菅野 仁
Department School of Medicine(Tokyo Women's Medical University Hospital), School of Medicine Position Professor (Fixed Term) |
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Article types | Original article |
Language | English |
Peer review | Peer reviewed |
Title | cDNA cloning of human R-type pyruvate kinase and identification of a single amino acid substitution (Thr384----Met) affecting enzymatic stability in a pyruvate kinase variant (PK Tokyo) associated with hereditary hemolytic anemia. |
Journal | Formal name:Proceedings of the National Academy of Sciences of the United States of America Abbreviation:Proc Natl Acad Sci U S A ISSN code:0027-8424(Print)0027-8424(Linking) |
Volume, Issue, Page | 88(18),pp.8218-21 |
Author and coauthor | Kanno H, Fujii H, Hirono A, Miwa S |
Authorship | Lead author |
Publication date | 1991/09 |
Summary | cDNA clones for human R-type pyruvate kinase (PK) were isolated from a human reticulocyte cDNA library, constructed by PCR with a single gene-specific primer. The full-length cDNA was 2060 base pairs long, and the cDNA encoded 574 amino acids, the same number as that by rat R-type PK. Compared with human L-type PK, R-type PK was 31 amino acids longer at the amino terminus. We also cloned and characterized R-type PK cDNA clones from patients with hereditary hemolytic anemia from a PK deficiency, PK Tokyo. A single nucleotide substitution (ACG to ATG) was found at nucleotide 1151 of the coding sequence of the R-type PK, which caused an amino acid substitution, Thr384----Met. Dot blot hybridization of PCR-amplified genomic DNA from patients and their parents by allele-specific oligonucleotide probes showed that the parents, who were second cousins, were heterozygous. To confirm that the nucleotide change was responsible for the variant phenotype, we expressed the L-type PK with the single amino acid change in Escherichia coli and characterized the enzyme. The variant PK was thermolabile and moved slowly in the polyacrylamide gel buffered in 10 mM Tris.HCl, pH 8.3; these characteristics were fully compatible with data obtained from the patient's PK. From these results, we concluded that enzymatic stability of the variant was affected by the point mutation of the PK-encoding gene. |
Document No. | 1896471 |