21] Analysis of two-dimensional crystals of Ca2+-ATPase in sarcoplasmic reticulum

Abstract

Publisher Summary This chapter focuses on the analysis of two-dimensional crystals of Ca 2+ -ATPase in sarcoplasmic reticulum. The Ca 2+ -transport ATPase of sarcoplasmic reticulum (SR) is an intrinsic membrane protein with a molecular weight of 109,000. It is asymmetrically distributed in the SR membrane with much of its mass exposed on the cytoplasmic surface, where it can be visualized by negative staining in the form of 40-A-diameter surface particles. The intramembranous portion of the Ca 2+ -ATPase is revealed by freeze-etching as particles of about 75 A diameter that are more numerous in the cytoplasmic than in the luminal fracture face of the membrane. During ATP-dependent Ca 2+ transport, the Ca 2+ -ATPase alternates between two distinct conformations, E l and E 2 . The Ca 2+ transport is initiated by interaction of the Ca 2+ -ATPase with Ca 2+ and ATP in the E l conformation. Phosphorylation of the enzyme by ATP is followed by conversion from the E l into the E 2 enzyme form. The Ca 2+ transport is completed by the release of Ca 2+ from the enzyme in the E2 conformation, and the subsequent hydrolysis of the phosphoenzyme intermediate. The E l conformation is stabilized by saturation of the high-affinity Ca 2+ binding site of the enzyme with Ca 2+ or lanthanides.