A selective colorimetric assay for cathepsin L using Z-Phe-Arg-4-methoxy-beta-naphthylamide.
Among the intracellular proteinases, the thiol proteinases such as cathepsin B (EC 3.4.22.1), cathepsin H (EC 3.4.22.16) and cathepsin L (EC 3.4.22.15) which act at slightly acidic pHs are more likely to play an important role in lysosomal protein catabolism. Out of these, cathepsin L plays a major role primarily because it has high degradative activity on cellular and matrix proteins. However, the studies on cathepsin L in crude homogenates and subcellular fractions have always been hampered by the lack of a specific substrate to exclusively measure the activity of this proteinase. The only synthetic substrate alpha-N-benzyloxycarbonyl-L-Phe-L-Arg-4-methoxy-beta-naphthylamide (Z-Phe-Arg-NNapOMe) which is hydrolysed by cathepsin L is hydrolysed equally well by cathepsin B. This substrate was manipulated to act as a selective substrate for cathepsin L. In presence of 4 M urea at pH 5.0, cathepsin B (the only other cathepsin which also hydrolyses Z-Phe-Arg-NNapOMe) was inactivated and, therefore, under these conditions, the enzyme activity quantitated by using this substrate is only due to cathepsin L. Using this newly-developed colorimetric assay method specific for cathepsin L, the subcellular and regional distribution of this proteinase were established in goat brain tissue. About 80% cathepsin L activity was recovered in the lysosomal fraction thus establishing its lysosomal nature. Among the various brain parts, highest activity was found in cerebrum followed by cerebellum, pituitary body, pons-varolli, thalamus, medulla-oblongata and hypothalamus.
Kamboj
R C
RC
Department of Chemistry, Kurukshetra University, Haryana, India.
Pal
S
S
Raghav
N
N
Singh
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001587:Benzoylarginine-2-Naphthylamide / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D002401:Cathepsin B / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000032:analysis* / Q000378:metabolism; D003124:Colorimetry; D003546:Cysteine Endopeptidases / Q000032:analysis* / Q000378:metabolism; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism*; D010450:Endopeptidases; D006041:Goats; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D014018:Tissue Distribution; D014508:Urea / Q000494:pharmacology
D004151:Dipeptides; D001587:Benzoylarginine-2-Naphthylamide; D014508:Urea; C085881:alpha-N-benzyloxycarbonyl-phenylalanyl-arginyl-4-methoxy-beta-naphthylamide; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D056668:Cathepsin L