Changes in the levels of human tropomyosins IEF 52,55, and 56 do not correlate with the loss of actin cables observed in SV 40 transformed MRC-5 fibroblasts

Summary A mouse monoclonal antibody (mAb 1D122G9) raised against human tropomyosin IEF 52 (HeLa protein catalogue number, Mr=35 kd) has been characterized both in terms of specificity and patterns of immunofluorescence staining in Triton extracted cultured cells. As determined by two dimensional gel immunoblotting of HeLa cell proteins the antibody recognized IEF 52 and two other acidic proteins (IEF 55, Mr=31.8 kd; IEF 56, Mr=31 kd) previously identified as putative tropomyosin-like proteins. Immunofluorescence staining of Triton extracted cultured cells revealed the striated or interrupted pattern on the actin cables characteristic of tropomyosin staining. Quantitation of the three tropomyosins in Triton cytoskeletons from normal and SV 40 transformed human MRC-5 fibroblasts showed that the latter contained significantly less of tropomyosin IEF's 52 (52%) and 56 (72%) as compared to their normal counterparts. The ratios of these two tropomyosins to actin however was very similar for both types of cytoskeletons. This was not the case for tropomyosin IEF 55, which was present in nearly twice the amount in the cytoskeletons from the SV 40 transformed cells. The ratio of actin to total tropomyosin for whole cells was found to be unchanged on transformation. This ratio however was 31% lower in the cytoskeletons from the transformed cells. These and other results presented here suggest that changes in the levels of these three tropomyosins are not enough to account for the magnitude of the loss of actin cables observed in the transformed cells.Abbreviations IEF isoelectric focusing - mAb monoclonal antibody - NEPHGE non equilibrium pH gradient electrophoresis