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Pyruvate kinase deficiency
Clin Biochem, Vol. 23, pp. 155-157, 1990 Printed in Canada. All rights reserved.
0009-9120/90 $3.00 + .00
Copyright © 1990 The Canadian Society of Clinical Chemists.
Pyruvate Kinase Deficiency S. MIWA1 and H. FUJII2 1Okinaka Memorial Institute for Medical Research, 2-2-2 Toranomon, Minato-ku, Tokyo 105, Japan; and 2Department of Blood Transfusion Medicine, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan Pyruvate kinase (PK) deficiency was initially described by Valentine et aL in 1961. Since then, more than 300 cases have been described, including 65 in Japan. PK deficiency is the most common hereditary nonspherocytic hemolytic anemia among several red cell enzyme defects of the Embden-Meyerhof glycolytic pathway. The clinical manifestations are highly variable. Splenectomy usually increases the hemoglobin level by about 2 g/100 mL. Standardization of methods for characterization of PK variants was achieved in 1979. There are four PK isozymes, M1, M2, L and R, in mammalian tissues. We have clarified the switch from M2-type to L-type PK during maturation of erythroid precursor cells. Recently we cloned and sequenced a full length human L-type PK cDNA. It will be useful to clarify the molecular basis of PK deficiency.
KEY WORDS: pyruvate kinase, deficiency; pyruvate kinase, isozymes; hemolytic anemia; hereditary nonspherocytic hemolytic anemia; red cell, enzyme anomalies; red cell, metabolism. Introduction
ereditary (congenital) nonspherocytic hemoH lytic anemias (HNSHA), without any particular red cell morphological abnormality other than anisocytosis and poikilocytosis, were first discussed by Dacie et al. (1) in 1953. Based on the results of the autohemolysis test, Selwyn and Dacie (2) divided HNSHA into two groups: in type I, autohemolysis was corrected by previous addition of glucose; in type II, glucose did not prevent hemolysis. Type II red cells also exhibited decreased consumption of glucose, suggesting a disturbance of glycolysis. In 1961, Valentine, Tanaka and Miwa (3) discovered that the red cells of three subjects with type II HNSHA were deficient in pyruvate kinase (PK). Other red cell enzyme deficiencies associated with HNSHA have been found since then. However, PK deficiency is the most common, and the best characterized, of the hemolytic anemias due to a defect in the Embden-Meyerhof glycolytic pathway; more than 300 cases have been reported from various
Correspondence:Dr. S. Miwa, Director, Okinaka Memorial Institute for Medical Research, 2-2-2 Toranomon, Minato-ku, Tokyo 105, Japan. Manuscript received April 11, 1989; revised July 26, 1989; accepted July 28, 1989. CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
parts of the world, including 65 in Japan. We describe clinical, biochemical and recent molecular biological advances, with special reference to the studies from our laboratory. For a comprehensive review on PK and other enzyme deficiency disorders of the erythrocyte, we recommend the article by Valentine et al. (4). Clinical features
PK deficiency is inherited as an autosomal recessive trait. Clinical symptoms are observed in the true homozygous or compound heterozygous state. The clinical manifestations of PK deficiency are highly variable, ranging from pronounced neonatal jaundice, requiring multiple exchange transfusions, to a fully compensated hemolytic anemia. The red cells are usually unremarkable morphologically. Echinocytes may be seen occasionally before splenectomy, but they increase in number and may become conspicuous after splenectomy. An increase in hemoglobin levels to 2-3 g/100 mL usually occurs after splenectomy, often accompanied by high reticulocyte counts >40%. Hence, splenectomy is indicated for PK deficient patients with severe anemia who require blood transfusions. Except for chronic hemolytic anemia, PK deficient patients do not manifest other symptoms. Biochemical genefics Most, if not all, cases of PK deficiency are caused by the production of mutant enzymes with functionally defective characteristics (5,6). Evidence for this was found in a case where the hemolysate had abnormal electrophoretic mobility (7). Since the 1970s, an increasing number of PK variants have been investigated biochemically in several laboratories. However, interpretation of the results was difficult, mainly because the methodologies used were different in each laboratory. Finally, standardization of methods for characterization of PK variants was achieved through the collaborative efforts of a working group of the International Committee for Standardization in Haematology (ICSH) Expert Panel on Red Cell Enzymes 155
MIWA AND F U J I I
(8). In these methods, red cell P K activity and thermostability are determined using crude hemolysate; whereas, the other eight parameters (Ko.ss for phosphoenolpyruvate (PEP), Hill coefficient, Ko.ss for ADP, fructose 1,6-diphosphate activation, ATP inhibition, nucleotide specificity using UDP, GDP and CDP in place of ADP, pH optimum and electrophoretic mobility) are determined using partially purified red cell PK. Seven variants from true homozygous PK deficient cases were characterized using these methods (9). Low substrate affinity for PEP, thermal instability and low inhibition constant (Ki) for ATP seem to play a major role in causing chronic hemolytic anemia. Note that four out of seven variants showed either normal or higher than normal red cell PK activity. Therefore, we may often overlook the patients because of normal PK activity if we assay red cell PK using only a conventional P E P concentration, which is very high, compared with a physiological concentration where low substrate affinity PK variants cannot function sufficiently. To discover such low affinity variants, it is necessary to perform either PK activity assay using a low P E P concentration or assay red cell glycolytic intermediates to detect the accumulation of intermediates that are located upstream of the PK step (PEP, 2-phosphoglycerate, 3-phosphoglycerate and 2,3-diphosphoglycerate). In another report, Shinohara and Tanaka (10) described a case that was not activated by fructose 1,6-diphosphate.
