{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T03:55:45Z","timestamp":1792036545329,"version":"4.4.0"},"reference-count":36,"publisher":"American Society of Hematology","issue":"10","content-domain":{"domain":["ashpublications.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2001,11,15]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Human erythrocyte pyruvate kinase plays an important role in erythrocyte metabolism. Mutation on the gene results in pyruvate kinase deficiency and is an important cause of hereditary nonspherocytic hemolytic anemia. Because of difficulties in isolating the mutant enzymes from patients, these mutations have not been fully studied. In this study, a complementary DNA (cDNA) encoding the human erythrocyte pyruvate kinase was generated. The cDNA was cloned into several expression vectors, and the protein was expressed and purified. The tetrameric protein exhibited properties characteristic of authentic human erythrocyte pyruvate kinase, including response to substrate, phosphoenolpyruvate, activation by fructose 1,6-bisphosphate, and inhibition by adenosine triphosphate (ATP). The N-terminal segment of the protein was highly susceptible to proteolysis, but only 2 of the 4 subunits were cleaved and lacked 47 N-terminal amino acid residues. A mutant protein, R510Q, which is the most frequently occurring mutation among Northern European population, was also generated and purified. The mutant protein retained its binding capacity to and could be activated by fructose 1,6-bisphosphate and showed similar kinetics toward phosphoenolpyruvate and adenosine diphosphate as for the wild-type enzyme. Conversely, the mutant protein has a dramatically decreased stability toward heat and is more susceptible to ATP inhibition. The enzyme instability decreases the enzyme level in the cell, accounting for the clinically observed \u201cpyruvate kinase deficiency\u201d of patients who are homozygous for this mutation. This study provides the first detailed functional characterization of human erythrocyte pyruvate kinase. These findings will allow the establishment of a fine correlation between molecular abnormalities and the clinical expression of the disease.<\/jats:p>","DOI":"10.1182\/blood.v98.10.3113","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T12:46:47Z","timestamp":1027774007000},"page":"3113-3120","update-policy":"https:\/\/doi.org\/10.1182\/blood.2019cm0000","source":"Crossref","is-referenced-by-count":46,"title":["Human erythrocyte pyruvate kinase: characterization of the recombinant enzyme and a mutant form (R510Q) causing nonspherocytic hemolytic anemia"],"prefix":"10.1182","volume":"98","author":[{"given":"Changqing","family":"Wang","sequence":"first","affiliation":[{"name":"From the Department of Biochemistry, Department of Genetics and Microbiology, University of Pavia, Pavia, Italy; Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA; and Hematology Division, I.R.C.C.S. Ospedale Maggiore, Milano, Italy."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laurent R.","family":"Chiarelli","sequence":"additional","affiliation":[{"name":"From the Department of Biochemistry, Department of Genetics and Microbiology, University of Pavia, Pavia, Italy; Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA; and Hematology Division, I.R.C.C.S. Ospedale Maggiore, Milano, Italy."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paola","family":"Bianchi","sequence":"additional","affiliation":[{"name":"From the Department of Biochemistry, Department of Genetics and Microbiology, University of Pavia, Pavia, Italy; Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA; and Hematology Division, I.R.C.C.S. Ospedale Maggiore, Milano, Italy."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Donald J.","family":"Abraham","sequence":"additional","affiliation":[{"name":"From the Department of Biochemistry, Department of Genetics and Microbiology, University of Pavia, Pavia, Italy; Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA; and Hematology Division, I.R.C.C.S. Ospedale Maggiore, Milano, Italy."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alessandro","family":"Galizzi","sequence":"additional","affiliation":[{"name":"From the Department of Biochemistry, Department of Genetics and Microbiology, University of Pavia, Pavia, Italy; Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA; and Hematology Division, I.R.C.C.S. Ospedale Maggiore, Milano, Italy."