The functioning of the lipids and lipoproteins of sarcotubular membranes in calcium transport

In the intact muscle cell, an internal tubular membrane system called the sarcoplasmic reticulum (SR) plays an important role in the contraction-relaxation cycle by controlling the Ca⁺⁺ of the myoplasm; release of Ca⁺⁺ from the SR to myoplasm initiates contractile activity and sequestring Ca⁺⁺ in the SR by means of a transport system causes muscle to relax. Fragments of the SR with a vesicular structure can be isolated from muscle homogenate and these vesicles are able to vigorously transport Ca⁺⁺ from incubation media into the intravesicular space thus enabling study of Ca⁺⁺ transport under precisely defined in vitro conditions. A highly purified fraction of SR vesicles called SF₁ were prepared from rat muscle by means of density gradient centrifugation procedures. The role of SR lipid in Ca⁺⁺ transport was studied. SF₁ was treated in vitro with either phospholipase A or C or D or polyene antibiotics. The effect of essential fatty acid deficiency, induced in vivo, was also investigated. It was concluded that the only structural feature of SF₁-lipid involved in Ca⁺⁺ transport and the associated adenosine triphosphatase is the phosphoryl moiety of the phospholipids. Evidence was obtained which inplicated histidine residues of the SF₁ protein in this transport function. To study the role of SF₁ protein in this process in depth, the membranes were solubilized by a sodium dodecylsulfate system and made free of their lipid components. More than 95% of this protein is soluble in dilute salt solution; of this, more than 90% is composed of a protein fraction which can be isolated by gel filtration (called protein fraction-2). Protein fraction-2 contains large molecular aggregates of small polypeptide subunits of identical or nearly identical molecular weight. They contain solely N-terminal glycine and probably only C-terminal alanine. The significance of such a high percentage of similar polypeptide subunits in SR is discussed. One of five papers to be published from the Symposium “Lipid Transport” presented at the AOCS Meeting, New Orleans, April 1970.