Reconstitution of 5'-nucleotidase of bull seminal plasma in spin-labeled liposomes.
Seminal plasma separated from freshly ejaculated bull semen contains vesicles with a 5'-nucleotidase activity incorporated as an ectoenzyme anchored by glycosyl phosphatidylinositol (GPI). After its extraction from bull seminal plasma vesicles, the protein was purified and reconstituted into hen egg yolk lecithin liposomes obtained through prolonged dialysis of buffered n-octylglucoside detergent solutions of lipid, protein and various effectors against detergent-free solutions. Gel filtration experiments showed that the enzyme incorporated into liposomes in a dimeric form with its two subunits linked by disulfide bridges. In the presence of reduced glutathione, the protein dissociated into monomers and failed to incorporate into liposomes. Electron spin resonance (ESR) experiments, performed with liposomes containing electron spin labels localized at the hydrophilic lipid headgroups (5-doxyl stearic acid) or in the hydrophobic lipid hydrocarbon chains (16-doxyl stearic acid), demonstrated that the incorporation of 5'-nucleotidase resulted in the immobilization of the spin probes. Furthermore, the spectral parameters obtained before and after treatment of 5'-nucleotidase-containing liposomes with phosphatidylinositol-specific phospholipase C (PI-PLC) indicated that the liposome membrane bilayer did not contain protein segments. This supports the well-known ecto-localization of 5'-nucleotidase and rules out a previously reported possibility of a proteic transmembrane anchoring of the enzyme.
Fini
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Dipartimento di Biologia Cellulare e Molecolare, Università di Perugia, Italy.
Thuong
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Aliante
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Floridi
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Cannistraro
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D015720:5'-Nucleotidase / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003553:Cystine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005978:Glutathione / Q000494:pharmacology; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D008051:Lipid Bilayers; D008081:Liposomes; D008297:Male; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D011487:Protein Conformation; D012661:Semen / Q000201:enzymology*
D017261:Glycosylphosphatidylinositols; D008051:Lipid Bilayers; D008081:Liposomes; D003553:Cystine; D015720:5'-Nucleotidase; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D005978:Glutathione