Presence of a Thiol Protease in Regenerating Rat-Liver Nuclei

Partial Purification and Some Properties

Authors


Abstract

1. Nuclei of regenerating rat liver washed with Triton X-100 were found to contain a new protease. Since the enzymatic activity for degrading ribosomal proteins was inhibited in vivo by administration of E-64, a thiol protease inhibitor, the enzyme may participate in the degradation of newly synthesized ribosomal proteins and histones in regenerating rat liver nuclei as reported previously by us [Biochem. Biophys. Res. Commun. 75, 525–531 (1977)]. The optimum pH was 5.5.

2. The enzyme was extracted from washed nuclei and partially purified by gel filtration through Sepharose 6B. Its molecular weight was about 40000. A maximal activity of partially purified enzyme was observed in the presence of 1 mM EDTA and 2 mM dithiothreitol at pH 5.5. It was inhibited by thiol reagents, E-64, leupeptin and heavy metal ions. The enzyme degraded ribosomal proteins endoproteolytically and degraded most proteins tested as substrates, although liver cell sap proteins and serum albumin were less degraded than ribosomal proteins and histones. α-N-Benzoylarginine-β-naphthylamide and benzoylarginine amide were not hydrolyzed.

Abbreviations
iPr2P-F

diisopropylfluorophosphate

BzArg-Nap

α-N-benzoylarginine-β-naphthylamide

PhMeSO2F

phenyl-methylsulfonyl fluoride

BzArgNH2

α-N-benzoylarginine amide

E-64

mixture of the optical isomers N-[N-(DL-3-transcarboxy-iran-carbonyl)-L-leucyl]-agmatine

Leupeptin

N-acetylleucyl-leucyl-arginal

Enzymes
 

Cathepsin B1 (EC 3.4.22.1)

 

rhodanese (EC 2.8.1.1)

 

glucose-6-phosphatase (EC 3.1.3.9)

 

acid phosphatase (EC 3.1.3.2)

 

deoxyribonuclease (EC 3.1.21.1)

Ancillary