was placed under the promotor of simian virus 40 and introduced into monkey COS cells. H u m a n L-type PK activity was detected in the extract of COS cells by classical PK electrophoresis, confirming t h a t this cDNA is the actual structural L-type PK gene. H u m a n full-length cDNA for L-type PK will be very useful to clarify the molecular basis of PK deficiency.
Change of PK isozymes during red cell maturation Leukocyte P K and platelet PK are M2-type, while red cell PK is R-type. As there are multipotent hematopoietic stem cells in bone marrow, and red cells, leukocytes and platelets differentiate from these common stem cells; therefore, there must be an isozyme switch from M2-type to R ( - L ) - t y p e P K during the differentiation and maturation steps of erythroid cells. The relationship between erythroid cell maturation and the change of P K isozymes was studied using anti-rat-L-type-PK fluorescent antibody techniques in normal subjects (23), in PK deficiency subjects (24), and in K562 cells (25). The results indicated that the precursor cells occurred in the early stage of maturation, and that compensatory M2-type PK production occurred in the erythroblasts of patients with PK deficiency. The experiment using K562 cells showed that after hemin induction, change from M2-type to L-type PK appeared with the appearance of hemoglobin F synthesis.
PK isozymes and molecular studies Acknowledgments Mammalian PKs have four isozymes (L, R, M1 and M2) that are encoded by two different genes. The L(liver)- and R(red cell)-PK differ from M 1 (muscle)and M2 (so-called prototype)-PK by their enzyme kinetics, electrophoretic and immunological properties; they are under control of different genes in the rat, and probably in humans. In the rat, L- and R-type PKs are produced from a single L gene by using different promotors (11); differential splicing was considered to be involved in the production of M1- and M2-type PKs from a single M gene (12). PK cDNA and genomic DNA from non-human species (yeast PK, chicken M~-PK, rat MI-, M 2 - , Land R-type PKs) have been isolated by several investigators (11-18). The structure of cat muscle PK has been extensively analyzed (19). In humans, structural abnormalities of the L(-R)-type P K are probably responsible for H N S H A due to PK deficiency. To clarify PK deficiency at the gene level, we isolated the partial L-type P K cDNA in 1987 (20), and assigned the h u m a n L-type P K gene to chromosome 1 at band q21 (20,21). Recently, we isolated and determined the full-length sequence of h u m a n L-type PK cDNA (22). The cDNA contains 1629 base pairs coding for 543 amino acids. The homology between h u m a n and rat L-type P K was 86.9% at the nucleotide sequence level, and 92.3% at the amino acid sequence level. The full-length L-type P K cDNA 156
Some of the studies cited in this manuscript were supported by research grants from the Ministry of Education, Science and Culture, and Ministry of Health and Welfare, Japan.
