Purification of rabbit skeletal muscle proteoglycogen: studies on the glucosyltransferase activity of polysaccharide-free and -bound glycogenin.
Proteoglycogen is the end product in the process of glycogen biogenesis. We have purified rabbit muscle proteoglycogen and studied the glucosyltransferase reactions catalyzed by its protein moiety, glycogenin, free or bound to the polysaccharide. The purification strategy involved dissolution of proteoglycogen and cosedimenting membrane vesicles in a Triton X-114/Triton X-45 mixture followed by partition in the aqueous phase, potassium iodide precipitation of accompanying proteins, and washing by high-speed centrifugation. Glycogenin or a proteoglycogen species of an average molecular mass of 200 kDa was isolated by ion-exchange chromatography after the purified proteoglycogen had been subjected to long or short amylolytic digestion, respectively. Besides autoglucosylation from UDP-glucose, glycogenin was capable of autogalactosylation from UDP-galactose. The autoglucosylation reaction was not inhibited by the simultaneous glucosylation of the exogenous acceptors N-(maltosyl-alpha-1-4-(1-deoxiglucitol))-peptide or n-dodecyl-beta-D-maltoside. The polysaccharide-bound glycogenin species of 200 kDa showed to be active for the glucosylation of exogenous acceptor and represented the isolated proteoglycogen of higher size having glucosyl transferase activity. This is the first description of the isolation of native proteoglycogen and a proteoglycogen species having glucosyltransferase activity.
Carrizo
M E
ME
Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), UNC-CONICET, Departamento de Química Biológica Dr. Ranwel Caputto, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina.
Miozzo
M C
MC
Goldraij
A
A
Curtino
J A
JA
D000818:Animals; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D003902:Detergents; D005964:Glucosyltransferases / Q000378:metabolism*; D006003:Glycogen / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006031:Glycosylation; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry*; D011134:Polysaccharides / Q000378:metabolism*; D011193:Potassium Iodide; D011817:Rabbits; D012995:Solubility; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D003902:Detergents; D006023:Glycoproteins; D009124:Muscle Proteins; D011134:Polysaccharides; C051887:glycogenin; D011193:Potassium Iodide; D006003:Glycogen; D005964:Glucosyltransferases; D014532:Uridine Diphosphate Glucose