Structural effects of substrate utilization on the ATPase chains of sarcoplasmic reticulum

Abstract

Addition of ATP to suspensions of fragmented [rabbit] sarcoplasmic reticulum (SR) containing low concentrations of a detergent that does not by itself produce major vesicular disruption was followed by a transient reduction in turbidity accompanied by solubilization of the vesicles. The effect of ATP was Ca2+-dependent and proceeded in parallel with utilization of the nucleotide as a substrate for the SR ATPase. Analogous effects were observed with other substrates producing enzyme phosphorylation at the catalytic site. The effects of ATP were also detected in studies of fluorescence energy transfer between enzyme chains. ATPase chains were labeled separately with N-(iodoacetyl)-N''-(5-sulfo-1-naphthyl)ethylenediamine (IAE-DANS) and 6-(iodoacetamido)fluorescein (IAF). Samples of vesicles uniformly labeled with either IAE-DANS or IAF, mixtures of 2 populations of vesicles uniformly labeled with either fluorophore and vesicles uniformly labeled with either fluorophore and vesicles containing randomized chains labeled with either fluorophore are used as experimental systems. In the last system, significant energy transfer from IAE-DANS (donor) to IAF (acceptor) was revealed by fluorescence spectra and measurements of donor fluorescence intensity and lifetime. This was attributed to close interactions between ATPase chains within the membrane bilayer. In the presence of low detergent concentrations, ATP changed the extent of energy transfer between labeled ATPase chains, consistent with destabilization of the interaction of chains. The observed effects were attributed to a reversible structural transition concomitant with enzyme phosphorylation and related to catalytic and transport function.