New method for the isolation of membranes from Mycoplasma gallisepticum.
Mycoplasma gallisepticum lysed readily in carbonate bicarbonate buffer at pH 9.2 to 10.5. The hemagglutination titer of the lysates was 2- to 16-fold greater than a cell suspension at the same protein concentration in buffered saline. Membranes prepared from cells lysed by this method at pH 10 were relatively free from cytoplasmic contaminants as shown by electron microscopy of thin sections. The membranes retained their hemagglutination activity, gave reactions in immunodiffusion tests identical to those obtained by osmotic lysis and sonic treatment, and showed a similar pattern of protein bands by polyacrylamide disk electrophoresis. When inoculated into rabbits, the membranes gave rise to antibodies active in growth-, metabolic- and hemagglutination-inhibition tests. On the average, membranes obtained by lysis at pH 10 contained 44% of the original cell protein. The method is simple, giving high yields of membranes, and may be adaptable to other mycoplasmas.
D000818:Animals; D001888:Boric Acids; D002021:Buffers; D002458:Cell Fractionation; D002462:Cell Membrane; D003584:Cytological Techniques; D005656:Fungal Proteins / Q000096:biosynthesis; D006386:Hemagglutination Tests; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007125:Immunogenetics; D008854:Microscopy, Electron; D009174:Mycoplasma / Q000166:cytology* / Q000254:growth & development; D011817:Rabbits / Q000276:immunology
D001888:Boric Acids; D002021:Buffers; D005656:Fungal Proteins