Membrane anchoring and surface distribution of glycohydrolases of human erythrocyte membranes

• Published in: FEBS letters Vol. 473; no. 1; pp. 89 - 94
• Main Authors: Goi, Giancarlo, Bairati, Chiara, Massaccesi, Luca, Lovagnini, Augusto, Lombardo, Adriana, Tettamanti, Guido
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 04-05-2000
• Subjects: AChE, acetylcholinesterase (EC 3.1.1.7); Adult; Detergents - pharmacology; Erythrocyte ghost; Erythrocyte Membrane - drug effects; Erythrocyte Membrane - enzymology; Erythrocyte Membrane - metabolism; Female; Fluorometry; Glucosides - pharmacology; Glycan phosphoinositide anchor; Glycohydrolase; Glycoside Hydrolases - analysis; Glycoside Hydrolases - metabolism; Glycosylphosphatidylinositols - metabolism; GPI, glycanphosphoinositide; Human erythrocyte; Humans; Hydrogen-Ion Concentration; Kinetics; Male; Membrane microdomain; Membrane Proteins - analysis; Membrane Proteins - metabolism; Middle Aged; MUB, methylumbelliferone; Octoxynol - pharmacology; Osmolar Concentration; Phosphatidylinositol Diacylglycerol-Lyase; Sodium Chloride - pharmacology; Solubility - drug effects; Solutions - pharmacology; Type C Phospholipases - metabolism; α-Fuc-ase, α-L-fucosidase (EC 3.2.1.51); α-Gal-ase, α-D-galactosidase (EC 3.2.1.22); α-Glc-ase, α-D-glucosidase (EC 3.2.1.20); α-Man-ase, α-D-mannosidase (EC 3.2.1.24); β-Gal-ase, β-D-galactosidase (EC 3.2.1.23); β-Glc-ase, β-D-glucosidase (EC 3.2.1.21); β-GlcA-ase, β-D-glucuronidase (EC 3.2.1.31); β-GlcNAc-ase, N-acetyl-β-D-glucosaminidase (EC 3.2.1.30); MUB, methylumbelliferone; Human erythrocyte; Membrane microdomain; α-Fuc-ase, α- L-fucosidase (EC 3.2.1.51); α-Man-ase, α- D-mannosidase (EC 3.2.1.24); β-Glc-ase, β- D-glucosidase (EC 3.2.1.21); β-GlcNAc-ase, N-acetyl-β- D-glucosaminidase (EC 3.2.1.30); β-Gal-ase, β- D-galactosidase (EC 3.2.1.23); Glycan phosphoinositide anchor; β-GlcA-ase, β- D-glucuronidase (EC 3.2.1.31); α-Glc-ase, α- D-glucosidase (EC 3.2.1.20); Erythrocyte ghost; AChE, acetylcholinesterase (EC 3.1.1.7); α-Gal-ase, α- D-galactosidase (EC 3.2.1.22); Glycohydrolase; GPI, glycanphosphoinositide
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(00)01504-0

The membrane anchoring of the following glycohydrolases of human erythrocyte plasma membranes was investigated: α- and β-
D-glucosidase, α- and β-
D-galactosidase, β-
D-glucuronidase,
N-acetyl-β-
D-glucosaminidase, α-
D-mannosidase, and α-
L-fucosidase. Optimized fluorimetric methods for the assay of these enzymes were set up. Treatment of the ghost preparation with 1.0 mol/l (optimal concentration) NaCl caused release ranging from 4.2% of α-
D-glucosidase to 70% of β-
D-galactosidase; treatment with 0.4% (optimal concentration) Triton X-100 liberated 5.1% of β-
D-galactosidase to 89% of α-
D-glucosidase; treatment with 1.75% (optimal concentration) octylglucoside yielded solubilization from 6.3% of β-
D-galactosidase to 85% of α-
D-glucosidase. Treatment with phosphoinositide-specific phospholipase C caused no liberation of any of the studied glycohydrolases. These results are consistent with the notion that the above glycohydrolases are differently anchored or associated with the erythrocyte plasma membrane, and provide the methodological basis for inspecting the occurrence of these enzymes in different membrane microdomains.