Xu Shengyuan, Zhao Linshu, Larsson Anders, Venge Per
Department of Medical Sciences, Clinical Chemistry, Uppsala University, Sweden.
FEBS J. 2009 Jan;276(1):175-86. doi: 10.1111/j.1742-4658.2008.06771.x. Epub 2008 Nov 19.
A phospholipase B (PLB) precursor was purified from normal human granulocytes using Sephadex G-75, Mono-S cation-exchange and hydroxyapatite columns. The molecular mass of the protein was estimated to be approximately 130 kDa by gel filtration and 22 and 42 kDa by SDS/PAGE. Tryptic peptide and sequence analyses by MALDI-TOF and tandem mass spectrometry (MS/MS) identified the protein as a FLJ22662 (Homo sapiens) gene product, a homologue of the amoeba Dictyostelium discoideum PLB. The native protein needed modifications to acquire deacylation activity against phospholipids including phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and lysophospholipids. Enzyme activity was associated with fragments derived from the 42 kDa fragment. The enzyme revealed a PLB nature by removing fatty acids from both the sn-1 and sn-2 positions of phospholipids. The enzyme is active at a broad pH range with an optimum of 7.4. Immunoblotting of neutrophil postnuclear supernatant using antibodies against the 42 kDa fragment detected a band at a molecular mass of 42 kDa, indicating a neutrophil origin of the novel PLB precursor. The existence of the PLB precursor in neutrophils and its enzymatic activity against phospholipids suggest a role in the defence against invading microorganisms and in the generation of lipid mediators of inflammation.
利用葡聚糖凝胶G-75、单磺酸阳离子交换柱和羟基磷灰石柱从正常人粒细胞中纯化出一种磷脂酶B（PLB）前体。通过凝胶过滤法估计该蛋白质的分子量约为130 kDa，通过SDS/PAGE法估计为22 kDa和42 kDa。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）和串联质谱（MS/MS）进行的胰蛋白酶肽段分析和序列分析确定该蛋白质为FLJ22662（智人）基因产物，是变形虫盘基网柄菌PLB的同源物。天然蛋白质需要进行修饰才能获得针对包括磷脂酰胆碱、磷脂酰肌醇、磷脂酰乙醇胺和溶血磷脂在内的磷脂的脱酰基活性。酶活性与源自42 kDa片段的片段相关。该酶通过从磷脂的sn-1和sn-2位去除脂肪酸显示出PLB的性质。该酶在较宽的pH范围内具有活性，最适pH为7.4。使用针对42 kDa片段的抗体对中性粒细胞核后上清液进行免疫印迹检测到一条分子量为42 kDa的条带，表明这种新型PLB前体起源于中性粒细胞。中性粒细胞中PLB前体的存在及其对磷脂的酶活性表明其在抵御入侵微生物和炎症脂质介质生成中发挥作用。
