Analysis of photoaffinity-labeled aryl hydrocarbon receptor heterogeneity by two-dimensional gel electrophoresis

Journal Article · Tue Jul 03 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00478a014· OSTI ID:6038581

Perdew, G H; Hollenback, C E ^[1]

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1. Purdue Univ., West Lafayette, IN (USA)

The level of charge heterogeneity in the aryl hydrocarbon receptor (AhR) was examined by high-resolution denaturing two-dimensional (2D) gel electrophoresis. Hepa 1c1c7 cell cytosolic fraction was photoaffinity-labeled with 2-azido-3-({sup 125}I)-iodo-7,8-dibromodibenzo-p-dioxin and applied to isoelectric focusing (IEF) tube gels. After optimization of focusing conditions a broad peak of radioactivity was detected in the apparent pI range of 5.2-5.7. IEF tube gels were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by visualization of the radiolabeled AhR by autoradiography; three distinct isoforms were detected. The same 2D electrophoretic isoform pattern was obtained when the AhR from Hepa 1c1c7 was photoaffinity-labeled in cell culture. BP{sup r}Cl cells, a mutant line derived from Hepa 1c1c7 cells, contain an AhR that is unable to bind to DNA. Photoaffinity-labeled BP{sup r}Cl cytosolic fractions were subjected to 2D gel electrophoretic analysis resulting in essentially the same molecular weight and isoform pattern as seen in Hepa 1c1c7 cytosol. This result would suggest that if a mutation is present in the BP{sup r}Cl AhR it has not caused a significant change in its IEF pattern, although a small shift in the pI values was observed. Two-dimensional gel electrophoresis of photoaffinity-labeled cytosolic fractions from HeLa cells, the rat liver tumor cell line McA-RH777, and buffalo rat thymus revealed three isoforms, essentially the same isoform pattern as in Hepa 1c1c7 cells. This would indicate that despite the considerable molecular weight polymorphism between species the level of charge heterogeneity is high conserved.

• OSTI ID: 6038581
• Journal Information: Biochemistry; (USA), Vol. 29:26; ISSN 0006-2960
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
POLYCYCLIC AROMATIC HYDROCARBONS
RADIORECEPTOR ASSAY
RECEPTORS
TWO-DIMENSIONAL ELECTROPHORESIS
DIOXIN
HELA CELLS
IODINE 125
PH VALUE
RATS
ANIMALS
AROMATICS
BETA DECAY RADIOISOTOPES
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
HETEROCYCLIC COMPOUNDS
HYDROCARBONS
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
MAMMALS
MEMBRANE PROTEINS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
PROTEINS
RADIOISOTOPES
RODENTS
TRACER TECHNIQUES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques