In vitro incorporation of label from (. gamma. /sup 32/P) ATP into isocitrate lyase of Escherichia coli

Conference · Thu May 01 00:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5333808

Robertson, F; Reeves, H C

A partially purified sonic extract of an E. coli mutant, constitutive for the glyoxylate by-pass enzymes, was incubated with (..gamma../sup 32/P) ATP in 50 mM MOPS buffer at pH 7.5 containing 2 mM 2-mercaptoethanol, 10 mM MgCl/sub 2/ and 10% glycerol at room temperature for 1 h. Incubation was continued for another hour following the addition of unlabeled ATP. The assay was terminated by the addition of 10 mM EDTA. The assay mixture was then analyzed, by several electrophoretic techniques and subsequent autoradiography, to determine which of the proteins in the extract had incorporated label. Isoelectric focusing was performed at pH 3-10 and pH 4-4.5. Specific enzyme staining of IEF gels revealed that active isocitrate lyase (ICL) co-migrated with a protein band which also was radioactive. Immuno blots of these IEF gels, using antibody raised in rabbits against ICL, also demonstrated incorporation of label. SDS-PAGE autoradiograms displayed a labeled protein which co-migrated with purified E. coli ICL, which has a subunit M/sub r/ of 48,000. The mixture was also analyzed by 2-D PAGE which further demonstrated that a labeled protein and authentic purified ICl co-migrate.

• Research Organization: Arizona State Univ., Tempe
• OSTI ID: 5333808
• Report Number(s): CONF-8606151-; TRN: 86-031435
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
LYASES
LABELLING
PROTEINS
AUTORADIOGRAPHY
ATP
ELECTROPHORESIS
ESCHERICHIA COLI
IN VITRO
PH VALUE
PHOSPHORUS 32
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
DAYS LIVING RADIOISOTOPES
ENZYMES
ISOTOPES
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS ISOTOPES
RADIOISOTOPES
550201* - Biochemistry- Tracer Techniques