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrea","family":"Mattevi","sequence":"additional","affiliation":[{"name":"From the Department of Biochemistry, Department of Genetics and Microbiology, University of Pavia, Pavia, Italy; Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA; and Hematology Division, I.R.C.C.S. Ospedale Maggiore, Milano, Italy."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alberto","family":"Zanella","sequence":"additional","affiliation":[{"name":"From the Department of Biochemistry, Department of Genetics and Microbiology, University of Pavia, Pavia, Italy; Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA; and Hematology Division, I.R.C.C.S. Ospedale Maggiore, Milano, Italy."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giovanna","family":"Valentini","sequence":"additional","affiliation":[{"name":"From the Department of Biochemistry, Department of Genetics and Microbiology, University of Pavia, Pavia, Italy; Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA; and Hematology Division, I.R.C.C.S. Ospedale Maggiore, Milano, Italy."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"234","reference":[{"key":"2020021218285914900_B1","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1016\/0079-6107(93)90001-Z","article-title":"Evolution in glycolysis.","volume":"59","author":"Fothergill","year":"1993","journal-title":"Prog Mol Biol Biophys."},{"key":"2020021218285914900_B2","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/0022-2836(75)90391-5","article-title":"Three-dimensional structure of cat muscle pyruvate kinase at 6 Angstrom resolution.","volume":"95","author":"Stammers","year":"1975","journal-title":"J Mol Biol."},{"key":"2020021218285914900_B3","doi-asserted-by":"crossref","first-page":"475","DOI":"10.1002\/j.1460-2075.1986.tb04236.x","article-title":"The structure of cat muscle pyruvate kinase.","volume":"5","author":"Muirhead","year":"1986","journal-title":"EMBO J."},{"key":"2020021218285914900_B4","doi-asserted-by":"crossref","first-page":"6301","DOI":"10.1021\/bi00186a033","article-title":"Structure of rabbit muscle pyruvate kinase complexed with Mn2+, K+, and pyruvate.","volume":"33","author":"Larsen","year":"1994","journal-title":"Biochemistry."},{"key":"2020021218285914900_B5","doi-asserted-by":"crossref","first-page":"729","DOI":"10.1016\/S0969-2126(01)00207-6","article-title":"Crystal structure of Escherichia coli pyruvate kinase type I: molecular basis of the allosteric transition.","volume":"3","author":"Mattevi","year":"1995","journal-title":"Structure."},{"key":"2020021218285914900_B6","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1016\/S0969-2126(98)00021-5","article-title":"The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate.","volume":"6","author":"Jurica","year":"1998","journal-title":"Structure."},{"key":"2020021218285914900_B7","doi-asserted-by":"crossref","first-page":"615","DOI":"10.1006\/jmbi.1999.2918","article-title":"The structure of pyruvate kinase from Leishmania mexicana reveals details of the allosteric transition and unusual effector specificity.","volume":"291","author":"Rigden","year":"1999","journal-title":"J Mol Biol."},{"key":"2020021218285914900_B8","first-page":"719","article-title":"Recombinant pyruvate kinase type I from Escherichia coli: overproduction and revised C-terminus of the polypeptide.","volume":"378","author":"Valentini","year":"1997","journal-title":"Biol Chem."},{"key":"2020021218285914900_B9","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1016\/0167-4838(89)90278-1","article-title":"A kinetic study of the pH effect on the allosteric properties of pyruvate kinase from Phycomyces blakesleeanus.","volume":"998","author":"Arriaga","year":"1989","journal-title":"Biochim Biophys Acta."},{"key":"2020021218285914900_B10","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/S0076-6879(82)90119-7","article-title":"Pyruvate kinases from human erythrocytes and liver.","volume":"90","author":"Kahn","year":"1982","journal-title":"Methods Enzymol."},{"key":"2020021218285914900_B11","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/0006-291X(79)90592-8","article-title":"Proteolytic processing of human erythrocyte pyruvate kinase: study of normal and deficient enzymes.","volume":"91","author":"Marie","year":"1979","journal-title":"Biochem Biophys Res Commun."