Coated vesicles from developing and adult rat skeletal muscles contain multiple molecular forms of acetylcholinesterase.
The main purpose of this work was to determine which of the multiple isoforms of acetylcholinesterase (AChE) are associated with clathrin-coated vesicles (CVs) from developing and adult rat skeletal muscles. CV-enriched preparations were obtained by subcellular fractionation/equilibrium sedimentation and further purified by immunoadsorption to anti-clathrin IgG-coated Staphylococcus aureus cells. Analysis of individual AChE isoforms by velocity sedimentation ultracentrifugation showed that a) while both globular (G-forms) and asymmetric (A-forms) AChE were detected in all subcellular fractions evaluated, the CV-enriched fraction contained a higher proportion of A-forms (mainly the A12 species); b) most of the AChE activity contained in such a CV fraction was recovered following immunoadsorption; c) alkaline extraction conditions (pH 8.5) which depolymerize clathrin were necessary to detect a large proportion of A-forms in both the CV-enriched and immunoprecipitated preparations, while most of the G-forms (especially G1 + G2 AChE) were detected following extraction at pH 6.8; and d) comparison of AChE isoform profiles from neonate and adult muscle CV-enriched fractions showed a greater concentration of A-forms in the former. These data suggest that considerable amounts of A12 and, to a lesser extent, G4 AChE are sequestered within muscle CVs which may be destined for the plasmalemma. Our findings also indicate that the relative proportions of AChE isoenzymes in rat muscle CVs vary according to the extent of muscle development and lend support to the contention that CVs participate in the externalization of functionally important AChE isoenzymes.
Hodges-Savola
C A
CA
Neuroscience Research Laboratory, Veterans Administration Medical Center, Kansas City, MO 64128.
Gregory
E J
EJ
Rummel
S A
SA
Fernandez
H L
HL
D000110:Acetylcholinesterase / Q000378:metabolism* / Q000502:physiology; D000375:Aging / Q000378:metabolism*; D000818:Animals; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D011992:Endosomes / Q000201:enzymology*; D008297:Male; D024510:Muscle Development; D009132:Muscles / Q000201:enzymology* / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000378:metabolism
D000110:Acetylcholinesterase