植物苯丙氨酸解氨酶的研究——1.鹰嘴豆PAL的纯化及其基本性质

Studies on Plant Phenylalanine Ammonia-Lyase(PAL)——Ⅰ.Isolation,Purification and Properties of PAL from Etiolated Seedlings of Gram Chickpea(cicer arietinum L.)

鹰嘴豆黄化苗苯丙氨酸解氨酶(PAL)经亲和层析等方法纯化.聚丙烯酰胺凝胶电泳和 SDS-聚丙烯酰胺凝胶电泳鉴定为单一蛋白带.用 SDS-聚丙烯酰胺凝胶电泳测得鹰嘴豆 PAL 的亚基分子量为55000.Sephadex G-200凝胶过滤法测得该酶的全酶分子量为205000.表明此植物 PAL 均由分子量相同的四个亚基构成.鹰嘴豆 PAL 具有一定的耐热性,最适温度为大约43℃.最适 PH 为9.4,酸碱稳定区域在碱侧.测得 PAL 的 K_m^(αpp)为1.8×10^(-4)M.

Gram chickpea PAL is isolated and purified to homogeneity byammonium sulphate fractionation,DEAE-cellulose chromatography andaffinity chromatography on L-phe-Sepharose 4B column.The purityis then identified by polyacrylamide gel electrophoresis in the presenceand absence of sodium dodecyl sulphate(SDS).The molecular weight of gram chickpea PAL subunit is 55000 daltonas assayed by SDS-polyacrylamide gel etectrophoresis.The molecularweight of gram chickpea PAL was found to be approximately 205 000dalton by Sephadex G-200 gel filtration.Thus,these findings showthat the native enzyme consists of four subunits of the same size.Gram chickpea PAL has certain thermostability.The PAL showscertain stability in the range pH 8～10.Optimum pH and temperaturefor activity are found to be pH 9.4 and 43℃,respectively.Theapparent Km value for L-phenylalanine is 1.8×10^(-4)M.