Nucleotide binding to autotaxin: crosslinking of bound substrate followed by lysC digestion identifies two labeled peptides.
Autotaxin (ATX) is a 125 kDa glycoprotein motility factor and exoenzyme which can catalyze the hydrolysis of either the alpha-beta or at the beta-gamma phosphodiester bond in ATP. Its motility stimulating activity requires an intact 5'-nucleotide phosphodiesterase (PDE) active site. Photolysis-dependent labeling of ATX with alpha-[32P]-8-N3-ATP, lysC digestion, and peptide HPLC resolved two radioactive fractions containing single peptides whose amino-terminal sequences were determined. Peptide A (T210FPNLYTLATG. . .) was derived from the PDE active site and peptide B (Y318GPFGPEMTNP. . .) was not previously known to be involved in any of the activities of ATX. The differential effect of NaCl concentration on the labeling of these two peptides, as well as on the two reaction types catalyzed by ATX, allows a classification of activities which predicts both the position of preferential peptide labeling by bound ATP and also the position of phosphodiester bond hydrolysis.
Clair
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T
Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Krutzsch
H C
HC
Liotta
L A
LA
Stracke
M L
ML
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D005956:Glucose-6-Phosphate Isomerase / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D010449:Peptide Mapping; D043264:Phosphodiesterase I; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D011994:Recombinant Proteins; D012965:Sodium Chloride / Q000494:pharmacology; D000099189:Lysophospholipase D
D000255:Adenosine Triphosphate; D000345:Affinity Labels; D005956:Glucose-6-Phosphate Isomerase; D006023:Glycoproteins; D008666:Metalloendopeptidases; D009097:Multienzyme Complexes; D043264:Phosphodiesterase I; D010726:Phosphodiesterase Inhibitors; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D011994:Recombinant Proteins; D012965:Sodium Chloride; D000099189:Lysophospholipase D; C039830:peptidyl-Lys metalloendopeptidase