Pisoni R L, Lindley E R
Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor 48109-2029.
J Biol Chem. 1992 Feb 25;267(6):3626-31.
When isolated human fibroblast lysosomes are incubated with 4 microM [32P]phosphate at pH 7.0, orthophosphate is transported into lysosomes and is rapidly incorporated into low and high molecular weight products. We have characterized the high molecular weight (HMW) lysosomal material into which [32P]phosphate is incorporated and have found it to consist of long chains of inorganic polyphosphate based on the following observations. 1) greater than 97% of HMW 32P-lysosomal material is converted to [32P]orthophosphate when incubated with 1 N HCl for 20 min at 100 degrees C. 2) Incubation of HMW 32P-lysosomal material at pH 7.0 and 65 degrees C for 96 h results in the formation of [32P]trimetaphosphate, which is known to be produced only from linear chains of polyphosphate under these conditions. 3) HMW 32P-lysosomal material is resistant to degradation by proteinase K, ribonuclease, and deoxyribonuclease and extracts into the aqueous phase during phenol/chloroform extractions. 4) HMW 32P-lysosomal material displays heterogeneous mobility on polyacrylamide gels with most chains ranging in length from 100 to at least 600 phosphate residues. 5) HMW 32P-lysosomal material is partially hydrolyzed under alkaline conditions to yield a continuous ladder of polyphosphate species differing by one or several residues in length on polyacrylamide gels.
当分离出的人成纤维细胞溶酶体在pH 7.0条件下与4微摩尔[32P]磷酸盐一起孵育时，正磷酸盐被转运到溶酶体中，并迅速掺入低分子量和高分子量产物中。我们已经对掺入[32P]磷酸盐的高分子量（HMW）溶酶体物质进行了表征，并基于以下观察结果发现它由无机多磷酸盐的长链组成。1）当在100℃下与1 N HCl孵育20分钟时，超过97%的HMW 32P-溶酶体物质转化为[32P]正磷酸盐。2）将HMW 32P-溶酶体物质在pH 7.0和65℃下孵育96小时会导致[32P]三偏磷酸盐的形成，已知在这些条件下只有多磷酸盐的线性链才能产生这种物质。3）HMW 32P-溶酶体物质对蛋白酶K、核糖核酸酶和脱氧核糖核酸酶的降解具有抗性，并且在苯酚/氯仿提取过程中会萃取到水相中。4）HMW 32P-溶酶体物质在聚丙烯酰胺凝胶上显示出异质迁移率，大多数链的长度范围为100至至少600个磷酸残基。5）HMW 32P-溶酶体物质在碱性条件下部分水解，在聚丙烯酰胺凝胶上产生一系列长度相差一个或几个残基的多磷酸盐物种的连续梯状图谱。
