Insect extramitochondrial glycerophosphate dehydrogenase I. Crystallization and physical properties of the enzyme from honeybee (Apis mellifera) thoraces

Ronald R. Marquardt, Ronald W. Brosemer
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引用次数: 44

Abstract

Extramitochondrial glycerophosphate dehydrogenase (L-glycerol-3-phosphate: DPN+ oxidoreductase, EC 1.1.1.8) was purified and crystallized from honeybee (Apis mellifera) thoraces. The isolation procedure is quite simple, since it consists merely of a series of (NH4)2SO4 precipitations.

The crystalline enzyme is homogeneous according to the following criteria: recrystallization to constant specific activity, electrophoresis on cellulose acetate strips, absence of 3 other glycolytic enzyme activities, chromatography on Sephadex G-200, and immunodiffusion on cellulose acetate strips.

Sedimentation velocity studies indicate that the native protein readily associates at moderate protein concentrations. The minimum molecular weight as determined by Sephadex G-200 chromatography is around 67 000.

Absorbance and fluorescence spectra show that neither DPNH nor adenosine diphosphate ribose is bound to the enzyme.

The bee enzyme differs from the crystalline rabbit-muscle enzyme in electrophoretic, ultracentrifugal, and immunological properties.

昆虫线粒体外甘油磷酸脱氢酶1 .蜜蜂胸脯酶的结晶和物理性质
从蜜蜂胸脯中纯化并结晶出线粒体外甘油磷酸脱氢酶(l-甘油-3-磷酸:DPN+氧化还原酶,EC 1.1.1.8)。分离过程很简单,因为它只包括一系列(NH4)2SO4沉淀。根据以下标准,结晶酶是均匀的:重结晶至恒定比活性,在醋酸纤维素条上电泳,不含其他3种糖酵解酶活性,在Sephadex G-200上层析,在醋酸纤维素条上免疫扩散。沉降速度研究表明,天然蛋白质在中等蛋白质浓度下很容易结合。Sephadex G-200色谱测定的最小分子量约为67 000。吸光度和荧光光谱显示,DPNH和二磷酸腺苷核糖都没有与酶结合。蜜蜂酶在电泳、超离心和免疫特性上不同于结晶兔肌酶。
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