Characterization of sphinganine kinase activity in corn shoot microsomes.
The activity of sphinganine kinase, the enzyme catalyzing the first step in the breakdown of the sphingoid long-chain base sphinganine by converting it to sphinganine 1-phosphate, was characterized in microsomes isolated from corn shoots. Activity was assayed by monitoring the conversion of [3H]sphinganine to [3H]sphinganine 1-phosphate, which was recovered in the aqueous phase following lipid extraction. Sphinganine kinase was found to utilize D-erythro-sphinganine and ATP as substrates. Maximum product formation required the presence of Mg2+. The apparent Km for ATP was 0.81 mM. GTP also served as a source of phosphate, whereas CTP and UTP were not effective substrates in this assay. Maximum product formation was observed at sphinganine concentrations of approximately 100 microM. Results of competition experiments suggested that the enzyme could also phosphorylate D-erythro-sphingosine but not DL-threo-sphinganine or D-phytosphingosine. Enzyme activity was greatest in the microsomal fraction obtained by differential centrifugation and was localized to the Golgi and endoplasmic reticulum using marker enzymes. The specific activity of the enzyme under optimal conditions was 1.08 nmol/min x mg protein, a value 25-fold higher than that reported for preparations from brain tissue. Fumonisin, a mycotoxin that disrupts sphingolipid metabolism, did not alter sphinganine kinase activity in vivo or in vitro. The results of this study demonstrate, for the first time, the presence of sphinganine kinase activity in plant tissue and suggest that the properties of the kinase from corn microsomes are distinct from those of the mammalian and protistan enzymes in some respects.
Crowther
G J
GJ
Department of Biology, Williams College, Williamstown, Massachusetts 01267, USA.
Lynch
D V
DV
D000255:Adenosine Triphosphate / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009183:Mycotoxins / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D018520:Plant Shoots / Q000201:enzymology; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
D009183:Mycotoxins; C060504:dihydrosphingosine 1-phosphate; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C022647:sphinganine kinase; D013110:Sphingosine; C005682:safingol