Membrane-bound choline-O-acetyltransferase in rat hippocampal tissue is anchored by glycosyl-phosphatidylinositol.
In an earlier study, we presented evidence to suggest that some of the particulate choline-O-acetyltransferase (ChAT) in rat hippocampal tissue might be linked to membranes by a glycosyl-phosphatidylinositol (GPI) anchor. In the present report, we attempted to determine if any of this GPI-anchored ChAT might be intracellular. Internalization of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis into rat hippocampal synaptosomes by the DMSO (dimethyl sulfoxide) freeze/thawing procedure caused an increase in cytosolic and a decrease in membrane-bound ChAT. Incubation of a plasma membrane enriched subcellular fraction at 16 degrees C relative to 4 degrees C led to a conversion of the membrane-bound, amphiphilic ChAT into hydrophilic ChAT. This conversion was blocked by zinc, an inhibitor of GPI-PLC. The cytosolic fraction of ChAT immunoreacted on western blots with an antibody directed against the cross-reacting determinant (CRD) of the GPI anchor. We suggest that some of the membrane-bound ChAT in rat hippocampal tissue is GPI-anchored intracellularly; also, that an endogenous GPI-PLC-like enzyme acts to release it into the cytosol.
Smith
L K
LK
Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock 79430.
Carroll
P T
PT
D000818:Animals; D001413:Bacillus thuringiensis / Q000201:enzymology; D015153:Blotting, Western; D002795:Choline O-Acetyltransferase / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D003902:Detergents; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D006624:Hippocampus / Q000201:enzymology*; D007120:Immunochemistry; D066298:In Vitro Techniques; D008297:Male; D008566:Membranes / Q000737:chemistry / Q000201:enzymology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes / Q000187:drug effects / Q000201:enzymology; D013696:Temperature; D010738:Type C Phospholipases / Q000378:metabolism
D003902:Detergents; D017261:Glycosylphosphatidylinositols; D002795:Choline O-Acetyltransferase; D010738:Type C Phospholipases