Heinz Winkler

Molecular basis of neurological dysfunction coupled with haemolytic anaemia in human glucose-6-phosphate isomerase (GPI) deficiency

Glucose-6-phosphate isomerase (GPI) deficiency, an autosomal recessive genetic disorder with the typical manifestation of nonspherocytic haemolytic anaemia, can be associated in some cases with neurological impairment. GPI has been found to be identical to neuroleukin (NLK), which has neurotrophic and lymphokine properties. To focus on the possible effects of GPI mutations on the central nervous system through an effect on neuroleukin activity, we analysed DNA isolated from two patients with severe GPI deficiency, one of them with additional neurological deficits, and their families. The neurologically affected patient (GPI Homburg) is compound heterozygous for a 59 A→C (H20P) and a 1016 T→C (L339P) exchange. Owing to the insertion of proline, the H20P and L339P mutations are likely to affect the folding and activity of the enzyme. In the second family studied, the two affected siblings showed no neurological symptoms. The identified mutations are 1166 A→G (H389R) and 1549 C→G (L517V), which are located at the subunit interface. We propose that mutations that lead to incorrect folding destroy both catalytic (GPI) and neurotrophic (NLK) activities, thereby leading to the observed clinical symptoms (GPI Homburg). Those alterations at the active site, however, that allow correct folding retain the neurotrophic properties of the molecule (GPI Calden).

On the diagnosis of erythrocyte enzyme defects in the presence of high reticulocyte counts

Summary. The separation of red blood cells into reticulocytes and young and old erythrocytes enables investigations of fractions with different contents of reticulocytes. Activities of hexokinase, glucose phosphate isomerase, phosphofructokinase, pyruvate kinase and glucose‐6‐phosphate dehydrogenase showed a linear relationship to reticulocyte counts. The dependence of these enzyme activities on the age of the red blood cells exhibited a strong decline from the reticulocyte to the young erythrocyte stage followed by only little further loss of activity, thus leading to a biphasic decay of enzyme activities.

Mutations in the R-type pyruvate kinase gene and altered enzyme kinetic properties in patients with hemolytic anemia due to pyruvate kinase deficiency

SummaryThe biochemical properties of erythrocyte pyruvate kinase (PK) together with mutations found in the coding sequence of the R-PK gene in five patients with severe hemolytic anemia due to PK deficiency are described. The enzyme variants were designated PK ‘Mosul’ (homozygote), PK ‘Bukarest1,2’, PK ‘Hamburg1’, PK ‘Köln1’, and PK ‘Essen’ (compound heterozygote). PK ‘Mosul’ showed normal positive cooperative substrate binding, PK ‘Bukarest1,2’ exhibited noncooperative behavior, and PK ‘Hamburg1’ and PK ‘Köln1’ displayed mixed cooperativity, whereas PK ‘Essen’ was negative cooperative. PK ‘Mosul’ was found to be homozygous for the mutation 1151 ACG to ATG, resulting in an amino acid substitution 384 Thr to Met. In one allele of PK ‘Bukarest1,2’ a single nucleotide substitution GAG-TAG was found at nucleotide 721, causing a change of 241 Glu to a chain termination codon (PK ‘Bukarest1’). Additionally, in the second allele of this patient a point mutation at position 1594 (CGG-TGG) occurs, changing 532 Arg to Trp (PK ‘Bukarest2’). Direct sequencing showed the heterozygosity of the patients mother (PK ‘Bukarest1’/normal) at position 721 and of the patients father (PK ‘Bukarest2’ /normal) at position 1594. A point mutation at position 1529 (CGA-CAA), causing an amino acid substitution 510 Arg-Gln, was identified in PK ‘Hamburg1’ and PK ‘Köln1’. The second mutation in these variants was not detected. In PK ‘Essen’ no mutation in the coding sequence was found at all. Screening for the mutation at position 1529 in further compound heterozygote patients and in normal subjects of Western European origin showed that this exchange is a common mutation responsible for PK deficiency in this population.

The binding of fluorescent 4,6,8(14)-triene-3-one steroids to cyclodextrins as a model for steroid—protein interactions

The 4,6,8(14)-triene-3-one steroids, highly fluorescent in aqueous solutions, lose their fluorescence power when binding occurs to hydrophobic regions of other molecules, such as the hydrophobic cavity in the ring system of cyclodextrins. The fluorescence intensity decreases almost completely when beta- and gamma-cyclodextrins are present in the solution. Scatchard plots derived from fluorescence titrations show that one or two molecules of steroid bind to one cyclodextrin molecule with KD,F-values of about 10(-4)-10(-5) mol/liter. Temperature-jump experiments show a single relaxation process, with rate constants for the decay of the beta-cyclodextrin-steroid complexes of about 10(4)-10(5) per s. For alpha- and gamma-cyclodextrins such relaxation processes are not observed.

