Human glucose-6-phosphate dehydrogenase: primary structure and cDNA cloning.
- 1 June 1986
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 83 (12), 4157-4161
- https://doi.org/10.1073/pnas.83.12.4157
Abstract
The X-chromosome-linked glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ oxidoreductase, EC 1.1.1.49) of humans and other mammals consists of a subunit with a molecular weight of about 58,000. The enzyme plays a key role in the generation of NADPH, particularly in matured erythrocytes, and the genetic deficiency of the enzyme is associated with chronic and drug- or food-induced hemolytic anemia in humans. The enzyme was purified to homogeneity from human erythrocytes. The complete amino acid sequence of the subunit, consisting of 531 amino acid residues, was determined by automated and manual Edman degradation of tryptic, chymotryptic, thermolytic, and cyanogen bromide peptides obtained from the enzyme. Based on the amino acid sequence data thus obtained, a 41-mer oligonucleotide with unique sequence was prepared. Two cDNA libraries constructed in phage .lambda.gt11.sbd.i.e., a human liver cDNA library and a human hepatoma Li-7 cDNA library.sbd.were screened with the synthetic nucleotide probe. Two positive clones, .lambda.G6PD-19 and .lambda.G6PD-25, were obtained from the hepatoma library. .lambda.G6PD-19 contained an insertion of 2.0 kilobase pairs (kbp), and encoded 204 amino acid residues that were completely compatible with the COOH-terminal portion of the enzyme. The insertion of the clone had a 3'' noncoding region of 1.36 kbp. The other clone, .lambda.-G6PD-25, had an insertion of 1.8 kbp and encoded 362 amino acid residues of G6PD. Southern blot analysis of DNA samples obtained from cells with and without the human X chromosome indicated that the cDNA hybridizes with a sequence in the X chromosome.This publication has 27 references indexed in Scilit:
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