title: Cystine/cysteine metabolism in cultured Sf9 cells: influence of cell physiology on biosynthesis, amino acid uptake and growth.
abstract: Spodoptera frugiperda (Sf9) insect cells proliferate in a cystine-free medium, with the same growth rate, reaching the same final cell density, as in a cystine-containing medium, provided that the inoculum is taken from a pre-culture sufficiently early, at 47-53 h. With an inoculum from a 103 h culture an extended lag phase accompanied by cell death was observed during the first 50 h of cystine-free culture, even though the culture had been adapted to cystine-free conditions for 10 passages. Cystine-free cultures seeded with a 103 h inoculum had lower growth rates and reached lower final cell densities than corresponding cystine-supplied cultures. Cysteine biosynthesis occurs from methionine via the β-cystathionine pathway. More methionine was consumed by the cells in cystine-free media, and cystathionine was secreted when methionine and cystine were supplied in excess. The data suggest that cysteine biosynthesis is up-regulated in proliferating cells but down-regulated when the cells enter the stationary phase.In cultures supplied with cystine (10-100 mg 1(-1)), the specific uptake rate and total consumption of cystine, as well as the uptake of glutamate, glutamine and glucose increased with increasing cystine concentrations. These results are interpreted in view of system x                   (c)                             (-)                 , a concentration dependent amino acid transporter. Similarly, the consumption of amino acids transported by system L (ile, leu, val, tyr) was enhanced in cystine-containing cultures, as compared to cystine-free cultures. Uptake of cystine, methionine and system L amino acids ceases abruptly in all cultures, even before growth ceased. The specific growth rate starts to decline early during the growth phase, but this growth behaviour could not be correlated to the depletion of nutrients. We therefore propose that the observed growth pattern is a result of (auto)regulatory events that control both proliferation and metabolism.
authors.affiliation: Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-100 44, Stockholm, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Doverskog
authors.forename: L
authors.initials: L
authors.lastname: Han
authors.forename: L
authors.initials: L
authors.lastname: Häggström