The C. reinhardtii CF1 with the mutation betaT168S has high ATPase activity.

We have generated the mutation T168S in the beta subunit of the chloroplast ATP synthase complex of Chlamydomonas reinhardtii by site directed mutagenesis and chloroplast transformation. CF1 and the alpha3beta3gamma complex of this mutant strain were isolated and their enzymatic activities were characterized and compared to those of the corresponding wild type complexes. Without activation the mutant CF1 exhibits MgATPase activity with at least 10 times higher rates than the wild type enzyme. The MgATPase activity could be stimulated to some extent by methanol, but less by ethanol and octylglucoside. The alpha3beta3gamma complex had an even higher MgATPase activity, which was only slightly enhanced by ethanol or methanol. The ATPase activities of the mutant complexes, like those of the wild type complexes, displayed a sharp concentration optimum for Mg2+. Free ADP inhibited neither the mutant nor the wild type ATPase significantly. Azide, which strongly inhibited the ATPase activity of the wild type enzyme, inhibited the mutant enzyme only at an about 30 times higher concentration suggesting that the mutation T168S prevents trapping of a tightly bound MgADP by a catalytic site that regulates chloroplast ATPase activity. The mutant cells grew photoautotrophically at a growth rate of about 50%. Similar to the wild type the cells survived on minimal medium in the dark. Under heterotrophic conditions with acetate as energy and carbon source the mutant cells grew much faster than the wild type cells, but the chlorophyll content per cell decreased dramatically.

Catalytic properties and sensitivity to tentoxin of Chlamydomonas reinhardtii ATP synthases changed in codon 83 of atpB by site-directed mutagenesis.

The participation of the amino acid beta83 in determining the sensitivity of chloroplast ATP synthases to tentoxin was reported previously. We have changed codon 83 of the Chlamydomonas reinhardtii atpB gene by site-directed mutagenesis to further examine the role of this amino acid in the response of the ATP synthase to tentoxin and in the mechanism of ATP synthesis and hydrolysis. Amino acid beta83 was changed from Glu to Asp (betaE83D) and to Lys (betaE83K), and the highly conserved tetrapeptide betaT82-E83-G84-L85 (DeltaTEGL) was deleted. Mutant strains were produced by particle gun transformation of atpB deletion mutants cw15DeltaatpB and FUD50 with the mutated atpB genes. The transformants containing the betaE83D and betaE83K mutant genes grew well photoautotrophically. The DeltaTEGL transformant did not grow photoautotrophically, and no CF1 subunits were detected by immunostaining of Western blots using CF1 specific antibodies. The rates of ATP synthesis at clamped DeltapH with thylakoids isolated from cw15 and the two mutants, betaE83D and betaE83K, were similar. However, only the phosphorylation activity of the mutant betaE83D was inhibited by tentoxin with 50% inhibition attained at 4 microM. These results confirm that amino acid beta83 is critical in determining the response of ATP synthase to tentoxin. The rates of the latent Mg-ATPase activity of the CF1s isolated from cw15, betaE83D, and betaE83K were similar and could be enhanced by heat, alcohols, and octylglucoside. As in the case of the membrane-bound enzyme, only CF1 from the betaE83D mutant was sensitive to tentoxin. A lower alcohol concentration was required for optimal stimulation of the ATPase of the betaE83K-CF1 than that of CF1 from the other two strains. Moreover, the optimal activity of the betaE83K-CF1 was also lower. These results suggest that introduction of an amino acid with a positively charged side chain in position 83 in the "crown" domain affects the active conformation of the CF1-ATPase.

Modification of domains of alpha and beta subunits of F1-ATPase from the thermophylic bacterium PS3, in their isolated and associated forms, by 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP).

