Jürgen Schumann

The primary structure of the γ‐subunit of the ATPase from Synechocystis 6803

The nucleotide sequence of the gene coding for the FoF1‐ATPase γ‐subunit (atpC) from the transformable cyanobacterium Synechocystis 6083 has been determined. The deduced translation product consists of 314 amino acid residues and is highly homologous (72% identical residues) to the sequences of other cyanobacterial γ‐subunits. The Synechocystis 6803 sequence is also homologous to the chloroplast γ‐sequence. Like in the other cyanobacterial subunits, only the first of the 3 cysteine residues, which are involved in energy‐linked functions of the γ‐subunit in spinach chloroplasts, is conserved in Synechocystis 6803.

Insertion of a ?chloroplast-like? regulatory segment responsible for thiol modulation into ?-subunit of F0F1-ATPase of the cyanobacterium Synechocystis 6803 by mutagenesis of atpC

A regulatory sequence in the γ subunit of the F0F1-ATPase complex of higher plant chloroplasts, responsible for so-called thiol modulation, is absent in the corresponding polypeptides of the cyanobacterial complexes analysed so far. We have modified the atpC gene encoding this γ subunit in Synechocystis 6803 by site-directed mutagenesis. A segment was introduced coding for nine additional amino acids, including the two functional cysteines, which constitutes the sequence of the respective element in the chloroplast γ subunit. The growth rate as well as the rate of photosynthesis of the transformant was comparable to that of the wild-type, but the transitory increase in respiration observed immediately after a period of illumination was significantly lower in the mutant than in the wild-type. The F1 subcomplex solubilized from thylakoid membranes of both the wild-type and the transformant can be activated by trypsin to yield Ca2+-dependent ATPase activity, but only the F1 from the transformant can be activated by the thiol reagent dithiothreitol.

A study on the exchange of tightly bound nucleotides on the membrane-associated chloroplast ATP synthase complex

Abstract Light-induced exchange of tightly bound ADP on the membrane-associated chloroplast coupling factor 1 (CF 1 ) was concluded to be a two-step mechanism involving a loose enzyme-ADP complex (Strotmann, H., Bickel-Sandkotter, S. and Shoshan, V. (1979) FEBS Lett. 101, 316–320). Rapid binding of [ 14 C]ADP to the coupling factor after deenergization of thylakoids which were illuminated in the presence of [ 14 C]ADP was suggested to reflect the conversion of loosely bound to tightly bound ADP. Experimental data of the present paper support the assumption of an intermediate enzyme form with loosely bound ADP: (a) the amplitude of the rapid binding phase is independent on the concentration of uncoupler added in the light; (b) the amplitude is virtually unaffected by dilution of the medium [ 14 ]CADP concentration; (c) high concentrations of unlabeled ADP are required to reduce the rapid binding phase while binding of medium [ 14 C]ADP is inhibited by unlabeled ADP in the micromolar range. These results exclude the possibility that the rapid initial formation of tightly bound [ 14 C]ADP on deenergization might be caused by an energized nucleotide-free enzyme form which is able to pick up [ 14 C]ADP from the medium at a higher rate than the deenergized nucleotide-free form. At saturating [ 14 C]ADP concentrations in the light, the amount of the loose enzyme-ADP complex is about 35%, while 65% of the coupling factors contain a tightly bound ADP. Dissociation of the loose complex is slow in the absence of medium nucleotides but accelerated if ADP is present, suggesting that ADP binding to another site of the enzyme promotes release of the former ADP molecule. The significance of the loosely bound nucleotide in the catalytic mechanism is discussed.

Expression and characterization of a Synechocystis PCC 6803 P-type ATPase in E. coli plasma membranes.

