Sarcoplasmic reticulum adenosine triphosphatase: Labeling of an essential lysyl residue with pyridoxal-5′-phosphate

Incubation of sarcoplasmic reticulum membranes (SR) with pyridoxal-5′-phosphate (PLP) followed by NaBH4 reduction results in a progressive loss of calcium-dependent ATPase activity. The rate constants and extents of inactivation are not linear functions of the PLP concentration; rather, saturation behavior is implied. The data are consistent with the scheme where K1 is the dissociation constant of the SR·PLP complex and k2 and k−2 are the rate constants for the formation and breakdown of the Schiff base SR = PLP, which can be irreversibly reduced to SR-PLP with NaBH4. K1, k2, and k−2 are, respectively, 1.5 m m , 1.8 min−1, and 0.37 min−1. In the presence of Ca-ATP, no decrease in activity occurs in 60 min; Mg-ATP exerts a similar but less pronounced protective effect. Pyridoxal is considerably less effective than PLP. The number of PLP/ATPase incorporated at early stages of the reaction is proportional to the loss of ATPase activity and extrapolates to about one at zero activity. Variation in the difference between the presence and absence of ATP is similar, also extrapolating to one PLP/ATPase. (The data also show that slower reaction at nonessential sites occurs.) That this single residue is a lysine is indicated by paper chromatography of acid hydrolysates. Tryptic digestion of PLP-NaB3H4-reacted SR followed by gel electrophoresis indicates that this lysyl residue is located in the ATPase fragment of 30,000 molecular weight.