (ES-PCR). The deduced peptide sequence of this enzyme is highly homologous to eukaryotic phosphoribulokinases but is not similar to known prokaryotic phosphoribulokinases. The mutation responsible for the phenotype of BRLS is a single nucleotide change that results in substitution of phenylalanine for Ser-222 in the phosphoribulokinase. The catalytic activity and the apparent affinity for ATP of the mutated kinase are about one-tenth and one-seventh those of the wild-type kinase, respectively. Furthermore, the mutated kinase is selectively degraded in BRLS cells in bright light. Degradation of the mutated kinase and cell death in bright light can be suppressed by inhibiting photosynthetic electron flow (PS-EF) with 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The data indicate that PS-EF is not impeded by an impaired ES-PCR although the ES-PCR activity is controlled by the rate of PS-EF. Continued PS-EF in the absence of the normal substrates for carbon reduction appears to result in damage to cellular components essential for life or in the generation of lethal components.

Comparison of the substrate dependence properties of the rat Na,K-ATPase alpha 1, alpha 2, and alpha 3 isoforms expressed in HeLa cells.

The role of multiple isoforms for the alpha subunit of Na,K-ATPase is essentially unknown. To examine the functional properties of the three alpha subunit isoforms, we developed a system for the heterologous expression of Na,K-ATPase in which the enzymatic activity of each isoform can be independently analyzed. Ouabain-resistant forms of the rat alpha 2 and alpha 3 subunits were constructed by site-directed mutagenesis of amino acid residues at the extracellular borders of the first and second transmembrane domains (L111R and N122D for alpha 2 and Q108R and N119D for alpha 3). cDNAs encoding the rat alpha 1 subunit, which is naturally ouabain-resistant, and rat alpha 2 and alpha 3, which were mutated to ouabain resistance (designated rat alpha 2* and rat alpha 3*, respectively) were cloned into an expression vector and transfected into HeLa cells. Resistant clones were isolated and analyzed for ouabain-inhibitable ATPase activity in the presence of 1 microM ouabain, which inhibits the endogenous Na,K-ATPase present in HeLa cells (I50 approximately equal to 10 nM). The remaining activity corresponds to Na,K-ATPase molecules containing the transfected rat alpha 1, rat alpha 2*, or rat alpha 3* isoforms. Utilizing this system, we examined Na+, K+, and ATP dependence of enzyme activity. Na,K-ATPase molecules containing rat alpha 1 and rat alpha 2* exhibited a 2-3-fold higher apparent affinity for Na+ than those containing rat alpha 3* (apparent KNa+ (millimolar): rat alpha 1 = 1.15 +/- 0.13; rat alpha 2* = 1.05 +/- 0.11; rat alpha 3* = 3.08 +/- 0.06). Additionally, rat alpha 3* had a slightly higher apparent affinity for ATP (in the millimolar concentration range) compared with rat alpha 1 or rat alpha 2* (apparent K0.5 (millimolar): rat alpha 1 = 0.43 +/- 0.12; rat alpha 2* = 0.54 +/- 0.15; rat alpha 3* = 0.21 +/- 0.04) and all three isoforms has similar apparent affinities for K+ (apparent KK+: rat alpha 1 = 0.45 +/- 0.01; rat alpha 2* = 0.43 +/- 0.004; rat alpha 3* = 0.27 +/- 0.01). This study represents the first comparison of the functional properties of the three Na,K-ATPase alpha isoforms expressed in the same cell type.

Functional consequences of alterations to amino acids located in the hinge domain of the Ca(2+)-ATPase of sarcoplasmic reticulum

Site-specific mutagenesis of the sarcoplasmic reticulum Ca(2+)-ATPase was used to investigate the functional roles of 18 amino acid residues located at or near the "hinge-domain," a highly conserved region of the cation-transporting ATPases. Mutation of Lys684 to arginine, alanine, histidine, and glutamine resulted in complete loss of calcium transport function and ATPase activity. For the Lys684----Ala, histidine, and glutamine mutants, this coincided with a loss of the ability to form a phosphorylated intermediate from ATP or Pi. The Lys684----Arg mutant retained the ability to phorphorylate from ATP with normal apparent affinity, demonstrating the importance of the positive charge. On the other hand, no phosphorylation was observed with Pi as substrate in this mutant. Examination of the partial reactions after phosphorylation from ATP in the Lys684----Arg mutant demonstrated a reduction of the rate of transformation of the ADP-sensitive phosphoenzyme intermediate (E1P) to the ADP-insensitive phosphoenzyme intermediate (E2P), which could account for the loss of transport function. Once accumulated, the E2P intermediate was able to decompose rapidly in the presence of K+ at neutral pH. These results may be interpreted in terms of a preferential destabilization of protein phosphate interactions in the E2P form of this mutant. The Asp703----Ala and Asn-Asp707----Ala-Ala mutants were completely inactive and unable to form phosphoenzyme intermediates from ATP or Pi. In these mutants as well as in the Lys684----Ala mutant, nucleotides were found to protect with normal affinity against intramolecular cross-linking induced with glutaraldehyde, indicating that the nucleotide binding site was intact. Mutation of Glu646, Glu647, Asp659, Asp660, Glu689, Asp695, Glu696, Glu715, and Glu732 to alanine did not affect the maximum rates of calcium transport and ATP hydrolysis or the apparent affinities for calcium and ATP. Mutation of the 2 highly conserved proline residues, Pro681 and Pro709, as well as Lys728, to alanine resulted in partially inhibited Ca(2+)-ATPase enzymes with retention of the ability to form a phosphoenzyme intermediate from ATP or Pi and with normal apparent affinities for ATP and calcium. The proline mutants retained the biphasic ATP concentration dependence of ATPase activity, characteristic of the wild-type, and therefore the partial inhibition of turnover could not be ascribed to a disruption of the low affinity modulatory ATP site.(ABSTRACT TRUNCATED AT 400 WORDS)

Modification of ATP regulatory function in sacroplasmic reticulum Ca2+-ATPase by hydrophobic molecules

The effects of the three hydrophobic molecules triphenylphosphine, trifluoperazine and 3-nitrophenol on Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles was investigated. When ATP was the substrate, triphenylphosphine (3 microM) increased the amount of Ca2+ accumulated by the vesicles. At high concentrations triphenylphosphine inhibited Ca2+ uptake. This effect varied depending on the ATP concentration and the type of nucleotide used. With ITP there was only inhibition and no activation of Ca2+ uptake by triphenylphosphine. On the other hand, trifluoperazine inhibited Ca2+ accumulation regardless of whether ATP or ITP was used as substrate. When 5 mM oxalate was included in the medium in order to avoid binding of Ca2+ to the low-affinity Ca2(+)-binding sites of the enzyme, both stimulation by triphenylphosphine and inhibition by trifluoperazine were reduced. In leaky vesicles at low Ca2+ concentrations, triphenylphosphine and 3-nitrophenol were competitive inhibitors of ATPase activity at the regulatory site of the enzyme (0.1-1 mM ATP). A striking difference was observed when both the high- and low-affinity Ca2(+)-binding sites were saturated. In this condition, triphenylphosphine and 3-nitrophenol promoted a 3-4-fold increase in the apparent affinity for ATP at its regulatory site.