Photoaffinity labeling by 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP) of the adenine nucleotide binding site(s) on isolated and complexed alpha and beta subunits of F1-ATPase from the thermophilic bacterium PS3 (TF1) is described. BzATP binds to both isolated alpha and beta subunits, to complexed beta subunit but not to complexed alpha subunit. Amino acid sequence determination of radiolabeled peptides obtained by proteolytic digestion of [gamma-32P]BzATP-labeled alpha subunit indicates that residues on both the amino-terminal (residues A41-E67) and carboxy-terminal (residues Q422-Q476) were modified by BzATP. One of the residues in the carboxy-terminal modified by BzATP is most probably alpha Q422. Although the binding stoichiometry of 1 mol of BzATP incorporated by either isolated or complexed beta subunit was maintained, the spatial conformation of the polypeptide determines which amino acid residue(s) is more accessible to the reactive radical. CNBr derived fragments beta G10-M64, beta E75-M233, and beta D390-M469 were labeled with the isolated beta subunit. With complexed beta subunit the label was found only in CNBr fragments: beta E75-M233 and beta G339-M389. The locations where the covalently bound BzATP was found, in the soluble and assembled subunits, indicate that different conformational states exist. In the isolated form of the alpha and beta subunits the amino- and carboxy-termini can fold and reach the central domain of the polypeptide, the domain containing the adenine nucleotide binding site. When alpha combines with beta to form the alpha 3 beta 3 core complex the new conformation of the subunits is such that covalent labeling by BzATP of alpha and of the amino terminal of beta subunit is excluded.

Isolation of CF0CF1 from Chlamydomonas reinhardtii cw15 and the N-terminal amino acid sequences of the CF0CF1 subunits.

CF0CF1 was isolated from chloroplasts of the cell wall-deficient Chlamydomonas reinhardtii strain cw15. The subunit pattern was analyzed by SDS-gel electrophoresis and the N-terminal amino acid sequences of all nine subunits were determined by microsequencing. The amino acid sequences of subunits alpha, beta, gamma and epsilon match with those derived from the corresponding Chlamydomonas DNA sequences. In variance with the previously assumed N-terminus of beta; however, it was found that the first 11 amino acids are lacking. The subunits delta, I, II, III and IV were identified by comparison with known sequences of homologous polypeptides of higher plant chloroplasts and cyanobacteria, respectively.

Covalent binding of 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP) to the isolated alpha and beta subunits and the alpha 3 beta 3 core complex of TF1. Covalent binding of BzATP prevents association of alpha and beta subunits and induces dissociation of the alpha 3 beta 3 core complex.

Binding of the photoreactive ATP analog, 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP), to the isolated alpha and beta subunits of TF1 and to the alpha 3 beta 3 "core" complex of the holoenzyme is described. About 1 mol of BzATP/mol of subunit was incorporated to isolated alpha and beta subunits. The incorporation of BzATP was prevented by ATP. Covalent binding of BzATP to the alpha subunit was in general somewhat lower than that observed with the beta subunit. No complex was formed upon mixing of either of the modified subunits with the complementary nontreated subunits. Covalent binding of 3 mol of BzATP/alpha 3 beta 3 complex completely inhibited ATPase activity and resulted in the dissociation of the complex. The labeled nucleotide analog was specifically incorporated into the beta subunit of the complex. The holoenzyme TF1, in contrast to the core complex, did not dissociate to the individual subunits upon covalent binding of BzATP. These results are discussed in relation to the location of the catalytic nucleotide binding site(s) and the conformation stability of the alpha 3 beta 3 core complex of TF1.

A trial of family therapy versus a relatives' group for schizophrenia. Two-year follow-up.

The results are reported of a two-year follow-up of a trial of family sessions in the home (including patients) (12 families) versus a relatives' group (excluding patients) (11 families). Subjects were patients with schizophrenia living in high face-to-face contact with high-EE relatives. Patients were maintained on neuroleptic drugs for two years where possible. Relatives' critical comments and hostility were significantly lowered by nine months, but no significant changes occurred subsequently. Relatives' overinvolvement reduced steadily throughout the trial, and reduction in relatives' EE, either alone or in combination with reduced face-to-face contact, appeared to be associated with a lower relapse rate. The relapse rates for patients in the family-therapy and relatives'-group streams were 33% and 36% at two years. When these data were combined with the results of a previous trial, it was found that patients in families assigned to any form of social intervention had a two-year relapse rate of 40%, significantly lower than the 75% relapse rate for patients whose families were offered no help. We therefore recommend that relatives' groups are established in conjunction with some family sessions in the home for patients at high risk of relapse.

Educating relatives of schizophrenic patients.

An education programme given to relatives of schizophrenic patients in the context of other social interventions is described. The findings show that although relatives remember relatively little one month after receiving the education, it is an important intervention. Several reasons are suggested, one of which is that education is a somewhat neutral but engaging beginning to the therapeutic relationship. At the later nine months follow up several positive changes in relatives' attitude were shown.

