LABELING THE CA2-ATPASE OF SKELETAL-MUSCLE SARCOPLASMIC-RETICULUM WITH THE CROSS-LINKER O-PHTHALALDEHYDE()

The Ca2+-ATPase in the sarcoplasmic reticulum of skeletal muscle react s with o-phthalaldehyde (OPA) to form a fluorescent isoindole product. The stoichiometry of labelling of the ATPase is 9 nmol of isoindole/m g of ATPase, corresponding to a 1:1 molar ratio of isoindole:ATPase. T here is no evidence for any intermolecular cross-linking. Isoindole fo rmation is faster in the presence of methylamine, but the stoichiometr y of labelling is unchanged, whereas in the presence of 2-mercaptoetha nol the level of labelling is much higher. It is concluded that OPA re acts with a single Cys residue (defining the specificity of the reacti on) in a fast step, subsequent reaction with a Lys residue to form the isoindole being rate-controlling. Labelling the ATPase with OPA in th e absence of methylamine leads to total loss of ATPase activity, where as in the presence of methylamine, the decrease in ATPase activity on reaction is small. We conclude that the loss of ATPase activity probab ly follows from formation of the intramolecular cross-link rather than from the initial modification of the Cys residue. Reaction with OPA i s not affected by the presence of ATP, ADP or Ca2+, so that the reacti ve Cys is not part of a ligand-binding site. The fluorescence emission spectrum of the labelled ATPase indicates a hydrophobic environment f or the isoindole ring.