Effect of K+, and other ligands on the thiol reactivity and tryptic cleavage pattern of scallop sarcoplasmic reticulum

Abstract

The kinetics of reaction of the thiol groups of both membranous and non-ionic detergent-solubilized Ca-ATPase of scallop sarcoplasmic reticulum towards 5,5'-dithiobis(2-nitrobenzoate) were greatly modified in different ways by the presence of the following combinations of ligands: Ca2+, EGTA (no Ca2+), (ATPMg2- + EGTA) and (ATP + Ca2+). K+ was found to influence greatly the pattern of reactivity of the thiol groups of the scallop Ca-ATPase, modifying the kinetics of reaction differently according to the types of other ligand present. While all the thiol groups on the non-ionic detergent-solubilized Ca-ATPase were available for reaction in the absence of K+, whatever the combination of ligands, in the presence of K+, several groups became completely unreactive towards the reagent. In some cases the rate of inactivation of Ca-ATPase activity could be related to the rates of reaction of different kinetic classes of thiol group, according to the ligands present. Large differences were also seen in the tryptic cleavage pattern in the presence of the different ligands, and K+ led to major modifications in the products of digestion in the absence of nucleotide. An 80 kDa tryptic fragment was observed, as with lobster Ca-ATPase but unlike rabbit skeletal muscle Ca-ATPase. A complete amino-acid analysis of the scallop Ca-ATPase was carried out. Differences in the non-polar amino-acid content from rabbit skeletal muscle Ca-ATPase may relate to the different lipid composition of the two membranes.

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

“…Based on phosphorylation levels of the scallop SR obtained with both ATP and P i (15,21) and on its specific Ca 2ϩ -activated ATPase activity (13,16), the Ca-ATPase must represent at least 90% of the 110-kDa material. None of the membrane-bound proteolytic fragments described in this report was derived from the Na-Ca exchanger, although soluble tryptic peptides originating in the exchanger have been identified (25).…”

“…Phosphorylation with P I -For digestions in the E 2 -P state, DOCextracted scallop FSR was phosphorylated with P i essentially as described previously (15,21) at room temperature for 15 min before addition of trypsin.…”

A Ca2+-dependent Tryptic Cleavage Site and a Protein Kinase A Phosphorylation Site Are Present in the Ca2+ Regulatory Domain of Scallop Muscle Na+-Ca2+ Exchanger

“…Preparation of Native Membranes Enriched in Fragmented Sarcoplasmic Reticulum-This was carried out essentially as described previously (25)(26)(27)(28)(29)(30). Total scallop muscle membranes were separated into fractions enriched in SL (B 1 fraction) and SR (B 2 fraction) by layering the crude total membranes, suspended in 0.32 M sucrose, 0.1 M KCl, 1 mM CaCl 2 , 20 mM MOPS-Na, pH 7.0, onto a discontinuous gradient comprised of a layer of 0.8 M sucrose on 1.3 M sucrose, both in 0.1 M KCl, 1 mM CaCl 2 , 20 mM MOPS-Na, pH 7.0 (29,30).…”

“…The Ca-ATPase of the scallop SR lacks a PKA consensus phosphorylation sequence (R(R/K)X(S/ T)) (36). As with the rabbit SERCA1a enzyme, self-phosphorylation of the Ca-ATPase using ATP has an absolute requirement for Ca 2ϩ (27,28) and so cannot occur under the conditions of the PKA treatment, which is carried out in the presence of excess EGTA. Thus, the phosphorylated peptides were likely to correspond to intact exchanger and the 60 -65-kDa membranebound C-terminal proteolytic fragment detected by Western blotting (see above).…”

“…In the past, a number of studies of the Ca-ATPase from the cross-striated adductor muscle of the deep sea scallop have used a deoxycholate-extracted membrane fraction enriched in fragmented SR (25)(26)(27)(28). In the course of examining the effect of trypsin on this preparation, it was observed that soluble polypeptides were released by the action of the protease in sufficient amounts for them to be detected by conventional * This work was supported by the Illinois Affiliate of the American Heart Association through Grant-in Aid C-04/GS-04.…”