Effect of phosphorylation on scallop sarcoplasmic reticulum

Abstract

Fragmented sarcoplasmic reticulum prepared from the cross-striated adductor muscle of the deep sea scallop (Placopecten magellanicus) was phosphorylated with inorganic phosphate to the E2P (ADP-insensitive) form. Negative staining of these preparations showed that the Ca-ATPase was organized into a quasi-crystalline array, which differed from the 'dimer ribbon' structure previously reported for the membrane under relaxing conditions (Castellani & Hardwicke, J. cell. Biol. 97 (1983) 557-61; Castellani et al., J. molec. Biol. 185 (1985) 579-94). In this new form there was only a single Ca-ATPase per unit cell. Dephosphorylation of the E2P membranes and incubation with substrate or substrate analogues in the absence of Ca2+ caused the 'dimer ribbon' structure to appear. These results imply that rotation of at least half of the Ca-ATPase subunits in the scallop sarcoplasmic reticulum may occur about an axis perpendicular to the plane of the membrane on conversion from the E2P state to the state corresponding to that existing in the relaxed muscle.

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.…”

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

“…Provided the E 2 form of the scallop Ca-ATPase is stabilized against loss of activity, it adopts a dimeric type of quaternary organization (13) that is absent in the E 1 form of the enzyme (14), and in both the E 1 ϳP and E 2 -P states the enzyme is arranged in parallel instead of antiparallel helical strands in the tubular vesicles with only a single asymmetric subunit in each unit cell (p1 lattice) (15).…”

“…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.…”