Raja K B, Smith G P, Peters T J
Clin Chim Acta. 1981 Nov 25;117(1):33-41. doi: 10.1016/0009-8981(81)90007-3.
A new rapid assay for inorganic pyrophosphatase has been developed and the procedure optimised for measurement of the enzyme in human neutrophils. Kinetic studies showed that the activity was optimal at pH 8.0 and was activated by Mg2+. No neutral or acid pyrophosphatase was detected. Neutrophils were homogenised in isotonic sucrose and, after low speed centrifugation the intracellular localization of pyrophosphatase was determined by analytical subcellular fractionation with sucrose density gradient centrifugation. Pyrophosphatase was shown to have a dual localization to mitochondria and cytosol. No activity could be attributed to either the endoplasmic reticulum or alkaline phosphatase-containing granules (phosphasomes). Inhibitor studies clearly show that the cytosolic and mitochondrial pyrophosphatases are due to distinct enzymes. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and subjects in the third trimester of pregnancy. The specific activity (mU/mg protein) of pyrophosphatase, in contrast to that of alkaline phosphatase was similar in the three groups. Levamisole, a potent inhibitor of alkaline phosphatase had no effect on pyrophosphatase activity, confirming that this activity is not attributable to neutrophil alkaline phosphatase.
已开发出一种用于无机焦磷酸酶的新型快速检测方法，并对该程序进行了优化，以测量人中性粒细胞中的该酶。动力学研究表明，该活性在pH 8.0时最佳，并被Mg2+激活。未检测到中性或酸性焦磷酸酶。将中性粒细胞在等渗蔗糖中匀浆，低速离心后，通过蔗糖密度梯度离心进行亚细胞分析分级来确定焦磷酸酶的细胞内定位。结果表明焦磷酸酶定位于线粒体和胞质溶胶。内质网或含碱性磷酸酶的颗粒（磷体）均无活性。抑制剂研究清楚地表明，胞质溶胶和线粒体焦磷酸酶是由不同的酶引起的。从对照受试者、慢性粒细胞白血病患者和妊娠晚期受试者中分离出中性粒细胞。与碱性磷酸酶相比，焦磷酸酶的比活性（mU/mg蛋白）在三组中相似。左旋咪唑是一种有效的碱性磷酸酶抑制剂，对焦磷酸酶活性没有影响，这证实了该活性并非源自中性粒细胞碱性磷酸酶。
