Crystalline structure of sarcoplasmic reticulum from scallop.

Abstract

Negatively stained sarcoplasmic reticulum from the scallop Placopecten magellanicus presented a variety of crystalline forms, the most common being tubular structures. These were characterized by paired rows of morphological units, spaced at ,--120 A, running diagonally across the tubules. The orthogonal unit cell (120 x 55 A) contained two units, related by a twofold axis, which probably represented the part of the Ca2+-ATPase molecule projecting from the outer surface of the membrane.Fragmented sarcoplasmic reticulum (FSR) 1 prepared from rabbit skeletal muscle and membranes reconstituted in vitro from SR Ca2÷-ATPase have been studied extensively by electron microscopy and x-ray diffraction. Negatively stained rabbit FSR shows vesicles of average diameter 0.15 um (1) covered on their outer surface with projections ~60 A long and 30-40 A diam which represent part of the Ca2+-ATPase molecule (2-5). Freeze-fracturing studies show 85-A-diam particles embedded in the concave fracture face, also interpreted as the CaE+-ATPase (6). A ratio of 3-4 surface projections per 85-A inner particle has led to the suggestion that the ATPase has a tetrameric structure in the membrane (7). Xray studies have confirmed the asymmetrical distribution of mass across the SR membrane and that the diameter of the projections is ~35 ,~ (8-10). Rabbit SR vesicles do not, however, show sufficient order to allow a detailed structure analysis, although treatment with vanadate has recently been reported to cause an ordered surface array of subunits (11).Scallop SR consists of cisternae connected by tubular elements, and is closely associated with the sarcolemma (12). We have prepared FSR from the cross-striated adductor muscle of Placopecten magellanicus and examined it by electron microscopy. Negatively stained vesicles showed a crystalline appearance, and images of one class of tubular SR have been analyzed by two-dimensional reconstruction techniques. MATERIALS AND METHODS Preparation of Sarcoplasmic Reticulum Vesicles: Live seascallops (Pecten magellanicus) were obtained from the Marine Biological Laboratory, Woods Hole, MA. Strips of the striated adductor muscle were excised from the animal and chemically skinned in a buffered solution containing 0.05-0.1% wl/vol saponin, 100 mM NaCI, 8 mM MgSO4, 5 mM EGTA, 5

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

“…Ca-ATPase assays for scallop FSR were carried out using a coupled assay as described previously (Castellani & Hardwicke, 1983;Kalabokis & Hardwicke, 1988) in the presence and absence of oligomycin af pli 7.4. Pyruvate kinase and lactate dehydrogenase activities were always at least 30-fold greater than the Ca-ATPase activity.…”

“…The FSR of the scallop cross-striated adductor muscle shows a highly ordered arrangement of Ca-ATPase in the membrane (Castellani & Hardwicke, 1983;Castellani, et al, 1985), which has prompted a general biochemical study of this interesting system. In the work reported here, the thiol content and general amino-acid composition of scallop SR Ca-ATPase have been determined.…”

Effect of phosphorylation on scallop sarcoplasmic reticulum

“…Ca-ATPase molecules in the E2P scallop FSR rnembranes were no longer in the 'dimer' anti-parallel arrangement previously described (Castellani & Hardwicke, 1983;Castellani et al, 1985), but in another sort of quasi-crystalline organization in which there was only a single Ca-ATPase per unit cell. When a substrate analogue was present, dephosphorylation of the E2P state of the scallop FSR in the absence of Ca 2+ led to the disappearance of the P1 lattice, and the appearance of the 'dimeric' P2 type of crystalline array of Ca-ATPase molecules.…”

“…The sarcoplasmic reticulum (SR) forms a system of tubular and cisternae-like structures, the latter being associated with the sarcolemma in couplings similar to that in the triads of vertebrate cross-striated muscle. On homogenization of the muscle, electron microscopy shows that many membrane vesicles with a tubular geometry are produced, and these may be purified and identified with the sarcoplasmic reticulum (Castellani & Hardwicke, 1983). In the absence of Ca 2+, and in the presence of substrate (ATPMg 2-, for example, Makinose & Boll, 1979), i.e., under conditions which relax the muscle, these SR vesicles exhibit a dimeric arrangement of Ca-ATPase molecules, the dimers being arranged in ribbons that wind in a right-handed helical fashion in 0142-4319/89 $03.00 + .12 9 1989 Chapman and Hall Ltd. the SR tube (Castellani & Hardwicke, 1983;Castellani et al, 1985).…”

Crystallization of the Ca2+‐ATPase of skeletal muscle sarcoplasmic reticulum Inhibition by myotoxin a

“…Decavanadate induces substantial immobilization or aggregation of the CaZ+-ATPase molecules in native membranes [13] and seems to act as a nonfunctional substrate analogue [14]. In scallop SR two-dimensional membrane crystals are found without the addition of vanadate and the crystalline Ca2+-ATPase is present as a dimer [15,16].…”