NEW CYTOCHEMICAL METHOD FOR ADENYLATE CYCLASE AND GUANYLATE CYCLASE WITH DIMETHYL SULFOXIDE

概要

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To assess the appropriateness of the lead nitrate method using dimethyl sulfoxide (DMSO) for the cytochemical demonstration of adenylate cyclase (ACLase) and guanylate cyclase (GCLase), we have carried out a biochemical assay of ACLase and GCLase in parallel with the cytochemical study. Tissues from rats were fixed in a mixture of 2% paraformaldehyde, 0.25% glutaraldehyde and 5% DMSO in 0.1 M cacodylate buffer, pH 7.4, for 30 min, and then washed with 5% DMSO in 0.1 M cacodylate buffer. Vibratome or Microslicer sections were incubated in the following medium for 15-60 min at 37℃. Medium for ACLase: 80 mM Tris-maleate buffer, pH 7.4; 4 mM MgSO_4; 10 mM NaF; 2 mM theophilline; 2 mM lead nitrate; 0.25 M sucrose; 5 % DMSO; 2.5 mM levamisole; 0.5 mM AMP-PNP. Medium for GCLase: 80 mM Tris maleate buffer, pH 7.4; 3 mM MnCl_2 or MgSO_4; 2 mM theophilline; 2 mM lead nitrate; 0.25 M sucrose; 5% DMSO; 2.5 mM levamisole; 0.5 mM GMP-PNP. Some reaction medium for GCLase contained NaN_3. ACLase and GCLase activities were biochemically and cytochemically enhanced by adding DMSO to the incubation medium as well as to fixatives. Cyclases were localized in the plasma membranes, gap junctions, endoplasmic reticulum and Golgi apparatus.
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