In a previous paper, we published the sequence of a P-type ATPase gene from Synechocystis 6803 [Geisler et al. (1993) J. Mol. Biol. 234, 1284] which showed significant homologies to eukaryotic calcium ATPases. To investigate the specificity and activities of this plasma membrane-bound enzyme, we expressed the slightly modified gene in an ATPase deficient E. coli strain. The expressed ATPase showed an apparent molecular mass of about 97kDa and is localized in the E. coli plasma membranes. The introduced 6xHis tag at the N-terminus allowed the purification of the Synechocystis 6xHis-ATPase by single-step affinity chromatography using a Ni2+-nitrilotriacetic acid resin. The ATPase activity of the enzyme is inhibited by vanadate (IC50 = 119 microM), N-ethylmaleimide, N,N-dicyclohexylcarbodiimide, and inhibitors of eukaryotic sarco(endo)plasmic reticulum Ca2+-ATPases; however, it is stimulated by thapsigargin. Formation of phosphorylated enzyme intermediates depends on calcium ions indicating that the Synechocystis P-ATPase acts as a calcium pump equivalent to eukaryotic sarco(endo)plasmic reticulum Ca2+-ATPases.

Characterization of nucleotide binding sites on the membrane-bound chloroplast ATP synthase (coupling factor CF1)

Abstract Phosphorylation of ADP and nucleotide exchange by membrane-bound coupling factor CF1 are very fast reactions in the light, so that a direct comparison of both reactions is difficult. By adding substrate ADP and phosphate to illuminated thylakoids together with the uncoupler FCCP, the phosphorylation time is limited and the amount of ATP formed can be reduced to less than 1 ATP per enzyme. Low concentrations of medium nucleotides during illumination increase the amount of ATP formed during uncoupling presumably by binding to the tight nucleotide binding site (further designated as ‘site A’) with an affinity of 1 to 7 μM for ADP and ATP. ATP formation itself shows half-saturation at about 30 μM. Loosely bound nucleotides are exchanged upon addition of nucleotides with uncoupler (Schumann, J. (1984) Biochim. Biophys. Acta 766, 334–342). Release depends binding of nucleotides to a second site. The affinity of this site for ADP (in the presence of phosphate) is about 30 μM. It is assumed that phosphorylation and induction of exchange both occur on the same site (site B). During ATP hydrolysis, an ATP molecule is bound to site A, while on another site, ATP is hydrolyzed rapidly. The affinity of ADP for the catalytic site (70 μM) is in the same range as the observed Michaelis constant of ADP during phosphorylation; it is assumed that site B is involved in ATP hydrolysis. Site A exhibits some catalytic activity; it might be that site A is involved in ATP formation in a dual-site mechanism. For ATP hydrolysis, however, direct determination of exchange rates showed that the exchange rate of ATP bound to site A is about 30-times lower than ATP hydrolysis under the same conditions.

Evidence for two tightly bound nucleotides on thylakoid-bound chloroplast coupling factor 1 (CF1): One ADP — Exchangeable upon illumination - And one non-exchangeable MgATP

Abstract Isolated chloroplast coupling factor 1 contains a tightly bound ADP (which is exchangeable for labeled medium ADP or ATP) but contains two tightly bound nucleotides (one ADP and one ATP) after incubation with radioactive ATP in the presence of magnesium ions (Bruist, M.F. and Hammes, G.G. (1981) Biochemistry 20, 6298–6305). On the membrane-bound enzyme, only one nucleotide (mainly ADP) was found to be exchangeable during incubation with labeled ATP in the light (Bickel-Sandkotter, S. and Strotmann, H. (1981) FEBS Lett. 125, 188–192). This paper demonstrates that membrane-bound CF 1 contains another tight binding site, occupied with non-exchangeable ATP in addition to the tight ADP site (exchangeable in the light). During isolation, the incubation of CF 1 with EDTA causes the loss of the tightly bound ATP so that the normal isolation procedure yields an enzyme species with only one tightly bound nucleotide molecule (ADP). If EDTA is omitted during isolation, the additional ATP can be detected easily by the luciferin-luciferase method. Reconstitution of isolated CF 1 containing two tightly bound nucleotides with CF 1 -free membranes results in a membrane-bound coupling factor with two radioactive nucleotides. Because the exchange of this ATP is very slow, it is assumed that the tightly bound ATP has structural rather than catalytic or regulatory functions.

