Crosslinking the active site of sarcoplasmic reticulum Ca(2+)-ATPase completely blocks Ca2+ release to the vesicle lumen.
Intramolecular crosslinking of the active site of the sarcoplasmic reticulum Ca(2+)-ATPase with glutaraldehyde results in substantial inhibition of ATPase activity and stabilization of the ADP-sensitive E1 approximately P(2Ca) intermediate (E, enzyme) with occluded Ca2+ [Ross, D. C., Davidson, G. A. & McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621]. We show here, using conditions of low passive vesicle permeability and absence of ADP, that Ca2+ "deoccludes" more rapidly than it leaks out of the vesicle lumen. Deocclusion is paralleled by dephosphorylation. Therefore, turnover of crosslinked E1 approximately P(Ca) (approximately 5 nmol/min per mg of protein at 25 degrees C) involves Ca2+ release to the vesicle exterior and concomitant phosphoenzyme hydrolysis. Ca2+ release to the lumen, the normal pathway, is apparently blocked completely. In the presence of ADP, Ca2+ is also released to the vesicle exterior, and this release is coupled to the synthesis of ATP. The results suggest that a tertiary structural change at the active site follows phosphorylation and is an absolute requirement for Ca2+ release from the native enzyme to the vesicle lumen.
McIntosh
D B
DB
Medical Research Council Biomembrane Research Unit, University of Cape Town Medical School, Observatory, South Africa.
Ross
D C
DC
Champeil
P
P
Guillain
F
F
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D005976:Glutaral / Q000494:pharmacology*; D007700:Kinetics; D008962:Models, Theoretical; D009132:Muscles / Q000201:enzymology; D010755:Organophosphates / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D003432:Cross-Linking Reagents; D010755:Organophosphates; C011632:acetyl phosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium; D005976:Glutaral