The Phosphorus Components of Solubilized Erythrocyte Membrane Protein

Abstract

Membrane protein preparations were obtained by n-butanol extraction of salt-free aqueous suspensions of human erythrocyte ghosts. The solubilized protein contained 4.5% carbohydrate, including glucosamine and galactosamine, 1.7% sialic acid, and 0.2% phosphorus. Gel electrophoresis indicated the presence of a large number of proteins in the solubilized fraction. Of the phosphorus present 15% could be extracted with chloroform–methanol (2/1) and was shown to consist of phosphatidylserine and some phosphatidylinositol. A further 65% of the phosphorus was extracted with chloroform–methanol–HCl (200/100/1) and this extract was shown to consist principally of diphosphoinositide and triphosphoinositide. The remaining protein-bound phosphorus, representing 0.03% of the protein, could not be separated from the protein. Following treatment with the organic solvents the protein was resolubilized. The carbohydrate and sialic acid concentrations and the gel electrophoretic pattern were not altered. Following incubation of erythrocytes with inorganic 32P, the polyphosphoinositides were rapidly labelled. The phosphoprotein was also rapidly labelled but to a lesser extent. The phosphatidylinositol and phosphatidylserine were very poorly labelled.

Relationship between proteolipids and calcium-phospholipid-phosphate complexes inBacterionema matruchotii calcification

“…Extraction of Ca-PL-P Complexes. To determine the presence of phosphatidylinositol (MPI), phosphatidylinositol-4phosphate (DPI), and/or phosphatidylinositol-4,5diphosphate (TPI), samples were chromatographed on silicai gel H containing 1% potassium oxalate according to the method of Palmer and Verpoorte [22]. Freeze-dried samples were sonicated in a biphasic solution consisting of redistilled chloroform:methanol (2:1, v/v) and Trishydroxymethylaminomethane-HCl (Tris) buffer (50 mM, pH 7.4).…”

Spectrin phosphorylation and shape change of human erythrocyte ghosts.

“…(59) suggested that the physical basis for the metabolic dependence of both erythrocyte shape and deformability might be ATP-dependent sot-gel transformations at the inner surface of the membrane. The spectrin-actin meshwork beneath the membrane was found to have some properties of an actomyosin-like system (33,34,49,53) and spectrin polypeptide 2 was shown to be phosphorylated (4,29,42,45,46). These findings raised the prospect that actomyosin-like ATPase 430 activity or protein kinase-mediated phosphorylation would prove to be a link between energy metabolism and control of shape in normal erythrocytes (21,30,33,37,43,44,50,53).…”

Alterations of Inositol Lipid Metabolism of Rat Sciatic Nerve in Streptozotocin‐Induced Diabetes

“…To each nerve 8 rnl of chloroform-methanoCHC1 (200: 100: 1, by vol.) (Palmer and Verpoorte, 1971; Shaikh and Palmer, 1976) was added and the lipids were extracted by homogenization in a Potter-Elvejhem homogenizer. The residue from the above extraction was reextracted with 8 ml of the same solvent.…”

“…Unlabeled phosphatidylinositol phosphate (DPI) and bisphosphate (TPI), prepared from rat brain (Michell et al, 1970), were added and the lipids were fractionated by TLC on layers of silica gel H containing 1% potassium oxalate, using chloroformlacetoneimethanoUacetic acid/ water (40: 15: 13: 12:8, by vol.) as developing solvent (Palmer and Verpoorte, 1971). Radioactivity in phospholipid fractions was determined in the presence of silica gel using 1 ml of water plus 10 ml of 3a70B counting cocktail (Research Products International Corp.).…”