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In an analysis of 12 human fibroblast cell lines by two-dimensional electrophoresis, one cell line (1493) was found to contain a major protein variant (Cytosk: 12; for the convention used in spot numbering, see Anderson, N. L. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2407-2411) not present in any of the other 11 cell lines. Biochemical characterization of the variant protein included determination of its subcellular location, partial amino acid composition, behavior on sodium dodecyl sulfate (SDS) versus SDS/urea gels, and partial proteolytic digestion patterns. All of these methods showed that Cytosk:12 is related to Cytosk:11. Both proteins are located in the cytoskeleton, contain little cysteine or proline and no detectable tryptophan, shift together to a higher apparent molecular weight when electrophoresed in the presence of SDS and 8 M urea versus SDS alone, and have identical products after partial proteolysis according to Cleveland et al. (Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. (1977) J. Biol. Chem. 252, 1102-1106). Preparation of nonmuscle tropomyosin resulted in the partial purification of both Cytosk:11 and :12. The data suggest that Cytosk:11 is fibroblast nonmuscle tropomyosin and that Cytosk:12 in cell line 1493 is a charge variant of that protein.

Tropomyosin preparations from human platelets, human peripheral blood leukocytes from normal individuals and from a patient with chronic lymphocytic leukemia, human lymphoblastoid cells (GM607), human epithelial cells, and human skin fibroblasts have all been found to contain more than one protein when analyzed by two-dimensional gel electrophoresis. Although the lymphoid cell preparations consistently contain two proteins of almost identical molecular weight (Mr = 30,000), the platelet, epithelial cell, and fibroblast preparations contain two or more major proteins with molecular weights between 31,000 and 36,000, in addition to a major protein at 30,000. All of these proteins have characteristics in common with tropomyosin including slightly acidic isoelectric point (approximately pH 4), stability to heat and organic solvents, association with the cytoskeleton, and reactivity with antibody against skeletal muscle tropomyosin. The nonmuscle tropomyosin-like proteins were compared with tropomysins from human skeletal, cardiac, and smooth muscle by peptide mapping after partial proteolysis. The results showed one of the non-muscle proteins to be identical to the major smooth muscle tropomyosin in human uterus (myometrium) and another to be similar but not identical to skeletal muscle alpha-tropomyosin. The remainder of the proteins with tropomyosin characteristics was unique to non-muscle cells. In all, nine distinct human proteins with characteristics of tropomyosin are described. Charge variants of two of these proteins have been described previously.

A variant of ornithine aminotransferase (OAT, EC 2.6.1.13) has been detected in an offspring of a male mouse treated with ethylnitrosourea. The evidence presented to support the identification of the protein variant (ENU 2) as altered OAT includes (a) a corresponding 50% decrease in the abundance of a protein, located one charge unit basic to the variant, which comigrates on two-dimensional gel patterns with purified mouse liver OAT; (b) the binding of anti-rat-OAT antibody to the variant; (c) the increased abundance of the variant protein in the livers of mice fed a high protein diet (85% casein); and (d) purification of the variant through an OAT purification protocol.

A computerized search for the appearance of heritable mutations (as indicated by changes in protein expression) was conducted on three sets of mice, whose sires had been either untreated, exposed to 3 gray units of gamma radiation, or treated with 150 mg/kg ethylnitrosourea. Proteins from the livers of approximately 800 mice were separated by two-dimensional electrophoresis, and abundances were measured by using image analysis techniques. Heritable mutations were detected by the appearance of new proteins or by the quantitative decrease in abundance of normally occurring proteins. Measurements of the variability of the protein abundance indicate that at least 48 proteins are consistent enough to be used in searches when mutations are expected to result in a 50% reduction in the normal amount of protein. New proteins were found in four offspring from ethylnitrosourea-treated mice, and in each case a nearby spot was found to be significantly diminished. These mutations were confirmed in subsequent generations. A computer-assisted search detected three of these mutations on the basis of the abundance decrease alone. These results indicate that two-dimensional electrophoresis can be used to detect mutations reflected as quantitative changes in protein expression, provided that the proteins to be monitored are quantitatively stable when samples from different individuals are compared.