Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody.
Vasoactive intestinal peptide (VIP) labeled with 125I, [Tyr10-125I]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 of the 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 X 10(-9) M) and the turnover number kcat (15.6 min-1) suggested relatively tight VIP binding and a moderate catalytic efficiency of the antibody.
Paul
S
S
Department of Pharmacology, University of Nebraska Medical Center, Omaha 68105.
Volle
D J
DJ
Beach
C M
CM
Johnson
D R
DR
Powell
M J
MJ
Massey
R J
RJ
D000595:Amino Acid Sequence; D001323:Autoantibodies; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006868:Hydrolysis; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D014660:Vasoactive Intestinal Peptide / Q000276:immunology*
D001323:Autoantibodies; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D010446:Peptide Fragments; D014660:Vasoactive Intestinal Peptide