Photoaffinity labeling of cottonseed microsomal N-acylphosphatidylethanolamine synthase protein with a substrate analogue, 12-[(4-azidosalicyl)amino]dodecanoic acid.
N-Acylphosphatidylethanolamine (NAPE), an unusual acylated derivative of phosphatidylethanolamine (PE), is synthesized from free fatty acids and PE in cotton seedlings (Chapman and Moore (1993) Plant Physiol. 102(3), 761-769). Here we use a photoreactive dodecanoic acid analogue, [12-(4-azidosalicy)amino]dodecanoic acid (ASD), and its 125I-labeled derivative to identify a protein subunit which corresponds to this cottonseed NAPE synthase activity. Dodecylmaltoside (DDM)-solubilized microsomal NAPE synthase enzyme was irreversibly and progressively inactivated by adding increasing concentrations of ASD and illuminating with UV254 light. Protection from this photoinactivation was afforded by the natural substrate, palmitic acid. In low light, microsomal NAPE synthase utilized ASD as a substrate to synthesize NAPE; palmitic acid competed for this activity. NAPE synthase activity was measured directly in gel slices following nondenaturing PAGE of DDM-solubilized microsomal membrane proteins. Two-dimensional electrophoresis (nondenaturing PAGE, followed by SDS-PAGE) of photoaffinity-labeled, DDM-solubilized microsomal proteins revealed a 64 kDa polypeptide that was associated with the active NAPE synthase enzyme. Also, a 64 kDa protein was photoaffinity labeled in all NAPE synthase isozyme fractions isolated by preparative isoelectric focusing; photoaffinity labeling of this 64 kDa polypeptide was diminished in the presence of exogenously supplied palmitic acid. Collectively, our results demonstrate that ASD specifically interacts with NAPE synthase in a manner analogous to its fatty acid substrate and indicate that a 64 kDa polypeptide is a component of cottonseed microsomal NAPE synthase. ASD will be a useful molecular probe in future studies aimed at understanding the physiological role of this NAPE synthase enzyme in membranes of plant cells.
McAndrew
R S
RS
University of North Texas, Department of Biological Sciences, Denton 76203-0218, USA.
Leonard
B P
BP
Chapman
K D
KD
D000217:Acyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D000345:Affinity Labels; D001386:Azides / Q000737:chemistry*; D003902:Detergents; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D003368:Gossypium / Q000201:enzymology*; D007457:Iodine Radioisotopes; D007850:Lauric Acids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008861:Microsomes / Q000201:enzymology; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D012639:Seeds / Q000201:enzymology
C094278:12-((4-azidosalicyl)amino)dodecanoic acid; D000345:Affinity Labels; D001386:Azides; D003902:Detergents; D007457:Iodine Radioisotopes; D007850:Lauric Acids; D008565:Membrane Proteins; D010169:Palmitic Acids; D010940:Plant Proteins; D019308:Palmitic Acid; D000217:Acyltransferases; C042139:phosphatidylethanolamine N-acyltransferase