Plasma membrane association and preliminary characterization of Drosophila sperm surface glycosidases.
Previous studies have identified beta-N-acetylglucosaminidase (GlcNAc'ase) and (alpha-mannosidase activities on the Drosophila melanogaster sperm surface which may have a role in fertilization. The aim of this study was to investigate their linkage to the sperm plasma membrane. We verified that glycosidases are not peripherally adsorbed to the cell surface by evaluating their resistance to release by KI, by buffered salt solutions of high ionic strength or alkaline buffers. Glycosidases were released from the sperm surface by detergents and, only to a minor extent, by mild proteolysis. Differential detergent solubilization pointed out that Triton X-114 was the most effective releasing agent for GlcNAc'ase and CHAPS for mannosidase. No activity was released from the membrane by a phosphatidylinositol-specific phospholipase C (PI-PLC). The released forms were quite hydrophilic in phase separation experiments with Triton X-114. This finding indicates the presence of a hydrophobic domain limited to a single transmembrane helix or/and the presence of an extensive glycosylation. The use of a Con-A binding assay demonstrated that both the enzymes are glycosylated. The molecular weight of the released glycosidases estimated by gel filtration was 158 kDa for GlcNAc'ase and 317 kDa for mannosidase. These results suggest that Drosophila melanogaster GlcNAc'ase and mannosidase are mannosylated integral membrane proteins that would function as exoenzymes with their active sites accessible in the extracellular space.
Pasini
M E
ME
Department of General Physiology and Biochemistry, University of Milan, Italy.
Cattaneo
F
F
Pinto
M R
MR
De Santis
R
R
Perotti
M E
ME
D000118:Acetylglucosaminidase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002793:Cholic Acids; D003902:Detergents; D004331:Drosophila melanogaster / Q000201:enzymology*; D008297:Male; D008361:Mannosidases / Q000302:isolation & purification / Q000378:metabolism*; D017830:Octoxynol; D013094:Spermatozoa / Q000201:enzymology* / Q000378:metabolism; D043323:alpha-Mannosidase
D002793:Cholic Acids; D003902:Detergents; D017830:Octoxynol; D008361:Mannosidases; D043323:alpha-Mannosidase; D000118:Acetylglucosaminidase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate