Mason R M, Sweeney C
Department of Biochemistry, Charing Cross and Westminster Medical School, London, United Kingdom.
Carbohydr Res. 1994 Mar 4;255:255-70. doi: 10.1016/s0008-6215(00)90983-2.
The effect of anaerobic culture conditions and various metabolic inhibitors on 35S-proteoglycan synthesis, UDP-sugar pools, and the ATP pool were investigated in confluent, primary, day 1 cultures of Swarm chondrosarcoma chondrocytes. (i) Incubation under a nitrogen atmosphere for 6 h did not affect 35S-proteoglycan synthesis or the pool size for UDP-glucuronate, other UDP-sugars, or ATP. Incubation with 5 mM sodium azide brought about a 40% reduction of proteoglycan synthesis in the first 30 min but no further change over the subsequent 90 min. UDP-Glucuronate, other UDP-sugar pools, and the ATP level were not affected by azide treatment. The results indicate that proteoglycan synthesis and its energy requirements can be supported entirely by anaerobic metabolism in these cells. (ii) 35S-Proteoglycan synthesis, UDP-sugar production, and nucleotide triphosphate pools were inhibited in a concentration-dependent fashion with sodium iodoacetate. A > 70% reduction of the ATP pool after 30 min treatment suggests that glycolysis is a major target for iodoacetate. Lactate production was inhibited by 40% after 3 h treatment with 10(-4) M iodoacetate. (iii) Glutamine deprivation resulted in a 60% contraction in the UDP-N-acetylhexosamine pool and markedly inhibited 35S-proteoglycan and 3H-protein synthesis. At the same time the UDP-glucose pool expanded to 200%, but the UDP-glucuronate pool was unchanged. The sum of the UDP-N-acetylhexosamine and UDP-hexose pools remained constant. Restoration of glutamine to previously depleted cultures resulted in excessive expansion of the UDP-N-acetylhexosamine pool and excessive contraction of the UDP-hexose pool before both adjusted to normal levels. The UDP-xylose pool was very small. No increases were observed during inhibition of proteoglycan synthesis induced by glutamine deprivation. (iv) 6-Diazo-5-oxo-L-norleucine (DON), a glutamine analogue and amino transferase inhibitor, induced a further contraction of the UDP-N-acetylhexosamine pool and a further decrease in proteoglycan synthesis in glutamine-deprived cultures. Thus cultures use endogenous glutamine during exogenous glutamine deprivation. DON unaccountably stimulated expansion of the UDP-glucuronate pool by 180%, irrespective of whether glutamine was present or not.
在融合的第1天原代群体软骨肉瘤软骨细胞培养物中，研究了厌氧培养条件和各种代谢抑制剂对35S-蛋白聚糖合成、UDP-糖库和ATP库的影响。(i) 在氮气气氛下孵育6小时不影响35S-蛋白聚糖合成或UDP-葡萄糖醛酸、其他UDP-糖或ATP的库大小。用5 mM叠氮化钠孵育在最初30分钟内使蛋白聚糖合成减少40%，但在随后的90分钟内没有进一步变化。叠氮化钠处理不影响UDP-葡萄糖醛酸、其他UDP-糖库和ATP水平。结果表明，这些细胞中的蛋白聚糖合成及其能量需求可以完全由无氧代谢支持。(ii) 碘乙酸钠以浓度依赖的方式抑制35S-蛋白聚糖合成、UDP-糖产生和三磷酸核苷酸库。处理30分钟后ATP库减少>70%，表明糖酵解是碘乙酸钠的主要作用靶点。用10(-4)M碘乙酸钠处理3小时后，乳酸产生受到40%的抑制。(iii) 谷氨酰胺缺乏导致UDP-N-乙酰己糖胺库收缩60%，并显著抑制35S-蛋白聚糖和3H-蛋白质合成。同时，UDP-葡萄糖库扩大到200%，但UDP-葡萄糖醛酸库不变。UDP-N-乙酰己糖胺和UDP-己糖库的总和保持不变。将谷氨酰胺恢复到先前耗尽的培养物中导致UDP-N-乙酰己糖胺库过度扩张和UDP-己糖库过度收缩，然后两者都调整到正常水平。UDP-木糖库非常小。在谷氨酰胺缺乏诱导的蛋白聚糖合成抑制期间未观察到增加。(iv) 6-重氮-5-氧代-L-正亮氨酸（DON），一种谷氨酰胺类似物和氨基转移酶抑制剂，在谷氨酰胺缺乏的培养物中诱导UDP-N-乙酰己糖胺库进一步收缩和蛋白聚糖合成进一步减少。因此，培养物在缺乏外源性谷氨酰胺期间使用内源性谷氨酰胺。无论是否存在谷氨酰胺，DON都意外地刺激UDP-葡萄糖醛酸库扩张180%。
