小鼠肿瘤性肥大细胞中大分子肝素的代谢

Metabolism of macromolecular heparin in mouse neoplastic mast cells.

作者信息

Ogren S, Lindahl U

出版信息

Biochem J. 1976 Mar 15;154(3):605-11. doi: 10.1042/bj1540605.

DOI:10.1042/bj1540605

PMID:821471

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1172761/

Abstract

Polysaccharide in a heparin-producing mouse mastocytoma was pulse-labelled in vivo with [35S] sulphate, and after various periods of time was isolated from subcellular fractions. Such fractions were recovered from tissue homogenates by consecutive centrifugations at 1000g for 10min, 20000g for 20min and 100000g for 1h. Initially the 35S-labelled polysaccharide formed occurred principally in the second centrifugal fraction (20000g precipitate), with small amounts in the first (granular) and third (microsomal) fractions. Analysis for glycosyltransferase activity confirmed that glycosaminoglycans were formed chiefly in particles sedimenting at 20000g. Molecules of this newly synthesized polysaccharide were considerably larger than those of commercially available heparin, as judged from gel chromatography. 2. Within the first hour after injection of [35S]sulphate, most of the labelled polysaccharide was redistributed from the second to the first centrifugal fraction. During, and possibly also after, this shift, the macromolecular polysaccharide was degraded, ultimately to the size of commercial heparin. The degradation process appeared complete 6h after injection of [35S]sulphate. 3. Particulate subcellular fractions were incubated with macromolecular [35S]heparin and the products were analysed by gel chromatography. Significant degradation of the substrate occurred only with the second centrifugal fraction. Further characterization of this fraction, by density-gradient centrifugation in iso-osmotic colloidal silica, revealed a single visible band of particles, at approximately the same density at lysosomes. This band contained all the beta-glucuronidase, 35S-labelled endogenous polysacchride and heparin-degrading enzyme present in the second fraction.

摘要

用[35S]硫酸盐对产生肝素的小鼠肥大细胞瘤中的多糖进行体内脉冲标记，在不同时间段后从亚细胞组分中分离出来。这些组分通过在1000g下离心10分钟、在20000g下离心20分钟和在100000g下离心1小时，从组织匀浆中回收。最初形成的35S标记多糖主要出现在第二个离心组分（20000g沉淀物）中，在第一个（颗粒）和第三个（微粒体）组分中含量较少。糖基转移酶活性分析证实，糖胺聚糖主要在以20000g沉降的颗粒中形成。从凝胶色谱判断，这种新合成的多糖分子比市售肝素的分子大得多。2. 在注射[35S]硫酸盐后的第一小时内，大部分标记多糖从第二个离心组分重新分布到第一个离心组分。在这种转移期间以及可能之后，大分子多糖被降解，最终降解到市售肝素的大小。在注射[35S]硫酸盐6小时后，降解过程似乎完成。3. 将亚细胞颗粒组分与大分子[35S]肝素一起孵育，并通过凝胶色谱分析产物。仅第二个离心组分使底物发生了显著降解。通过在等渗胶体二氧化硅中进行密度梯度离心对该组分进行进一步表征，发现了一条单一的可见颗粒带，其密度与溶酶体的密度大致相同。该带包含第二个组分中存在的所有β-葡萄糖醛酸酶、35S标记的内源性多糖和肝素降解酶。