Covalent labeling of the cytoplasmic or luminal domains of the sarcoplasmic reticulum Ca2+-ATPase with fluorescent azido dyes

摘要: Sarcoplasmic reticulum (SR) vesicles were incubated with azido derivatives of Cascade blue (ACB), Lucifer yellow (ALY), 2,7-naphthalene-disulfonic acid (ANDS), and fluorescein (AF) for 0.1-24 h at 2 degrees C. All four dyes gave intense reaction the cytoplasmic domain Ca(2+)-ATPase on photoactivation after brief incubation. The penetration into luminal space SR was determined centrifugation through Sephadex microcolumns to remove external dye, followed by photolabeling gel electrophoresis photolabeled proteins. ACB ANDS calsequestrin increased progressively during incubation up 24 indicating their slow accumulation in space, while ALY AF did not show significant vesicles. distribution covalently attached tested tryptic proteolysis labeling exclusively from outside (OS), inside (IS) or both sides (BS). In all cases fluorescence seen A fragment inhibition ATPase activity. OS preparations A1, IS A2 more intensely labeled. There no incorporation region B identified FITC fluorescence. crystallization EGTA + decavanadate completely inhibited BS samples either Ca2E1 E2V conformation. preparations. labeled state impaired, there only slight disorganization crystals. total amount photoincorporated proteins 1.75 nmol/mg protein; 2/3 this occurred 1/3 inside. Similar level obtained media that stabilize E1 E2 conformation Ca(2+)-ATPase.