Stabilization and purification of glucosamine 6-phosphate isomerase from rat kidney.
1. Glucosamine 6-phosphate (GlcN-6-P) isomerase of rat kidney was resistant to heating at 50--55 degrees in crude extract but not after several purification steps. GlcN-6-P and N-acetylglucosamine 6-phosphate were found to stabilize the isomerase under these conditions. They also protected the enzyme from tryptic digestion, but only GlcN-6-P was effective against inactivation by p-chloromercuribenzoate. 2. When GlcN-6P isomerase was purified from fresh kidney and kidney stored at -20 degrees, separately and under GlcN-6-P, the two preparations were different in elution profile from a hydroxyapatite column. It was subsequently found that storage of crude extract at -20 degrees resulted in molecular alterations of the enzyme. Prolonged purification appeared to affect the enzyme similarly. The molecular alterations, however, were suppressed if the extract was stored at -70 degrees. 3. These findings have been utilized to develop a procedure, which enables us to purify rat kidney GlcN-6-P isomerase without any molecular alteration and in good yield.
D019747:Aldose-Ketose Isomerases; D000818:Animals; D002238:Carbohydrate Epimerases / Q000302:isolation & purification*; D004355:Drug Stability; D005944:Glucosamine / Q000031:analogs & derivatives / Q000302:isolation & purification; D006358:Hot Temperature; D007668:Kidney / Q000201:enzymology*; D008297:Male; D009926:Organ Preservation; D051381:Rats
C020582:glucosamine-6-phosphate isomerase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; D005944:Glucosamine