Mary Osborn

Forty proteins with polypeptide chains of well characterized molecular weights have been studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate following the procedure of Shapiro, Vinuela, and Maizel (Biochem. Biophys. Res. Commun., 28, 815 (1967)). When the electrophoretic mobilities were plotted against the logarithm of the known polypeptide chain molecular weights, a smooth curve was obtained. The results show that the method can be used with great confidence to determine the molecular weights of polypeptide chains for a wide variety of proteins.

Abstract The cell envelope of Salmonella typhimurium has been separated into cytoplasmic and outer membrane fractions of relatively high purity by a new method based on isopycnic sucrose density gradient centrifugation of the total membrane fraction obtained by lysis of lysozyme-EDTA spheroplasts. The outer membrane fraction contained approximately 60% of the protein of the total membranes, 50% of the phospholipid, and 90% of the lipopolysaccharide. The cytoplasmic and outer membrane fractions differed markedly in over-all protein composition as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate as well as in specific enzyme activities. Total cytochromes, DPNH oxidase, succinate dehydrogenase, d-lactate dehydrogenase, and Enzyme II of the α-methylglucoside phosphotransferase system were recovered essentially exclusively in cytoplasmic membrane; the specific activities of these in the outer membrane were 1 to 5% of the values obtained for cytoplasmic membrane fractions. In contrast, a phospholipase activity tentatively identified as a mixture of phospholipase A and lysophospholipase, appeared to be localized primarily in the outer membrane. UDP-sugar hydrolase, ribonuclease I and endonuclease I activities were associated with both membrane fractions. Preliminary analysis of the phospholipid composition of the isolated fractions also showed significant quantitative differences in the relative distribution of the major glycerophosphatides. The ratios of phosphatidylglycerol to phosphatidylethanolamine and of cardiolipin to phosphatidylethanolamine in the outer membrane were approximately one-half and one-quarter those of the cytoplasmic membrane.

The major protein of intermediate-sized filaments in mouse 3T3 cells, for which the name vimentin is proposed, has a molecular weight of 57,000. Antibodies against vimentin and antibodies against prekeratin have been used in parallel in immunofluorescence microscopy on a variety of cultured cells as well as on frozen tissue sections. Both antibodies decorate extended wavy arrays of filaments that are different from microfilaments and microtubules. Intermediate filament bundles decorated by antibodies against prekeratin are predominant in many epithelial cells, including epithelia-derived tumor cells, and are not decorated by antibodies to vimentin. In contrast, intermediate filaments decorated by antibodies against vimentin are widespread among nonmuscle cells of mesenchymal origin, including transformed cells, and also occur in other cells. Perinuclear whorls of aggregates of intermediate filaments induced by prolonged treatment with Colcemid generally show strong decoration with antibodies against vimentin. No significant reaction with either antiserum has been observed in muscle structures or in brain nerve tissue. These observations show that intermediate filaments with similar ultrastructure and solubility characteristics can be distinguished immunologically.

In most cell types intermediate or 10-mm filaments (IF) are a major cytoskeletal organization and, thus, directly or indirectly influence the structural appearance of the cytoplasm. In line with the cell type-specific expression patterns of different IF proteins in normal animal and human tissue, IF typing distinguishes the major tumor groups, as documented by results with several hundred human tumors classified by conventional histologic methods. Carcinomas are characterized by cytokeratins, sarcomas of muscle cells by desmin, nonmuscle sarcomas by vimentin, and gliomas by glial fibrillary acidic protein. Furthermore, certain tumors originating from the sympathetic nervous system, e.g., ganglioneuroblastoma, pheochromocytoma, and at least some neuroblastomas, are characterized by the presence of neurofilaments. Carcinomas can often be further subdivided with regard to their possible derivation by examining their cytokeratin profiles. The IF type characteristic of the cell of origin seems to be kept not only in the primary tumor but usually also in solid metastases. In general, tumors do not acquire additional IF types. Therefore, IF typing can provide an unambiguous and rapid characterization in certain cases, that are difficult to diagnose by conventional techniques. Some useful examples are the small cell tumors of childhood and the discrimination between undifferentiated carcinoma and lymphoma. IF typing of a few tumors has already led to a revision or reconsideration of the original light microscopic diagnosis. The combined results indicate that at least certain carcinomas, as well as certain other tumor types, seem to arise by the selective multiplication of a particular and identifiable cell type present in the normal tissue. The procedure is not restricted to tumor material. IF typing of Mallory bodies, Alzheimer's disease tangles, certain myopathies, and the cells of the amniotic fluid offers further interesting applications. Thus, IF typing should become a valuable new tool both in histology and surgical pathology.