Human glucose-6-phosphate dehydrogenase: Primary structure and cDNA cloning

T. Takizawa, I. Y. Huang, T. Ikuta, A. Yoshida

Research output: Contribution to journalArticle

82 Citations (Scopus)

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 λgt11 - i.e., a human liver cDNA library and a human hepatoma Li-7 cDNA library - were screened with the synthetic nucleotide probe. Two positive clones, λG6PD-19 and λG6PD-25, were obtained from the hepatoma library. λG6PD-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, λG6PD-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.

Original languageEnglish (US)
Pages (from-to)4157-4161
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume83
Issue number12
DOIs
StatePublished - Jan 1 1986
Externally publishedYes

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Glucosephosphate Dehydrogenase
Organism Cloning
Complementary DNA
Gene Library
Enzymes
Clone Cells
X Chromosome
NADP
Amino Acids
Amino Acid Sequence
Hepatocellular Carcinoma
Erythrocytes
Chromosomes, Human, X
Cyanogen Bromide
Glucose-6-Phosphate
Hemolytic Anemia
Southern Blotting
Oligonucleotides
Bacteriophages
Libraries

ASJC Scopus subject areas

  • General

Cite this

Human glucose-6-phosphate dehydrogenase : Primary structure and cDNA cloning. / Takizawa, T.; Huang, I. Y.; Ikuta, T.; Yoshida, A.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 83, No. 12, 01.01.1986, p. 4157-4161.

Research output: Contribution to journalArticle

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