Determination of the phosphorylation, uncovering of mannose 6-phosphate groups and targeting of lysosomal enzymes.
There are at least three stages in the targeting of soluble lysosomal enzymes: transfer of N-acetylglucosaminyl 1-phosphate to high-mannose oligosaccharide side chains, removal of N-acetylglucosamine and recognition of the "uncovered" mannose 6-phosphate residues. Defects in the transfer reaction cause mucolipidoses II and III. Those in the subsequent stages of the targeting may result in similar clinical disorders. To differentiate between possible defects of the targeting in cultured cells we have developed a procedure for a combined detection of the phosphorylation, uncovering of the transferred phosphate residues and the targeting of lysosomal enzymes. For this purpose cultured cells are metabolically labelled with [32P]phosphate and a lysosomal enzyme, such as cathepsin D, is isolated from the labelled cells and the medium by immunoprecipitation. The immunoprecipitates are dissolved with sodium dodecylsulphate and incubated in the presence and absence of calf intestine alkaline phosphatase. We show that the treatment of the denatured protein results in hydrolysis of phosphomonoester groups and that the phosphodiester and the peptide bonds remain intact. The initial and the residual radioactivity associated with the lysosomal enzyme which represent the total phosphate and the phosphodiester groups, respectively, are determined by gel-electrophoresis, fluorography and densitometry. This procedure extends one of the previously established methods for the diagnosis of mucolipidoses II and III.
Isidoro
C
C
Institut für Physiologische Chemie und Pathobiochemie, Universität, Münster, Germany.
Grässel
S
S
Baccino
F M
FM
Hasilik
A
A
D002117:Calcitriol / Q000494:pharmacology; D002402:Cathepsin D / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D008360:Mannosephosphates / Q000032:analysis*; D008715:Methionine / Q000378:metabolism; D009844:Oligosaccharides / Q000302:isolation & purification*; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011865:Radioisotope Dilution Technique; D013462:Sulfur Radioisotopes
D008360:Mannosephosphates; D009844:Oligosaccharides; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes; C027693:mannose-6-phosphate; D008715:Methionine; D002402:Cathepsin D; D002117:Calcitriol