PLANT PHYSIOLOGY , Vol 107, Issue 1 155-160, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase
J. M. Shockey, R. Rajasekharan and J. D. Kemp
Plant Genetic Engineering Laboratory, New Mexico State University, Las Cruces, New Mexico 88003
Jojoba (Simmondsia chinensis, Link) is the only plant known that
synthesizes liquid wax. The final step in liquid wax biosynthesis is
catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty
alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a
fatty alcohol to form the wax ester. To purify the acyltransferase, we have
labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA,
12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts
as an inhibitor of acyltransferase activity in the dark and as an
irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA
protects enzymatic activity in a concentration-dependent manner. Photolysis
of microsomal membranes with labeled ASD-CoA resulted in strong labeling of
two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA
reduced the labeling of the 57-kD polypeptide dramatically, whereas the
labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA.
Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide
was enhanced considerably when photolyzed in the presence of dodecanol.
These results suggest that a 57-kD polypeptide from jojoba microsomes may
be the acyl-CoA:fatty alcohol acyltransferase.
