Red Cell Pyruvate Kinase Research Articles

Retrovirus-Mediated Gene Transfer of Human Pyruvate Kinase (PK) cDNA Into Murine Hematopoietic Cells: Implications for Gene Therapy of Human PK Deficiency

AbstractWith the advent of recent molecular studies, nonspherocytic hemolytic anemia caused by red blood cell pyruvate kinase (PK) deficiency is now considered to be caused by a structural mutation of the PK-LR gene. Because PK deficiency is a monogenic disorder, the introduction of the normal PK gene into a patient’s bone marrow stem cells should cure the disorder. To study the feasibility of gene therapy for PK deficiency, we first constructed the PK retrovirus pMNSM-hPK using human liver-type PK (LPK) cDNA and obtained a producer cell line of E86/AmPK. By using the supernatant of this virus-producer cell, we transduced NIH/3T3 cells, mouse leukemic cells (NFS60, FDCP-2), and human leukemic cells (K562, HEL). The expression of human LPK enzyme activity was ascertained from the retrovirally transduced NIH/3T3 cells. Northern blot analysis demonstrated the expression of the human LPK mRNA in each transduced cell line. Furthermore, bone marrow stem cells (c-kit+, Lin−, Thy-1lo) sorted by fluorescence-activated cell sorting were also transduced by the producer cells in the presence of interleukin-3 and interleukin-6, and were transplanted into lethally irradiated C57BL/6 mice. Polymerase chain reaction analysis demonstrated the expression of human LPK mRNA in both the peripheral blood and hematopoietic organs on day 30 and on day 135 of bone marrow transplantation.

Iron status in red cell pyruvate kinase deficiency: study of Italian cases.

We investigated the iron status of 33 pyruvate kinase (PK) deficient patients, most of the cases reported in Italy. Serum ferritin (SF) was higher than the upper limit of the range of matched controls in 15/25 (60%) non-transfused patients (median 228 micrograms/l, range 58-3160 v 43, 22-310). Liver siderosis and fibrosis were found in 8/9, and cirrhosis in two who died at age 39 and 42 of complications of iron overload. SF was independent of age, sex, or severity of haemolysis. The prevalence of HLA-A3 antigen in PK deficient patients was not significantly different from that of our healthy population (29.6% v 23%). The HLA-A3 positive, non-transfused patients had significantly higher SF values than the HLA-A3 negative ones (median 675 micrograms/l, range 340-3160 v 145, 58-400). A pedigree study of six high SF-probands indicated that iron overload has a multifactorial pathogenesis. In particular, the association of PK deficiency-induced haemolysis, splenectomy and an additional factor (heterozygosity for idiopathic haemochromatosis, ineffective erythropoiesis) leads to severe iron accumulation. We suggest that monitoring iron status would be useful in PK deficient patients, particularly in splenectomized and HLA-A3 positive ones, to identify those at risk of iron overload and prevent the clinical consequences of iron accumulation.

Glycolytic and Associated Enzymes of Rainbow Trout (Oncorhynchus Mykiss) Red Cells: In Vitro And In Vivo Studies

ABSTRACT Studies were undertaken in vitro and in vivo to assess the maximal activities of 26 glycolytic and associated enzymes of rainbow trout (Oncorhynchus mykiss) red cells. The red cells possess a complete sequence of glycolytic enzymes capable of anaerobic oxidation of glucose to lactate. Red cell pyruvate kinase (PK) was inhibited by ATP (I50 ≈ 5 mmol I 1−1), but was not sensitive to alanine inhibition or fructose-1,6-bisphosphate activation. The properties of red cell PK were similar to those of the muscle-type enzyme. Lactate dehydrogenase (LDH) from the trout erythrocyte resembled LDH from trout heart in terms of pyruvate inhibition in vitro. Enzymes associated with phosphagen and amino acid metabolism as well as the pentose phosphate shunt were also present. However, enzyme indicators of glycogenolytic and gluconeogenic potential were either absent or present at very low levels. The capacity for aerobic respiration via the tricarboxylic acid (TCA) cycle was suggested by the presence of citrate synthase activity. The association of glycolytic enzymes with the particulate fraction of the red cell was assessed for six enzymes. No binding was detected for hexokinase, PK and LDH; low levels of binding by phosphofructokinase (5%), aldolase (1%) and glyceraldehyde-3-phosphate dehydrogenase (3%) were not altered by strenuous exercise. Glycolytic enzyme binding, therefore, does not appear to be an important regulator of energy metabolism in these cells. Intracellular glucose levels, in contrast, appeared to be regulated following exercise stress. An increase in intra-erythrocytic lactate levels at this time may reflect the importance of exogenously produced lactate as a substrate for ATP production. The description of trout red cell metabolism presented here provides a basis for further study of the relationships between organismic gas exchange and molecular-level adaptation of nucleated red cell function.

