Photoinactivation of the F1-ATPase from spinach chloroplasts by dequalinium is accompanied by derivatization of methionine beta183.
In contrast to the F1-ATPases from bovine mitochondria and the thermophilic Bacillus PS3, which are reversibly inhibited by dequalinium in the absence of irradiation, the Mg2+-ATPase activity of heat- or dithiothreitol-activated chloroplast F1 (CF1) from spinach chloroplasts is slightly stimulated by dequalinium. Conversely, dequalinium is a partial inhibitor (maximal inhibition is 85-90%) of the Ca2+-ATPase of CF1 activated by heat, dithiothreitol, or octylglucoside. The Mg2+- and Ca2+-ATPase activities of CF1 respond differently in the presence of lauryl dimethylamine oxide (LDAO) in the assay medium. Whereas the Mg2+-ATPase activity of heat- or dithiothreitol-activated CF1 is stimulated up to 14-fold by increasing concentrations of LDAO, the Ca2+-ATPase is inhibited in a biphasic manner by increasing concentrations of LDAO. In the presence of LDAO, dequalinium does not stimulate the heat-activated Mg2+-ATPase over that promoted by LDAO alone. That dequalinium slightly stimulates Mg2+-ATPase activity although it inhibits Ca2+-ATPase activity can be reconciled by assuming that dequalinium binds to two sites in CF1, a stimulatory site that also binds LDAO and an inhibitory site. By acting as a partial inhibitor of the Mg2+-ATPase activity that it activates, the combined effect of dequalinium is modest stimulation. Irradiation of heat- or dithiothreitol-activated CF1 or the alpha3beta3gamma subcomplex of CF1 in the presence of 12 microM dequalinium led to rapid photoinactivation. ATP and ADP, separately or in combination with Mg2+, protect against photoinactivation. After photoinactivating the alpha3beta3gamma subcomplex of CF1 with [14C]dequalinium, tryptic and peptic digests of the isolated, derivatized beta subunit were fractionated by high performance liquid chromatography. Sequencing of the isolated, radioactive tryptic and peptic peptides revealed that Metbeta183, which is at or near the catalytic site, is derivatized in a single beta subunit when CF1 is photoinactivated with [14C]dequalinium.
Ren
H M
HM
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0601, USA.
Allison
W S
WS
D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002250:Carbon Radioisotopes; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology / Q000528:radiation effects*; D003868:Dequalinium / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006358:Hot Temperature; D008715:Methionine / Q000737:chemistry*; D015394:Molecular Structure; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000528:radiation effects; D018724:Spinacia oleracea
D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D008715:Methionine; D003868:Dequalinium; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases