Acid Phosphatase in Rat Liver Lysosomal Membranes: Purification and Characterization1

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Abstract
Acid phosphatase associated with rat liver lysosomal membranes (M-APase) was purified about 4,200-fold over the homogenate with 10% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included; preparation of lysosomal membranes, solubilization of the membranes with 1% Triton X-100, immunoaffinity chromatography, and gel filtration with FPLC equipped with a Sephacryl S-300HR column. The molecular weight, estimated by gel filtration through TSK SW 3000G, was approximately 320K and SDS gel electrophoresis showed that the enzyme is composed of four identical subunits with an apparent molecular weight of 67K. The enzyme contains about 24.3% carbohydrate consisting of mannose, galactose, fucose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetyl-neuraminic acid in a molar ratio of 38 : 20 : 5 : 36 : 4 : 11, respectively. In addition, three soluble forms of acid phosphatase (C-APase I, II, and III) in lysosomal contents were separated from rat liver lysosomal contents with DEAE-Sephacel. These three enzymes were also purified using immunoaffinity chromatography followed by gel filtration. C-APase I, II, III, and M-APase have isoelectric points of 7.7–8.2, 6.6–7.0, 5.7–6.7, and 3.4–3.8, respectively. All four APases are sensitive to endo-β;-N-acetylglucosaminidase H. However, only C-APase III and M-APase are digestible with neuraminidase. Susceptibility of M-APase to neuraminidase in intact tritosomes was examined to study the topography of M-APase in tritosomal membranes. Neuraminidase susceptibility of M-APase was not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock. This result indicated that the oligosaccharide chains containing sialic acid in M-APase are on the inside surface of the tritosomal membranes. The N-terminal 18 residues of M-APase and C-APase I were the same, that is: Arg-Ser-Leu-Arg-Phe-Val-Thr-Leu-Leu-Tyr-Arg-His-Gly-Asp-Arg-X-Pro-Val-. Electron microscopic immunocytochemistry clearly localized acid phosphatase, which is associated with the interior of lysosomal membranes and with flocculating materials present in the lysosomal matrix.