CMP-N-acetylneuraminic acid: isolation from and penetration into mouse liver microsomes

Abstract

We present three lines of evidence for the existence of an intramicrosomal pool of CMP-NeuNAc in liver slices incubated with 3H-NeuNAc: first, enrichment in the microsomal fraction over the cell homogenate of CMP-3H-NeuNAc relative to CMP-14C-NeuNAc, the latter added as marker for sugar nucleotide trapped between and adsorbed onto vesicles; second, removal from microsomes by isotonic sucrose washing of only half of the CMP-3H-NeuNAc synthesized in vivo under conditions which remove all the CMP-14C-NeuNAc; and third, complete loss of both sugar nucleotides upon treatment of microsomes by hypotonic shock or with low concentrations of detergents. Incubation of CMP-NeuNAc with microsomes in vitro showed that the intact sugar nucleotide was penetrating the vesicles. When the sugar nucleotide is labeled in the sugar moiety, microsomal vesicles accumulate soluble radiolabeled products. This accumulation was saturable and dependent on time, temperature and concentration of sugar nucleotide, and was also inhibited by substrate analogues and by pretreatment of microsomes with proteases. Together, these results provide strong evidence for the existence of a specific transport system for CMP-NeuNAc in microsomes.