Hodinka R L, Modrzakowski M C
Infect Immun. 1983 Apr;40(1):139-46. doi: 10.1128/iai.40.1.139-146.1983.
Granule contents from rat polymorphonuclear neutrophils were prepared by extraction with 0.2 M acetate (pH 4), dialyzed against phosphate-buffered saline (pH 7), and tested for bactericidal activity. Bactericidal assays consisted of mixing rat granule extract with 1 x 10(3) to 3 x 10(3) bacterial cells per ml at 37 degrees C for 1 h in a medium suited for bacterial growth. The granule extract demonstrated a distinctive dose-dependent bactericidal activity against outer membrane lipopolysaccharide mutants of Salmonella typhimurium LT-2, independent of added hydrogen peroxide or other active oxygen derivatives. The rough bacterial mutants showed an ordered increase in sensitivity to the rat lysosomal extracts inversely related to the length of their lipopolysaccharide carbohydrate side chains. Fractionation of the rat polymorphonuclear neutrophil granule extract with Sephadex G-100 column chromatography revealed an elution profile containing three major areas (peaks) of protein. Polyacrylamide gel electrophoresis and examination of enzymatic activity showed that these peaks contained myeloperoxidase (peak A), neutral protease (peak B), and lysozyme (peak C) activities. Also observed in peak C were cationic protein species whose cathodal electrophoretic migration was faster than that for lysozyme. Only peak C exhibited a bactericidal activity against the rough mutants of S. typhimurium LT-2 similar to that obtained for the unfractionated granule extract, with susceptibility of the bacterial mutants increasing with a progressive loss of carbohydrate residues in the lipopolysaccharide of the cell wall. The bactericidal activity of the peak C protein fraction was dose dependent. Boiling the unfractionated granule extract or peak C for 30 min had little affect on their antimicrobial activity when reacted against a deep-rough lipopolysaccharide mutant. However, trypsin pretreatment of these fractions significantly reduced their antimicrobial activity for the same mutant chemotype.
通过用0.2M乙酸盐（pH 4）提取制备大鼠多形核中性粒细胞的颗粒内容物，用磷酸盐缓冲盐水（pH 7）透析，并测试其杀菌活性。杀菌试验包括在适合细菌生长的培养基中，于37℃将大鼠颗粒提取物与每毫升1×10³至3×10³个细菌细胞混合1小时。颗粒提取物对鼠伤寒沙门氏菌LT-2的外膜脂多糖突变体表现出独特的剂量依赖性杀菌活性，与添加的过氧化氢或其他活性氧衍生物无关。粗糙型细菌突变体对大鼠溶酶体提取物的敏感性呈有序增加，与它们脂多糖碳水化合物侧链的长度呈负相关。用Sephadex G-100柱色谱对大鼠多形核中性粒细胞颗粒提取物进行分级分离，得到的洗脱图谱包含三个主要蛋白质区域（峰）。聚丙烯酰胺凝胶电泳和酶活性检测表明，这些峰含有髓过氧化物酶（峰A）、中性蛋白酶（峰B）和溶菌酶（峰C）活性。在峰C中还观察到阳离子蛋白种类，其阴极电泳迁移速度比溶菌酶快。只有峰C对鼠伤寒沙门氏菌LT-2的粗糙突变体表现出与未分级颗粒提取物相似的杀菌活性，细菌突变体的敏感性随着细胞壁脂多糖中碳水化合物残基的逐渐丧失而增加。峰C蛋白组分的杀菌活性是剂量依赖性的。将未分级颗粒提取物或峰C煮沸30分钟，当与深粗糙型脂多糖突变体反应时，对其抗菌活性影响很小。然而，用胰蛋白酶预处理这些组分可显著降低它们对相同突变体化学型的抗菌活性。
