In this paper, we present the results of an investigation into the catalytic properties of CMPNeuSAc hydroxylase (NeuSAc: N-acetylneuraminic acid) in high-speed supernatants of mouse liver. The enzyme was most active in Hepes/NaOH pH 7.4 and was markedly inhibited by relatively small increases in ionic strength, though the inhibition was abolished by desalting procedures. Several nonionic detergents could activate the hydroxylase to various degrees in a concentration-dependent manner. Ionic detergents and a number of phospholipids were, however, generally inert or inhibitory.The lack of inhibitory influence of a wide range of nucleotides revealed that CMP-NeuSAc hydroxylase binds its sugar-nucleotide substrate with a high degree of specificity. Thus, even millimolar concentrations of several cytidine nucleotides elicited virtually negligible inhibition, though the reaction product, CMP-NeuSGc (Neu5Gc: N-glycoloylneuraminic acid), was a weak inhibitor. The results also indicate that the enzyme is not regulated by any nucleotides or sugar -nucleotides.Dilution of high-speed supernatants with buffer gave rise to a decrease in the specific activity of the hydroxylase, implicating the involvement of more than one component in catalysis. Activity could be restored by the addition of a heat extract of the supernatant. The active principle in this extract was found to be a heat-stable protein with a molecular mass of about 17 kDa. Immunochemical studies allowed this protein to be identified as cytochrome bs and it was shown that this electron carrier is essential for the activity of CMP-Neu5Ac hydroxylase.Inhibition studies using iron ligands and activation by exogenous iron salts suggest the involvement of a non-haem iron cofactor in the catalytic cycle of this hydroxylase. Cytochrome bs may thus serve as an electron donor for this postulated cofactor.
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.
In this report, the nature of the protein components involved in the functioning of cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-NeuSAc) hydroxylase in high-speed supernatants of mouse liver has been investigated. Fractionation and reconstitution experiments showed that this enzyme system consists of NADH-cytochrome b, reductase, cytochrome b, and a 56-kDa terminal electron acceptor having the CMP-Neu5 Ac hydroxylase activity. This enzyme system is extracted in a soluble protein fraction ; however, the amphipathic, usually membrane-associated, forms of cytochrome b, and the reductase were found to predominate and are presumably the forms which support the turnover of the hydroxylase in vivo. Although the majority of cellular cytochrome b, and cytochrome b, reductase is membrane-bound, the addition of intact microsomes elicited no significant increase in the hydroxylase activity of supernatants. Detergent-solubilised microsomes, however, potently activated the hydroxylase, probably due to the greater accessibility of the cytochrome b,. Accordingly, in reconstitution experiments, pure hydrophilic cytochrome b, interacts more effectively with the hydroxylase than isolated amphipathic cytochrome b,.Studies on the CMP-Neu5 Ac hydroxylase system in fractionated porcine submandibular glands and bovine liver suggest that the composition of this enzyme system is conserved in all mammals possessing sialoglycoconjugates containing N-glycoloylneuraminic acid.The sialic acid N-glycoloylneuraminic acid (NeuSGc) occurs in sialoglycoconjugates of most animal groups throughout the deuterostomate lineage [l, 21. The level of sialylation with Neu5Gc is highly characteristic for each tissue of a particular organism, and can be subject to further variation, depending on age and diseased state. Humans and chickens are notable, since Neu5Gc is completely absent from normal tissues and has only been detected in small amounts in cancerous tissues [3, 41. Neu5Gc is synthesised as its CMP-glycoside by the action of a hydroxylase specific for the sugar nucleotide cytidine-5'-monophosphate-N-acetylneuraminic acid (CMPNeuSAc) [S, 61. Several metabolic studies suggest that the rate of CMP-Neu5Gc production by CMP-Neu5 Ac hydroxylase is important in regulating the extent of sialylation with NeuSGc [7-101. Investigations into the properties of CMP-Neu5 Ac hydroxylase from pig [5, 111, mouse [6, 12, 131 and starfish [14] reveal that the enzyme is an NADH-dependent, cytoCorrespondence to
N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-glycoside by the action of CMP-N-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase. This enzyme is a soluble cytochrome b5-dependent monooxygenase and has been purified to apparent homogeneity from pig submandibular glands by precipitation with N-cetyl-N,N,N-trimethylammonium bromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose, Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12. This procedure resulted in an 8960-fold purification of the hydroxylase with a recovery of 0.8%. The molecular mass of this protein was shown to be 65 kDa on SDS-PAGE and approximately 60 kDa as determined by gel filtration on Superose S.12, which suggests that the enzyme is a monomer. The purified CMP-Neu5Ac hydroxylase is activated by FeSO4 and inhibited by iron-binding reagents such as o-phenanthroline, KCN, Tiron and ferrozine. An apparent Km of 11 microM was determined for the substrate CMP-Neu5Ac using purified hydroxylase in the presence of Triton X-100-solubilized microsomes. In a reconstituted system consisting of purified hydroxylase, cytochrome b5, cytochrome b5 reductase and catalase, an apparent Km of 3 microM was measured. The apparent Km for cytochrome b5 in this system was 0.24 microM. Immunization of a rabbit with enriched and purified hydroxylase led to an antiserum that inhibited CMP-Neu5Ac hydroxylase activity and reacted with the purified 65 kDa protein on a Western blot after SDS-PAGE. Antibodies specific for this 65 kDa protein were isolated and showed a strong reaction with the purified CMP-Neu5Ac hydroxylase from mouse liver after immunoblotting.(ABSTRACT TRUNCATED AT 250 WORDS)
CMP-N-acetylneuraminate hydroxylase was isolated from mouse liver high speed supernatant with a yield of 0.4% and an apparent 1000-fold purification. The enzyme is a monomeric protein with a molecular weight of 66 kDa, as determined by gel filtration and SDS-PAGE. The hydroxylase system was reconstituted with Triton X-100-solubilized mouse liver microsomes and purified soluble or microsomal forms of cytochrome b5 reductase and cytochrome b5. The systems were characterized in detail and kinetic parameters for each system were determined.
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