Please use this identifier to cite or link to this item: http://nopr.niscair.res.in/handle/123456789/3807
Title: Purification and characterization of cathepsin L-like proteinase from goat brain
Authors: Sadana, Rachna
Mittal, Ashwani
Khurana, Shiwani
Singh, Hari
Kamboj, Ramesh C
Keywords: cathepsin L-like proteinase
purification
characterization
goat brain
Issue Date: Oct-2003
Publisher: CSIR
Abstract: Cathepsin L-like proteinase was purified ~1708-fold with 40% activity yield to an apparent electrophoretic homogeneity from goat brain by homogenization, acid-autolysis at pH 4.2, 30-80% (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> 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 ~65,000 Da, by gel-filtration chromatography. The pH optima were 5.9 and 4.5 for the hydrolysis of Z-Phe-Arg-4mβNA (benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methoxy-β-naphthylamide) and azocasein, respectively. Of the synthetic chromogenic substrates tested, Z-Phe-Arg-4mβNA was hydrolyzed maximally by the enzyme (K<sub>m</sub> value for hydrolysis was 0.06 mM), followed by Z-Val-Lys-Lys-Arg-4mβNA, Z-Phe-Val-Arg-4mβNA, Z-Arg-Arg-4mβNA and Z-Ala-Arg-Arg-4mβNA. The proteinase was activated maximally by glutathione in conjunction with EDTA, followed by cysteine, dithioerythritol, thioglycolic acid, dithiothreitol and β-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 44x10<sup>-9</sup> M. The enzyme was strongly inhibited by 4 M urea. Metal ions, Hg<sup>2+</sup>, Ca<sup>2+</sup>, Cu<sup>2+</sup>, Li<sup>2+</sup>, K<sup>+</sup>, Cd<sup>2+</sup>, Ni<sup>2+</sup>, Ba<sup>2+</sup>, Mn<sup>2+</sup>, Co<sup>2+</sup> and Sn<sup>2+</sup> also inhibited the activity of the enzyme. The enzyme was stable between pH 4.0-6.0 and up to 40ºC. The optimum temperature for the hydrolysis of Z-Phe-Arg-4mβNA was ~50-55ºC with an activation energy E<sub>a</sub> of ~6.34 KCal mole<sup>-1 </sup>.
Description: 315-323
URI: http://hdl.handle.net/123456789/3807
ISSN: 0301-1208
Appears in Collections:IJBB Vol.40(5) [October 2003]

Files in This Item:
File Description SizeFormat 
IBB 40(5) 315-323.pdf276.9 kBAdobe PDFView/Open


Items in NOPR are protected by copyright, with all rights reserved, unless otherwise indicated.