In vitro functional characterization of bacterially expressed human fibroblast tropomyosin isoforms and their chimeric mutants
- 1 January 1993
- journal article
- research article
- Published by Wiley in Cell Motility
- Vol. 26 (3), 248-261
- https://doi.org/10.1002/cm.970260308
Abstract
At least eight tropomyosin isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsmα) are expressed from four distinct genes in human fibroblasts. In order to elucidate isoform properties, we have subcloned hTM3 and hTM5 full‐length cDNAs, as well as their chimeric cDNAs into the bacterial expression pET8C system. Bacterially expressed tropomyosin isoforms (called PEThTM3, PEThTM5. PEThTM5/3, and PEThTM3/5) were purified and characterized. Under optimal binding conditions, the binding of PEThTM5 isoform to F‐actin was stronger than the PEThTM3 isoform. However, analysis of actin‐binding by the McGhee and von Hippel equation revealed that PEThTM3 exhibits higher cooperativity in binding than PEThTM5 does. Furthermore, the chimera PEThTM5/3 which possessed the N‐terminal fragment of hTM5 fused to the C‐terminal fragment of hTM3 had even stronger actin binding ability. The reverse chimera PEThTM3/5 which possessed the N‐terminal fragment of hTM3 fused to the C‐terminal fragment of hTM5 demonstrated greatly reduced affinity to actin filaments. In addition, both chimeras had different KCl requirements for optimal binding to F‐actin than their parental tropomyosins. A bacterially made C‐terminal fragment of human fibroblast caldesmon (PETCaD39) and native chicken gizzard caldesmon were both able to enhance the actin‐binding of these bacterially expressed tropomyosins. However, PETCaD39′s enhancement of binding to F‐actin was greater for PEThTM5 than PEThTM3. Under 30 mM KCl and 4 mM MgCl2, the low Mr isoform PEThTM5 appeared to be able to amplify the actin‐activated HMM ATPase activity by 4.7 fold, while the high Mr isoform PEThTM3 stimulated the activity only 1.5 fold. The higher enhancement of ATPase activity by PEThTM5 than by PEThTM3 suggested that the low Mr isoform hTM5 may be more involved in modulating nonmuscle cell motility than hTM3. These results further suggested that different isoforms of tropomyosin might have finite differences in their specific functions (e.g., cytoskeletal vs. motile) inside the cell.Keywords
This publication has 55 references indexed in Scilit:
- Generation of skeletal, smooth and low molecular weight non-muscle tropomyosin isoforms from the chicken tropomyosin 1 geneJournal of Molecular Biology, 1990
- Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extensionGene, 1989
- Differential localization of tropomyosin isoforms in cultured nonmuscle cells.The Journal of cell biology, 1988
- Myosin subfragment-1 is sufficient to move actin filaments in vitroNature, 1987
- Characterization of a cDNA defining a gene family encoding TM30pl, a human fibroblast tropomyosinJournal of Molecular Biology, 1987
- Tropomyosin isoforms in chicken embryo fibroblasts: purification, characterization, and changes in Rous sarcoma virus-transformed cells.The Journal of cell biology, 1985
- Monoclonal Antibodies Against Chicken Tropomyosin Isoforms: Production, Characterization, and ApplicationHybridoma, 1985
- Amino acid sequence of equine platelet tropomyosinFEBS Letters, 1983
- Equilibrium of the actin-tropomyosin interactionJournal of Molecular Biology, 1979
- Theoretical aspects of DNA-protein interactions: Co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous latticeJournal of Molecular Biology, 1974