Acid Phosphatase in Rat Liver Lysosomal Membranes: Purification and Characterization

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-3001IR column. The molecular weight, estimated by gel filtration through TSK SW 3000G, was approximately 320K and SDS gel electropho-resis 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.