Detection of serum proteins in the electrophoretic patterns of total proteins of mycoplasma cells.
The contamination of mycoplasma cell preparations by serum proteins originating from culture medium was studied. A. laidlawii and M. arthritidis cells were grown in the presence of [14C]-aminoacids, and the cells were washed with 0-9% NaC1 by threefold centrifugation. Total proteins of the washed cells were analysed by SDS gel electrophoresis. Coomassie-stained electrophoretic patterns were compared with autoradiographs of the same gels. The stained electrophoretic pattern of washed A. laidlawii grown without serum was identical with autoradiographs of the same cells grown without or with serum. That of washed A. laidlawii grown with serum differed from the corresponding autoradiography by the presence of extra protein bands I, II, III, and IV with molecular weights of over 160,000, 80,000-87,000, 55,000 and 25,000, respectively. The same extra bands were found in stained electrophoretic patterns of washed: (a) A. laidlawii cells grown without serum and mixed with serum in the stationary phase, (b) M. arthritidis cells, as compared with their autoradiographs, (c) serum precipitate. The bands III and IV may be due to the heavy and light chains of gamma-globulin, the band II might belong to transferrin or to some component of complement. Acidification of serum to pH 5 brought about 100-fold rise of amount of serum precipitate, the number of bands in the electrophoretic pattern of the precipitate being also increased. Stained electrophoretic patterns of cells purified by twofold centrifugation in step sucrose density gradient (1-20-1-27 g./cm.3 for A. laidlawii, and 1-15-1-25 for M. arthritidis) contained no extra bands and matched completely with their autoradiographs. It was concluded that contamination of washed mycoplasma cells by serum proteins is mainly due to co-precipitation of aggregated serum proteins together with cells during centrifugation rather than to adsorption of serum proteins on the cell surface.
Detection of serum proteins in the electrophoretic patterns of total proteins of mycoplasma cells. The contamination of mycoplasma cell preparations by serum proteins originating from culture medium was studied. A. laidlawii and M. arthritidis cells were grown in the presence of [14C]-aminoacids, and the cells were washed with 0-9% NaC1 by threefold centrifugation. Total proteins of the washed cells were analysed by SDS gel electrophoresis. Coomassie-stained electrophoretic patterns were compared with autoradiographs of the same gels. The stained electrophoretic pattern of washed A. laidlawii grown without serum was identical with autoradiographs of the same cells grown without or with serum. That of washed A. laidlawii grown with serum differed from the corresponding autoradiography by the presence of extra protein bands I, II, III, and IV with molecular weights of over 160,000, 80,000-87,000, 55,000 and 25,000, respectively. The same extra bands were found in stained electrophoretic patterns of washed: (a) A. laidlawii cells grown without serum and mixed with serum in the stationary phase, (b) M. arthritidis cells, as compared with their autoradiographs, (c) serum precipitate. The bands III and IV may be due to the heavy and light chains of gamma-globulin, the band II might belong to transferrin or to some component of complement. Acidification of serum to pH 5 brought about 100-fold rise of amount of serum precipitate, the number of bands in the electrophoretic pattern of the precipitate being also increased. Stained electrophoretic patterns of cells purified by twofold centrifugation in step sucrose density gradient (1-20-1-27 g./cm.3 for A. laidlawii, and 1-15-1-25 for M. arthritidis) contained no extra bands and matched completely with their autoradiographs. It was concluded that contamination of washed mycoplasma cells by serum proteins is mainly due to co-precipitation of aggregated serum proteins together with cells during centrifugation rather than to adsorption of serum proteins on the cell surface.