Abstract
We have previously shown that rat cultured cells contain five isoforms of tropomyosin (Matsumura, F., Yamashiro-Matsumura, S., and Lin, J. J.-C. (1983) J. Biol. Chem. 258, 6636-6644) and that these tropomyosins are differentially expressed upon cell transformation (Matsumura, F., Lin, J. J.-C., Yamashiro-Matsumura, S., Thomas, G. P., and Topp, W. C. (1983) J. Biol. Chem. 258, 13954-13964). To examine functions of tropomyosin in microfilament organization, we have purified and partially separated the multiple isoforms of tropomyosin by chromatography on hydroxylapatite. Analyses of cross-linked dimers produced by air oxidation have revealed that all isoforms except the tropomyosin isoform with apparent Mr of 35,000 form homodimers. Although these tropomyosins share many properties characteristic of tropomyosin, structural analyses at a peptide level and immunological analyses have shown that the five isoforms can be classified into two groups, i.e. tropomyosins with higher apparent Mr (Mr = 40,000, 36,500, and 35,000) and tropomyosins with lower apparent Mr (Mr = 32,400 and 32,000). The low Mr tropomyosins show less ability for head-to-tail polymerization and lower affinity to actin than the high Mr tropomyosins. We suggest that these differences in properties may be related to the changes in microfilament organization observed in transformed cells.
Highlights
We havepreviouslyshownthatratcultured cells positions, isoelectric points,anda-helixcontentsare very contain five isoforms of tropomyosin
The studies (4-7,40,42; see Ref.43forreview) of TM isolated from nonmuscle tissues such as platelets and brain haverevealed several major contrasts betweenmuscle and nonmuscle TMs
Multiple Isoforms oCfultured Cell Tropomyosin was directly proven by the complete amino acid sequence where equine platelet TM has a M, of 28,552(35) while rabbit skeletal muscle TM has a M, of 32,800 [34]
Summary
0 1985 byThe American Society of Biological Chemists, Inc. Vol 260, Nof. 25, Issue November 5, pp. 13851-13859.1985 Printed in U.S.A. MIsuolftoiprmles of Cultured Cell Tropomyosin in 500 ml of low salt solution containing 0.1M NaC1, 2.5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM P-mercaptoethanol, and 20 mM Tris/HCl (pH8.0). In order to get partial cleavage,the digestion was performed under two different conditions (one (described in a)was milder than the other (described in b)).(a) Reaction a t 0 "C in methanol: 20 pl of T M samples (-200 pg/ml) were mixed with 160 pl of formic acid and 20 pl of methanol To these mixtures were added 30 pl of CNBr (100 mg/ml in distilled water) and they were incubated overnight on ice. Duplicates of samples were first analyzed on slab gels of the Tris/glycine buffer system [21] with a low concentration of bisacrylamide [22]. Antibody Production-Antibodies to the lowM, TMs (TM-4 and TM-5) were made by the subcutaneous immunization of rabbits with the protein purified by preparative SDS-gel electrophoresis of crude T M from REF-WT4A cells. Rabbit skeletaml uscle actin and T M were prepared as described previously [11]
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