High performance liquid chromatography resolution of ubiquitin pathway enzymes from wheat germ. [Triticum vulgare]

Journal Article · Mon Oct 01 04:00:00 EDT 1990 · Plant Physiology; (USA)

DOI:https://doi.org/10.1104/pp.94.2.710· OSTI ID:5852840

Sullivan, M L; Callis, J; Vierstra, R D ^[1]

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1. Univ. of Wisconsin, Madison (USA)

The highly conserved protein ubiquitin is involved in several cellular processes in eukaryotes as a result of its covalent ligation to a variety of target proteins. Here, we describe the purification of several enzymatic activities involved in ubiquitin-protein conjugate formation and disassembly from wheat germ (Triticum vulgare) by a combination of ubiquitin affinity chromatography and anion-exchange high performance liquid chromatography. Using this procedure, ubiquitin activating enzyme (E1), several distinct ubiquitin carrier proteins (E2s) with molecular masses of 16, 20, 23, 23.5, and 25 kilodaltons, and a ubiquitin-protein hydrolase (isopeptidase) were isolated. Purified E1 formed a thiol ester linkage with {sup 125}I-ubiquitin in an ATP-dependent manner and transferred bound ubiquitin to the various purified E2s. The ubiquitin protein hydrolase fraction was sensitive to hemin, and in an ATP-independent reaction, was capable of removing the ubiquitin moiety from both ubiquitin {sup 125}I-lysozyme conjugates ({epsilon}-amino or isopeptide linkage) and the ubiquitin 52-amino acid extension protein fusion ({alpha}-amino or peptide linkage). Using this procedure, wheat germ represents an inexpensive source from which enzymes involved in the ubiquitin pathway may be isolated.

• OSTI ID: 5852840
• Journal Information: Plant Physiology; (USA), Journal Name: Plant Physiology; (USA) Vol. 94:2; ISSN 0032-0889; ISSN PLPHA
• Country of Publication: United States
• Language: English

Related Subjects

551001* -- Physiological Systems-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ATP
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL FUNCTIONS
BIOLOGICAL PATHWAYS
CEREALS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ENZYME ACTIVITY
FUNCTIONS
GRASS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LILIOPSIDA
MAGNOLIOPHYTA
NUCLEI
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PLANTS
PROTEINS
RADIOISOTOPES
REACTION KINETICS
SEPARATION PROCESSES
TRACER TECHNIQUES
WHEAT