Conversion of coupling factor 1 of Rhodospirillum rubrum from a Ca2+-ATPase into a Mg2+-ATPase

Isolation of F1-ATPase from Rhodospirillum rubrum by chloroform extraction of chromatophores, followed by purification on a glycerol gradient, results in a very pure enzyme preparation containing five subunits with high Ca2+-ATPase activity (15 mumol per min per mg protein). Furthermore, conditions are reported under which the purified F1 exhibits Mg2+-dependent ATPase activity of about 35 mumol per min per mg protein. NaHCO3 stimulates the Mg2+-activity from 1.5 mumol per min per mg protein to 5 mumol per min per mg protein giving a maximal activity at a concentration of about 60 mM NaHCO3. Lauryl dimethylamine oxide (LDAO), octyl glucoside and nonanoyl N-methylglucamide enhance the Mg2+-ATPase activity from 1.5 to 14, 22 and 35 mumol per min per mg protein, respectively, in the absence of NaHCO3, and from 5 to 34, 30 and 37 mumol per min per mg protein, respectively, in the presence of 50 mM NaHCO3. The Vmax is increased, but the Km for ATP remains the same, about 0.22 mM, both in the absence of activators and in the presence of NaHCO3, LDAO or NaHCO3 plus LDAO. Ca2+-dependent ATPase activity is slightly stimulated by NaHCO3 but strongly inhibited by octyl glucoside.     

Interaction of a coupling factor from Rhodospirillum rubrum with coupling factor deficient chromatophores.

A coupling factor necessary for the photophosphorylation and Mg2+-ATPase activities in Rhodospirillum rubrum chromatophores has been separated from these particles. Although the redox potential of coupling factor deficient chromatophores is slightly more oxidized than of the control, the addition of the coupling factor for reconstitution does not alter the redox potential. Phenazine methosulfate cannot restore or significantly enhance the photophosphorylation activities of uncoupled or reconstituted chromatophores compared to the control. The coupling factor can bind to coupling factor deficient membranes without addition of magnesium ions and thus restore the photophosphorylation and Mg2+-ATPase activities of these vesicles. The Ca2+-ATPase in the coupling factor preparation shows binding characteristics similar to those of the coupling factor.     

Interaction of a coupling factor from Rhodospirillum rubrum with coupling factor deficient chromatophores

A coupling factor necessary for the photophosphorylation and Mg²⁺-ATPase activities of Rhodospirillum rubrum chromatophores has been separated from these particles. Although the redox potential of coupling factor deficient chromatophores is slightly more oxidized than of the control, the addition of the coupling factor for reconstitution does not alter the redox potential. Phenazine methosulfate cannot restore or significantly enhance the photophosphorylation activities of uncoupled or reconstituted chromatophores compared to the control. The coupling factor can bind to coupling factor deficient membranes without addition of magnesium ions and thus restore the photophosphorylation and Mg²⁺-ATPase activities of these vesicles. The Ca²⁺-ATPase in the coupling factor preparation shows binding characteristics similar to those of the coupling factor.     

Heterogeneity of the coupling factor isolated from Rhodospirillum rubrum chromatophores by means of lithium chloride

By means of 2.0 M lithium chloride we have been able to extract a coupling factor protein complex from membranes of Rhodospirillum rubrum strain W. The subunit composition analyzed by SDS-gel electrophoresis showed the presence of both the and subunits.The coupling factor protein analyzed by nondissociating gel electrophoresis indicated the presence of two protein bands (Protein I and II), of which only one showed Ca-ATPase activity.     

Involvement of an essential arginyl residue in the coupling activity of Rhodospirillum rubrum chromatophores.

The arginine reagents phenylglyoxal and 2,3-butanedione in borate buffer completely inhibited photophosphorylation and Mg-ATPase of Rhodospirillum rubrum chromatophores. The inactivation rates followed apparent first order kinetics. Oxidative phospho-rylation and the light-dependent ATP-P_i exchange reactions ofR. rubrum chromatophores and the Ca-ATPase activity of the soluble coupling factor were similarly inhibited by 2,3-butanedione in borate buffer. The apparent order of reaction with respect to inhibitor concentrations for all these reactions gave values of near 1 suggesting that inactivation was the consequence of modifying one arginine per active site. ATP synthesis and hydrolysis by R. rubrum chromatophores were strongly protected against inactivation by ADP and ATP, respectively, and by other nucleotides that are substrates of the reactions but not by the products. Similarly, the Ca-ATPase of the soluble coupling factor was protected by ATP but not by ADP. Inactivation of chromatophores reactions by butanedione in borate buffer was more rapid in the light than in the dark. The results suggest that the catalytic sites for ATP synthesis and hydrolysis on the chromatophore coupling factor are different and both contain an essential arginine.     

