CYSTINE CYSTEINE METABOLISM IN CULTURED SF9 CELLS - INFLUENCE OF CELLPHYSIOLOGY ON BIOSYNTHESIS, AMINO-ACID-UPTAKE AND GROWTH/

Spodoptera frugiperda (Sf9) insect cells proliferate in a cystine-free medium, with the same growth rate, reaching the same final cell densi ty, as in a cystine-containing medium, provided that the inoculum is t aken from a pre-culture sufficiently early, at 47-53 h. With an inocul um from a 103 h culture an extended lag phase accompanied by cell deat h was observed during the first 50 h of cystine-free culture, even tho ugh the culture had been adapted to cystine-free conditions for 10 pas sages. Cystine-free cultures seeded with a 103 h inoculum had lower gr owth rates and reached lower final cell densities than corresponding c ystine-supplied cultures. Cysteine biosynthesis occurs from methionine via the beta-cystathionine pathway. More methionine was consumed by t he cells in cystine-free media, and cystathionine was secreted when me thionine and cystine were supplied in excess. The data suggest that cy steine biosynthesis is up-regulated in proliferating cells but down-re gulated when the cells enter the stationary phase. In cultures supplie d with cystine (10-100 mg l(-1)), the specific uptake rate and total c onsumption of cystine, as well as the uptake of glutamate, glutamine a nd glucose increased with increasing cystine concentrations. These res ults are interpreted in view of system x(c)(-), a concentration depend ent amino acid transporter. Similarly, the consumption of amino acids transported by system L (ile, leu, val, tyr) was enhanced in cystine-c ontaining cultures, as compared to cystine-free cultures. Uptake of cy stine, methionine and system L amino acids ceases abruptly in all cult ures, even before growth ceased. The specific growth rate starts to de cline early during the growth phase, but this growth behaviour could n ot be correlated to the depletion of nutrients. We therefore propose t hat the observed growth pattern is a result of (auto)regulatory events that control both proliferation and metabolism.