Are Z-Arg-Gly-Phe-Phe-Leu-MNA and Z-Arg-Gly-Phe-Phe-Pro-MNA suitable substrates for the demonstration of cathepsin D activity?

Summary

The suitability of Z-Arg-Gly-Phe-Phe-Leu-MNA and Z-Arg-Gly-Phe-Phe-Pro-MNA for the assessment of cathepsin D activity was tested in biochemical and histochemical experiments. Substrates were dissolved in dimethylformamide and used at 0.1–0.5 mM in various buffers over a pH range of 3.5–7.4. Homogenates of various rat organs and isolated purified enzymes [cathepsin D from bovine spleen, dipeptidyl peptidase (DPP) I.V from porcine kidney and rat lung] were used as enzyme sources. Pepstatin, di-isopropylfluorophosphate (DFP),p-chloromercuribenzoate,o-phenanthroline and a series of DPP IV inhibitors were used in inhibitor experiments. At pH 3.5 and 5.0, substrates were used in a two-step postcoupling procedure with aminopeptidase M and dipeptidyl peptidase IV as auxiliary enzymes and Fast Blue BB as coupling agent. Results were compared with those obtained with haemoglobin. Above pH 5.0 substrates were used in a one-step postcoupling procedure.

Cryostat sections of snap-frozen or cold aldehyde-fixed tissue pieces of various rat organs and biopsies of human jejunal mucosa were used in histochemical experiments. As in biochemical tests a two-step procedure was used in the pH range 3.5–5.0, but Fast Blue B was used in the second step for the simultancous coupling. Above pH 5.0 a onestep simultaneous azo coupling procedure was used with Fast Blue B as coupling agent.

At pH 3.5 the hydrolysis rate of both synthetic substrates was about 100 x lower than that of haemoglobin when cathepsin D from bovine spleen was used. The activity was inhibited by pepstatin. With increasing pH the hydrolysis rate of Z-Arg-Gly-Phe-Phe-Pro-MNA increased, while that of Z-Arg-Gly-Phe-Phe-Leu-MNA decreased when organ homogenates were used as enzyme sources. However, the activity was not inhibited by pepstatin. It was inhibited by DFP. The extent of the inhibition with other substances was species and organ dependent. Z-Arg-Gly-Phe-Phe-Pro-MNA was also cleaved by isolated and purified DPP IV of porcine kidney and rat lung and the activity was inhibited by DFP and DPP IV inhibitors.

In histochemical experiments the staining obtained with both synthetic substrates at pH 3.5 was weak and rather diffuse, with only slight accentuation or none at all in the lysosomal region of cells. In the pH range 5.5–7.4 a very distinct reaction was observed with Z-Arg-Gly-Phe-Phe-Pro-MNA only. The reaction product was localized in the brush border of enterocytes and of cells of the proximal kidney tubules. Endothelial cells of glomeruli and capillaries of the propria of the human jejunum also displayed a positive reaction. Lymphocytes in the propria of rat small intestine reacted to some extent. The reaction was inhibited by DFP. The extent of the inhibition with other substances varied.

Z-Arg-Gly-Phe-Phe-Leu-MNA and Z-Arg-Gly-Phe-Phe-Pro-MNA are not efficient substrates for the assessment of cathepsin D activity. In histochemical studies diffusion artifacts must always be considered. In the pH range 5.5–7.4, Z-Arg-Gly-Phe-Phe-Pro-MNA is cleaved by a serine endopeptidase and by a metalloendopeptidase. It remains to be established whether prolyl endopeptidase or DPP IV (or both) and which metalloendopeptidase are responsible for the cleavage. In the evaluation of enterobiopsies the demonstration of this activity is a sensitive means for the assessment of the state of the brush border.