Characterization and purification of carbohydrate response element-binding protein of the rat L-type pyruvate kinase gene promoter.
The L-III transcriptional regulatory element of the rat pyruvate kinase L gene is located between -170 and -150 base pairs upstream of the hepatocyte-specific transcription initiation site. As the L-III element is not only necessary for cell type-specific expression but also for transcriptional stimulation by carbohydrates, it is also referred to as a carbohydrate-response element. Electrophoretic mobility shift assays using rat liver nuclear extract showed that L-III element-binding protein (L-IIIBP) was observed as multiple bands. These bands disappeared when the nuclear extract was preincubated at 60 degrees C for 5 min and were competed with unlabeled L-III oligonucleotide but not with unlabeled adenovirus major late promoter E box oligonucleotide. In addition, these bands were not affected in the presence of antiserum against upstream stimulating factor (USF). Thus, we conclude that L-IIIBP is different from USF. Then, heat-labile L-IIIBP was purified from rat liver nuclear extracts. Purified L-IIIBP exhibited two bands on sodium dodecyl sulfate/polyacrylamide gel electrophoresis by silver staining. Ultraviolet crosslinking experiment showed that both bands had binding activity to the L-III oligonucleotide.
Yamada
K
K
Department of Biochemistry, Fukui Medical University, Shimoaizuki, Matsuoka, Fukui, 910-1193, Japan.
Tanaka
T
T
Noguchi
T
T
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002241:Carbohydrates / Q000494:pharmacology*; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006358:Hot Temperature; D006801:Humans; D007106:Immune Sera; D008099:Liver / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011770:Pyruvate Kinase / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020218:Response Elements / Q000235:genetics*; D014157:Transcription Factors / Q000276:immunology / Q000378:metabolism; D051720:Upstream Stimulatory Factors
D002241:Carbohydrates; D004268:DNA-Binding Proteins; D007106:Immune Sera; D009841:Oligonucleotides; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; D011770:Pyruvate Kinase