Effect of Orthophosphate, Nucleotide Analogues, ADP, and Phosphorylation on the Cytoplasmic Domains of Ca2+-ATPase from Scallop Sarcoplasmic Reticulum

Abstract

The effects of orthophosphate, nucleotide analogues, ADP, and covalent phosphorylation on the tryptic fragmentation patterns of the E 1 and E 2 forms of scallop Ca-ATPase were examined. Sites preferentially cleaved by trypsin in the E 1 form of the Ca-ATPase were detected in the nucleotide (N) and phosphorylation (P) domains, as well as the actuator (A) domain. These sites were occluded in the E 2 (Ca 2؉ -free) form of the enzyme, consistent with mutual protection of the A, N, and P domains through their association into a clustered structure. Similar protection of cytoplasmic Ca 2؉ -dependent tryptic cleavage sites was observed when the catalytic binding site for substrate on the E 1 form of scallop Ca-ATPase was occupied by P i , AMP-PNP, AMP-PCP, or ADP despite the presence of saturating levels of Ca 2؉ . These results suggest that occupation of the catalytic site on E 1 can induce condensation of the cytoplasmic domains to yield a unique structural intermediate that may be related to the form of the enzyme in which the active site is prepared for phosphoryl transfer. The effect of P i on the E 2 form of the scallop Ca-ATPase was also investigated, when it was found that formation of E 2 -P led to extreme resistance toward secondary cleavage by trypsin and stabilization of enzymatic activity for long periods of time.The sarco(endo)plasmic reticulum ATPases (SERCAs) 1 transport Ca 2ϩ against an electrochemical gradient from the cytoplasm into the intracellular membranous compartment of the sarcoplasmic or endoplasmic reticulum and play a major role in Ca 2ϩ homeostasis in both muscle and non-muscle cells (1, 2). In the course of transporting Ca 2ϩ , these enzymes pass through a number of relatively well defined intermediate biochemical states that can be isolated and studied. These include forms in which the ␤-carboxyl group of a specific aspartyl residue (Asp 351 in the case of rabbit SERCA1a) is present as an acylphosphate mixed anhydride. Much of the experimental data has been interpreted in terms of the enzyme being able to exist in two basic forms: the E 1 state, in which the enzyme possesses high affinity binding sites for Ca 2ϩ and can be phosphorylated by ATP but not by P i , and the E 2 state, which possesses low affinity Ca 2ϩ sites and can be phosphorylated by P i but not by ATP (3).Comparison of the tryptic digestion patterns of rabbit SERCA1a in its Ca 2ϩ -bound and -free forms has provided some of the strongest direct evidence supporting such a model where the enzyme can exist in two fundamentally different conformational states (4 -6). Recent studies have found that the E 1 (Ca 2ϩ ) 2 form of rabbit SERCa1a shows clear structural differences to the enzyme in its vanadate-stabilized Ca 2ϩ -free (E 2 ) state (7-10). In the E 1 form, the Actuator (A) domain is isolated from the nucleotide (N) and phosphorylation (P) domains, whereas in the E 2 form all three domains are clustered together.The Ca-ATPase from the cross-striated part of the adductor muscle of the sea scallop has been the subject of a ...

Regulation of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) in turtle muscle and liver during acute exposure to anoxia

“…Transiently phosphorylated catalytic intermediates have been established for SERCA (Ryan et al, 2004). While liver SERCA is not typically known to have sites that are targets for stable, covalently bound phosphate with regulatory properties, our findings do have a precedent in muscle (Hawkins et al, 1994).…”

“…While liver SERCA is not typically known to have sites that are targets for stable, covalently bound phosphate with regulatory properties, our findings do have a precedent in muscle (Hawkins et al, 1994). Additionally, the presence of covalently bound phosphate on bay scallop SERCA was shown to increase stability of both structure and enzymatic activity (Ryan et al, 2004), comparable to the results seen in anoxic turtle tissues at low pH and low temperature.…”