Rosenblum D, Petzold S J
J Clin Invest. 1973 Jul;52(7):1665-72. doi: 10.1172/JCI107347.
To characterize the biological changes which result in increased granulocyte alkaline p-nitrophenyl phosphatase activity in patients with polycythemia vera, the enzyme was purified from granule fractions of sucrose homogenates made from dextran-sedimented leukocytes of normal subjects and patients with polycythemia vera. Polycythemic blood yielded 3-10 times as much granulocyte alkaline phosphatase per 10(9) leukocytes as did normal blood. Sodium dodecyl sulfate extracts of granules were purified by DEAE-cellulose chromatography and sucrose gradient centrifugation to apparent homogeneity as judged by polycarylamide disk gel electrophoresis. Granulocyte alkaline phosphatase from normal subjects was purified 6910-fold with a 60% yield and a specific activity of 47 U/mg. Granulocyte alkaline phosphatase from polycythemic patients was purified 1.166-fold with a 50% yield and a specific activity of 70 U/mg. The two enzymes did not differ in molecular weight; both appeared to be about 160,000 daltons by sucrose gradient centrifugation. Both appeared to be zinc metalloenzymes, in that they were specifically inhibited by o-phenanthroline. Their elution requirements when adsorbed to DEAE-cellulose suggested they were lipoproteins although the content of phosphorus was below the threshold of detection. The identity of the two enzymes was suggested by immunological studies in which antibody prepared against purified polycythemia vera enzyme gave a precipitation reaction of identity with another polycythemia vera enzyme and two pools of normal enzyme. It is possible to account for the difference in alkaline phosphatase activity between the granulocytes of patients with polycythemia vera and normal subjects by differences in the quantity of enzyme synthesized.
为了描述真性红细胞增多症患者粒细胞碱性对硝基苯磷酸酶活性增加所导致的生物学变化，从正常人和真性红细胞增多症患者经葡聚糖沉淀的白细胞制成的蔗糖匀浆颗粒组分中纯化该酶。每10⁹个白细胞，真性红细胞增多症患者的血液产生的粒细胞碱性磷酸酶是正常血液的3至10倍。颗粒的十二烷基硫酸钠提取物通过DEAE - 纤维素色谱和蔗糖梯度离心纯化至表观均一，这通过聚丙烯酰胺圆盘凝胶电泳判断。正常受试者的粒细胞碱性磷酸酶纯化了6910倍，产率为60%，比活性为47 U/mg。真性红细胞增多症患者的粒细胞碱性磷酸酶纯化了1166倍，产率为50%，比活性为70 U/mg。这两种酶的分子量没有差异；通过蔗糖梯度离心，两者似乎都约为160,000道尔顿。两者似乎都是锌金属酶，因为它们被邻菲罗啉特异性抑制。当吸附到DEAE - 纤维素上时，它们的洗脱要求表明它们是脂蛋白，尽管磷含量低于检测阈值。免疫研究表明这两种酶具有同一性，其中针对纯化的真性红细胞增多症酶制备的抗体与另一种真性红细胞增多症酶和两池正常酶产生了同一性沉淀反应。真性红细胞增多症患者和正常受试者粒细胞之间碱性磷酸酶活性的差异可能是由于合成的酶量不同。
