Protein interactions of SGP, an essential Streptococcus mutans GTPase, revealed by biochemical and yeast two-hybrid system analyses

SGP, a Streptococcus mutans essential GTPase, plays a role in the stress response of the organism. Recently, we proposed that one of the physiological functions of the SGP is the modulation of the GTP/GDP ratio under different growth conditions. In order to further determine the functions of SGP and its possible interactions with other molecules, we carried out immunoprecipitation, SGP binding, and the yeast two-hybrid system analyses. These approaches suggest that SGP may oligomerize and such interactions could be important for the function of this regulatory protein.     

A remodeling system for the oligosaccharide chains on glycoproteins with microbial endo-β-N-acetylglucosaminidases

Endo-M, endo-β-N-acetylglucosaminidase from Mucor hiemalis, transferred the complex type oligosaccharide of sialoglycopeptide to partially deglycosylated proteins (N-acetylglucosamine-attached proteins), which were prepared by excluding high-mannose type oligosaccharides from glycoproteins with Endo-H, endo-β-N-acetylglucosaminidase from Streptomyces plicatus. This finding indicated that the high-mannose type oligosaccharides on glycoproteins can be changed to complex type ones by the transglycosylation activity of Endo-M. This is the first report of the establishment of a remodeling system for the different types of oligosaccharides on glycoproteins with microbial endo-β-N-acetylglucosaminidases having different substrate specificities. Endo-M is a powerful tool for the in vitro synthesis of glycoproteins containing complex type oligosaccharides from glycoproteins produced by yeast.     

Inactivation of yeast ornithine decarboxylase by polyamines in vivo does not result from the incorporation of polyamines into enzyme protein

The peptide mixture obtained from controlled proteolytic digestion of ligandin with proteinase K or subtilisin retained 40% of glutathione-S-transferase and steroid isomerase activities, immunological reactivity and lower affinity bilirubin binding but binding at the primary site was abolished. When these limited proteolytic digests, which had no intact ligandin as determined by SDS gel electrophoresis, were subjected to Sephadex G-75 column chromatography, 40–50% of the peptide fragments were recovered in fractions where intact ligandin eluted. The results suggest that intact ligandin is not required for enzymatic activities, binding of bilirubin at the secondary site, or immunological reactivity; steroid isomerase and glutathione-S-transferase activities are modulated in a parallel manner and may be mediated by the same region of the protein, and primary and secondary binding sites for bilirubin are distinct and independent, despite nicks introduced by proteolysis in ligandin's subunits, some of the fragments remain associated under non-denaturing conditions and the susceptibility of the two subunits to the proteases is different.