References 1. Dacie JV, Mollison PL, Richardson N, Selwyn JG, Shapiro L. Atypical congenital haemolytic anaemia. Quart J Med 1953; 22: 6-15. 2. Selwyn JG, Dacie JV. Autohemolysis and other changes resulting from the incubation in vitro of red cells from patients with congenital hemolytic anemia. Blood 1954; 9: 414-38. 3. Valentine WN, Tanaka KR, Miwa S. A specific erythrocyte glycolytic enzyme defect (pyruvate kinase) in three subjects with congenital non-spherocytic hemolytic anemia. Trans Ass A m e r Physicians 1961; 74: 100-10. 4. Valentine WN, Tanaka KR, Paglia DE. Pyruvate kinase and other enzyme deficiency disorders of the erythrocyte. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease. 6th ed. Pp. 2341-65. New York: McGraw-Hill, 1989. 5. Paglia DE, Valentine WN, Baughan MA, Miller DR, Reed CF, McIntyre OR. An inherited molecular lesion of erythrocyte pyruvate kinase. Identification of a kinetically aberrant isozyme associated with premaCLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
PYRUVATE KINASE DEFICIENCY ture hemolysis. J Clin Invest 1968; 47: 1929-46. 6. Miwa S, Nakashima K, Ariyoshi K, Shinohara K, Oda E, Tanaka T. Four new pyruvate kinase (PK) variants and a classical PK deficiency. Br J Haematol 1975; 29: 157-69. 7. I m a m u r a 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 (Tokyo) 1973; 74: 1165-77. 8. Miwa S, Boivin P, Blume KG et al. International Committee for Standardization in Haematelogy: Recommended methods for the characterization of red cell pyruvate kinase variants. Br J Haematol 1979; 43: 275-86. 9. Miwa S, Fujii H, Takegawa S et al. Seven pyruvate kinase variants characterized by the ICSH recommended methods. Br J Haematol 1980; 45: 575-83. 10. Shinohara K, Tanaka KR. Pyruvate kinase deficiency hemolytic anemia: enzymatic characterization studies in twelve patients. Hemoglobin 1980; 4: 611-25. 11. 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 promotors. J Biol Chem 1987; 262: 14366-71. 12. Noguchi T, Inoue H, Tanaka T. The M1- and Me-type isozymes of rat pyruvate kinase are produced from the same gene by alternative splicing. J Biol Chem 1986; 261: 13807-12. 13. Lonberg N, Gilbert W. Primary structure of chicken muscle pyruvate kinase mRNA. Proc Natl Acad Sci USA 1983; 80: 3661-5. 14. Lonberg N, Gilbert W. Intron/exon structure of the chicken pyruvate kinase gene. Cell 1985; 40: 81-90. 15. Inoue H, Noguchi T, Tanaka T. Complete amino acid sequence of rat L-type pyruvate kinase deduced from
CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
the cDNA sequence. Eur JBiochem 1986; 154: 465-9. 16. Lone Y-C, Simon M-P, Kahn A, Marie J. Complete nucleotide and deduced amino acid sequence of rat L-type pyruvate kinase. FEBS Lett 1986; 195: 97-100. 17. Burke RL, Tekemp-Olaon P, Najarian R. The isolation, characterization and sequence of the pyruvate kinase of Saccharomyces cervisiae. J Biol Chem 1983; 258: 2193-201. 18. Cognet M, Lone Y-C, Vaulont S, Kahn A, Marie J. Structure of the rat L-type pyruvate kinase gene. J Mol Biol 1987; 196: 11-25. 19. Muirhead H, Clayden DA, Barford D et al. The structure of cat muscle pyruvate kinase. EMBO J 1986; 5: 475-81. 20. Tani K, Fujii H, Tsutsumi H et al. H u m a n liver type pyruvate kinase: cDNA cloning and chromosomal assignment. Biochem Biophys Res Commun 1987; 143: 431-38. 21. Satoh H, Tani K, Yoshida MC et al. The human liver-type pyruvate kinase (PKL) gene is on chromosome 1 at band q21. Cytogenet Cell Genet 1988; 47: 132-3. 22. Tani K, Fujii H, Nagata S e t al. H u m a n liver type pyruvate kinase: complete amino acid sequence and the expression in mammalian cells. Proc Natl Acad Sci USA 1988; 85: 1792-5. 23. Takegawa S, Fujii H, Miwa S. Change of pyruvate kinase isozymes from M2- to L-type during development of the red cell. Br J Haematol 1983; 54: 467-74. 24. Takegawa S, Miwa S. Change ofpyruvate kinase (PK) isozymes in classical type PK deficiency and other PK deficiency cases during red cell maturation. A m J Hematol 1984; 16: 53-8. 25. Takegawa S, Shinohara T, Miwa S. Hemin-induced conversion of pyruvate kinase isozymes in K562 cells. Blood 1984; 64: 754-7.
