Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes

l h e activity of serine palmitoyltransferase (palmitoyl-coenzyme A [CoA]:i-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 microsoma1 membrane fraction from summer squash fruit (Cucurbita pepo 1. cv Early Prolific Straightneck) was assayed by monitoring the incorporation of i-r3H]Ser into the chloroform-soluble produd, 3-ketosphinganine. Addition of NADPH to the assay system resulted in the conversion of 3-ketosphinganine to sphinganine. The apparent K,,, for Ser was approximately 1.8 mM. l h e enzyme exhibited a strong preference for palmitoyl-COA, with optimal activity at a substrate concentration of 200 j ~ . Pyridoxal 5'phosphate was required as a coenzyme. l h e pH optimum was 7.6, and the temperature optimum was 36 to 40'C. Enzyme activity was greatest in the microsomal fraction obtained by differential centrifugation and was localized to the endoplasmic reticulum using marker enzymes. l w o known mechanism-based inhibitors of the mammalian enzyme, L-cycloserine and 8-chloro-L-alanine, were effective inhibitors of enzyme activity in squash microsomes. Changes in enzyme activity with size (age) of squash fruit were observed. l he 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. l h e specific activity of the enzyme in squash microsomes ranged from 0.57 to 0.84 nmol min-' mg-' of protein, values 2to 20-fold higher than those previously reported for preparations from animal tissues.