Purification and properties of a 4-methylene-L-glutamine amidohydrolase from peanut leaves

Abstract

A 4-methylene-L-glutamine amidohydrolase has been partially purified from leaf extracts of 2-week germinated peanuts (Arachis hypogaea). Purification steps include DEAE-Sephacel and gel filtration chromatography as well as chromatofocusing; amidohydrolase purified 300- to 400-fold is obtained. The enzyme has an approximate molecular weight of 45,000 as determined by gel filtration chromatography and polyacrylamide gel electrophoresis, a broad activity optimum between pH 8.0 and 9.0, and is highly specific toward 4-methylene-L-glutamine. Of a number of amides tested as substrate, only L-glutamine serves 20% as effectively as the methylene-substituted analog. Multiple bands of activity, seen when enzyme samples are electrophoresed on polyacrylamide gels, are not completely resolved but appear to be very similar in molecular weight, pK values, and subcellular localization. Activity is not affected either by added thiols, metal ions, sulfhydryl-reacting reagents, or metal-ion chelators; it is inhibited by borate ions but stimulated by sodium dodecyl sulfate. This amidohydrolase activity is absent in imbibed seeds, but on germination increases rapidly in the cotyledons and leaves for 3 weeks followed by a gradual decline as the plant matures. Activity is almost completely (95%) localized in the leaves and cotyledons. Differential centrifugation studies indicate that the enzyme is found solely in the soluble fraction of peanut leaves.