Ubiquitin-dependent proteolytic pathway in wheat germ: Isolation of multiple forms of ubiquitin-activating enzyme, E1

Journal Article · Tue Jan 24 04:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00428a048· OSTI ID:5405703

Hatfield, P M; Vierstra, R D ^[1]

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1. Univ. of Wisconsin, Madison (USA)

Ubiquitin is a highly conserved protein involved in several important regulatory processes through its ATP-dependent, covalent ligation to a variety of eukaryotic target proteins. The authors describe here the characterization of ubiquitin conjugation in wheat germ extracts and the subsequent isolation of enzymes involved in conjugation. With {sup 125}I-ubiquitin as a substrate, wheat germ extracts form conjugates with either endogenous or added proteins. Ubiquitin-activating enzyme (E1) was purified from wheat germ extracts by using a modification of the covalent affinity chromatography procedure of Ciechanover et al. E1 from wheat germ, like that from rabbit reticulocytes, formed thiol ester intermediates with ubiquitin in the presence of ATP. Purified E1 preparations contained three polypeptides of apparent molecular masses of 117, 123, and 126 kDa after NaDodSO{sub 4}-PAGE. Under nondenaturing conditions, these proteins have native molecular masses of {approx}115 kDa, indicating that they exist as monomers. They concluded that all three species were E1 on the basis of their coelution with E1 activity, by immunorecognition by anti-human E1 antibodies, and by the similarity of their peptide maps. Furthermore, antibodies prepared against wheat germ E1's recognized E1 from rabbit reticulocytes. All three wheat germ E1's were detected in crude extracts prepared under conditions that minimized proteolysis, suggesting that the heterogeneity of the purified E1 preparations was not the result of posthomogenization breakdown. The immunological similarity of animal and plant E1's indicates that this conjugation enzyme, like ubiquitin, has been conserved through evolution.

• OSTI ID: 5405703
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:2; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ATP
BETA DECAY RADIOISOTOPES
BIOLOGICAL PATHWAYS
CEREALS
CHEMICAL ACTIVATION
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENZYMATIC HYDROLYSIS
ENZYMES
GRAMINEAE
HYDROLYSIS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
LABELLED COMPOUNDS
LILIOPSIDA
LYSIS
MAGNOLIOPHYTA
NUCLEI
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PLANTS
PROTEINS
PURIFICATION
RADIOISOTOPES
SOLVOLYSIS
WHEAT