NUCLEOTIDE-BINDING TO AUTOTAXIN - CROSS-LINKING OF BOUND SUBSTRATE FOLLOWED BY LYSC DIGESTION IDENTIFIES 2 LABELED PEPTIDES

Autotaxin (ATX) is a 125 kDa glycoprotein motility factor and exoenzym e which can catalyze the hydrolysis of either the alpha-beta or at the beta-gamma phosphodiester bond in ATP. Its motility stimulating activ ity requires an intact 5'-nucleotide phosphodiesterase (PDE) active si te, Photolysis-dependent labeling of ATX with alpha[P-32]-8-N-3-ATP, l ysC digestion, and peptide HPLC resolved two radioactive fractions con taining single peptides whose amino-terminal sequences were determined . Peptide A (T(210)FPNLYTLATG...) was derived from the PDE active site and peptide B (Y(318)GPFGPEMTNP...) was not previously known to be in volved in any of the activities of ATX. The differential effect of NaC l concentration on the labeling of these two peptides, as well as on t he two reaction types catalyzed by ATX, allows a classification of act ivities which predicts both the position of preferential peptide label ing by bound ATP and also the position of phosphodiester bond hydrolys is. (C) 1997 Academic Press.