Biochemical and Molecular Characterization of Variant Pyruvate Kinase Enzymes and Genes from Three Patients with Red Blood Cell Pyruvate Kinase Deficiency

Pyruvate kinase (PK) from red blood cells (RBC) of three patients with nonspherocytic hemolytic anemia due to PK deficiency was characterized according to internationally standardized methods. The variant enzymes, which were designated PK 'Memphis', PK 'Bartlett', and PK 'Pontotoc', had 11, 60, and 61%, respectively, of the normal enzyme activity. All variant PK enzymes had increased thermolability. Compared with control, Km (PEP) were 200-300% greater for PK 'Memphis', 50% less for PK 'Bartlett' and 300-400% greater for PK 'Pontotoc'. The Km (ADP) were 40 and 300% greater than normal for PK 'Bartlett' and PK 'Pontotoc', respectively. All variants required higher than normal concentrations of the allosteric modifier, fructose-1,6-diphosphate, to achieve 50% activation of maximal enzyme activity. To define the molecular basis of the gene defect, DNA samples from these patients were examined for restriction-fragment-linked polymorphisms. No differences were observed in the structure of the patients' PK genes compared with a normal control. These results are consistent with a mutation in coding sequences, rather than a large insertion, deletion or rearrangement of genetic information, as the underlying genetic defect that accounts for the altered enzyme properties in these PK-deficient patients.

Inherited persistence of immature type pyruvate kinase and hexokinase isozymes in dog erythrocytes.

1. Red cell pyruvate kinase (EC 2.7.1.40) and hexokinase (EC 2.7.1.1) in high and low potassium (K) dogs were shown to exist as multiple forms which were separable by electrophoresis and ion-exchange chromatography. The R2-type pyruvate kinase, which was determined to be a young type enzyme in canine red cells, was shown to be the predominant form of pyruvate kinase in high K cells. 2. The M2-type pyruvate kinase, a prototype isozyme in erythroid cells, existed in high K dog erythrocytes as well as in high K and low K dog reticulocytes. 3. Isozyme analysis of high K red cell hexokinase also showed a profile similar to that obtained for low K reticulocytes. 4. These results seem to reflect the immaturity of high K erythrocytes, which suggest that an abnormal cell differentiation or maturation may occur at an early stage of erythroid cell proliferation in high K dogs.

Comparative Study of Human M2-Type Pyruvate Kinases Isolated from Human Leukocytes and Erythrocytes of a Patient with Red Cell Pyruvate Kinase Hyperactivity

M2-type pyruvate kinases (M2-PK) have been isolated from human leukocytes and from the erythrocytes of a patient with erythrocyte PK hyperactivity. The kinetic characteristics of the patient erythrocyte M2-PK were similar to those of leukocyte M2-PK except for the Hill coefficient of phosphoenol pyruvate kinetics that showed little difference in the values. The patient erythrocyte M2-PK displayed complete immunological identity with leukocyte M2-PK in immunodiffusion, immunoblotting and immunoneutralization. The sensitivity to proteolysis by trypsin and the electrophoretic migration in different conditions were similar for the M2-PK of both origins. These results suggest an identity between this M2-PK abnormally present in erythrocytes and the M2-PK from leukocytes.

