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Title: Purification of two high molecular weight proteases from rabbit reticulocyte lysate

Abstract

The authors have purified two large proteases from rabbit reticulocyte lysate. The enzymes are so similar in their chromatographic behavior that each is the only significant contaminant of the other during the final stages of purification. At pH 7.8, both hydrolyze /sup 125/I-..cap alpha..-casein and 4-methylcoumaryl-7-amide (MCA) derivatives with tyrosine, phenylalanine or arginine at the P/sub 1/ position. The larger, ATP-dependent enzyme degrades ubiquitin-lysozyme conjugates, but it does not degrade unmodified lysozyme. Hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA by this enzyme is also stimulated two-fold in the presence of ATP. The protease has a molecular weight of 950,000 based on sedimentation, gel filtration and non-denaturing PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the protease is composed of a number of subunits with molecular masses between 32 and 110 kDa. Densitometric analysis showed equivalent amounts of the two larger chains, and the presence of one copy of each in the native enzyme would be consistent with an M/sub r/ of 950,000. The smaller protease has a molecular weight of 700,000 and is composed of 8 to 10 subunits ranging from 21,000 to 32,000. It cleaves ubiquitin-lysozyme conjugates only slightly, and hydrolysis of conjugates or fluorogenic peptide substrates is not stimulated by ATP. Thismore » protease appears similar, if not identical, to the multicatalytic protease complex first purified by Wilk and Orlowski.« less

Authors:
; ;
Publication Date:
Research Org.:
Univ. of Utah, Salt Lake City
OSTI Identifier:
6126538
Report Number(s):
CONF-870644-
Journal ID: CODEN: FEPRA; TRN: 87-034013
Resource Type:
Conference
Resource Relation:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States); Journal Volume: 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; PEPTIDE HYDROLASES; FRACTIONATION; ATP; ELECTROPHORESIS; IODINE 125; MOLECULAR WEIGHT; RABBITS; TRACER TECHNIQUES; ANIMALS; BETA DECAY RADIOISOTOPES; DAYS LIVING RADIOISOTOPES; ELECTRON CAPTURE RADIOISOTOPES; ENZYMES; HYDROLASES; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; MAMMALS; NUCLEI; NUCLEOTIDES; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; RADIOISOTOPES; SEPARATION PROCESSES; VERTEBRATES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Hough, R., Pratt, G., and Rechsteiner, M. Purification of two high molecular weight proteases from rabbit reticulocyte lysate. United States: N. p., 1987. Web.
Hough, R., Pratt, G., & Rechsteiner, M. Purification of two high molecular weight proteases from rabbit reticulocyte lysate. United States.
Hough, R., Pratt, G., and Rechsteiner, M. Fri . "Purification of two high molecular weight proteases from rabbit reticulocyte lysate". United States.
@article{osti_6126538,
title = {Purification of two high molecular weight proteases from rabbit reticulocyte lysate},
author = {Hough, R. and Pratt, G. and Rechsteiner, M.},
abstractNote = {The authors have purified two large proteases from rabbit reticulocyte lysate. The enzymes are so similar in their chromatographic behavior that each is the only significant contaminant of the other during the final stages of purification. At pH 7.8, both hydrolyze /sup 125/I-..cap alpha..-casein and 4-methylcoumaryl-7-amide (MCA) derivatives with tyrosine, phenylalanine or arginine at the P/sub 1/ position. The larger, ATP-dependent enzyme degrades ubiquitin-lysozyme conjugates, but it does not degrade unmodified lysozyme. Hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA by this enzyme is also stimulated two-fold in the presence of ATP. The protease has a molecular weight of 950,000 based on sedimentation, gel filtration and non-denaturing PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the protease is composed of a number of subunits with molecular masses between 32 and 110 kDa. Densitometric analysis showed equivalent amounts of the two larger chains, and the presence of one copy of each in the native enzyme would be consistent with an M/sub r/ of 950,000. The smaller protease has a molecular weight of 700,000 and is composed of 8 to 10 subunits ranging from 21,000 to 32,000. It cleaves ubiquitin-lysozyme conjugates only slightly, and hydrolysis of conjugates or fluorogenic peptide substrates is not stimulated by ATP. This protease appears similar, if not identical, to the multicatalytic protease complex first purified by Wilk and Orlowski.},
doi = {},
journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 46:6,
place = {United States},
year = {Fri May 01 00:00:00 EDT 1987},
month = {Fri May 01 00:00:00 EDT 1987}
}

Conference:
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