Coupling of ATP hydrolysis to phosphate uptake in Rhodospirillum rubrum chromatophores under the influence of Ca2+ and Mg2+

The Pi-ATP exchange and ATP hydrolytic reactions, by the F0F1 complex, were studied in Rhodospirillum rubrum chromatophores in the dark. An optimal pH between 7.0 and 8.5 was determined for the hydrolytic and exchange reactions. Under these conditions, the hydrolysis/exchange ratio was approximately 2. The kinetic analysis of the hydrolytic and exchange reactions using Mg-ATP as substrate showed a change in the hydrolysis/exchange ratio that varied between 2.0 and 2.8 as the substrate concentration was increased. With Ca-ATP, hydrolysis was not saturated up to a substrate concentration of 5.0 mM, and the hydrolysis/exchange ratios changed from 2 to 240 as the substrate concentration was increased from 0.06 to 5.0 mM. Free Mg2+ inhibited hydrolysis and phosphate uptake without altering the hydrolysis/exchange ratio. Nigericin induced an increase in the hydrolysis/exchange ratio from 2.7 to 130, whereas in the presence of valinomycin, this ratio increased from 2.7 to 21. From these results, it can be concluded that Ca-ATP hydrolysis is loosely coupled to phosphate uptake given that Pi-ATP exchange activity is extremely low, even at high rates of ATP hydrolysis.     

Coupling factor ATPase complex of Rhodospirillum rubrum. Purification and characterization of an oligomycin and N,N'-dicyclohexylcarbodiimide-sensitive (Ca+ + Mg2+)-ATPase.

An ATPase complex sensitive to the energy transfer inhibitors oligomycin, dicyclohexylcarbodiimide and venturicidin has been solubilized from Rhodospirillum rubrum chromatophores with Triton X-100 and further purified by centrifugation on a glycerol gradient. The partially purified RrFo . F1 contains 13 distinct polypeptide subunits, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, including the subunits of the oligomycin-sensitive, water-soluble RrF1 ATPase. The ATPase activity of RrF0 . F1 as that of the membrane-bound enzyme complex depends on Ca2+ or Mg2+ and from detailed kinetic studies it is concluded that the divalent cation-ATP complex is the substrate for both ATPase complexes. Free ATP and free Mg2+ act as competitive inhibitors, with Ki values of 1 mM and 7 muM, respectively. The subunit composition of the purified RrFo . F1 and its similarity to the membrane-bound ATPase with respect to cation dependence and sensitivity to energy transfer inhibitors suggests that it contains all the subunits of the R. rubrum coupling factor-ATPase complex.     

Action of chlorophyllase purified from rye seedlings on light-harvesting bacteriochlorophyll of chromatophores and spheroplasts from Rhodospirillum rubrum

1. The chlorophyllase [EC 3.1.1.14] purified from greened rye seedlings hydrolyzed the bacteriochlorophyll isolated from Rhodospirillum rubrum, but not the pigment bound to the membrane of chromatophores or spheroplasts from the bacterium. 2. Acetone, if added at such concentrations that the bound bacteriochlorophyll would not be solubilized, enabled the enzyme to hydrolyze the bound pigment. The acetone concentrations required for half the maximum hydrolysis rates were 16% with chromatophores and 7% with spheroplasts. 3. The enzymic hydrolysis of the bound bacteriochlorophyll in the presence of acetone removed bacteriochlorophyllide from the membrane, leaving its esterifying alcohol, possibly all-trans-geranylgeraniol, in situ. 4. Washing of chromatophores with 30% acetone removed about 10% of the bound bacteriochlorophyll. The bound pigment remaining after washing was not hydrolyzed by the enzyme unless acetone was added. 5. It seems possible that light-harvesting bacteriochlorophyll was mostly, if not all, bound to the inner surface of chromatophores (the outer surface of spheroplasts), having its esterifying alcohol residue buried in the membrane and its porphyrin residue emerging from the membrane into the inside solution; thus, chlorophyllase could not make contact with the ester linkage between the esterifying alcohol and porphyrin moieties of the pigment unless the esterifying alcohol residue was partly exposed.     

