ERYTHROSIN ISOTHIOCYANATE SELECTIVELY LABELS LYSINE(464) WITHIN AN ATP-PROTECTABLE BINDING-SITE ON THE CA-ATPASE IN SKELETAL SARCOPLASMIC-RETICULUM MEMBRANES

Conditions that permit the selective modification of an ATP-protectabl e site on the Ca-ATPase in skeletal sarcoplasmic reticulum (SR) membra nes using erythrosin isothiocyanate (Er-ITC) have been identified. The major labeling site for Er-ITC has been identified using reversed-pha se HPLC and positive FAB mass spectrometry after exhaustive tryptic di gestion of the Er-ITC-modified Ca-ATPase. An ATP-protectable peptide c orresponding to M452NVFNTEVRNLSK464VER467 is modified by Er-ITC, the a verage mass of which is 2830.1 +/- 0.3 Da. The exclusive modification of lysine residues indicates Lys(464) as the site of Er-ITC modificati on. Derivatization with Er-ITC diminishes the secondary activation of steady-state ATPase activity and the rate of dephosphorylation by mill imolar concentrations of ATP, In contrast, in the presence of micromol ar ATP concentrations Er-ITC modification of the Ca-ATPase does not af fect (i) the apparent affinity of ATP, (ii) the maximal extent of phos phoenzyme formation by ATP, (iii) the rate of steady-state ATP hydroly sis, or (iv) the rate of dephosphorylation of the Ca-ATPase. Furthermo re, ATP utilization by the Ca-ATPase is unaffected by detergent solubi lization, irrespective of Er-ITC modification, indicating that the sec ondary activation of ATP hydrolysis involves a single Ca-ATPase polype ptide chain. Therefore, Er-ITC does not interfere with the normal stru ctural transitions associated with phosphoenzyme decay. Rather, these results indicate that Er-ITC bound to Lys(464) interferes with either ATP binding to a low-affinity site or the associated structural transi tions that modulate the rate of enzyme dephosphorylation.