Pyruvate kinase Greensboro. A four-generation study of a high K0.5s (phosphoenolpyruvate) variant [published erratum appears in Blood 1988 Dec;72(6):2082

The proband with lifelong hemolytic anemia has a high K0.5s phosphoenolypyruvate (PEP) erythrocyte pyruvate kinase (PK) variant substantially but incompletely normalized by the allosteric modifier fructose-1,6-diphosphate (F-1,6-P2) with conversion of sigmoidal to hyperbolic kinetics. Heterozygotes in four generations express qualitatively identical but less severely abnormal kinetics and lack overt hemolysis. Kinetic abnormalities are closely mimicked by sulfhydryl modification of normal PK. Three distinct clinical and metabolic phenotypes characterize the proband and two sisters: variant PK and hemolytic anemia, variant PK without clinical manifestations or hemolysis, and complete normality. Their mother, whose red cell PK is entirely normal except for a questionably slightly low Vmax, is postulated to express the gene products of nonidentical alleles, one encoding a product with mildly less favorable catalytic characteristics. At low PEP concentrations, the proband and heterozygotes for the PK mutant express only a very small fraction of normal PK activity despite apparent inheritance of one normal allele in the latter. Evidence suggests that disproportionately lowered PK activity may be a property of a heterotetrameric PK. Illusory abnormalities in nucleotide specificity are artifacts of diminished substrate affinity characterizing the mutant PK.

Pyruvate Kinase Greensboro. A Four-Generation Study of a High K0.5s (Phosphoenolypyruvate) Variant

AbstractThe proband with lifelong hemolytic anemia has a high K0.5s phosphoenolypyruvate (PEP) erythrocyte pyruvate kinase (PK) variant substantially but incompletely normalized by the allosteric modifier fructose-1,6-diphosphate (F-1,6-P2) with conversion of sigmoidal to hyperbolic kinetics. Het-erozygotes in four generations express qualitatively identical but less severely abnormal kinetics and lack overt hemolysis. Kinetic abnormalities are closely mimicked by sulfhydryl modification of normal PK. Three distinct clinical and metabolic phenotypes characterize the proband and two sisters: variant PK and hemolytic anemia, variant PK without clinical manifestations or hemolysis, and complete normality. Their mother, whose red cell PK is entirely normal except for a questionably slightly low Vmax, is postulated to express the gene products of nonidentical alleles, one encoding a product with mildly less favorable catalytic characteristics. At low PEP concentrations, the proband and heterozygotes for the PK mutant express only a very small fraction of normal PK activity despite apparent inheritance of one normal allele in the latter. Evidence suggests that disproportionately lowered PK activity may be a property of a heterotetrameric PK. Illusory abnormalities in nucleotide specificity are artifacts of diminished substrate affinity characterizing the mutant PK.1988 by Grune & Stratton, Inc. 0006-4971/88/7203-0028$3.00/0

Erythrocyte pyruvate kinase deficiency: 11 new cases.

Eleven new cases of red cell pyruvate kinase (PK) deficiency with congenital haemolytic disease from 10 unrelated Italian families were characterized using the methods recommended by the International Committee for Standardization in Haematology (ICSH). All patients were double heterozygotes for the PK gene. The 10 variants were designated PK 'Lecce,' 'Parma,' 'Verona,' 'Milano,' 'Soresina,' 'Macerata,' 'Sassari,' 'Genova,' 'Mantova' and 'Brescia.' PK 'Sassari' was associated with glucose-6-phosphate dehydrogenase deficiency in two siblings. All mutants displayed multiple biochemical abnormalities except for PK 'Lecce' that only showed decreased red cell PK activity. No relation was found between the severity of anaemia and either the residual PK activity or specific biochemical enzyme abnormalities. Increased serum ferritin levels were detected in most of the patients, suggesting the need for systematically monitoring iron status in this disease.