Inhibition of the transthylakoid gradient of electrochemical proton potential by the local anesthetic dibucaine

The effects of the local anesthetic dibucaine on coupling between electron transport and ATP synthesis-hydrolysis by the coupling-factor complex (CF0CF1 ATPase) were investigated in thylakoid membranes from Spinacia oleracea L. cv. Monatol. Evidence is presented that inhibition of ATP synthesis was produced by a specific uncoupling mechanism which was based on dibucaine-membrane surface interactions rather than on the interaction of dibucaine with the ATPase complex. Dibucaine reduced the osmotic space of thylakoid vesicles. At low pH of the medium it stimulated ATP hydrolysis beyond the rates obtained with optimum concentrations of ‘classical’ uncouplers. After addition of dibucaine, there was displacement of membrane-bound Mg2+ and strong thylakoid stacking in the presence of only low Mg2+ concentrations. Inhibition of ATP synthesis and transmembrane pH gradient increased with medium pH. Hydrolysis of ATP by isolated CF1 and the CF0CF1 complex was only slightly affected by dibucaine. The data are discussed assuming the involvement of localized proton channels on the membrane surface in protonic coupling of electron transport and ATP synthesis. A hypothesis for the mechanisms of action of local anesthetics at the thylakoid membrane is presented.

Cotranscription of a GTPase gene from the cyanobacterium Synechocystis PCC 6803 and a P-type Ca2+-ATPase gene☆

A GTPase gene adjacent to the Ca(2+)-ATPase gene from Synechocystis PCC 6803 has been sequenced. It encodes for a protein of 456 amino acids revealing high homology to so-called 50K proteins of Bacillus subtilis and Pseudomonas putida. Cotranscription of GTPase and Ca(2+)-ATPase genes has been shown by reverse transcription PCR.

Inhibition of ATP Hydrolysis in Isolated Chloroplasts by Lipophilic Tertiary Amines and the Mechanism of ‘Selective Uncoupling’

Lipophilic, tertiary amines (t-amines) are effective inhibitors of ATP synthesis (1–3). Following light-dependent 9-aminoacridine (9-AA) fluorescence quenching and (l4C)-methylamine uptake, inhibition of ATP synthesis is correlated with a decline of the light-induced transthyla-koid proton gradient (ΔpH). In this, t-amines resemble ‘classical’ uncouplers. Nevertheless, deliminating from uncoupling is the absence of t-amine effects on the proton-dependent control of electron flux (3). In the presence of t-amines, flux control may be strong even when 9-AA and (l4C)-methylamine indicate a low ΔpH (1,2). Considering this, the effects of t-amines resemble energy transfer inhibition. Evidence that protons still mediated flux control in the presence of t-amines came from the observation that flux control was released by addition of a ‘classical’ uncoupler plus dibucaine. t-amines at concentrations required for an inhibition of ATP synthesis and ΔpH do not inhibit electron transport per se (3). With regard to the selective inhibition of ATP-synthesis, t-amines were termed ‘selective uncouplers’ (1).

Kinetics of nucleotide exchange and of ATP hydrolysis by isolated chloroplast coupling factor CF1 in the presence of inhibitors

Abstract Isolated chloroplast coupling factor CF1 contains a tightly bound ADP which is exchangeable upon addition of medium ADP or ATP. In the presence of CaCl2 and ATP, the activated CF1 catalyzes ATP hydrolysis during substitution of tightly bound ADP by medium ATP. It was found that exchange of bound nucleotides is fast enough to be a catalytic intermediate during ATP hydrolysis (Feldman, R.I. and Boyer, P.D. (1985) J. Biol. Chem. 260, 13088–13094). In this paper, the influence of various substances on binding of nucleotides to the tight site and on hydrolysis of medium ATP was investigated. Under normal conditions, the exchange of bound nucleotides was found to be faster than hydrolysis of 10 μM ATP. After addition of thiosulfate, however, exchange of tightly bound ADP was nearly completely inhibited, while hydrolysis of ATP was slightly activated. It was observed that preincubation of CF1 with MgATP inhibited the exchange reaction, while ATP hydrolysis under the same conditions was only slightly inhibited. It is concluded that exchange of tightly bound ADP for medium ATP, and CaATP hydrolysis by activated CF1 are independent reactions, at least at low medium ATP concentrations.