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.

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.

[Main branch stenosis after bypass operation. Indications for elective PTCA]. / Hauptstammstenose nach Bypass-Operation. Indikation zur elektiven PTCA.

The unprotected left main stenosis still represents one of the contraindications of PTCA; recently developed concepts using percutaneous bypass techniques have not changed this fact so far. However, following bypass grafting the procedure can be done with low risk and may improve prognosis in case of later bypass occlusion. This study should clarify whether a higher rate of bypass occlusion is caused by postsurgical left main PTCA. From October 1981 to January 1991 a left main stenosis was dilated in 41 patients, 2 weeks to 12 years (mean 3.5 years) after bypass grafting. To date, 17/65 venous bypass grafts were already occluded, and 72.4% of the patients suffered from typical angina. In 34/41 patients (82.9%) PTCA was successful, severe complications (death, emergency surgery or myocardial infarction) did not occur and clinical improvement was achieved in 80% of symptomatic patients. Four months later, 26/34 patients (76.5%) had angiographic follow-up. Fifteen restenoses were found and a second PTCA was performed in 9/15. None of the venous bypass grafts, open at the time of the first PTCA, was occluded at follow-up. In one case PTCA of the left main stenosis turned out to be life-saving 7 years later because an occlusion of RCA- and LCX-bypasses occurred and the LAD graft showed a subtotal thrombosis. It is concluded that PTCA of left main stenosis after bypass grafting is a safe procedure and does not lead to a higher rate of venous bypass occlusions. A prognostic indication seems to be justified.