Erythrocyte pyruvate kinase (PK): the variable significance of "nucleotide specificity" in the characterization of mutant variants.

The half-saturation constant (K0.5s) phosphoenolpyruvate (PEP) for red cell pyruvate kinase (PK) with co-factors UDP and GDP is less than one-half that with ADP with or without additions of the allosteric modifier, fructose-1, 6-dephosphate (F-1, 6-P2) to the assay. The Vmax is markedly greater with ADP than with UDP or GDP, but with (PEP) at 0.5 mM, activity with all co-factors is about equal and at lower concentrations greater with UDP and GDP. With high K0.5s (PEP) mutant enzymes, and at the usual test concentration (lmM) for PEP when nucleotide specificity is assessed, the abnormally low saturation of variant enzymes may result in higher activity with UDP and GDP than with ADP--the opposite of the "normal situation." The apparent aberration in nucleotide specificity may thus be illusory and secondary to the abnormal K0.5s (PEP) of the mutant. Example data are recorded. Variations in K0.5s (PEP) may also be introduced during enzyme preparation for assay, particularly when partial purification is employed.

Elevated pyruvate kinase activity in patients with hemolytic anemia due to red cell pyruvate kinase “deficiency”

Two patients with non-spherocytic hemolytic anemia were found to have elevated red blood cell pyruvate kinase activities commensurate with the decreased mean red cell age, but the residual pyruvate kinase had marked kinetic abnormalities. Accumulation of metabolic intermediates before pyruvate kinase and reduced levels of activity of the red blood cells of the parents of both patients supported the diagnosis of an inherited abnormal pyruvate kinase causing hemolytic anemia. Although it was observed in two unrelated persons, review of enzyme assays performed on the red blood cells of 651 patients with hereditary non-spherocytic hemolytic anemia suggests that this occurrence is rare.

Characteristics of red cell pyruvate kinase (PK) and pyrimidine 5'nucleotidase (P5N) abnormalities in acute leukaemia and chronic lymphoid diseases with leukaemic expression.

Red cell pyruvate kinase (PK), pyrimidine 5'nucleotidase (P5N) and reduced glutathione content (GSH) were studied in 126 untreated patients with acute leukaemia (AL, 80 cases), chronic lymphocytic leukaemia (B-CLL, 38 cases) and B-cell lymphoma with leukaemic expression (LSCL, eight cases). Acute leukaemias were classified into lymphoblastic (ALL) and non-lymphoblastic (ANLL), the latter have been further sub-divided into four different variants according to FAB morphological criteria (1976). A significant decrease of PK activity was observed only in the ANLL group, leading to a clear-cut difference with the ALL group where a normal value was obtained. The decrease of P5N activity was similar in all the morphological variants of ANLL and no abnormalities in the low PEP assay system or after fructose 1,6-bisphosphate (Fru 1,6-P2) activation were observed. P5N activity was found to be significantly decreased in all groups of patients except in B-CLL, where it was normal. In regards to the different morphological groups of ANLL, a striking decrease of P5N activity was observed in the M3 variant. Although red cell GSH content was significantly increased in all groups of patients, no correlation was demonstrated between the raised GSH levels and the decreased P5N activities.

Red cell pyruvate kinase in acute leukemia.

The red cell pyruvate kinase (PK) activity, KM for phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), and thermostability properties were studied in patients with acute leukemias. The acquired PK defect was found in patients with acute myeloid leukemia, while it was normal in acute non-myeloid leukemia enzyme kinetic tests. PK abnormality was expressed in 17 patients as deficient PK activity, in 15 patients as decreased enzyme thermostability, and in 11 patients as altered PK affinity for ADP.

