Purification, properties and subunit structure of arginase from Iris bulbs.
Arginase (L-arginine amidinohydrolase, EC 3.5.3.1) from Iris hollandica bulbs has been purified approximately 20 000-fold. The purification procedure involved extraction from a particulate fraction (probably mitochondria), DEAE-Sephacel chromatography, aminohexyl-Sepharose 4B chromatography and gel filtration on Ultrogel AcA 34. Optimum assay conditions were determined, i.e. pH 9.0, 0.5 mM Mn2+, 0.5 mM dithiothreitol. The arginase is a slightly acidic protein (pI approximately equal to 5.6) highly specific for L-arginine. The arginase was almost completely inactivated by dialysis against a Mn2+-free buffer, 70% of the activity was then recovered after a treatment with 0.1 mM Mn2+ and 1 mM dithiothreitol at 40 degrees C for 45 min. The Mn2+ was found essential for the preservation of the arginase activity during the enzyme assay and for activation. However, the optimum Mn2+ concentration for activation was reduced to one-tenth and a better activity recovery was obtained in the presence of 1 mM dithiothreitol. The same dithiothreitol and Mn2+ requirement was observed to preserve arginase activity on storage. The native arginase showed an apparent Mr of about 191 000 as estimated by gel filtration and polyacrylamide gradient electrophoresis in the presence of 1 mM Mn2+. The subunit apparent Mr was 36 500 as estimated by dodecylsulfate electrophoresis. The arginase dissociated into reactivatable oligomers (apparent Mr = 59 000 +/- 5000 and 120 000) during electrophoresis on polyacrylamide gradient gels, carried out in a Mn2+-free electrophoresis buffer. The intramolecular cross-linkage of subunits by glutaraldehyde treatment showed that the purified arginase dissociated into dimers when losing its Mn2+. These results suggest that the iris bulb arginase is a hexamer, which can be dissociated into activatable dimers when losing its Mn2+. Such a structure has never been shown with other arginases.
D001119:Arginase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D008345:Manganese / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology; D010944:Plants / Q000201:enzymology*
D008345:Manganese; D001119:Arginase