Immunological and fluorescence studies with the coupling factor ATPase from Rhodospirillum rubrum.

1. Purification of the coupling factor ATPase from Rhodospirillum rubrum has been achieved by a combination of a previously described procedure with chromatography on DEAE-Sephadex A50. 2. Identification of the coupling factor ATPase during purification, and estimation of the relative amount of the enzyme in each fraction was greatly simplified by utilization of its unusual fluorescence. 3. Preparations of R. rubrum coupling factor ATPase injected into rabbits yielded antisera which were suitable for following the course of purification. 4. Judged by immunoelectrophoretic analysis and polyacrylamide gel electrophoresis the final preparation was pure. Under standardized conditions, apparently pure preparations showed fluorescence ratios at 300/350 nm of 3-6, which are considerably higher than those reported for pure CF1 from chloroplasts. 5. The enzyme lost its activity and changed its immunological identity during prolonged storage and by treatment with urea. Antisera against urea-treated enzyme showed the presence of two distinct antigens in the modified preparations.     

The interaction of 4-chloro-7-nitrobenzofurazan with Rhodospirillum rubrum chromatophores, their soluble F1-ATPase, and the isolated purified beta-subunit

ATP synthesis and hydrolysis by Rhodospirillum rubrum chromatophores as well as the soluble RrF1-ATPase activity are inhibited by 4-chloro-7-nitrobenzofurazan (NBD-C1) in a dithiothreitol-reversible manner. Using the method earlier developed in these chromatophores to remove specifically the beta-subunit from their membrane-bound RrF1 leaving all other subunits attached to the resulting inactive beta-less chromatophores (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977) J. Biol. Chem. 252, 8747-8752), we have tested the effect of NBD-Cl also on the isolated beta-subunit and on the beta-less chromatophores before and after their reconstitution with the missing beta-subunit. The isolated purified beta-subunit as well as the RrF1-ATPase bind covalently [14C]NBD-Cl with an accompanying increase in absorbance at 385 nm, indicative of a tyrosyl-O-NBD bond. But, unlike the inactive RrF1-NBD complex, the beta-NBD adduct is as capable as the native beta-subunit to reconstitute beta-less chromatophores and restore their ATP synthesis and hydrolysis activities. On the other hand, incubation of beta-less chromatophores with NBD-Cl before or after their reconstitution with either native beta or the NBD-saturated beta adduct results in complete inhibition of their restored activities. It is, therefore, concluded that there are different binding sites for NBD-Cl on the isolated beta-subunit and on the beta-less chromatophores or on chromatophores reconstituted with the beta-NBD adduct, where the beta-site is already occupied. Furthermore, the site responsible for inactivation by NBD-Cl of the coupled and reconstituted chromatophores and of the soluble RrF1 is different from the site modified by NBD-Cl on the isolated beta-subunit. Its subunit location is as yet unknown.     

Reconstitution of the purified calmodulin-dependent (Ca2+ + Mg2+)-ATPase from smooth muscle

The purified calmodulin dependent (Ca2+ + Mg2+)-ATPase (CaMg ATPase) from porcine antral smooth muscle transports Ca2+ after reconstitution in lipid vesicles indicating that this enzyme is indeed a Ca2+-transport ATPase. For CaMg ATPase reconstituted in asolectin vesicles a good correlation was found between the time course of Ca2+ accumulation and the corresponding changes in CaMg ATPase activity. The ATPase activity was stimulated 8-fold by A23187, which further indicates a tight coupling between ATP hydrolysis and Ca2+ transport. Asolectin vesicles with incorporated enzyme accumulated Ca2+ with a ratio approaching one Ca2+ ion transported for each ATP hydrolyzed. For CaMg ATPase reconstituted in phosphatidylcholine vesicles on the other hand, Ca2+ transport and CaMg ATPase were poorly coupled as is shown by the approximately 3.5 fold stimulation by A23187. The activity of the CaMg ATPase when reconstituted in asolectin vesicles was stimulated 1.25 fold by calmodulin while in phosphatidylcholine a value of 4.25 was obtained. The CaMg ATPase activity of the enzyme reconstituted either in asolectin or phosphatidylcholine was, after its stimulation by A23187, still further stimulated by detergent by a factor of 5.     