157
0009-9120/90 $3.00 + .00
Copyright © 1990 The Canadian Society of Clinical Chemists.
Pyruvate Kinase Deficiency S. MIWA1 and H. FUJII2 1Okinaka Memorial Institute for Medical Research, 2-2-2 Toranomon, Minato-ku, Tokyo 105, Japan; and 2Department of Blood Transfusion Medicine, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan Pyruvate kinase (PK) deficiency was initially described by Valentine et aL in 1961. Since then, more than 300 cases have been described, including 65 in Japan. PK deficiency is the most common hereditary nonspherocytic hemolytic anemia among several red cell enzyme defects of the Embden-Meyerhof glycolytic pathway. The clinical manifestations are highly variable. Splenectomy usually increases the hemoglobin level by about 2 g/100 mL. Standardization of methods for characterization of PK variants was achieved in 1979. There are four PK isozymes, M1, M2, L and R, in mammalian tissues. We have clarified the switch from M2-type to L-type PK during maturation of erythroid precursor cells. Recently we cloned and sequenced a full length human L-type PK cDNA. It will be useful to clarify the molecular basis of PK deficiency.
KEY WORDS: pyruvate kinase, deficiency; pyruvate kinase, isozymes; hemolytic anemia; hereditary nonspherocytic hemolytic anemia; red cell, enzyme anomalies; red cell, metabolism. Introduction
ereditary (congenital) nonspherocytic hemoH lytic anemias (HNSHA), without any particular red cell morphological abnormality other than anisocytosis and poikilocytosis, were first discussed by Dacie et al. (1) in 1953. Based on the results of the autohemolysis test, Selwyn and Dacie (2) divided HNSHA into two groups: in type I, autohemolysis was corrected by previous addition of glucose; in type II, glucose did not prevent hemolysis. Type II red cells also exhibited decreased consumption of glucose, suggesting a disturbance of glycolysis. In 1961, Valentine, Tanaka and Miwa (3) discovered that the red cells of three subjects with type II HNSHA were deficient in pyruvate kinase (PK). Other red cell enzyme deficiencies associated with HNSHA have been found since then. However, PK deficiency is the most common, and the best characterized, of the hemolytic anemias due to a defect in the Embden-Meyerhof glycolytic pathway; more than 300 cases have been reported from various
Correspondence:Dr. S. Miwa, Director, Okinaka Memorial Institute for Medical Research, 2-2-2 Toranomon, Minato-ku, Tokyo 105, Japan. Manuscript received April 11, 1989; revised July 26, 1989; accepted July 28, 1989. CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
parts of the world, including 65 in Japan. We describe clinical, biochemical and recent molecular biological advances, with special reference to the studies from our laboratory. For a comprehensive review on PK and other enzyme deficiency disorders of the erythrocyte, we recommend the article by Valentine et al. (4). Clinical features
PK deficiency is inherited as an autosomal recessive trait. Clinical symptoms are observed in the true homozygous or compound heterozygous state. The clinical manifestations of PK deficiency are highly variable, ranging from pronounced neonatal jaundice, requiring multiple exchange transfusions, to a fully compensated hemolytic anemia. The red cells are usually unremarkable morphologically. Echinocytes may be seen occasionally before splenectomy, but they increase in number and may become conspicuous after splenectomy. An increase in hemoglobin levels to 2-3 g/100 mL usually occurs after splenectomy, often accompanied by high reticulocyte counts >40%. Hence, splenectomy is indicated for PK deficient patients with severe anemia who require blood transfusions. Except for chronic hemolytic anemia, PK deficient patients do not manifest other symptoms. Biochemical genefics Most, if not all, cases of PK deficiency are caused by the production of mutant enzymes with functionally defective characteristics (5,6). Evidence for this was found in a case where the hemolysate had abnormal electrophoretic mobility (7). Since the 1970s, an increasing number of PK variants have been investigated biochemically in several laboratories. However, interpretation of the results was difficult, mainly because the methodologies used were different in each laboratory. Finally, standardization of methods for characterization of PK variants was achieved through the collaborative efforts of a working group of the International Committee for Standardization in Haematology (ICSH) Expert Panel on Red Cell Enzymes 155
MIWA AND F U J I I
(8). In these methods, red cell P K activity and thermostability are determined using crude hemolysate; whereas, the other eight parameters (Ko.ss for phosphoenolpyruvate (PEP), Hill coefficient, Ko.ss for ADP, fructose 1,6-diphosphate activation, ATP inhibition, nucleotide specificity using UDP, GDP and CDP in place of ADP, pH optimum and electrophoretic mobility) are determined using partially purified red cell PK. Seven variants from true homozygous PK deficient cases were characterized using these methods (9). Low substrate affinity for PEP, thermal instability and low inhibition constant (Ki) for ATP seem to play a major role in causing chronic hemolytic anemia. Note that four out of seven variants showed either normal or higher than normal red cell PK activity. Therefore, we may often overlook the patients because of normal PK activity if we assay red cell PK using only a conventional P E P concentration, which is very high, compared with a physiological concentration where low substrate affinity PK variants cannot function sufficiently. To discover such low affinity variants, it is necessary to perform either PK activity assay using a low P E P concentration or assay red cell glycolytic intermediates to detect the accumulation of intermediates that are located upstream of the PK step (PEP, 2-phosphoglycerate, 3-phosphoglycerate and 2,3-diphosphoglycerate). In another report, Shinohara and Tanaka (10) described a case that was not activated by fructose 1,6-diphosphate.
