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Some properties of human and bovine brain cathepsin B

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Abstract

Cathespin B has been purified 750-fold to apparent homogeneity from human and bovine brain cortex using ammonium sulfate fractionation (30–70%), chromatography on Sephadex G-100, CM-Sephadex C-50, and concanavalin A-Sepharose. Enzyme was assayed fluorometrically at pH 4.0 with pyridoxyl-hemoglobin in the presence of 1 mM DTT and 1 mM EDTA. Properties of the enzyme from the two sources proved to be similar. On disc PAGE the purified preparation produced two bands associated with proteinase activity that are due to existence of two multiple forms of brain cathepsin B with pI 6.1 and 6.8. The enzyme is completely inactivated by thiol-blocking reagents, leupeptin, E-64, and demands thiol compounds for its ultimate activity. Z-Phe-Ala-CHN2 is a potent inhibitor of the enzyme (K 2nd=1280 M−1s−1) in contrast to Z-Phe-Phe-CHN2 (K 2nd=264 M−1s−1). pH optimum in the reaction of hydrolysis of Pxy-Hb is 4.0–6.0,K M(app.) =10−5 M. Cathepsin B splits azocasein: pH optimum 5.0–6.0,K M(app.)=2.2·10−5 M, but inclusion of urea in the incubation medium depresses the azocaseinolytic activity of the enzyme 1.5-fold. It does not split Lys-NNap, Arg-NMec and is not inhibited by bestatin. The specific activity of brain cathepsin B with Z-Arg-Arg-NNapOMe at pH 6.0 is 10-fold higher than with Bz-Arg-NNap, Z-Gly-Gly-Arg-NNap is a poor substrate. With Z-Arg-Arg-NMec and Bz-Phe-Val-Arg-NMec the specific acitivity is 80 and 35%, respectively of that with Z-Phe-Arg-NMec.

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Abbreviations

Pxy-Hb:

pyridoxyl-hemoglobin

LMW:

low molecular weight

HMW:

high molecular weight

BSA:

bovine serum albumin

SDS:

sodium dodecyl sulfate

PAGE:

polyacrylamide gel electrophoresis

DFP:

diisopropyl-fluorophosphate

DTT:

dithiothreitol

Bz:

benzoyl

Z:

benzyloxycarbonyl

-NMec:

7-(4-methyl)coumarylamide

-NNap:

2-naphthylamide

-NNapOme:

2-(4-methoxy)naphthylamide

CHN2 :

diazomethane

E-64:

1,3-carboxy-trans-2,3-epoxypropyl-L-leucyl-amido(4-guanidino)buthane

Boc:

t-butoxycarbonyl

N-EM:

N-ethylmaleimide

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Authors and Affiliations

  1. Institute of Biochemistry, Armenian SSR Academy of Sciences, 375044, Yerevan, USSR

    Anahit Azaryan, Nina Barkhudaryan & Armen Galoyan

Authors
  1. Anahit Azaryan
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  2. Nina Barkhudaryan
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  3. Armen Galoyan
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Special Issue dedicated to Dr. Eugene Kreps.

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Azaryan, A., Barkhudaryan, N. & Galoyan, A. Some properties of human and bovine brain cathepsin B. Neurochem Res 10, 1511–1524 (1985). https://doi.org/10.1007/BF00988863

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