Purification and characterization of a heterogeneous glycosylated invertase from the rumen holotrich ciliate Isotricha prostoma.
The invertase (beta-fructofuranosidase, EC 3.2.1.26) of the rumen holotrich ciliate Isotricha prostoma has been purified. This is the first report of an enzyme purification from a known species of rumen protozoon. Cells were disrupted by ultrasonic treatment and the enzyme was purified from the cell-free extract by three successive liquid column chromatographies (Sepharose CL4B/octyl-Sepharose CL4B, DE52 DEAE-cellulose and concanavalin A-Sepharose 4B). This resulted in a 160-fold purification and a 15% yield. The major form of the purified enzyme was a tetramer with Mr about 350,000 that was readily dissociated by electrophoresis. The invertase was heterogeneous, as five types of monomers were shown by SDS/polyacrylamide-gel electrophoresis after denaturation. Part of this heterogeneity was due to different glycosylated forms of one of the polypeptides present in the purified enzyme. Isotricha prostoma invertase exhibited maximum activity at pH 5.5-6.0 and 50 degrees C. The kinetic properties of the purified enzyme were very similar to those of invertases from other sources such as yeast or plants (substrate and product inhibition, transferase activity).
Dauvrin
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Unité de Biochimie Physiologique, Université Catholique de Louvain, Belgium.
Thinès-Sempoux
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D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D016798:Ciliophora / Q000201:enzymology* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005632:Fructose / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006023:Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D008970:Molecular Weight; D012417:Rumen; D012756:Sheep; D043324:beta-Fructofuranosidase
D002241:Carbohydrates; D006023:Glycoproteins; D005632:Fructose; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D005947:Glucose