The nature and location of Acholeplasma laidlawii membrane proteins investigated by two-dimensional gel electrophoresis.
The high resolution, two-dimensional electrophoresis system for the separation of proteins described by O'Farrell, (O'Farrell, P.H. (1975) J. Biol. Chem. 250, 4007--4021) has been modified for the separation of Acholeplasma laidlawii proteins. Reproducible protein patterns have been obtained from A. laidlawii cell, membrane and soluble protein preparations. The isoelectric focusing of membrane proteins was greatly improved by removing the bulk of the membrane lipid before solubilizing the protein. A. laidlawii peripheral membrane proteins were removed from the membrane by low ionic strength washing and by treatment with EDTA. The effect of an exhaustive EDTA treatment and a rapid, warm EDTA treatment were compared. By comparing the protein patterns obtained in these ways it was possible to distinguish two separate groups of peripheral membrane proteins and one integral membrane protein group. The peripheral membrane proteins which were removed from the membrane at low ionic strength (group I) were also insoluble in Triton X-100, whereas additional peripheral membrane proteins extractable by subsequent EDTA treatment (group II) were soluble in Triton X-100. Exterior-facing membrane proteins were distinguished from the interior-facing ones by lactoperoxidase-catalyzed iodination of intact cells and membranes. Group I peripheral membrane proteins faced the cell interior whereas group II proteins faced the cell exterior. We counted approximately 320 individual whole cell proteins. Of these, about 140 were membrane associated and a maximum of 40 proteins were iodinated after iodinating intact cells. A. laidlawii was also grown in the presence of NaH232PO4 and whole cell proteins were separated by two-dimensional gel electrophoresis. One membrane protein and two soluble proteins were labelled.
D000128:Acholeplasma laidlawii / Q000648:ultrastructure*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D002462:Cell Membrane / Q000648:ultrastructure; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011092:Polyethylene Glycols; D012995:Solubility
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011092:Polyethylene Glycols; D004492:Edetic Acid