Jensen K F, Nygaard P
Eur J Biochem. 1975 Feb 3;51(1):253-65. doi: 10.1111/j.1432-1033.1975.tb03925.x.
The purine nucleoside phosphorylases from Escherichia coli and from Salmonella typhimurium have been purified to electrophoretic homogeneity and crystallized. Comparative studies revealed that the two enzymes are very much alike. They obey simple Michaelis-Menten kinetics for their substrates with the exception of phosphate for which they show negative cooperativity. Gel filtration on Sephadex G-200 of the native enzymes revealed a molecular weight for both enzymes of 138000 plus or minus 10%. By use of dodecylsulphate gel electrophoresis a subunit molecular weight of 23700 plus or minus 5% was determined, suggesting that both enzymes consist of six subunits of equal molecular weight. When the subunits were partially crosslinked with dimethyl suberimidate before dodecylsulphate electrophoresis six protein bands were observed in agreement with the proposed oligomeric state of the enzyme, consisting of six subunits of equal molecular weight. Analysis of the amino acid composition also indicates that the subunits are identical. 6M guanidinium chloride dissociates the enzymes; association experiments with native and succinylated enzymes suggested that only the hexameric form is active. Both enzymes could be dissociated into subunits by p-chloromercuribenzoate; this dissociation is prevented by the substrates: the nucleosides, the pentose 1-phosphates, and mixtures of phosphate and purine bases.
已将来自大肠杆菌和鼠伤寒沙门氏菌的嘌呤核苷磷酸化酶纯化至电泳纯并使其结晶。比较研究表明，这两种酶非常相似。除了对磷酸盐表现出负协同性外，它们对底物遵循简单的米氏动力学。用葡聚糖凝胶G - 200对天然酶进行凝胶过滤，结果显示两种酶的分子量均为138000，正负误差10%。通过十二烷基硫酸钠凝胶电泳测定亚基分子量为23700，正负误差5%，这表明两种酶均由六个分子量相等的亚基组成。在进行十二烷基硫酸钠电泳之前，用辛二酸亚胺二甲酯使亚基部分交联，观察到六条蛋白带，这与所提出的由六个分子量相等的亚基组成的寡聚体状态一致。氨基酸组成分析也表明亚基是相同的。6M氯化胍可使这些酶解离；对天然酶和琥珀酰化酶进行的缔合实验表明只有六聚体形式具有活性。对氯汞苯甲酸可使两种酶解离成亚基；底物（核苷、戊糖1 - 磷酸以及磷酸盐和嘌呤碱的混合物）可阻止这种解离。
