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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Sep 15;88(18):8218–8221. doi: 10.1073/pnas.88.18.8218

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.

H Kanno 1, H Fujii 1, A Hirono 1, S Miwa 1
PMCID: PMC52478  PMID: 1896471

Abstract

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.

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Selected References

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  1. Burke R. L., Tekamp-Olson P., Najarian R. The isolation, characterization, and sequence of the pyruvate kinase gene of Saccharomyces cerevisiae. J Biol Chem. 1983 Feb 25;258(4):2193–2201. [PubMed] [Google Scholar]
  2. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  3. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Imamura K., Tanaka T. Multimolecular forms of pyruvate kinase from rat and other mammalian tissues. I. Electrophoretic studies. J Biochem. 1972 Jun;71(6):1043–1051. doi: 10.1093/oxfordjournals.jbchem.a129852. [DOI] [PubMed] [Google Scholar]
  5. Imamura K., Tanaka T., Nishina T., Nakashima K., Miwa S. Studies on pyruvate kinase (PK) deficiency. II. Electrophoretic, kinetic and immunological studies on pyruvate kinase of erythrocytes and other tissues. J Biochem. 1973 Dec;74(6):1165–1175. doi: 10.1093/oxfordjournals.jbchem.a130344. [DOI] [PubMed] [Google Scholar]
  6. Inoue H., Noguchi T., Tanaka T. Complete amino acid sequence of rat L-type pyruvate kinase deduced from the cDNA sequence. Eur J Biochem. 1986 Jan 15;154(2):465–469. doi: 10.1111/j.1432-1033.1986.tb09420.x. [DOI] [PubMed] [Google Scholar]
  7. Levitt M. Conformational preferences of amino acids in globular proteins. Biochemistry. 1978 Oct 3;17(20):4277–4285. doi: 10.1021/bi00613a026. [DOI] [PubMed] [Google Scholar]
  8. Lonberg N., Gilbert W. Primary structure of chicken muscle pyruvate kinase mRNA. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3661–3665. doi: 10.1073/pnas.80.12.3661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lone Y. C., Simon M. P., Kahn A., Marie J. Complete nucleotide and deduced amino acid sequences of rat L-type pyruvate kinase. FEBS Lett. 1986 Jan 20;195(1-2):97–100. doi: 10.1016/0014-5793(86)80138-7. [DOI] [PubMed] [Google Scholar]
  10. Miwa S., Fujii H., Takegawa S., Nakatsuji T., Yamato K., Ishida Y., Ninomiya N. Seven pyruvate kinase variants characterized by the ICSH recommended methods. Br J Haematol. 1980 Aug;45(4):575–583. doi: 10.1111/j.1365-2141.1980.tb07181.x. [DOI] [PubMed] [Google Scholar]
  11. Miwa S. Molecular basis of red cell enzymopathies associated with hereditary nonspherocytic hemolytic anemia. Haematologia (Budap) 1989;22(4):215–231. [PubMed] [Google Scholar]
  12. Muirhead H., Clayden D. A., Barford D., Lorimer C. G., Fothergill-Gilmore L. A., Schiltz E., Schmitt W. The structure of cat muscle pyruvate kinase. EMBO J. 1986 Mar;5(3):475–481. doi: 10.1002/j.1460-2075.1986.tb04236.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nakashima K., Miwa S., Oda S., Tanaka T., Imamura K. Electrophoretic and kinetic studies of mutant erythrocyte pyruvate kinases. Blood. 1974 Apr;43(4):537–548. [PubMed] [Google Scholar]
  14. Noguchi T., Inoue H., Chen H. L., Matsubara K., Tanaka T. Molecular cloning of DNA complementary to rat L-type pyruvate kinase mRNA. Nutritional and hormonal regulation of L-type pyruvate kinase mRNA concentration. J Biol Chem. 1983 Dec 25;258(24):15220–15223. [PubMed] [Google Scholar]
  15. Noguchi T., Inoue H., Tanaka T. The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. J Biol Chem. 1986 Oct 15;261(29):13807–13812. [PubMed] [Google Scholar]
  16. Noguchi T., Yamada K., Inoue H., Matsuda T., Tanaka T. The L- and R-type isozymes of rat pyruvate kinase are produced from a single gene by use of different promoters. J Biol Chem. 1987 Oct 15;262(29):14366–14371. [PubMed] [Google Scholar]
  17. Oyama H., Kumatori T., Nishina T., Miwa S. Functionally abnormal pyruvate kinase in congenital hemolytic anemia. Nihon Ketsueki Gakkai Zasshi. 1969 Apr;32(2):330–335. [PubMed] [Google Scholar]
  18. Paglia D. E., Valentine W. N., Baughan M. A., Miller D. R., Reed C. F., McIntyre O. R. An inherited molecular lesion of erythrocyte pyruvate kinase. Identification of a kinetically aberrant isozyme associated with premature hemolysis. J Clin Invest. 1968 Aug;47(8):1929–1946. doi: 10.1172/JCI105883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  20. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tanaka T., Harano Y., Sue F., Morimura H. Crystallization, characterization and metabolic regulation of two types of pyruvate kinase isolated from rat tissues. J Biochem. 1967 Jul;62(1):71–91. doi: 10.1093/oxfordjournals.jbchem.a128639. [DOI] [PubMed] [Google Scholar]
  22. Tani K., Fujii H., Nagata S., Miwa S. Human liver type pyruvate kinase: complete amino acid sequence and the expression in mammalian cells. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1792–1795. doi: 10.1073/pnas.85.6.1792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tani K., Yoshida M. C., Satoh H., Mitamura K., Noguchi T., Tanaka T., Fujii H., Miwa S. Human M2-type pyruvate kinase: cDNA cloning, chromosomal assignment and expression in hepatoma. Gene. 1988 Dec 20;73(2):509–516. doi: 10.1016/0378-1119(88)90515-x. [DOI] [PubMed] [Google Scholar]
  24. Tremp G. L., Boquet D., Ripoche M. A., Cognet M., Lone Y. C., Jami J., Kahn A., Daegelen D. Expression of the rat L-type pyruvate kinase gene from its dual erythroid- and liver-specific promoter in transgenic mice. J Biol Chem. 1989 Nov 25;264(33):19904–19910. [PubMed] [Google Scholar]
  25. VALENTINE W. N., TANAKA K. R., MIWA S. A specific erythrocyte glycolytic enzyme defect (pyruvate kinase) in three subjects with congenital non-spherocytic hemolytic anemia. Trans Assoc Am Physicians. 1961;74:100–110. [PubMed] [Google Scholar]
  26. Valentini G., Iadarola P., Somani B. L., Malcovati M. Two forms of pyruvate kinase in Escherichia coli. A comparison of chemical and molecular properties. Biochim Biophys Acta. 1979 Oct 11;570(2):248–258. doi: 10.1016/0005-2744(79)90145-1. [DOI] [PubMed] [Google Scholar]
  27. de Boer H. A., Comstock L. J., Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. doi: 10.1073/pnas.80.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]

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