Subject(s)
Blotting, Western/methods , Proteins/isolation & purification , Biotechnology , Biotin/chemistry , Cell Line , HeLa Cells , Humans , NF-kappa B/chemistry , NF-kappa B/isolation & purification , Proteins/chemistry , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/isolation & purification , Proto-Oncogene Proteins c-rel , StreptavidinABSTRACT
Denver, Tokyo, and Salt Lake City investigators recently published different complimentary deoxyribonucleic acid (cDNA) sequences for human liver xanthine dehydrogenase/xanthine oxidase (XD/XO). The gene encoding the Denver cDNA was subsequently linked to juvenile familial amyotrophic lateral sclerosis (JFALS) at chromosome 2q33 and has been proposed as the ALS2 locus. The present investigation was undertaken to elucidate the differences between the three cDNA sequences, and we provide evidence that the Denver cDNA encodes aldehyde oxidase (AO): first, the Denver cDNA sequence diverged significantly from the Tokyo and Salt Lake City cDNA sequences which were very similar; second, the deduced protein sequence from the Denver cDNA was very similar to the amino acid sequence of purified rabbit liver AO protein; third, the deduced Denver protein sequence was 76% identical to the encoded 101 amino acid long peptides from partial cDNAs for rabbit and rat AO and 81.7% identical to 300 amino acids from an incomplete cDNA encoding bovine AO; fourth, the Denver gene was expressed in liver, kidney, lung, pancreas, prostate, testes, and ovary while the Tokyo XD gene was expressed predominantly in liver and small intestine; fifth, the Denver gene was previously mapped to chromosome 2q33 which is syntenic to the mouse AO locus on chromosome 1. Our results have revealed dramatic similarities in protein and DNA sequence in the human molybdenum hydroxylases, have uncovered unanticipated complexity in the human molybdenum hydroxylase genes, and advance the potential for AO derived oxygen radicals in JFALS and other human diseases.
ABSTRACT
We isolated cDNAs encoding xanthine dehydrogenase (XD; xanthine:NAD+ oxidoreductase, EC 1.1.1.204) from a human liver cDNA library. The complete nucleotide sequence of human XD was determined; the deduced amino acid sequence encoded a protein of 1336 amino acid residues of M(r) 147,782. Human XD possessed many of the signature sequences typical of XDs from flies and rodents, including an unusual cysteine distribution, a potential 2Fe/2S binding site, and a putative molybdopterin cofactor binding domain. Analysis of potential NAD binding sites suggested a simple hypothesis for the conversion of human XD into the oxygen metabolite forming xanthine oxidase (XO; xanthine:oxygen oxidoreductase, EC 1.1.3.22). Using a human XD complementary RNA hybridization probe, we found a 5100-base RNA in human liver by RNA blot-hybridization analysis. This RNA exhibited tissue-specific distribution that may be pertinent to XD- and XO-mediated oxygen radical injury in ischemia/reperfusion and inflammation. A second 4500-base RNA was detected in some tissues and may arise through differential transcription termination.