Erythrocyte pyruvate kinase- and glucose phosphate isomerase deficiency: Perturbation of Glycolysis by structural defects and functional alterations of defective enzymes and its relation to the clinical severity of chronic hemolytic anemia

The pathogenesis of two metabolic disorders caused by enzyme defects in the red blood cell leading to hemolytic anemia, and in some cases of glucose phosphate isomerase (GPI) deficiency additionally to neurological impairment was investigated. Rheological studies were performed to determine the influence of a shortage of energy on the deformability of the erythrocytes. The functions of the enzymes were determined by studying the enzyme kinetics, the temperature dependence of the enzyme activity and the migration of the proteins in an electric field. A detailed molecular genetic analysis of the gene encoding for the given protein allowed the detection of mutations involving amino acid exchanges which cause alterations of the protein structure. For both enzyme deficiencies, a good correlation was found between the structural changes (usually caused by single point mutations in the gene), the altered function of the enzymes and the severity of the clinical picture. The exchange of amino acids close to either the active site or the regulatory domain results in a decreased turnover as well as an alteration of the regulatory properties of the enzymes; this usually leads to an increased severity of the disease. Increased concentrations of glucose-6-phosphate (G-6-P), found in all red blood cells of patients suffering from hemolytic anemia caused by pyruvate kinase (PK) and GPI deficiency, correlate well with the severity of the clinical picture, apparently reflecting the degree of the perturbation of glycolysis. This results in a lack of the energy donor adenosine triphosphate (ATP); this leads then to a destabilization of the red cell membrane which causes earlier lysis of the red blood cell, which in turn gives rise to hemolytic anemia of variable degrees. One patient with neurological symptoms has been studied so far biochemically and at the molecular genetic level. The point mutations found in this patients GPI gene support the idea that GPI may have a neurological function in addition to its role in the carbohydrate metabolism; this is due to the presence of a monomeric sequence analogue called neuroleukin (NLK). The mutations apparently lead to the incorrect folding of this neurotrophic factor, and thus destroy the neurological activity.

Erythrocyte pyruvate kinase deficiency: Relations of residual enzyme activity, altered regulation of defective enzymes and concentrations of high-energy phosphates with the severity of clinical manifestation3

Abstract: The defective enzymes of 54 patients with pyruvate kinase (PK) deficiency were characterized according to the recommendations of the International Committee for Standardization in Haematology (ICSH). The erythrocyte PK activity in whole blood was calculated considering the 16‐fold higher activity of the reticulocyte enzyme (AR) compared to the erythrocyte enzyme (AE). The following parameters turned out to give a good correlation to the degree of haemolytic anaemia and can therefore serve as a prognostic tool: All patients with a severe course of the disease had residual erythrocyte PK activities less than 33% of the normal enzymes (percentage activity), and patients with mild haemolytic anaemia exhibited residual activity values below and above this threshold value. Studies of enzyme cooperativity showed that positive cooperative or mixed cooperative phosphoenolpyruvate (PEP) binding with a predominant positive cooperative part appeared in all cases with a mild clinical course, and about one‐third of the severe ones. Negative cooperativity or mixed cooperativity with predominant negative cooperative part was observed only with severe haemolytic anaemia. Furthermore, the determination of glucose‐6‐phosphate (G‐6‐P) turned out to be a good prognostic criterion, i.e. all patients with mild clinical course exhibited G‐6‐P‐concentrations lower than 0.11 μmol/l red blood cells. In the case of patients with severe haemolytic anaemia, about 80% showed values higher than 0.11 μmol/l RBC.

Erythrocyte pyruvate kinase deficiency: Characterization of a new variant (PK “Aarau”)

SummaryA new PK variant with moderate hemolytic anemia is described. The enzymes of the nonanemic parents show sigmoidal reaction kinetics, with normal kinetic parameters, but differ with respect to nucleotide specificity, thermostability, and the concentrations of the glycolytic intermediates in the erythrocytes.The most characteristic features of the patients (daughter) enzyme are a 30% activity, hyperbolic reaction kinetics and only two bands in the SDS-gel electrophoresis instead of three bands observed with the parental enzymes. Moreover, the pH-optimum is shifted to the acidic range, the affinity for PEP and ADP is decreased, ATP inhibition is negligible and FDP-activation is roughly ten times smaller than with controls. The concentrations of 2.3-DPG, 2-PG and PEP in the erythrocyte are increased, but ATP decreased.As there is no consanguinity in the parents and their enzymes are different this PK mutant can be considered to be compound-heterozygous for two different mutant PK alleles.

