Cytochemical observations on mannose-specific binding sites for horseradish peroxidase in liver sinusoidal cells

Summary

Paraformaldehyde-fixed, frozen sections of the liver of rats were processed for the detection of mannose-specific binding sites of horseradish peroxidase (HRP) by a method reported previously, with some modifications resulting in a more intense binding reaction. Before staining for peroxidase activity, the sections were held in buffered solutions of physiological saline at different temperatures and pH's, and in the presence or absence of added Ca²⁺, mannose or galactose. The gradual decrease and final disappearance of the binding reaction were observed. The release of HRP from the binding sites as determined by the disappearance of the cytochemical reaction was 50–100 times faster at 22°C than at 4°C and was 5–10 times faster at 37°C than at 22°C. The release was approximately twice as fast at pH 7.0 than at pH 9.0 and 20–30 times faster at pH 6.0 than at pH 7.0. The release of HRP was 10–15 times faster in the absence of 1 mM Ca²⁺ in the buffer solution and was approximately 100times faster in the presence of 0.1 M d-mannose as compared to 0.1 M d-galactose. Pretreatment of the sections with trypsin abolished the binding reaction whereas neuraminidase, phospholipases A₂ and C, and chondroitinase ABC were without effect. An acidic isoenzyme of HRP, Sigma type VIII, was bound more intensely and more widely to liver sinusoidal cells than another acidic isoenzyme, Sigma type VII, a basic isoenzyme, Sigma type IX, and the routinely used preparation,Sigma type VI. The effect of the temperature on the binding reaction was re-examined with an improved procedure. In contradistinction to the previous finding, strong binding of HRP after 2–4 h incubation at 4°C was observed.