An antibody probe to determine the native species of glycinamide ribonucleotide transformylase in chicken liver

Antibody probes of Western blots [Renart, J., Reiser, J., & Stark, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3116] of chicken liver homogenates under various conditions revealed that glycinamide ribonucleotide transformylase can be rapidly proteolyzed in such homogenates. These findings, along...

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Bibliographic Details
Published in:Biochemistry (Easton) 1984-08, Vol.23 (17), p.3979-3986
Main Authors: Young, M, Sammons, R. Douglas, Mueller, W. Thomas, Benkovic, S. J
Format: Article
Language:English
Subjects:
Acyltransferases - isolation & purification
Acyltransferases - metabolism
Analytical, structural and metabolic biochemistry
Animals
Biological and medical sciences
Chickens
Chymotrypsin - metabolism
Enzymes and enzyme inhibitors
Fundamental and applied biological sciences. Psychology
Hydroxymethyl and Formyl Transferases
Immunosorbent Techniques
Kinetics
Liver - enzymology
Macromolecular Substances
Molecular Weight
Peptide Hydrolases - metabolism
Phosphoribosylglycinamide Formyltransferase
Transferases
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Summary:Antibody probes of Western blots [Renart, J., Reiser, J., & Stark, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3116] of chicken liver homogenates under various conditions revealed that glycinamide ribonucleotide transformylase can be rapidly proteolyzed in such homogenates. These findings, along with molecular weight measurements by ultracentrifugation, identify the true form of glycinamide ribonucleotide transformylase as a monomeric protein of 117000 daltons. This protein has been purified 400-fold in 44% yield from chicken liver in one step on an affinity column of 10-formyl-5,8-dideazafolate-Sepharose. Native glycinamide ribonucleotide transformylase retains full activity after proteolytic cleavage to a form (Mr 55000) similar to fragments seen in the Western blot of the homogenates. This phenomenon may be responsible for the previous identification of glycinamide ribonucleotide (GAR) transformylase as a dimer of 55000-dalton subunits. Similar analyses using antibodies to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase [Mueller, W. T., & Benkovic, S. J. (1981) Biochemistry 20, 337] and trifunctional enzyme [Smith, G. K., Mueller, W. T., Wasserman, G. F., Taylor, W. D., & Benkovic, S. J. (1980) Biochemistry 19, 4313] confirm that these two proteins were isolated in their native forms.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00312a027