Purification and properties of Cellvibrio gilvus cellobiose phosphorylase.
The cellobiose phosphorylase (EC 2.4.1.20) of Cellvibrio gilvus, which is an endocellular enzyme, has been purified 196-fold with a recovery of 11% and a specific activity of 27.4 mumol of glucose 1-phosphate formed/min per mg of protein. The purification procedure includes fractionation with protamine sulphate, and hydroxyapatite and DEAE-Sephadex A-50 chromatography. The enzyme appears homogeneous on polyacrylamide-gel electrophoresis, and a molecular weight of 280 000 was determined by molecular-sieve chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed a single band and mol.wt. 72 000, indicating that cellobiose phosphorylase consists of four subunits. The enzyme had a specificity for cellobiose, requiring Pi and Mg2+ for phosphorylation, but not for cellodextrin, gentibiose, laminaribiose, lactose, maltose, kojibiose and sucrose. The enzyme showed low thermostability, an optimum pH of 7.6 and a high stability in the presence of 2-mercaptoethanol or dithiothreitol. The Km values for cellobiose and Pi were 1.25 mM and 0.77 mM respectively. Nojirimycin acted as a powerful pure competitive inhibitor (with respect to cellobiose) of the enzyme (Ki = 45 microM). Addition of thiol-blocking agents to the enzyme caused 56% inhibition at 500 microM-N-ethylmaleimide and 100% at 20 microM-p-chloromercuribenzoate.
D002384:Catalysis; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D005947:Glucose / Q000031:analogs & derivatives / Q000494:pharmacology; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011548:Pseudomonadaceae / Q000201:enzymology*; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D020245:p-Chloromercuribenzoic Acid; D005964:Glucosyltransferases; C034714:cellobiose phosphorylase; D005947:Glucose; D005033:Ethylmaleimide