With the substrate DNP-α-GalNAc (2,4-dinitrophenyl-N-acetyl-α-d-galactosaminide) three α-N-acetylgalactosaminidase-like activities could be distinguished in serum, in addition to the classical lysosomal enzyme (Naga, EC 3.2.1.49, pH optimum at 4). Two activities had optima in the pH 5 to 6 region and one peaked around pH 8. Like the Naga activity at pH 4, the activity at pH 8 was detectable under standard assay conditions. However, the two activities in the pH 5 to 6 range were not readily apparent in such assays. They could be unmasked as separate activities only when low serum concentrations were used. Addition of 1% saturated ammonium sulphate to the assay medium stimulated these activities. All activities in the pH 5 to 8 range decreased with increasing serum concentration in the assay, suggesting the presence of endogenous inhibitors. The activities between pH 5 and 6 might be similar to an activity described in 1996, which was considerably elevated in serum of patients with great variety of cancers (N. Yamamoto, V.R. Naraparaju, and S.O. Asbell (1996). Deglycosylation of serum vitamin D3-binding protein leads to immunosuppression in cancer patients. Cancer Res. 56, 2827-2811).
Keywords: A380, optical absorbance at 380 nm; DMF, dimethylformamide; DNP, 2,4-dinitrophenol; DNP-α-GalNAc; DNP-α-GalNAc, 2,4-dinitrophenyl-N-acetyl-α-d-galactosaminide; General tumour marker; Human serum; MU, 4-methylumbelliferone; Naga, α-N-acetylgalactosaminidase (EC 3.2.1.49); Naga6, the collective Naga-like activities in the pH 5 to 6 region; Naga8, the Naga-like activity in the pH 6.5 to 8 region; RT, room temperature; S.A., specific activity in nmol per min per mL serum (nmol min− 1 mL− 1), using 2 mM DNP-α-GalNAc; TM, tumour marker; pNP-α-GalNAc, para-nitrophenyl-α-GalNAc; α-GalNAc, N-acetyl-α-d-galactosaminide; α-N-Acetylgalactosaminidase.