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A Selective Colorimetric Assay for Cathepsin L Using Z-Phe-Arg-4-methoxy-beta-naphthylamide

A Selective Colorimetric Assay for Cathepsin L Using Z-Phe-Arg-4-methoxy-beta-naphthylamide

R C Kamboj, S Pal, N Raghav, H Singh

Biochimie. 1993;75(10):873-8.

PMID: 8312391

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP100930061	Phe-Pro-Ala-Met 4-methoxy-β-naphthylamide		100930-06-1	Price

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