},{"key":"2020021218285914900_B12","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/S0021-9258(19)68350-X","article-title":"Influence of phosphorylation on the interaction of effectors with rat liver pyruvate kinase.","volume":"257","author":"El-Maghrabi","year":"1982","journal-title":"J Biol Chem."},{"key":"2020021218285914900_B13","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1111\/j.1432-1033.1980.tb04718.x","article-title":"Phosphorylation of human erythrocyte pyruvate kinase by soluble cyclic-AMP-dependent protein kinases: comparison with human liver L-type enzyme.","volume":"108","author":"Marie","year":"1980","journal-title":"Eur J Biochem."},{"key":"2020021218285914900_B14","doi-asserted-by":"crossref","first-page":"7105","DOI":"10.1021\/bi00243a010","article-title":"An in vitro novel mechanism of regulating the activity of pyruvate kinase M2 by thyroid hormone and fructose 1,6-bisphosphate.","volume":"30","author":"Ashizawa","year":"1991","journal-title":"Biochemistry."},{"key":"2020021218285914900_B15","doi-asserted-by":"crossref","first-page":"14366","DOI":"10.1016\/S0021-9258(18)47947-1","article-title":"The L- and R-type isozymes of rat pyruvate kinase are produced from a single gene by use of different promoters.","volume":"262","author":"Noguchi","year":"1987","journal-title":"J Biol Chem."},{"key":"2020021218285914900_B16","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1006\/bcmd.2000.0276","article-title":"Hematologically important mutations: red cell pyruvate kinase (third update).","volume":"26","author":"Bianchi","year":"2000","journal-title":"Blood Cells Mol Dis."},{"key":"2020021218285914900_B17","doi-asserted-by":"crossref","first-page":"1793","DOI":"10.1182\/blood.V89.5.1793","article-title":"Molecular analysis of 29 pyruvate kinase-deficient patients from central Europe with hereditary hemolytic anemia.","volume":"89","author":"Lenzner","year":"1997","journal-title":"Blood."},{"key":"2020021218285914900_B18","doi-asserted-by":"crossref","first-page":"4213","DOI":"10.1182\/blood.V89.11.4213","article-title":"Frame shift mutation, exon skipping, and a two-codon deletion caused by splice site mutations account for pyruvate kinase deficiency.","volume":"89","author":"Kanno","year":"1997","journal-title":"Blood."},{"key":"2020021218285914900_B19","doi-asserted-by":"crossref","first-page":"3847","DOI":"10.1182\/blood.V89.10.3847","article-title":"Molecular characterization of PK-LR gene in pyruvate kinase-deficient Italian patients.","volume":"89","author":"Zanella","year":"1997","journal-title":"Blood."},{"key":"2020021218285914900_B20","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1111\/j.1365-2141.1979.tb03751.x","article-title":"Recommended methods for the characterization of red cell pyruvate kinase.","volume":"43","author":"Miwa","year":"1979","journal-title":"Br J Haematol."},{"key":"2020021218285914900_B21","doi-asserted-by":"crossref","first-page":"4324","DOI":"10.1073\/pnas.90.9.4324","article-title":"Analysis of pyruvate kinase-deficiency mutations that produce non-spherocytic hemolytic anemia.","volume":"90","author":"Baronciani","year":"1993","journal-title":"Proc Natl Acad Sci U S A."},{"key":"2020021218285914900_B22","doi-asserted-by":"crossref","first-page":"45","DOI":"10.3109\/10425179709020884","article-title":"Complete genomic sequence of the human PK-L\/R-gene includes four intragenic polymorphisms defining different haplotype backgrounds of normal and mutant PK-genes.","volume":"8","author":"Lenzner","year":"1997","journal-title":"DNA Seq."},{"key":"2020021218285914900_B23","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1053\/beha.1999.0057","article-title":"Red cell pyruvate kinase deficiency: from genetics to clinical manifestations.","volume":"13","author":"Zanella","year":"2000","journal-title":"Baillieres Best Pract Res Clin Haematol."},{"key":"2020021218285914900_B24","doi-asserted-by":"crossref","first-page":"805","DOI":"10.1016\/0076-6879(81)76159-7","article-title":"DNA analysis in the diagnosis of hemoglobin disorders.","volume":"76","author":"Goossens","year":"1981","journal-title":"Methods Enzymol."},{"key":"2020021218285914900_B25","doi-asserted-by":"crossref","first-page":"883","DOI":"10.1182\/blood.V47.6.883.883","article-title":"Enrichment of erythrocytes of fetal origin from adult-fetal blood mixtures via selective hemolysis of adult blood cells: an aid to antenatal diagnosis of hemoglobinopathies.","volume":"47","author":"Boyer","year":"1976","journal-title":"Blood."