Relaxation kinetic studies of coenzyme binding to glutamate dehydrogenase from beef liver

Fluorescence temperature-jump experiments were performed to study the binding of coenzyme to glutamate dehydrogenase from beef liver in 0.1 M sodium phosphate buffer, pH 7.4, T = 25.0°C. 1. 1) NADPH shows a single second-order relaxation process, indicating coenzyme binding to one site per enzyme subunit. The dissociation constant of the reaction was calculated from the rate constants (k21 = 92 sec−1, k12 = 2.13 .106 M−1 sec−1) to be 43 μM. No deviation from a straight line was observed over the whole concentration range. 2. 2) With NADH two well separated second-order processes are detected. 3. 3) The observed concentration dependence of these two relaxation processes is consistent with a reaction scheme assuming two completely independent but non-equivalent NADH binding sites per enzyme subunit. 4. 4) From the rate constants of the faster process (k21 = 235 sec−1 k12 = 3.83 .106 M−1 sec−1) and of the slower process (k32 = 4.2 sec−1, k23 = 0.18 .106 M−1 sec−1) the dissociation constants are calculated to be K21 = k21k12 = 61 μM and K32 = k32k23 = 23 μM, respectively. 5. 5) Small amounts of ADP (<10 μM) abolish the slower relaxation process, while the faster (but weaker binding) process remains uninfluenced. Therefore, the weaker binding site must be the active site.

Kinetic measurements on the pyruvate kinase reaction in erythrocytes

Pyruvate kinase (PK, EC 2.7.1.40, ATP-pyruvate phosphotransferase) is a key enzyme of glycolysis controlling its rate and direction. It exhibits the well-known properties of a rate-controlling enzyme, showing sigmoidal reaction kinetics, FDP acting as allosteric effector and PEP being the substrate. Marie et al. [1] had proved that PK from liver (L-type enzyme) and from red cells were antigenically identical. Hereditary erythrocyte PK deficiency is the most frequent of the enzymopathies with congenital non-spherocytic hemolytic anemia. Considerable heterogeneity in clinical severity and in biochemical abnormability has been described. There is no distinct correlation between the degree of reduced enzyme activity and the severity of clinical manifestation. Unfortunately, from the very beginning a bewildering variety of assay techniques had been used to characterize the kinetic properties of the enzyme. Consequently, rather different rate values in different and surprisingly even in a given laboratory were obtained. For reasons of better comparability, the International Committee for Standardization in Hematology recently convened an expert panel to examine the technical problems involved in the performance of red cell enzyme assays [2]. PK activity is measured in an optical assay in which the PK reaction is coupled with that of LDH. PK l. PEP + ADP ~ ~ Pyruvate + ATP Mg2+ K +

Bestimmung von Enzym-Coenzym-Dissoziationskonstanten durch Verdrängung von Tetrajodfluorescein aus den Enzym-Farbstoff-Komplexen

Tetraiodofluorescein (Erythrosin B) is bound to several dehydrogenases. This binding becomes measureable by an absorption difference spectrum in the wavelength range between 500 nm and 600 nm. Apparently the dye is bound to the adenosine moiety of the active site of nicotineamide dependent dehydrogenases. Thus, displacement of the dye should occur by several coenzymes and nucleotides and hence gives valuable information about the binding properties of these sites.The dissociation constant of enzyme-coenzyme complexes is given byKD,EL=K · KD,EF whereKD,EF is the dissociation constant of the enzyme-dye complex andK is the equilibrium constant between dye and coenzyme during the displacements.The method described is used to determine the dissociation constants of NAD+ and NADH on alcoholdehydrogenase from yeast and horse liver and on lactate dehydrogenase from beef and rabbit sceletal muscle. The results are compared with known values.ZusammenfassungDie Bindung des Farbstoffs Tetrajodfluorescein an verschiedene Dehydrogenasen wird herangezogen, um die Dissoziationskonstanten von Enzym-Coenzym-Komplexen zu bestimmen, da der Farbstoff und pyridinhaltige Coenzyme um die gleiche Bindungsstelle konkurrieren. Durch Zugabe der interessierenden Coenzyme läßt sich somit der Farbstoff aus seiner Bindungsstelle am Enzym verdrängen.Die Bindung und die Verdrängung des Farbstoffs läßt sich durch Differenzspektrophotometrie untersuchen, wobei die Eigenschaften der Enzym-Farbstoff- und der Enzym-Coenzym-Komplexe aus den Differenzspektren entnehmbar sind. Dabei ergibt sich folgender Zusammenhang:KD,EL=K · KD,EF, d.h. die gesuchte Dissoziationskonstante des Enzym-Coenzym-Komplexes ist das Produkt aus der Dissoziationskonstanten des Enzym-Farbstoff-Komplexes und der „Kopplungskonstanten“K zwischen Farbstoff und Coenzym bei der Verdrängungsreaktion.Um Anwendbarkeit und Genauigkeit dieser Methodik zu zeigen, werden die Dissoziationskonstanten von NAD+ bzw. NADH an Alkoholdehydrogenase aus Hefe und Pferdeleber und an Lactatdehydrogenase aus Rinder- und Kaninchenmuskel bestimmt und mit den bekannten Werten verglichen.