REGULATION OF BIOSYNTHESIS OF N-GLYCOLYLNEURAMINIC ACID-CONTAINING GLYCOCONJUGATES - CHARACTERIZATION OF FACTORS REQUIRED FOR NADH-DEPENDENT CYTIDINE 5'MONOPHOSPHATE-N-ACETYLNEURAMINIC ACID HYDROXYLATION

The hydroxylation of CMP-NeuAc has been demonstrated to be carried out by several factors including the soluble form of cytochrome b5. In th e present study, mouse liver cytosol was subjected to ammonium sulfate fractionation and cellulose phosphate column chromatography for the s eparation of two other essential fractions participating in the hydrox ylation. One of the fractions, which bound to a cellulose phosphate co lumn, was able to reduce the soluble cytochrome b5, using NADH as an e lectron donor. The other fraction, which flowed through the column, wa s assumed to contain the terminal enzyme which accepts electrons from cytochrome b5, activates oxygen, and catalyses the hydroxylation of CM P-NeuAc. Assay conditions for the quantitative determination of the te rminal enzyme were established, and the activity of the enzyme in seve ral tissues of mouse and rat was measured. The level of the terminal e nzyme activity is associated with the expression of N-glycolylneuramin ic acid in these tissues, indicating that the expression of the termin al enzyme possibly regulates the overall velocity of CMP-NeuAc hydroxy lation.