Selectivity of modification when latent and activated forms of the chloroplast F1-ATPase are inactivated by 7-chloro-4-nitrobenzofurazan.
The characteristics and specificity of inactivation of the chloroplast F1-ATPase (CF1) with 7-chloro-4-nitrobenzofurazan (Nbf-Cl) have been investigated. Inactivation of the octylglucoside-dependent Mg2+-ATPase activity of latent CF1 by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. Following inactivation of CF1 with [14C]Nbf-Cl, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the majority of the radioactive reagent incorporated is present in the beta subunit. Treatment of the enzyme with [14C]Nbf-Cl following dithiothreitol heat activation, led to similar labeling of the beta subunit and substantial incorporation of 14C into the gamma subunit. On complete inactivation, about 4 mol of Nbf-S-Cys is formed per mole of dithiothreitol-heat-activated CF1. Incorporation of 14C into the gamma subunit is prevented by prior treatment of the latent CF1 or of the dithiothreitol-heat-activated CF1 with iodoacetamide. Following incubation of the dithiothreitol-heat-activated CF1 with iodoacetamide, complete inactivation of the octylglucoside-dependent Mg2+-ATPase activity by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. After stabilization of the [14C]Nbf-O-Tyr derivative by treatment with sodium dithionite, a labeled peptide was purified. Automatic Edman degradation of this peptide revealed the sequence V-X-V-P-A-D-(D). The majority of the radioactivity was cleaved in the second cycle, the position occupied in CF1 by Tyr-beta-328, which is homologous to Tyr-beta-311, the residue reactive with Nbf-Cl in the beef heart mitochondrial F1-ATPase. When CF1, modified at Tyr-beta-328 with Nbf-Cl, is incubated at pH 9.0, the Nbf-O-Tyr adduct is hydrolyzed, leading to concomitant recovery of the ATPase activity. In double labeling experiments, two-dimensional isoelectric focusing in the presence of urea followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that 2-azido-ADP, covalently bound at the tight ADP binding site, and the tyrosine modified by [14C]Nbf-Cl are located in different beta subunits.
Ceccarelli
E A
EA
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Verburg
J G
JG
Zhuo
S Q
SQ
Allison
W S
WS
D001572:Benzofurans / Q000494:pharmacology*; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000378:metabolism; D046911:Macromolecular Substances; D010944:Plants; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
D001572:Benzofurans; D046911:Macromolecular Substances; C043708:7-chloro-4-nitrobenzofuran; D017301:Ca(2+) Mg(2+)-ATPase; D020558:GTP Phosphohydrolases; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases