Purification and characterization of cathepsin L-like proteinase from goat brain.
Cathepsin L-like proteinase was purified approximately 1708-fold with 40% activity yield to an apparent electrophoretic homogeneity from goat brain by homogenization, acid-autolysis at pH 4.2, 30-80% (NH4)2SO4 fractionation, Sephadex G-100 column chromatography and ion-exchange chromatography on CM-Sephadex C-50 at pH 5.0 and 5.6. The molecular weight of proteinase was found to be approximately 65,000 Da, by gel-filtration chromatography. The pH optima were 5.9 and 4.5 for the hydrolysis of Z-Phe-Arg-4mbetaNA (benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methoxy-beta-naphthylamide) and azocasein, respectively. Of the synthetic chromogenic substrates tested, Z-Phe-Arg-4mbetaNA was hydrolyzed maximally by the enzyme (Km value for hydrolysis was 0.06 mM), followed by Z-Val-Lys-Lys-Arg-4mbetaNA, Z-Phe-Val-Arg-4mbetaNA, Z-Arg-Arg-4mbetaNA and Z-Ala-Arg-Arg-4mbetaNA. The proteinase was activated maximally by glutathione in conjunction with EDTA, followed by cysteine, dithioerythritol, thioglycolic acid, dithiothreitol and beta-mercaptoethanol. It was strongly inhibited by p-hydroxymercuribenzenesulphonic acid, iodoacetic acid, iodoacetamide and microbial peptide inhibitors, leupeptin and antipain. Leupeptin inhibited the enzyme competitively with Ki value 44 x 10(-9) M. The enzyme was strongly inhibited by 4 M urea. Metal ions, Hg(2+), Ca(2+), Cu(2+), Li(2+), K(+), Cd(2+), Ni(2+), Ba(2+), Mn(2+), Co(2+) and Sn(2+) also inhibited the activity of the enzyme. The enzyme was stable between pH 4.0-6.0 and up to 40 degrees C. The optimum temperature for the hydrolysis of Z-Phe-Arg-4mbetaNA was approximately 50-55 degrees C with an activation energy Ea of approximately 6.34 KCal mole(-1).
Department of Biochemistry, Kurukshetra University, Kurukshetra 136 119, Haryana, India.
Rachna
R
Sadana
Ashwani
A
Mittal
Shiwani
S
Khurana
Hari
H
Singh
Ramesh C
RC
Kamboj
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D056668:Cathepsin L / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005591:Chemical Fractionation / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006041:Goats; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D019163:Reducing Agents / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D011480:Protease Inhibitors; D019163:Reducing Agents; D014508:Urea; D056668:Cathepsin L