Generation of subunit‐specific antibody probes for Torpedo acetylcholinesterase: Cross‐species reactivity and use in cell‐free translations

Abstract

The assembly of the collagen tailed A12 form of acetylcholinesterase (AChE) is regulated by muscle contraction. To begin to study this regulation, we derived antibody probes for the three subunits (100 kd, catalytic, and collagen tail) of AChE purified from Torpedo californica electric tissue. These included a polyclonal antiserum that recognizes all 3 subunits and 19 monoclonal antibodies; 16 of the monoclonals recognized the catalytic subunit, 2 recognized the tail subunit, and 1 recognized the 100 kd subunit on Western blots. We used immunohistochemical procedures to show that several of the anticatalytic and one of the antitail monoclonals cross-reacted with frog muscle AChE and Western blotting to show that several of the anticatalytic monoclonals cross-react with rat brain AChE. These antibodies were then used to immunoprecipitate AChE precursors from a cell-free translation system. There were generally three primary translation products, corresponding to the three enzyme subunits. Therefore, each subunit is probably derived from a separate mRNA. Occasionally there were two translation products corresponding to the catalytic subunit alone. The catalytic subunit was glycosylated following addition of canine microsomal membranes to the translation mix. The mRNA coding for this subunit appeared to be present in the poly(A)- RNA pool.

A molecular defect in virally transformed muscle cells that cannot cluster acetylcholine receptors.

“…The appropriate second antibody-horseradish peroxidase (HRP) conjugate (Hyclone Laboratories, Logan, UT) was diluted in Buffer A, incubated with the blots at room temperature for 2 h, and washed as above. Blots were rinsed in water or 10 mM sodium citrate, pH 4.5, and then reacted with 0.01% hydrogen peroxide and 0.5 mg/ml 4-chloroi-naphthol in the citrate buffer (24,30). The reaction was stopped by rinsing the blots in water.…”

“…One day before the fusion, macrophage feeder layers were prepared (40). Fusion of spleen cells with NS-1 mouse myeloma cells was done using slight modifications (30) of standard procedures (40) with polyethylene glycol in 75 mM Hepes buffer (Boehringer Mannheim, Indianapolis, IN). HAT selection was carried out according to standard protocols.…”

Cloning of a cDNA encoding the rat high molecular weight neurofilament peptide (NF-H): developmental and tissue expression in the rat, and mapping of its human homologue to chromosomes 1 and 22.

“…NFs were purified from rat brain (Sprague-Dawley, Charles River Breeding Laboratories) as described (1). Total rat brain protein was prepared by direct homogenization of tissue into NaDodSO4 sample buffer (15). Proteins were separated on 7.5% NaDodSO4/polyacrylamide gels and blotted electrically (15).…”

“…Total rat brain protein was prepared by direct homogenization of tissue into NaDodSO4 sample buffer (15). Proteins were separated on 7.5% NaDodSO4/polyacrylamide gels and blotted electrically (15). Probing, washing, and visualizing of immunoblots were performed as described (15).…”

“…Proteins were separated on 7.5% NaDodSO4/polyacrylamide gels and blotted electrically (15). Probing, washing, and visualizing of immunoblots were performed as described (15).…”