was placed under the promotor of simian virus 40 and introduced into monkey COS cells. H u m a n L-type PK activity was detected in the extract of COS cells by classical PK electrophoresis, confirming t h a t this cDNA is the actual structural L-type PK gene. H u m a n full-length cDNA for L-type PK will be very useful to clarify the molecular basis of PK deficiency.
Change of PK isozymes during red cell maturation Leukocyte P K and platelet PK are M2-type, while red cell PK is R-type. As there are multipotent hematopoietic stem cells in bone marrow, and red cells, leukocytes and platelets differentiate from these common stem cells; therefore, there must be an isozyme switch from M2-type to R ( - L ) - t y p e P K during the differentiation and maturation steps of erythroid cells. The relationship between erythroid cell maturation and the change of P K isozymes was studied using anti-rat-L-type-PK fluorescent antibody techniques in normal subjects (23), in PK deficiency subjects (24), and in K562 cells (25). The results indicated that the precursor cells occurred in the early stage of maturation, and that compensatory M2-type PK production occurred in the erythroblasts of patients with PK deficiency. The experiment using K562 cells showed that after hemin induction, change from M2-type to L-type PK appeared with the appearance of hemoglobin F synthesis.
PK isozymes and molecular studies Acknowledgments Mammalian PKs have four isozymes (L, R, M1 and M2) that are encoded by two different genes. The L(liver)- and R(red cell)-PK differ from M 1 (muscle)and M2 (so-called prototype)-PK by their enzyme kinetics, electrophoretic and immunological properties; they are under control of different genes in the rat, and probably in humans. In the rat, L- and R-type PKs are produced from a single L gene by using different promotors (11); differential splicing was considered to be involved in the production of M1- and M2-type PKs from a single M gene (12). PK cDNA and genomic DNA from non-human species (yeast PK, chicken M~-PK, rat MI-, M 2 - , Land R-type PKs) have been isolated by several investigators (11-18). The structure of cat muscle PK has been extensively analyzed (19). In humans, structural abnormalities of the L(-R)-type P K are probably responsible for H N S H A due to PK deficiency. To clarify PK deficiency at the gene level, we isolated the partial L-type P K cDNA in 1987 (20), and assigned the h u m a n L-type P K gene to chromosome 1 at band q21 (20,21). Recently, we isolated and determined the full-length sequence of h u m a n L-type PK cDNA (22). The cDNA contains 1629 base pairs coding for 543 amino acids. The homology between h u m a n and rat L-type P K was 86.9% at the nucleotide sequence level, and 92.3% at the amino acid sequence level. The full-length L-type P K cDNA 156
Some of the studies cited in this manuscript were supported by research grants from the Ministry of Education, Science and Culture, and Ministry of Health and Welfare, Japan.
