Expression of smooth-muscle and nonmuscle tropomyosins in Escherichia coli and characterization of bacterially produced tropomyosins

The cDNA encoding the beta-tropomyosin isoform of chicken smooth muscle (CSMbeta) was constructed and expressed in Escherichia coli to produce recombinant, unacetylated beta-tropomyosin (rCSMbeta) and a mutant (rCSMbeta-7) with a 7-residue deletion at its amino-terminus. Furthermore, the cDNA coding for human fibroblast tropomyosin isoform 3 (hTM3) was also used to produce unacetylated hTM3 (called PEThTM3). All of bacterially-made tropomyosins were high alpha-helical in structure as judged by CD analysis and resistant to heat denaturation. Both the rCSMbeta and PEThTM3 exhibited saturable binding to F-actin with apparent binding constants of 1.14 . 10(6) and 2.78 . 10(6) M-1, respectively. The bacterially made, unacetylated smooth muscle tropomyosin (rCSMbeta) appeared to have a comparable actin-binding affinity to that of gel-purified CSMbeta homodimer (1.25 . 10(6) M-1) but significantly lower than that for native gizzard tropomyosin (CSM-TM) heterodimer (1.28 . 10(7) M-1). The amino-terminal deletion mutant rCSMbeta-7 failed to bind to F-actin. Effects of gizzard caldesmon on the actin binding of these bacterially made tropomyosins were also examined. Under the binding condition containing 0.5 mM MgCl2 and 30 mM KCl, caldesmon greatly enhanced the binding of rCSMbeta to F-actin. However, under the same condition, there was a slight enhancement in the actin-binding for gel-purified CSMbeta or PEThTM3 (1.2-1.6-fold stimulation) and no enhancement for native gizzard tropomyosin. Neither the presence of caldesmon nor native gizzard tropomyosin induced detectable binding of the amino-terminal deletion mutant rCSMbeta-7 to F-actin. These results clearly imply the importance of the amino-terminal 7 amino-acid residues of CSMbeta in the actin binding and the caldesmon enhancement.
Publisher
ELSEVIER SCIENCE BV
Issue Date
1993-03
Language
ENG
Keywords

COMPLETE NUCLEOTIDE-SEQUENCE; CHICKEN-EMBRYO FIBROBLASTS; ACTIN-BINDING PROPERTIES; RAT CULTURED-CELLS; ALPHA-TROPOMYOSIN; F-ACTIN; BETA-TROPOMYOSIN; MESSENGER-RNAS; MONOCLONAL-ANTIBODIES; MULTIPLE ISOFORMS

Citation

BIOCHIMICA ET BIOPHYSICA ACTA, v.1162, no.3, pp.255 - 265

ISSN
0006-3002
DOI
10.1016/0167-4838(93)90289-4
URI
http://hdl.handle.net/10203/63743
Appears in Collection
BS-Journal Papers(저널논문)
Files in This Item
There are no files associated with this item.
  • Hit : 108
  • Download : 0
  • Cited 0 times in thomson ci
This item is cited by other documents in WoS
⊙ Detail Information in WoSⓡClick to seewebofscience_button
⊙ Cited 24 items in WoSClick to see citing articles inrecords_button

qr_code

  • mendeley

    citeulike


rss_1.0 rss_2.0 atom_1.0