Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody

Sudhir Paul, Deanna J. Volle, Carol M. Beach, Donald R. Johnson, Michael J. Powell, Richard J. Massey

Research output: Contribution to journalArticle

353 Scopus citations

Abstract

Vasoactive intestinal peptide (VIP) labeled with 125I, [Tyr l0_125I]VIP, can be hydrolyzed by immunoglobulin G (IgG) purified from a human subject, as judged by trichloroacetic acid precipitation and reversed-phase high-performance liquid chromatography (HPLC). The hydrolytic activity was precipitated by antibody to human IgG, it was bound by immobilized protein G and showed a molecular mass close to 150 kilodaltons by gel filtration chromatography, properties similar to those of authentic IgG. The Fab fragment, prepared from IgG by papain treatment, retained the VIP hydrolytic activity ofthe IgG. Peptide fragments produced by treatment of VIP with the antibody fraction were purified by reversed-phase HPLC and identified by fast atom bombardment-mass spectrometry and peptide sequencing. The scissile bond in VIP deduced from these experiments was Gln16-Met17. The antibody concentration (73.4 fmol per milligram of IgG) and the Kd (0.4 nM) were computed from analysis of VIP binding under conditions that did not result in peptide hydrolysis. Analysis of the antibody-mediated VIP hydrolysis at varying concentrations of substrate suggested conformity with Michaelis-Menton kinetics (Km). The values for Km (37.9 × 10-9M) and the turnover number kcat (15.6 min -1) suggested relatively tight VIP binding and a moderate catalytic efficiency of the antibody.

Original languageEnglish (US)
Pages (from-to)1158-1162
Number of pages5
JournalScience
Volume244
Issue number4909
DOIs
StatePublished - Jan 1 1989

    Fingerprint

ASJC Scopus subject areas

  • General

Cite this

Paul, S., Volle, D. J., Beach, C. M., Johnson, D. R., Powell, M. J., & Massey, R. J. (1989). Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody. Science, 244(4909), 1158-1162. https://doi.org/10.1126/science.2727702