Incorporation of [32P]orthophosphate into long chains of inorganic polyphosphate within lysosomes of human fibroblasts.
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.
Pisoni
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RL
Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor 48109-2029.
Lindley
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ER
D001692:Biological Transport; D003851:Deoxyribonucleases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D012260:Ribonucleases / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism
D010710:Phosphates; D010761:Phosphorus Radioisotopes; D003851:Deoxyribonucleases; D012260:Ribonucleases; D012697:Serine Endopeptidases; D019286:Endopeptidase K