Control of Oxygen Delivery from the Erythrocyte by Modification of Pyruvate Kinase

This chapter discusses how modification of pyruvate kinase activity can control oxygen delivery from the erythrocyte. Hemoglobins of a variety of animals fall into two classes: low-0 2 -affinity hemoglobins insensitive to P 2 GA and high-O 2 -affinity, P 2 GA-sensitive hemoglobins. How the levels of 2,3-P 2 GA in the red cell are regulated is an important question that has not yet been fully answered. The discovery that the human red cell contains phosphoglycolate, a powerful activator of the phosphatase may hint at a previously unsuspected mechanism for control of 2, 3-P 2 GA concentration. The best established correlation with 2,3-P 2 GA concentration in red cells is the activity of the enzyme pyruvate kinase. Red cell pyruvate kinase activity can be affected by a variety of factors. The enzyme is an allosteric enzyme showing cooperative homotropic interactions with its substrate phosphoenolpyruvate (PEP) but not with adenosine diphosphate (ADP). The enzyme is activated by fructose bisphosphate (FBP) that appears to stabilize the PEP-binding conformation, decreasing the K m for PEP and changing the PEP saturation curve to a rectangular hyperbola. Activation is not specific to FBP because mannose bisphosphate and glucose bisphosphate show the same activating effects although at high affector concentrations. Erythrocyte pyruvate kinase, in the absence of FBP, is very sensitive to oxidation. Oxidation raises the K m for PEP but not for ADP and causes increased sigmoidicity of PEP saturation. No evidence has been obtained that oxidation of pyruvate kinase in vivo is a reversible control mechanism but it has been suggested that abnormal pyruvate kinase found in some genetic diseases may be an oxidized form of a normal enzyme rather than a mutant enzyme. The chapter presents the role of phosphorylation in the red cell as a determinant of the oxygen delivery capacity of that cell. Cyclic adenosine monophosphate (cAMP) added externally is clearly capable of stimulating phosphorylation of pyruvate kinase and decreasing its affinity for PEP. This necessarily causes an increase in the concentration of 2,3-P 2 GA and a decreased affinity of hemoglobin for oxygen.

Studies with human erythrocyte pyruvate kinase (PK): effects of modification of sulfhydryl groups.

Cysteinyl residues of red cell pyruvate kinase (PK; ATP: pyruvate phosphotransferase, EC 2.7.1.40) were modified with methylmethanethiosulfonate (MMTS), p-nitrophenoxycarbonyl methyl disulfide (NPCMD), and sodium tetrathionate (NaTT). At pH greater than 7 . 0, K0.5 s phosphoenol-pyruvate (PEP) was markedly increased. Fructose-1,6-diphosphate (FDP) increased affinity for PEP, but K0.5 s (PEP) remained elevated and hyperbolic kinetics were not achieved. Inhibition by negative effectors ATP and alanine was not reversed by PEP and FDP concentrations far greater than those abolishing inhibition of unmodified enzyme. At pH less than 7 . 0, PEP affinity was reduced, and FDP markedly increased Vmax and diminished K0.5 s (PEP). MMTS greatly impaired the thermostability of PK. Acid pH alone and the simultaneous presence of Mg++, K+ and PEP prior to MMTS treatment protected against the effects on PEP kinetics, but did not alter the induction of thermolability. No MMTS effect on the FDP binding site, on ADP kinetics or on the relative effectiveness of GDP, UDP or CDP cofactors was demonstrated. The MMTS-induced alterations closely resembled those observed with certain PK mutants associated with haemolytic anaemia.

Metabolism of exogenous adenine in a pyruvate kinase-deficient patient

The metabolism of adenine, administered intravenously and orally, was studied in a patient with red cell pyruvate kinase (PK)-deficiency. Intravenous infusion of 58.5 mumol of adenine per kg containing 6475 Bq of 8-14C adenine caused an initial decrease in red cell ATP level and the gradual return to the pre-infusion level coincided with an increase in radiolabelled ATP. Of the infused adenine, 1.3% had entered the red cell pool after 2 h. The route of administration affected the urinary excretion of adenine and its oxymetabolites; after 6 h, 10.7% of the infused adenine had been excreted, compared with 7.4% after ingestion of a similar dose. In 10 control subjects given 45.1-55.5 mumol/kg, excretion of adenine plus oxymetabolites accounted for 13.5 +/- 2.9% of the ingested dose. Differences were also observed in the urinary purine excretion patterns for the two routes of administration. This study has shown that adenine can be incorporated into ATP in the red cells of a PK-deficient patient.

