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?
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) IV 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 simultaneous coupling. Above pH 5.0 a one-step 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 100x lower than that of haemoglobin when cathepsin D from bovine spleen was used.(ABSTRACT TRUNCATED AT 250 WORDS)
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000818:Animals; D002402:Cathepsin D / Q000378:metabolism*; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000378:metabolism; D006651:Histocytochemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D066298:In Vitro Techniques; D009281:Naphthalenes; D009842:Oligopeptides; D051381:Rats; D013379:Substrate Specificity
D009281:Naphthalenes; D009842:Oligopeptides; C055836:benzyloxycarbonyl-arginyl--glycyl-phenylalanyl-phenylalanyl-proline-4-methoxy-2-naphthylamine; C055840:benzyloxycarbonyl-arginyl-glycyl-phenylalanyl-phenylalanyl-leucine-4-methoxy-2-naphthylamine; D015081:2-Naphthylamine; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4; D002402:Cathepsin D