A trial of family therapy v. a relatives group for schizophrenia.

Schizophrenic patients living in high contact with relatives having high expressed emotion (EE) were recruited for a trial of social interventions. The patients were maintained on neuroleptic medication, while their families were randomly assigned to education plus family therapy or education plus a relatives group. Eleven out of 12 families accepted family therapy in the home, whereas only six out of 11 families were compliant with the relatives group. Non-compliance was associated with a poorer outcome for the patients in terms of the relapse rate. The relapse rate over nine months in the family therapy stream was 8%, while that in compliant families in the relatives group stream was 17%. Patients' social functioning showed small, non-significant, gains. The data from the current trial were compared with data from a previous trial. The lowering of the relapse rate in schizophrenia appears to be mediated by reductions in relatives' EE and/or face-to-face contact, and is not explained by better compliance with medication. Reduction in EE and/or contact was associated with a minuscule relapse rate (5%). Very little change occurred in families who were non-compliant with the relatives group. On the basis of these findings, we recommend that the most cost-effective procedure is to establish relatives groups in conjunction with family education and one or more initial family therapy sessions in the home. It is particularly important to offer home visits to families who are unable to or refuse to attend the relatives groups.

Characterization of nucleotide-binding sites on the chloroplast coupling factor 1 using two photolabile analogs.

Nucleotide-binding sites on the chloroplast coupling factor 1 (CF1) have been probed using two photoreactive ADP analogs: 2-azido-ADP (2-N3-ADP) and 2',3'-O-(4-benzoyl)benzoyl-ADP (Bz-ADP). Photolabeling of the isolated CF1 with 2-N3-ADP results in incorporation of the analog exclusively into the beta-subunit of the enzyme. The location of the nucleotide-binding site(s) within the beta-subunit of the CF1 was investigated using peptide mapping. Within the discrimination limits of this technique, it is concluded that the azido- and benzoyl-modified analogs both bind to the same conformation of the nucleotide-binding site(s) of soluble CF1. Bz-ADP, however, labels the binding site(s) on membrane-bound CF1 in a slightly different manner.

Photoaffinity labeling of the TF1-ATPase from the thermophilic bacterium PS3 with 3'-O-(4-benzoyl)benzoyl ADP.

3'-O-(4-Benzoyl)benzoyl ADP (BzADP) was used as a photoaffinity label for covalent binding of adenine nucleotide analogs to the nucleotide binding site(s) of the thermophilic bacterium PS3 ATPase (TF1). As with the CF1-ATPase (Bar-Zvi, D. and Shavit, N. (1984) Biochim. Biophys. Acta 765, 340-356) noncovalently bound BzADP is a reversible inhibitor of the TF1-ATPase. BzADP changes the kinetics of ATP hydrolysis from noncooperative to cooperative in the same way as ADP does, but, in contrast to the effect on the CF1-ATPase, it has no effect on the Vmax. In the absence of Mg2+ 1 mol BzADP binds noncovalently to TF1, while with Mg2+ 3 mol are bound. Photoactivation of BzADP results in the covalent binding of the analog to the nucleotide binding site(s) on TF1 and correlates with the inactivation of the ATPase. Complete inactivation of the TF1-ATPase occurs after covalent binding of 2 mol BzADP/mol TF1. Photoinactivation of TF1 by BzADP is prevented if excess of either ADP or ATP is present during irradiation. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the Bz[3H]ADP-labeled TF1-ATPase shows that all the radioactivity is incorporated into the beta subunit.

Evaluation by steady-state enzyme kinetics of the role of tightly bound nucleotides during photophosphorylation.

The ATP synthetase of chloroplast membranes binds ADP and ATP with high affinity, and the binding becomes quasi-irreversible under certain conditions. One explanation of the function of these nucleotides is that they are transiently tightly bound during ATP synthesis as part of the catalytic process, and that the release of tightly bound ATP from one catalytic site is promoted when ADP and P(i) bind to a second catalytic site on the enzyme. Alternatively, it is possible that the tightly bound nucleotides are not catalytic, but instead have some regulatory function. We developed steady-state rate equations for both these models for photophosphorylation and tested them with experiments where two alternative substrates, ADP and GDP, were phosphorylated simultaneously. It was impossible to fit the results to the equations that assumed a catalytic role for tightly bound nucleotides, whether we assumed that both ADP and GDP, or only ADP, are phosphorylated by a mechanism involving substrate-induced release of product from another catalytic site. On the other hand, the equations derived from the regulatory-site model that we tested were able to fit all the results relatively well and in an internally consistent manner. We therefore conclude that the tightly bound nucleotides most likely do not derive from catalytic intermediates of ATP synthesis, but that substrate (and possibly also product) probably bind both to catalytic sites and to noncatalytic sites. The latter may modulate the transition of the ATP-synthesizing enzyme complex between its active and inactive states.

