The mechanism of inhibition of the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum by the cross-linker o-phthalaldehyde.
Labelling the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum with o-phthalaldehyde (OPA) results in loss of ATPase activity at a 1:1 molar ration of label to ATPase. The affinity of the ATPase for CA2+ is unaffected, as is the E1/E2 equilibrium constant. The rate of dissociation of Ca2+ from the Ca(2+)-bound ATPase is also unaffected and Mg2+ increases the rate of dissociation, as for the unlabelled ATPase. Effects of Mg2+ on the fluorescence intensity of the ATPase labelled with 4-(bromo-methyl)-6,7-dimethoxycoumarin are also unaffected by labelling with OPA, consistent with the fluorescence change reporting on Mg2+ binding at the gating site on the ATPase. The affinity of the ATPase for ATP is reduced by labelling, as is the rate of phosphorylation. The rate of phosphorylation is independent of the concentration of ATP above 25 microM ATP, so that the slow step is the first-order rate constant for phosphorylation by bound ATP. The rate of the back reaction between phosphorylated ATPase and ADP is little affected, suggesting that the slow step in phosphorylation could be the slow conformation step before phosphoryl transfer. The rate of dephosphorylation of the phosphorylated ATPase is also decreased, suggesting that a similar conformation change could be involved in the dephosphorylation step. The rate of the Ca(2+)-transport step appears to be unaffected by labelling. The net result of these changes is that the labelled ATPase is present predominantly in a Ca(2+)-free, phosphorylated form at steady state in the presence of ATP.
Khan
Y M
YM
Department of Biochemistry, University of Southampton, UK.
Starling
A P
AP
East
J M
JM
Lee
A G
AG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008956:Models, Chemical; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D009764:o-Phthalaldehyde / Q000494:pharmacology*
D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D009764:o-Phthalaldehyde; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium