G.E.J. Staal

Human erythrocyte pyruvate kinase. Its purification and some properties

Abstract 1. 1. Human erythrocyte pyruvate kinase (EC 2.7.1.40) was purified 30 000-fold by successive (NH4)2SO4 precipitation and column chromatography with blue dextran 2000. The resulting enzyme preparation had a specific activity of 150 μmoles NADH · min −1 · mg −1 at 25°. 2. 2. The molecular weight as determined by gel filtration with Sephadex G-200 was 205 000 ± 5000 . 3. 3. With starch gel electrophoresis only one band was observed after-detection of the enzyme activity with the fluorescent technique. 4. 4. The variation of activity of pyruvate kinase at various ADP and phosphoenolpyruvate (PEP) concentrations was studied. 5. 5. The stimulatory effect of Fru -1,6-P 2 as well as the inhibitory effect of ATP on the activity of pyruvate kinase were pH dependent. 6. 6. Phosphorylated hexoses ( Fru -1,6-P 2 and Glc -6-P ), P1 and the substrate PEP overcame the inhibition of the activity of pyruvate kinase by ATP at physiological pH. 7. 7. Phosphorylated hexoses and P1 stimulated the activity of pyruvate kinase.

On the molecular basis of pyruvate kinase deficiency II. Role of thiol groups in pyruvate kinase from pyruvate kinase-deficient patients

Abstract 1. 1. Human erythrocyte pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) from the class of pyruvate kinase-deficient patients, characterized by an increased affinity towards phosphoenolpyruvate and a loss of cooperative interaction towards this substrate, shows less affinity for the allosteric inhibitor ATP, when compared to pyruvate kinase from control persons. From the obtained kinetic data we can conclude that the loss of cooperativity towards phosphoenolpyruvate is a consequence of a shift in the R ⇌ T equilibrium to the R state. 2. 2. Incubation of pyruvate kinase, obtained from this class of pyruvate kinase-deficient deficient patients with mercaptoethanol, changes the abnormal kinetics into normal kinetics, as can be conclded from the change in phosphoenolpyruvate dependency and ATP inhibition. 3. 3. The effect of mercaptoethanol on the kinetics of pyruvate kinase from pyruvate kinase-deficient patients suggests that the alteration in the enzyme is a consequence of a modification of the -SH groups. It is suggested that pyruvate kinase deficiency is a secondary defect and that the process which causes the change in the -SH groups of pyruvate kinase, may also be responsible for the increased rate of haemolysis, found in these patients.

Human erythrocyte phosphofructokinase: its purification and some properties.

Abstract 1. 1. Human erythrocyte phosphofructokinase (ATP: d -fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) was purified 15 000-fold by (NH 4 ) 2 SO 4 precipitation, heating and column chromatography on Sepharose 6-B. The resulting enzyme preparation had a specific activity of 60 μmoles Fru-1,6- P 2 formed per min per mg protein at 25 °C. 2. 2. With cellulose-acetate electrophoresis only one band was observed after detection of the enzyme activity with the fluorescent technique. 3. 3. Citrate and 2,3-diphosphoglycerate do not inhibit. The inhibition by ATP is pH dependent. Cyclic AMP is able to reverse the inhibition by ATP to some extent. 4. 4. With GTP, ITP and UTP no inhibition is observed. At saturating concentrations of GTP, ATP still inhibits phosphofructokinase. 5. 5. The variation of the activity of phosphofructokinase at various ATP and Fru-6- P concentrations was studied. In the reaction mechanism a ternary complex is involved.

A NEW VARIANT OF GLUCOSEPHOSPHATE ISOMERASE DEFICIENCY (GPI-UTRECHT)

A new case of glucosephosphate isomerase deficiency is described in a Dutch family. The activity of the enzyme was decreased to 20-25% of the normal value. Characterization of the defect enzyme showed a pronounced thermolability. Heating of the enzyme at 45 degrees C showed a loss of activity of 90% after one hour. The pH-optimum and the electrophoretic migration were normal. The Km-value for F-6-P, the Ki for the competitive inhibitors 2,3-DPG and 6-PG were in the normal range. The variant described here differs from all known variants. Therefore we propose to give to this new variant the name of GPI-Utrecht.

Normalisation of red blood cell pyruvate kinase in pyruvate kinase deficiency by riboflavin treatment

A patient with an erythrocyte glutathione reductase activity of 50% of the normal value and an abnormal pyruvate kinase (PK) was given 36 mg riboflavin daily for 6 months. The glutathione reductase activity was restored and the abnormal pyruvate kinase was converted to normal. The clinical state of the patient improved. It can be concluded that the abnormality of PK, at least with this patient, is a secondary effect. Therefore, it is suggested that other abnormalities be searched for when an altered PK is detected. Treatment of this abnormality will help the patients more efficiently.