References 1. Dacie JV, Mollison PL, Richardson N, Selwyn JG, Shapiro L. Atypical congenital haemolytic anaemia. Quart J Med 1953; 22: 6-15. 2. Selwyn JG, Dacie JV. Autohemolysis and other changes resulting from the incubation in vitro of red cells from patients with congenital hemolytic anemia. Blood 1954; 9: 414-38. 3. Valentine WN, Tanaka KR, Miwa S. A specific erythrocyte glycolytic enzyme defect (pyruvate kinase) in three subjects with congenital non-spherocytic hemolytic anemia. Trans Ass A m e r Physicians 1961; 74: 100-10. 4. Valentine WN, Tanaka KR, Paglia DE. Pyruvate kinase and other enzyme deficiency disorders of the erythrocyte. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease. 6th ed. Pp. 2341-65. New York: McGraw-Hill, 1989. 5. Paglia DE, Valentine WN, Baughan MA, Miller DR, Reed CF, McIntyre OR. An inherited molecular lesion of erythrocyte pyruvate kinase. Identification of a kinetically aberrant isozyme associated with premaCLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
PYRUVATE KINASE DEFICIENCY ture hemolysis. J Clin Invest 1968; 47: 1929-46. 6. Miwa S, Nakashima K, Ariyoshi K, Shinohara K, Oda E, Tanaka T. Four new pyruvate kinase (PK) variants and a classical PK deficiency. Br J Haematol 1975; 29: 157-69. 7. I m a m u r a 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 (Tokyo) 1973; 74: 1165-77. 8. Miwa S, Boivin P, Blume KG et al. International Committee for Standardization in Haematelogy: Recommended methods for the characterization of red cell pyruvate kinase variants. Br J Haematol 1979; 43: 275-86. 9. Miwa S, Fujii H, Takegawa S et al. Seven pyruvate kinase variants characterized by the ICSH recommended methods. Br J Haematol 1980; 45: 575-83. 10. Shinohara K, Tanaka KR. Pyruvate kinase deficiency hemolytic anemia: enzymatic characterization studies in twelve patients. Hemoglobin 1980; 4: 611-25. 11. 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 promotors. J Biol Chem 1987; 262: 14366-71. 12. Noguchi T, Inoue H, Tanaka T. The M1- and Me-type isozymes of rat pyruvate kinase are produced from the same gene by alternative splicing. J Biol Chem 1986; 261: 13807-12. 13. Lonberg N, Gilbert W. Primary structure of chicken muscle pyruvate kinase mRNA. Proc Natl Acad Sci USA 1983; 80: 3661-5. 14. Lonberg N, Gilbert W. Intron/exon structure of the chicken pyruvate kinase gene. Cell 1985; 40: 81-90. 15. Inoue H, Noguchi T, Tanaka T. Complete amino acid sequence of rat L-type pyruvate kinase deduced from
CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
the cDNA sequence. Eur JBiochem 1986; 154: 465-9. 16. Lone Y-C, Simon M-P, Kahn A, Marie J. Complete nucleotide and deduced amino acid sequence of rat L-type pyruvate kinase. FEBS Lett 1986; 195: 97-100. 17. Burke RL, Tekemp-Olaon P, Najarian R. The isolation, characterization and sequence of the pyruvate kinase of Saccharomyces cervisiae. J Biol Chem 1983; 258: 2193-201. 18. Cognet M, Lone Y-C, Vaulont S, Kahn A, Marie J. Structure of the rat L-type pyruvate kinase gene. J Mol Biol 1987; 196: 11-25. 19. Muirhead H, Clayden DA, Barford D et al. The structure of cat muscle pyruvate kinase. EMBO J 1986; 5: 475-81. 20. Tani K, Fujii H, Tsutsumi H et al. H u m a n liver type pyruvate kinase: cDNA cloning and chromosomal assignment. Biochem Biophys Res Commun 1987; 143: 431-38. 21. Satoh H, Tani K, Yoshida MC et al. The human liver-type pyruvate kinase (PKL) gene is on chromosome 1 at band q21. Cytogenet Cell Genet 1988; 47: 132-3. 22. Tani K, Fujii H, Nagata S e t al. H u m a n liver type pyruvate kinase: complete amino acid sequence and the expression in mammalian cells. Proc Natl Acad Sci USA 1988; 85: 1792-5. 23. Takegawa S, Fujii H, Miwa S. Change of pyruvate kinase isozymes from M2- to L-type during development of the red cell. Br J Haematol 1983; 54: 467-74. 24. Takegawa S, Miwa S. Change ofpyruvate kinase (PK) isozymes in classical type PK deficiency and other PK deficiency cases during red cell maturation. A m J Hematol 1984; 16: 53-8. 25. Takegawa S, Shinohara T, Miwa S. Hemin-induced conversion of pyruvate kinase isozymes in K562 cells. Blood 1984; 64: 754-7.
157