},{"key":"2020021218285914900_B26","doi-asserted-by":"crossref","first-page":"8218","DOI":"10.1073\/pnas.88.18.8218","article-title":"cDNA cloning of human R-type pyruvate kinase and identification of a single amino acid substitution (Thr384\u2013-Met) affecting enzymatic stability in a pyruvate kinase variant (PK Tokyo) associated with hereditary hemolytic anemia.","volume":"88","author":"Kanno","year":"1991","journal-title":"Proc Natl Acad Sci U S A."},{"key":"2020021218285914900_B27","first-page":"455","article-title":"Site-directed mutagenesis of double-stranded plasmids, domain substitution, and marker rescue by comutagenesis of restriction enzyme sites.","volume":"58","author":"Nickoloff","year":"1996","journal-title":"Methods Mol Biol."},{"key":"2020021218285914900_B28","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/S0021-9258(19)52451-6","article-title":"Protein measurement with the Folin phenol reagent.","volume":"193","author":"Lowry","year":"1951","journal-title":"J Biol Chem."},{"key":"2020021218285914900_B29","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of structural proteins during the assembly of the head of bacteriophage T4.","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature."},{"key":"2020021218285914900_B30","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1002\/9780470110539.ch4","article-title":"Isoelectric focusing in immobilized pH gradients: theory and newer methodology.","volume":"32","author":"Righetti","year":"1987","journal-title":"Methods Biochem Anal."},{"key":"2020021218285914900_B31","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1006\/bcmd.1995.0008","article-title":"Study of the molecular defects in pyruvate kinase deficient patients affected by non-spherocytic hemolytic anemia.","volume":"21","author":"Baronciani","year":"1995","journal-title":"Blood Cell Mol Dis."},{"key":"2020021218285914900_B32","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1016\/0006-291X(65)90711-4","article-title":"Thermodynamic considerations on erythrocyte glycolysis.","volume":"18","author":"Minakami","year":"1965","journal-title":"Biochem Biophys Res Comm."},{"key":"2020021218285914900_B33","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1046\/j.1365-2141.2001.02711.x","article-title":"Molecular characterization of the PK-LR gene in sixteen pyruvate kinase-deficient patients.","volume":"113","author":"Zanella","year":"2001","journal-title":"Br J Haematol."},{"key":"2020021218285914900_B34","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/0005-2744(78)90009-8","article-title":"The genetic system of the L-type pyruvate kinase forms in man: subunit structure, interrelation and kinetic characteristics of the pyruvate kinase enzymes from erythrocytes and liver.","volume":"523","author":"Kahn","year":"1978","journal-title":"Biochim Biophys Acta."},{"key":"2020021218285914900_B35","doi-asserted-by":"crossref","first-page":"656","DOI":"10.1016\/S0021-9258(17)38152-8","article-title":"Stimulation of glucagon of in vivo phosphorylation of rat hepatic pyruvate kinase.","volume":"253","author":"Riou","year":"1978","journal-title":"J Biol Chem."},{"key":"2020021218285914900_B36","doi-asserted-by":"crossref","first-page":"862","DOI":"10.1016\/0006-291X(79)92037-0","article-title":"Endogenous, cyclic 3\u2032-5\u2032 AMP-dependent phosphorylation of human red cell pyruvate kinase.","volume":"87","author":"Marie","year":"1979","journal-title":"Biochem Biophys Res Commun."}],"container-title":["Blood"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/ashpublications.org\/blood\/article-pdf\/98\/10\/3113\/1678369\/h8220103113.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"http:\/\/ashpublications.org\/blood\/article-pdf\/98\/10\/3113\/1678369\/h8220103113.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,6]],"date-time":"2024-01-06T06:45:27Z","timestamp":1704523527000},"score":1,"resource":{"primary":{"URL":"https:\/\/ashpublications.org\/blood\/article\/98\/10\/3113\/52991\/Human-erythrocyte-pyruvate-kinase-characterization"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2001,11,15]]},"references-count":36,"journal-issue":{"issue":"10","published-print":{"date-parts":[[2001,11,15]]}},"URL":"https:\/\/doi.org\/10.1182\/blood.v98.10.3113","relation":{},"ISSN":["1528-0020","0006-4971"],"issn-type":[{"value":"1528-0020","type":"electronic"},{"value":"0006-4971","type":"print"}],"subject":[],"published":{"date-parts":[[2001,11,15]]}}}