The effect of urea and temperature on red blood cell pyruvate kinase

Abstract The effects of urea and temperature on the catalytic activity of red blood cell pyruvate kinase have been studied. Urea inactivates the enzyme rapidly at pH 8.0 and 0°, while at pH 5.9 and 25° the enzyme is quite stable to urea. At pH 5.9 and 25° Fru-1, 6-P2 do not stimulate the enzyme; however, at pH 5.9 and 5° a stimulation occurs, while the Hill coefficient remains unchanged. At pH 8.2 and 5° the enzyme exhibits a somewhat larger Hill coefficient than pH 8.2 and 25°. The obtained data can be explained by the hypothesis that pyruvate kinase exists in two conformations which are in equilibrium with each other.

A new variant of glucosephosphate isomerase deficiency

SummaryA new variant of glucose-6-phosphate isomerase deficiency is described. The enzyme kinetics and properties were studied. Genetic and electrophoretic data pointed to a double heterozygous state in the patient. These data are compared to the other variants described in the literature until now.

Genetic and molecular mechanisms of the congenital defects in glucose phosphate isomerase activity: studies of four families.

Summary: Four patients with hereditary glucose phosphate isomerase (GPI) deficiency and their parents have been studied by means of various enzymatic, immunologic, electrophoretic, and stability methods. The four defective mutants enzymes (identified here as “GPI Barcelona,” “GPI Utrecht,” “GPI Nijmegen,” and “GPI Kortrijk”) were antigenically identical with normal enzyme as judged by double immunodiffusion and microcomplement fixation tests. Immunologic specific activity (i.e., the ratio of enzyme activity to antigen concentration) was slightly lowered for three variants (between 60 and 75% of normal). The various methods used allowed us to establish that all of the patients were heterozygous for two different mutated alleles inherited from each parent. One of them was a silent allele which did not seem to code for any enzymatic cross-reacting material; the other mutated gene coded for a structurally modified glucose phosphate isomerase subunit.In two parents heterozygous for a structurally modified gene, only two enzymatic forms were detected, even in the young cells synthesizing actively proteins such as granulocytes: one was the normal homodimer, and the other corresponded to the heterodimer “normal subunit-mutant subunit”; the mutant homodimer was not detected. The assumption that, in these heterozygotes, the mutant subunit dimerized more easily with a normal subunit than with another mutant subunit could be proposed. In contrast, the three expected enzyme forms were detected in the leukocytes from a woman heterozygous for GPI Nijmegen.From these results the defect in activity observed in the patients could be explained in the following way: 50% of the defect was due to the inheritance of a silent gene. The decrease below 50% of the residual activity in red cells was due to the molecular instability of the mutant enzyme associated in three cases (GPI Barcelona, GPI Utrecht, and GPI Kortrijk) with the decreased immunologic specific activity.Speculation: In spite of the relatively high frequency of the mutations which are responsible for a silent GPI structural gene, GPI deficiency due to the inheritance of two silent genes has never been described. It can be speculated that the homozygous state of such a mutation, which would lead to null enzyme activity in all the tissues, could be lethal. As in most cases of genetic disorders due to silent genes, the nature of the mutation involved is unknown. A deletion, a polar mutation, a structural mutation leading to a highly labile product, or a cis-dominant regulator mutation are the different genetic hypotheses which could be put forward.

A New variant of glucosephosphate isomerase deficiency: GPI-kortrijk

A new case of glucosephosphate isomerase deficiency in a Belgian family is described. The activity of the enzyme was decreased to about 25-30% of the normal value. Characterization of the defect enzyme showed a decreased thermostability. Heating of the enzyme at 45 degrees C showed a loss of activity of 50% after 90 min. The pH-optimum and the KM-value for fructose 6-phosphate were normal. The electrophoretic pattern showed a faster migration. The variant described here differs from all known variants. Therefore we propose to give to this new variant the name GPI-Kortrijk.

Excessive hepatic glycogen storage in glucosephosphate isomerase deficiency.

Abstract. Excessive amounts of glycogen were found in liver and erythrocytes of a patient suffering from generalized glucosephosphate isomerase deficiency. A low carbohydrate diet, frequent meals and avoidance of peak carbohydrate challenges resulted in a significant decrease of liver volume without affecting the haematological condition. The possible mechanism of these findings are discussed.