Modulation of the chloroplast ATPase by tight ADP binding. Effect of uncouplers and ATP.

Inactivation of the membrane-bound ATPase by tight ADP binding was studied under nonenergized conditions. The energy state of the system was controlled either by omitting MgCl2, preventing ATP hydrolysis, or by addition of an uncoupler which dissipates the delta mu H+. In the absence of Mg2+, ATP prevents the inactivation of the enzyme by ADP, in a competitive manner. This effect of ATP resembles that of GDP with Mg2+ present. In the presence of nigericin, Mg2+, and ATP, inactivation occurs after a 10-15-sec interval, during which the enzyme is able to hydrolyze ATP at a relatively rapid rate. The degree of inactivation is proportional to the level of bound ADP detected. This behavior is different from that of the coupled ATPase (no uncoupler added), where inactivation is attained only upon exhaustion of the ATP by its hydrolysis, despite the finding that ADP binds tightly to the active ATPase at all stages of the reaction. Higher levels of tightly bound ADP were detected in the presence of an uncoupler. We suggest that the interval during which the enzyme becomes inactive is that required for the enzyme to generate and bind ADP, and to change from the active to the inactive conformation. These results support the mechanism suggested previously for the modulation of the ATPase by tight nucleotide binding.

Source of rapidly labeled ATP tightly to non-catalytic sites on the chloroplast ATP synthetase.

Bound [32P]ATP is found on deenergized, washed chloroplast thylakoids which were illuminated in the presence of ADP and [32P]Pi. Tight binding of [32P]ATP occurred both during and after energization. Different classes of bound [32P]ATP were distinguished on the basis of their rates of formation, susceptibility to hexokinase and displacement by unlabeled ATP. 1. The rates of formation and discharge of the rapidly labeled tightly bound ATP class were much lower than that of ATP formation. The level of this bound ATP saturates at lower concentrations of substrates than does the rate of phosphorylation. Unlabeled ATP, present in the reaction medium, displaces the rapidly labeled tightly bound ATP without affecting the rate of phosphorylation. 2. We therefore conclude that the rapidly labeled bound ATP class does not fulfill the requirements expected for a catalytic intermediate and that the nucleotide tight binding site(s) on the ATP synthetase differ from the catalytic site(s) for ATP formation. 3. Since the rapidly labeled tightly bound [32P]ATP is not abolished by high concentrations of hexokinase, but is nevertheless displaced by exogenous ATP, we propose that tight binding of ATP to non-catalytic sites occurs via a free species of newly synthesized ATP which diffuses slowly to the medium from a space accessible to ATP but not to hexokinase.

Quercetin interaction with the chloroplast ATPase complex.

1. Quercetin, a flavonoid which acts as an energy transfer inhibitor in photophosphorylation is shown to inhibit the P-ATP exchange activity of membrane-bound CF1 and the ATPase activity of isolated CF1. Quercetin, affects also the proton uptake in chloroplasts in a manner similar to that of dicyclohexylcarbodiimide. 2. The light-dependent proton uptake in EDTA-treated chloroplasts is stimulated by quercetin. In untreated chloroplasts quercetin has a dual effect: it enhances at pH above 7.5 while at lower pH values it decreases the extent of H+ uptake. Similar effects were obtained with dicyclohexylcarbodiimide. 3. Like quercetin, dicyclohexylcarbodiimide was also found to inhibit the ATPase activity of isolated CF1. 4. Quercetin inhibits uncoupled electron transport induced by either EDTA-treatment of chloroplasts or by addition of uncouplers. Quercetin restores H+ uptake in both types of uncoupled chloroplasts. 5. The mode of action of quercetin and dicyclohexylcarbodiimide in photophosphorylation is discussed, and interaction with both CF1 and F0 is suggested.