Extraction of peripheral proteins is accompanied by selective depletion of certain glycerophospholipid classes and changes in the phosphorylation pattern of acetylcholine-receptor-rich-membrane proteins.
The widely used alkaline treatment of acetylcholine-receptor (AChR)-rich membranes from Torpedo marmorata (electric fish) and Discopyge tschudii (a marine ray) results not only in the extraction of non-receptor peripheral proteins but also in that of glycerophospholipids (approximately 13%). Minor acidic phospholipids, notably phosphatidic acid and polyphosphoinositides, are particularly enriched in the NaOH extracts. When electrocytes or receptor-rich membranes are incubated with [32P]Pi or [gamma-32P]ATP, polyphosphoinositides accumulate most of the label (approximately 45% in D. tschudii; 96% in T. marmorata) and exhibit the highest specific radioactivity. Furthermore, more than 50% of these phosphorylated lipids are extracted by NaOH together with the peripheral membrane proteins. NaOH treatment also results in modification of the phosphorylation pattern of AChR membrane proteins. Phosphorylation decreases in the Mr-43,000 group of peripheral proteins and in the gamma-subunit of the receptor. The results indicate that polyphosphoinositides constitute a metabolically very active lipid pool in the postsynaptic membrane, and that a substantial proportion of these phospholipids are preferentially released from the membrane together with other acidic phospholipids upon peripheral-protein extraction. The conclusion is drawn that membranes submitted to the above treatments can no longer be considered equivalent to native ones in terms of their phospholipid composition and phosphorylation characteristics.
Bonini de Romanelli
I C
IC
Instituto de Investigaciones Bioquimicas, Universidad Nacional del Sur-Consejo Nacional de Investigaciones Científicas y Técnicas, Bahía Blanca, Argentina.
Roccamo de Fernández
A M
AM
Barrantes
F J
FJ
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D005399:Fishes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D010766:Phosphorylation; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000378:metabolism*; D012972:Sodium Hydroxide / Q000494:pharmacology; D014101:Torpedo / Q000378:metabolism
D008565:Membrane Proteins; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010743:Phospholipids; D011950:Receptors, Cholinergic; D012972:Sodium Hydroxide