Lemkin M C, Farquhar M G
Proc Natl Acad Sci U S A. 1981 Mar;78(3):1726-30. doi: 10.1073/pnas.78.3.1726.
The biosynthesis of glycosaminoglycans (GAG) and glycopeptides was studied in rat kidney cortex, glomeruli, and isolated glomerular basement membranes (GBM). Rats were given four intraperitoneal injections of [(35)S]sulfate and [(3)H]glucosamine (over 10 hr) and sacrificed 14 hr after the last injection. Fractions of kidney glomeruli and purified GBM were prepared. The percent of the label incorporated into specific GAG or into glycopeptides was determined by selective degradative techniques in conjunction with gel filtration chromatography using the methods of Hart [Hart, G. W. (1976) J. Biol. Chem. 251, 6513-6521; Hart, G. W. (1978) Dev. Biol. 62, 78-98]. After digestion with Pronase and chromatography on Sephadex G-50, approximately 68% of the total (35)S radioactivity and 10-15% of the total (3)H radioactivity incorporated into cortex, glomeruli, or GBM was found in the GAG fraction, and the remainder ( approximately 32% of (35)S radioactivity and 85-90% of the (3)H radioactivity) was found in glycopeptide fractions. Treatment of GAG fractions isolated from the three sources (cortex, glomeruli, and GBM) with nitrous acid (which degrades heparan sulfates) indicated that the majority (85%, 65%, and 87%) of the (35)S radioactivity as well as the majority (60%, 50%, and 91%) of the (3)H radioactivity from all three sources was degraded by this treatment. When nitrous acid-resistant GAG from GBM were subjected to digestion with Streptomyces hyaluronidase (which degrades hyaluronic acid), approximately 6% of the (3)H-labeled material was sensitive to this treatment. The remaining (35)S- and (3)H-labeled GAG isolated from GBM were digested with chondroitinase ABC (which degrades chondroitin sulfates A and C and dermatan sulfate). Although the ratios of the types of GAG synthesized by all three sources were similar, in GBM the ratios of (35)S- to (3)H-labeled GAG and of (3)H-labeled glycopeptides to (3)H-labeled GAG were higher (2.5 times) than those found for glomeruli. The data demonstrate the synthesis of both sulfated and nonsulfated GAG by rat kidney cortex and glomeruli and their transport to and incorporation into the GBM. Heparan sulfate is the major GAG synthesized by glomeruli, but the glomeruli also synthesize smaller amounts of hyaluronic acid and chondroitin sulfates, which are in part incorporated into GBM. In addition, the renal cortex and the glomeruli synthesize glycopeptides, some of which are sulfated, and incorporate them into GBM.
在大鼠肾皮质、肾小球及分离出的肾小球基底膜（GBM）中研究了糖胺聚糖（GAG）和糖肽的生物合成。给大鼠腹腔内注射四次[³⁵S]硫酸盐和[³H]葡萄糖胺（历时10小时），并在末次注射后14小时处死大鼠。制备肾小球部分和纯化的GBM。采用Hart [Hart, G. W. (1976) J. Biol. Chem. 251, 6513 - 6521; Hart, G. W. (1978) Dev. Biol. 62, 78 - 98] 的方法，通过选择性降解技术结合凝胶过滤色谱法测定掺入特定GAG或糖肽中的标记物百分比。用链霉蛋白酶消化并在Sephadex G - 50上进行色谱分析后，发现在掺入皮质、肾小球或GBM的总³⁵S放射性中约68%以及总³H放射性中10 - 15%存在于GAG部分，其余部分（约32%的³⁵S放射性和85 - 90%的³H放射性）存在于糖肽部分。用亚硝酸（降解硫酸乙酰肝素）处理从三个来源（皮质、肾小球和GBM）分离出的GAG部分表明，来自所有三个来源的³⁵S放射性的大部分（85%、65%和87%）以及³H放射性的大部分（60%、50%和91%）经此处理后被降解。当用透明质酸酶（降解透明质酸）消化GBM中耐亚硝酸的GAG时，约6%的³H标记物质对此处理敏感。从GBM分离出的其余³⁵S和³H标记的GAG用软骨素酶ABC（降解硫酸软骨素A和C以及硫酸皮肤素）消化。尽管所有三个来源合成的GAG类型比例相似，但在GBM中，³⁵S标记的GAG与³H标记的GAG之比以及³H标记的糖肽与³H标记的GAG之比比在肾小球中高（2.5倍）。数据表明大鼠肾皮质和肾小球合成了硫酸化和非硫酸化的GAG，并将它们转运至GBM并掺入其中。硫酸乙酰肝素是肾小球合成的主要GAG，但肾小球也合成少量透明质酸和硫酸软骨素，它们部分掺入GBM。此外，肾皮质和肾小球合成糖肽，其中一些是硫酸化的，并将它们掺入GBM。