The role of Mg2+ and Mn2+ in the enzyme-catalysed activation of nitrogenase Fe protein from Rhodospirillum rubrum.

The activation of the Fe protein of nitrogenase (Rr2) from glutamate-grown Rhodospirillum rubrum by activating enzyme (AE) was investigated. AE is confirmed to have Mr about 20 000 and is shown to operate catalytically. There is a role in activation for metal-ion-ATP, which can be met by either MnATP or MgATP. There is also a site of action for free metal ions. This site prefers Mn2+ (apparent Kd approx. 20 microM) over Mg2+ (apparent Kd approx. 20 mM) by a factor of 1000-fold. Non-activated Rr2 does not contain this binding site. MnATP is an inhibitor of C2H2 reduction, and excess Mg2+ inhibits both AE activity and C2H2 reduction, when each is studied independently under otherwise optimal conditions. The activity of AE is increased in normal reaction mixtures (in which AE activity and nitrogenase activity occur simultaneously) by Mg2+ concentrations in excess of ATP concentrations; this occurs because the excess Mg2+ prevents ATP from chelating the free Mn2+ necessary for optimal AE activity.     

Catalytic properties of purified Ca2+,Mg2+-ATPase from the myometrium sarcolemma

The catalytic properties of myometrium sarcolemmal Ca2+, Mg2(+)-ATPase purified from plasma membrane solubilizate by affinity chromatography on calmodulin-Sepharose were investigated. The enzyme isolated in the presence of azolectin revealed a calmodulin-independent affinity for Ca2+ (Km = 0.17 microM). Purified Ca2+, Mg2(+)-ATPase displayed a strict substrate specificity, was inhibited by low concentrations of o-vanadate and was insensitive to oxytocin and prostaglandins E2 and F2 alpha. The enzyme activity was maximal at 45 degrees C, pH 7.5-8.0, and at Mg-ATP and Ca2+ concentrations of 1.5-2.5 mM and 5-20 microM, respectively.     

Agents inducing high Mg2+-ATPase activity of isolated coupling factor 1 from spinach chloroplasts

Conditions are reported under which purified coupling factor 1 (CF1) from spinach chloroplasts exhibits Mg2+-dependent ATPase activity of about 120 mumoles/min/mg protein. It is shown that CF1, partially activated by treatment with heat and dithiothreitol (DTT), can be further activated by octyl glucoside. The Mg2+-dependent ATPase activity increases linearly as a function of the concentration of octyl glucoside from about 20 mumoles/min/mg protein in the absence of detergent to 120 mumoles/min/mg protein in the presence 15 mM octyl glucoside. This concentration is below the critical micellar concentration (CMC) of the detergent, indicating that the monomeric form is responsible for the activation. Without treatment with heat and DTT, the Mg2+-dependent ATPase activity of CF1 is virtually zero, but can be stimulated by octyl glucoside. In this case, however, only concentrations around CMC give a substantial increase in activity (about 50 mumoles/min/mg at 28 mM octyl glucoside). Concentrations higher than CMC inhibit both latent and heat-activated CF1.     

Effect of spermine on activity of purified Ca2+, Mg2+-ATPase from plasma membranes of myometrium cells

Effect of endogenous polyamine spermine, a relaxant of smooth muscle, on the activity of myometrium cell plasma membrane Ca2+, Mg(2+)-ATPase was studied. It was observed a tendency to activation of enzyme at the spermine concentrations 0.1-0.5 mM, the increase of the polyamine concentrations up to 10 mM inhibited. ATPase by 80% (I50 = 5.5 +/- 0.3 mM). Spermine inhibited enzyme decreasing its turnover rate and affinity for Ca2+. The ATPase affinity for Mg2+ increased in the presence of spermine. It was revealed, that the inhibitory effect of spermine is changed by the stimulatory effect under the increase of Ca2+ concentration (up to 2.6 microM), that correlates with the relaxing effect of this polyamine on the smooth muscle.