The H+-ATPase from chloroplasts: effect of different reconstitution procedures on ATP synthesis activity and on phosphate dependence of ATP synthesis.
The H+-ATP synthase from chloroplasts, CF0F1, was isolated, reconstituted into liposomes and ATP synthesis activity was measured after energization of the proteoliposomes with an acid-base transition. The ATP yield was measured as a function of the reaction time after energization, the data were fitted by an exponential function and the initial rate was calculated from the fit parameters. CF0F1 was reconstituted by detergent dialysis in asolectin liposomes and phosphatidylcholine/phosphatidic acid (PtdCho/PtdAc from egg yolk) liposomes. In asolectin liposomes, high initial rates of ATP synthesis (up to 400 s(-1)) were observed with a rapid decline of the rate; in PtdCho/PtdAc liposomes the initial rate is smaller (up to 200 s(-1)), but the decline of the activity is slower. CF0F1 was reconstituted into PtdCho/PtdAc liposomes either by detergent dialysis or into reverse phase liposomes. The dependence of the rate of ATP synthesis on the phosphate concentration was measured with both types of proteoliposomes. The data can be described by Michaelis-Menten kinetics with a K(M) value of 350 microM for reverse phase liposomes and a K(M) value of 970 microM for dialysis liposomes. Both K(M) values depend neither on the magnitude of DeltapH nor on the electric potential difference, whereas V(max) decreases strongly with decreasing energization. At low phosphate concentration, there are small deviations from Michaelis-Menten kinetics. The measured rates are higher than those calculated from the fitted Michaelis-Menten parameters. This effect is interpreted as evidence that more than one phosphate binding site is involved in ATP synthesis.
Grotjohann
Ingo
I
Institut für Klinische Physiologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, D-12203, Berlin, Germany.
Gräber
Peter
P
D000255:Adenosine Triphosphate / Q000096:biosynthesis* / Q000378:metabolism; D000818:Animals; D025222:Chloroplast Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology*; D003902:Detergents / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D010084:Oxidation-Reduction; D010710:Phosphates / Q000378:metabolism; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000737:chemistry; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D018724:Spinacia oleracea / Q000201:enzymology
D003902:Detergents; D008081:Liposomes; D010710:Phosphates; D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids; C020485:proteoliposomes; C024990:asolectin; D000255:Adenosine Triphosphate; D025222:Chloroplast Proton-Translocating ATPases