Kawano T, Kozutsumi Y, Takematsu H, Kawasaki T, Suzuki A
Department of Membrane Biochemistry, Tokyo Metropolitan Institute of Medical Science, Japan.
Glycoconj J. 1993 Feb;10(1):109-15. doi: 10.1007/BF00731194.
The hydroxylation of CMP-NeuAc has been demonstrated to be carried out by several factors including the soluble form of cytochrome b5. In the present study, mouse liver cytosol was subjected to ammonium sulfate fractionation and cellulose phosphate column chromatography for the separation of two other essential fractions participating in the hydroxylation. One of the fractions, which bound to a cellulose phosphate column, was able to reduce the soluble cytochrome b5, using NADH as an electron donor. The other fraction, which flowed through the column, was assumed to contain the terminal enzyme which accepts electrons from cytochrome b5, activates oxygen, and catalyses the hydroxylation of CMP-NeuAc. Assay conditions for the quantitative determination of the terminal enzyme were established, and the activity of the enzyme in several tissues of mouse and rat was measured. The level of the terminal enzyme activity is associated with the expression of N-glycolylneuraminic acid in these tissues, indicating that the expression of the terminal enzyme possibly regulates the overall velocity of CMP-NeuAc hydroxylation.
CMP-唾液酸的羟基化已被证明可由多种因素完成，包括细胞色素b5的可溶性形式。在本研究中，对小鼠肝脏胞质溶胶进行硫酸铵分级分离和磷酸纤维素柱色谱，以分离参与羟基化的其他两种必需组分。其中一个与磷酸纤维素柱结合的组分，能够以NADH作为电子供体还原可溶性细胞色素b5。另一个流经柱子的组分，被认为含有从细胞色素b5接受电子、激活氧气并催化CMP-唾液酸羟基化的末端酶。建立了定量测定末端酶的分析条件，并测定了该酶在小鼠和大鼠几种组织中的活性。末端酶活性水平与这些组织中N-羟乙酰神经氨酸的表达相关，表明末端酶的表达可能调节CMP-唾液酸羟基化的整体速度。
