The effect of castanospermine on the synthesis of synaptic glycoproteins by rat brain slices.
Slices were prepared from rat forebrains and the incorporation of [3H]mannose and [35S]methionine into proteins and glycoproteins determined. The incorporation of methionine continued to increase for up to 8 hours whereas mannose incorporation was maximal between 2 and 4 hours and declined thereafter. Glycopeptides prepared by pronase digestion of [3H]mannose-labeled glycoproteins were digested with endoglucosaminidase H (endo H) and analysed by gel filtration. The major endo H-sensitive oligosaccharide eluted in a position similar to standard Man8GlcNAc. In the presence of castanospermine, which inhibits glucosidase I, the first enzymatic step in the processing of N-linked oligosaccharides, a new endo H-sensitive glycan similar in size to standard Glc3Man9GlcNAc2 accumulated. Synaptic membranes (SMs) were isolated from slices which had been incubated with either [3H]mannose or [35S]methionine in the presence and absence of castanospermine. In the presence of inhibitor the relative incorporation of [3H]mannose into high-mannose glycans of synaptic glycoproteins was increased. The incorporation of newly synthesized, [35S] methionine-labeled, Con A-binding glycoproteins into SMs was not affected by the addition of inhibitor. Many of the glycoproteins synthesized in the presence of castanospermine exhibited a decreased electrophoretic mobility indicative of the presence of altered oligosaccharide chains. The results indicate that changes in oligosaccharide composition produced by castanospermine had little effect on the subsequent transport and incorporation of glycoproteins into synaptic membranes.
Department of Biochemistry, University of Toronto, Ontario, Canada.
S
S
Howes
N
N
Bissoon
M
M
Ito
P W
PW
Beesley
J W
JW
Gurd
D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D006023:Glycoproteins / Q000096:biosynthesis*; D065089:Glycoside Hydrolase Inhibitors; D006596:Hexosaminidases / Q000378:metabolism; D007212:Indolizines; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D011402:Pronase / Q000378:metabolism; D051381:Rats; D013570:Synaptic Membranes / Q000187:drug effects / Q000378:metabolism*; D000520:alpha-Glucosidases; D054836:Indolizidines
D000470:Alkaloids; D006023:Glycoproteins; D065089:Glycoside Hydrolase Inhibitors; D006596:Hexosaminidases; D007212:Indolizines; D008358:Mannose; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008715:Methionine; D009419:Nerve Tissue Proteins; D011402:Pronase; D000520:alpha-Glucosidases; C042059:glucosidase I; C037806:castanospermine; D054836:Indolizidines