1981 年 14 巻 6 号 p. 687-704
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 0.1M cacodylate buffer, pH 7.4, for 30min, and then washed with 5% DMSO in 0.1M cacodylate buffer. Vibratome or Microslicer sections were incubated in the following medium for 15-60min at 37°C. Medium for ACLase: 80mM Tris-maleate buffer, pH 7.4; 4mM MgSO4; 10mM NaF; 2mM theophilline; 2mM lead nitrate; 0.25M sucrose; 5% DMSO; 2.5mM levamisole; 0.5mM AMP-PNP. Medium for GCLase: 80mM Tris maleate buffer, pH 7.4; 3mM MnCl2 or MgSO4; 2mM theophilline; 2mM lead nitrate; 0.25M sucrose; 5% DMSO; 2.5mM levamisole; 0.5mM GMP-PNP. Some reaction medium for GCLase contained NaN3.
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.