UDP-sugar metabolism in swarm rat chondrosarcoma chondrocytes

Abstract

UDP-sugars and adenine nucleotides were extracted from freshly isolated chondrocytes and primary cell cultures and analysed by anion-exchange h.p.l.c. The pool sizes of UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, UDP-glucosegalactose, UDP-glucuronate and UDP-xylose were 2.9, 1.2, 2.5, 0.6 and 0.03 nmol/106 freshly isolated chondrocytes. When chondrocytes were maintained in Dulbecco's modified Eagle medium supplemented with 15% foetal-bovine serum, synthesis of [35S]proteoglycan and [3H]protein decreased over the first 48 h in culture, as did the pools of UDP-glucuronate and ATP. In contrast, the size of the UDP-N-acetylhexosamine pools underwent little change during culture. [35S]Proteoglycan and [3H]protein syntheses were stimulated in cultures supplemented with serum or insulin compared with those maintained in medium alone, in agreement with previous results. However, the UDP-sugar pool sizes were the same in both supplemented and nonsupplemented cultures. In cultures maintained in the presence of [1-3H]glucose, the UDP-sugars were labelled to a constant 3H specific radioactivity which was very similar to that of the labelling medium. UDP-N-acetylhexosamines were labelled to constant 3H specific radioactivity with [6-3H]glucosamine as precursor, but only about 1 in 375 of these UDP-sugars was derived from the amino sugar in the presence of glucose. The half-life (t( 1/2 )) for UDP-hexoses, UDP-glucuronate and UDP-N-acetylhexosamines was about 12, 12 and 50 min respectively.