Molecular cloning of a proteolytic antibody light chain

Q. S. Gao, M. Sun, S. Tyutyulkova, D. Webster, A. Rees, A. Tramontano, R. J. Massey, S. Paul

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

The cDNA for an antibody light chain raised by immunization against vasoactive intestinal peptide (VIP) was cloned in a bacterial expression vector, and the recombinant light chain was purified to electrophoretic homogeneity. The light chain catalyzed the hydrolysis of VIP efficiently owing to its comparatively high affinity for the substrate. In control experiments, the catalytic activity was preserved at a constant level after further chromatography of the light chain on anion-exchange and gel- filtration fast protein liquid chromatography columns, and it was removed by immunoadsorption with immobilized anti-mouse light chain antibody. The amide bond linking methylcoumarinamide (MCA) and arginine in a tripeptide unrelated in sequence to VIP was cleaved by the light chain with lower affinity and kinetic efficiency (k(cat)/K(m)). Hydrolysis of the peptidyl-MCA conjugate was inhibited competitively by the alternate substrate, VIP. The K(i) and K(m) values for VIP were in the same range, indicating that peptide-MCA and VIP hydrolysis occurs at a common catalytic site in the light chain. Molecular modeling suggested the presence of a serine protease-like site in the light chain. This was supported by inhibition of the hydrolytic activity by serine protease inhibitors, but not by inhibitors of other classes of proteases. These observations suggest a poorly discriminatory catalytic site, with specificity for VIP arising chiefly by means of the antigen recognition function of the light chain combining site.

Original languageEnglish (US)
Pages (from-to)32389-32393
Number of pages5
JournalJournal of Biological Chemistry
Volume269
Issue number51
StatePublished - 1994
Externally publishedYes

Fingerprint

Cloning
Molecular Cloning
Vasoactive Intestinal Peptide
Light
Antibodies
Hydrolysis
Catalytic Domain
Immunization
Serine Proteinase Inhibitors
Molecular modeling
Liquid chromatography
Serine Proteases
Substrates
Chromatography
Amides
Liquid Chromatography
Gel Chromatography
Anions
Arginine
Catalyst activity

ASJC Scopus subject areas

  • Biochemistry

Cite this

Gao, Q. S., Sun, M., Tyutyulkova, S., Webster, D., Rees, A., Tramontano, A., ... Paul, S. (1994). Molecular cloning of a proteolytic antibody light chain. Journal of Biological Chemistry, 269(51), 32389-32393.

Molecular cloning of a proteolytic antibody light chain. / Gao, Q. S.; Sun, M.; Tyutyulkova, S.; Webster, D.; Rees, A.; Tramontano, A.; Massey, R. J.; Paul, S.

In: Journal of Biological Chemistry, Vol. 269, No. 51, 1994, p. 32389-32393.

Research output: Contribution to journalArticle

Gao, QS, Sun, M, Tyutyulkova, S, Webster, D, Rees, A, Tramontano, A, Massey, RJ & Paul, S 1994, 'Molecular cloning of a proteolytic antibody light chain', Journal of Biological Chemistry, vol. 269, no. 51, pp. 32389-32393.
Gao QS, Sun M, Tyutyulkova S, Webster D, Rees A, Tramontano A et al. Molecular cloning of a proteolytic antibody light chain. Journal of Biological Chemistry. 1994;269(51):32389-32393.
Gao, Q. S. ; Sun, M. ; Tyutyulkova, S. ; Webster, D. ; Rees, A. ; Tramontano, A. ; Massey, R. J. ; Paul, S. / Molecular cloning of a proteolytic antibody light chain. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 51. pp. 32389-32393.
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