Lidholt K, Riesenfeld J, Jacobsson K G, Feingold D S, Lindahl U
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala.
Biochem J. 1988 Sep 1;254(2):571-8. doi: 10.1042/bj2540571.
The formation of heparin-precursor polysaccharide (N-acetylheparosan) was studied with a mouse mastocytoma microsomal fraction. Incubation of this fraction with UDP-[3H]GlcA and UDP-GlcNAc yielded labelled macromolecules that could be depolymerized, apparently to single polysaccharide chains, by alkali treatment, and thus were assumed to be proteoglycans. Label from UDP-[3H]GlcA (approx. 3 microM) is transiently incorporated into microsomal polysaccharide even in the absence of added UDP-GlcNAc, probably owing to the presence of endogenous sugar nucleotide. When the concentration of exogenous UDP-GlcNAc was increased to 25 microM the rate of incorporation of 3H increased and proteoglycans carrying polysaccharide chains with an Mr of approx. 110,000 were produced. Increasing the UDP-GlcNAc concentration to 5 mM led to an approx. 4-fold decrease in the rate of 3H incorporation and a decrease in the Mr of the resulting polysaccharide chains to approx. 6000 (predominant component). When both UDP-GlcA and UDP-GlcNAc were present at high concentrations (5 mM) the rate of polymerization and the polysaccharide chain size were again increased. The results suggest that the inhibition of polymerization observed at grossly different concentrations of the two sugar nucleotides, UDP-GlcA and UDP-GlcNAc, may be due either to interference with the transport of one of these precursors across the Golgi membrane or to competitive inhibition of one of the glycosyltransferases. The maximal rate of chain elongation obtained, under the conditions employed, was about 40 disaccharide units/min. The final length of the polysaccharide chains was directly related to the rate of the polymerization reaction.
利用小鼠肥大细胞瘤微粒体部分研究了肝素前体多糖（N-乙酰肝素）的形成。该部分与UDP-[3H]GlcA和UDP-GlcNAc一起温育，产生了标记的大分子，经碱处理后可解聚，显然解聚为单条多糖链，因此被认为是蛋白聚糖。即使在没有添加UDP-GlcNAc的情况下，UDP-[3H]GlcA（约3 microM）中的标记也会短暂掺入微粒体多糖中，这可能是由于内源性糖核苷酸的存在。当外源性UDP-GlcNAc的浓度增加到25 microM时，3H的掺入速率增加，产生了带有Mr约为110,000的多糖链的蛋白聚糖。将UDP-GlcNAc浓度增加到5 mM导致3H掺入速率约降低4倍，并且所得多糖链的Mr降低到约6000（主要成分）。当UDP-GlcA和UDP-GlcNAc都以高浓度（5 mM）存在时，聚合速率和多糖链大小再次增加。结果表明，在两种糖核苷酸UDP-GlcA和UDP-GlcNAc浓度差异很大时观察到的聚合抑制，可能是由于干扰了这些前体之一穿过高尔基体膜的转运，或者是由于对一种糖基转移酶的竞争性抑制。在所采用的条件下获得的链伸长的最大速率约为40个二糖单位/分钟。多糖链的最终长度与聚合反应的速率直接相关。
