Studies on the specificity of Pisum glycosyltransferases towards polyprenyl acceptors

Total polyprenols prepared from Pisum stem and analyzed by HPLC, possessed chain-lengths equivalent to 15-17 isoprene units and were (alpha)-saturated. Pea membranes were supplied with GDP- ('14)C mannose, UDP- ('14)C GlcNAc and UDP- ('14)C glucose as substrates for glycosyltransfer in the presence or absence of added polyprenyl phosphates. Mono- and pyro-phosphorylated fractions were characterized using chromatographic and hydrolytic criteria. Glucosyl and mannosyl transfer took place to form endogenous polyprenyl-P-saccharide. Dolichyl-P was the only added polyprenyl phosphate which acted as an acceptor for these transferases. In contrast, incorporation of all the supplied sugars into polyprenyl-PP-saccharide was stimulated greatly in the presence of relatively short (alpha)-saturated polyprenyl phosphates, e.g., dihydro-heptaprenyl phosphate (7HP). A major product was 7H-PP-monosaccharide.Oligosaccharide-lipids were also synthesized, all of which could be digested by treatment with endo H, indicating the presence of chitobiose. When UDP- ('14)C GlcNAc was used as substrate in the presence of dolP, labelled dol-PP-chitobiose was formed and then lengthened in the presence of unlabelled GDP-mannose. The same oligosaccharides were formed in experiments where the label was present in mannose. Evidence is presented for the further addition of terminal glucose to form "G" oligosaccharide. Endogenous levels of polyprenyl phosphates clearly limited the activities of pea transglycosylases, which were capable of recognizing isoprenoids of particular chain lengths and saturation patterns

Baculovirus expression of mammalian G protein α subunits

AbstractComplementary DNAs encoding three subtypes of the α subunit (αi−1, αo and αx) of rat guanyl nucleotide regulatory proteins were used to construct recombinant baculoviruses which direct high-level expression of the corresponding proteins in cultured Sf9 insect cells. The expressed proteins were recognized by polyclonal antisera specific for the different α chains, and co-migrated with the native proteins from rat brain membranes in immunoblotting analyses. Soluble and particulate forms of all three immunoreactive α chains were observed following ultracentrifugation of cell lysates. Biosynthetic radiolabelling of infected cells with [35S]methionine or [3H]myristate showed that both soluble and particulate forms of αi−1 and αo were myristoylated; in contrast, α, did not incorporate myristate. The soluble fractions from cells expressing α chains showed high levels of GTP-binding activity over that observed in uninfected cells, or in cells infected with wild-type virus. The peak expression levels observed at 72 h post-infection were highest for αo at ca, 400 pmol of GTP-γ-35S/mg protein, or roughly 2% of the total soluble protein. The results of this work show that the baculovirus system can be employed for high-level production of mammalian G protein α chains which retain GTP-binding activity and are appropriately modified by myristoylation