PLANT PHYSIOLOGY , Vol 103, Issue 4 1421-1429, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes)
D. V. Lynch and S. R. Fairfield
Department of Biology, Williams College, Williamstown, Massachusetts 01267
The activity of serine palmitoyltransferase (palmitoyl-coenzyme A
[CoA]:L-serine [Ser]-C-palmitoyltransferase [decarboxylating], EC
2.3.1.50), the enzyme catalyzing the first step in the synthesis of the
long-chain base required for sphingolipid assembly, has been characterized
in a plant system. Enzyme activity in a microsomal membrane fraction from
summer squash fruit (Cucurbita pepo L. cv Early Prolific Straightneck) was
assayed by monitoring the incorporation of L-[3H]Ser into the
chloroform-soluble product, 3-ketosphinganine. Addition of NADPH to the
assay system resulted in the conversion of 3-ketosphinganine to
sphinganine. The apparent Km for Ser was approximately 1.8 mM. The enzyme
exhibited a strong preference for palmitoyl-CoA, with optimal activity at a
substrate concentration of 200 [mu]M. Pyridoxal 5[prime]-phosphate was
required as a coenzyme. The pH optimum was 7.6, and the temperature optimum
was 36 to 40[deg]C. Enzyme activity was greatest in the microsomal fraction
obtained by differential centrifugation and was localized to the
endoplasmic reticulum using marker enzymes. Two known mechanism-based
inhibitors of the mammalian enzyme, L-cycloserine and
[beta]-chloro-L-alanine, were effective inhibitors of enzyme activity in
squash microsomes. Changes in enzyme activity with size (age) of squash
fruit were observed. The results from this study suggest that the
properties and catalytic mechanism of Ser palmitoyltransferase from squash
are similar to those of the animal, fungal, and bacterial enzyme in most
respects. The specific activity of the enzyme in squash microsomes ranged
from 0.57 to 0.84 nmol min-1 mg-1 of protein, values 2- to 20-fold higher
than those previously reported for preparations from animal tissues.
