Biochemical and histochemical evidence for lysosomal proteases in rodent lenses.
The lysosomal proteases cathepsin B and dipeptidyl peptidase II (DPP II) were identified biochemically in the supernatant fraction of rat and guinea-pig lens homogenates, using as substrates CBZ-Arg-Arg-4-methylcoumarin-7-amide (AMC) (5 microM, pH 6.0, 37 degrees C for cathepsin B, and Lys-Ala-AMC or Lys-Pro-AMC (5 microM, pH 5.5, 37 degrees C) for DPP II. Cathepsin B-like activity was totally inhibited by the specific inhibitor lepeptin (0.1 mM); while DPP-II-like activity was partially inhibited by Tris (10 mM) or puromycin (0.1 mM), which are specific for DPP II, and by phenylmethylsulphonyl fluoride (PMS-F), an inhibitor of serine class proteases. DPP-II-like activity also was demonstrated histochemically. The histochemical preparations consisted of cryostat sections (6 micron) of glutaraldehyde-fixed rat lenses, incubated in medium containing cacodylic acid-NaOH (0.05 M), Lys-Pro-4-methoxy-2-naphthylamide (MNA) or Lys-Ala-MNA (1 mM), and 0.1% w:v Fast Blue B salt (pH 5.5, 37 degrees C). Control sections were treated with medium in which the substrates were replaced by Lys-Ser-MNA (1 mM), which is not acted upon by DPP II; or medium containing PMS-F (1 mM). Sections treated with experimental medium containing Lys-Pro-MNA showed intense enzymatic activity throughout the epithelium and bow region. In the bow region and superficial cortex activity was mostly granular, suggesting a lysosomal localization. Histochemical activity was reduced partially by inclusion of PMS-F, and totally by substituting Lys-Ser-MNA for Lys-Pro-MNA.
D000818:Animals; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006168:Guinea Pigs; D006651:Histocytochemistry; D007908:Lens, Crystalline / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence
D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100151:dipeptidyl peptidase II; D002401:Cathepsin B