The relative contribution of N-glycoloyl-P-D-neuraminic acid (NeuSGc) to total sialic acids expressed in mouse and rat liver glycoconjugates was found to be 95% and 11%, respectively. This considerable difference in sialic acid composition made these two tissues suitable models for a comparative investigation into the regulation of NeuSGc biosynthesis and utilization.An examination of the CMP-glycoside specificity of Golgi-associated sialyltransferases using CMP-N-acetylp-D-neuraminic acid (CMP-NeuSAc) and CMP-NeuSGc revealed no significant tissue-dependent differences. The Golgi membrane CMP -sialic acid transport system from rat liver did, however, exhibit a slightly higher internalisation rate for CMP-NeuSAc, though no preferential affinity for this sugar nucleotide over CMP-NeuSGc was observed.In experiments, where Golgi membrane preparations were incubated with an equimolar mixture of labelled CMP-NeuSAc and CMP-NeuSGc, no significant tissue-dependent differences in [14C]sialic acid composition were observed, either in the luminal soluble sialic acid fraction or in the precipitable sialic acid fraction, results which are consistent with the above observations. From this experiment, evidence was also obtained for the presence of a Golgi-lumen-associated CMP -sialic acid hydrolase which exhibited no apparent specificity for either CMPNeuSAc or CMP-NeuSGc.The specific activity of the CMP-NeuSAc hydroxylase, the enzyme responsible for the biosynthesis of NeuSGc, was found to be 28-fold greater in high-speed supernatants of mouse liver than of rat liver. No hydroxylase activity was detected in the Golgi membrane preparations. It is therefore proposed that the cytoplasmic ratio of CMPNeuSAc and CMP-NeuSGc produced by the hydroxylase, remains largely unmodified after CMP-glycoside uptake into the Golgi apparatus and transfer on to growing glycoconjugate glycan chains. The close relationship between the total sialic acid composition and the sialic acid pattern in the CMP-glycoside pools of the tissues lends considerable weight to this hypothesis.

CMP-NeuSGc has been shown to be transported into mouse liver Golgi vesicles by a specific carrier the characteristics of which were investigated in detail. In the system employed, CMP-NeuSGc enters the Golgi vesicles within 2 mitt; transport was saturable with high concentrations of the sugar-nucleotide and was dependent on temperature. A kinetic analysis gave an apparent K,,, of 1.3 PM and a maximal transport velocity of 335 pmol/mg protein per min. Almost identical values were obtained with CMP-NeuSAc, under the same incubation conditions. Furthermore, the uptake of CMPNeuSGc was inhibited by CMP-NeuSAc, a substrate analogue. Conversely, the uptake of CMP-NeuSAc was inhibited by CMP-NeuSGc to the same extent, leading to the conclusion that the transport of CMP-Neu5Ac and CMP-NeuSGc is mediated by the same carrier molecule. This transport system for CMP-NeuSGc involves both CMP and CMP-NeuSGc since intravesicular CMP induced the entry of external CMP-NeuSGc.

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