On the temperature- and salt-dependent conformation change in human erythrocyte pyruvate kinase.

The influence of temperature, K+, Mg2+ and fructose 1,6-bisphosphate on human red cell pyruvate kinase was investigated. Kinetic measurements between 4 degrees C and 43 degrees C revealed a remarkable influence of the temperature on the allosteric behaviour of the enzyme. Below a transition region between 15 degrees C and 20 degrees C (as obtained from an Arrhenius plot) the enzyme shows non-cooperative behaviour, as can be deduced from Michaelis-Menten, Hill and Scatchard plots. At temperatures above 20 degrees C cooperativity increases with rising temperature. This effect becomes even more pronounced at higher temperatures upon addition of increasing amounts of K+ and Mg2+ accompanied by a slight decrease of the reaction velocity. Fructose 1,6-bisphosphate, however, abolishes cooperativity at every temperature and salt concentration measured. Difficulties which arise in evaluating the correct values of V, Km and the Hill coefficient nH with cooperative systems are met by using a computer program of Wieker, Johannes and Hess, especially designed for the determination of kinetic parameters obtained from sigmoidal steady-state kinetics.

Erythrocyte Pyruvate Kinase Deficiency

Investigations on the activity and the kinetic behavior of red cell pyruvate kinase in the erythrocyte membrane and hemolysate of 6 patients with pyruvate kinase (PK) deficiency are presented and compared with those of normal individuals. Hemolysate: The PK in the hemolysate of normal individuals showed the well-known allosteric properties, reflected by sigmoidal reaction kinetics (Hill-coefficient > 1). In contrast to these findings the enzymes of 4 of the patients which had not been splenectomised and which were all in satisfactory condition showed between 15 and 35% of the PK activity of normal individuals in the hemolysates, whereas in the case of the 2 splenectomised patients only 5 and 15 % of this activity could be measured. All of the patients’ enzymes showed non allosteric (hyperbolic) reaction kinetics (Hill-coefficient ~ 1.0). Red cell membranes: Our studies confirmed earlier reports that not only the hemolysate of the patients, but also their red cell membranes (‘ghosts’) exhibit PK activity. In normal individuals the activity of the membrane-localised PK is about 5% of the cytoplasmatic one. In case of the membrane-localised enzyme of all patients - irrespective if they were splenectomised or not - the activity is decreased to 30-50%, compared to the activity of ghosts of the control persons. Reaction kinetic studies on the PK isolated from red cell membranes from normal individuals as well as from all 6 patients exhibited hyperbolic Michaelis-Menten kinetics with Hill coefficients around 1.0.

A Simple Technique for Preparation of Young Red Cells for Transfusion From Ordinary Blood Units

Abstract We have developed a rapid and simple method for the isolation of relatively young cells from ordinary fresh or previously frozen blood units, using the IBM-2991 Blood Cell Processor. Based on in vitro measurements of red cell pyruvate kinase activity, the resolution of the method is excellent, comparable to that of the stractan discontinuous gradient system. In splenectomized patients with thalassemia major, the administration of the “youngest” 50% of blood units yielded 51Cr survival times of 40 and 42 days for fresh packed red cells and 41 and 43 days for previously frozen red cells.

A simple technique for preparation of young red cells for transfusion from ordinary blood units

We have developed a rapid and simple method for the isolation of relatively young cells from ordinary fresh or previously frozen blood units, using the IBM-2991 Blood Cell Processor. Based on in vitro measurements of red cell pyruvate kinase activity, the resolution of the method is excellent, comparable to that of the stractan discontinuous gradient system. In splenectomized patients with thalassemia major, the administration of the "youngest" 50% of blood units yielded 51Cr survival times of 40 and 42 days for fresh packed red cells and 41 and 43 days for previously frozen red cells.