[3H]saxitoxin as a marker for canine deep muscular plexus neurons.
The objectives of this study are 1) to examine the potential of [3H]saxitoxin binding as a marker for the neuronal membranes in canine small intestinal muscle membrane preparations, 2) to develop a synaptosomal preparation from deep muscular plexus, and 3) to partially characterize [3H]saxitoxin binding to this fraction. A purified synaptosomal fraction, relatively low in the smooth muscle plasma membrane marker enzyme 5'-nucleotidase but enriched in [3H]saxitoxin binding (2,592 fmol/mg), was obtained on sucrose density gradient. Vasoactive intestinal peptide immunoreactivity was also highest (51.82 pmol/mg protein) in this fraction. The binding was rapid at 20 degrees C with quick and complete dissociation after the addition of excess unlabeled tetrodotoxin (TTX). Scatchard analysis of the saturation binding data revealed a single population of binding sites (Bmax = 5,705 fmol/mg protein). The affinity constants calculated from the kinetic and saturation data were in close agreement (Kd = 0.26 and 0.69 nM, respectively). TTX competed for the binding (Ki = 2.1 nM), whereas veratridine and guanidinium hydrochloride did not. Monovalent and divalent cations had differential effects on the binding.
Ahmad
S
S
Department of Neurosciences, McMaster University Medical Center, Hamilton, Ontario, Canada.
Allescher
H D
HD
Manaka
H
H
Manaka
Y
Y
Daniel
E E
EE
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000294:innervation*; D009130:Muscle, Smooth / Q000294:innervation* / Q000378:metabolism; D009197:Myenteric Plexus / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D012530:Saxitoxin / Q000378:metabolism*; D014660:Vasoactive Intestinal Peptide / Q000032:analysis
D012530:Saxitoxin; D014660:Vasoactive Intestinal Peptide