Biomedical subjects
Publications and source records attributed to J Marie.
The defective PK variant of a patient with a severe form of hemolytic anemia was characterized by its inability to undergo a normal 'proteolytic maturation.' In obligatory heterozygotes it could be proved that red cells contained different PK species, some of them sensitive and the others partially resistant to the action of trypsin.
Six new defective pyruvate kinase variants have been characterized in patients suffering from chronic hemolysis. Partially purified enzyme variants exhibited various anomalies from immunological, kinetic, stability and electrophoretic points of view. The significance of the electrophoretic anomalies has been interpreted in view of the normal post-synthetic maturation of the precursor enzyme L'4 into L2L'2 and L4, and the ability of trypsin to induce in vitro the transition L'4 leads to L4 has been tested. One defective enzyme existed in a single L'4 form and could not be transformed by trypsin into L4. In three cases slow-moving L'4 and L2L'2 forms were transformed by trypsin into an abnormal slow-moving L4 form. In the last two observations the L'4 and L2L'2 forms exhibited normal mobility and were normally transformed by trypsin into L4. The relevance of these data to the functional anomalies of the defective variants and to the nature of the primary genetic anomaly giving rise to the congenital defects in erythrocyte pyruvate kinase is discussed.
Pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from human liver and red cells has been purified to homogeneity; its subunit structure and some of its kinetic characteristics have been studied. The influence of a partial proteolysis by trypsin on the subunit structure, the isozymic pattern and the kinetic characteristics of red cell and liver enzyme have been investigated. From the results of this study we may conclude that: 1. Liver (L-type) pyruvate kinase is composed of 4 identical L subunits while the major form of erythrocyte enzyme (PK-R2) is a heterotetramer designated as L2L2', the molecular weight of L' being slightly higher than that of L subunits (63 000 and 58 000 respectively). Pyruvate kinase PK-R1, predominant in the erythroblasts and the young red cells, is composed of four identical L' subunits. 2. A mild tryptic attack is able to transform PK-R1 into PK-R2, then PK-R2 into pyruvate kinase L (PK-L). The same proteolytic treatment transforms the L' subunits into L ones. 3. Consequently L-type pyruvate kinase seems to be initially synthesized in the erythroid precursors as an L4' enzyme secondarily partially proteolysed into L2L2'. In liver a very active proteolytic system would be responsible for the total transformation into L4 pyruvate kinase. 4. L4' enzyme exhibits Michaelis-Menten kinetic behaviour with an apparent Michaelis constant of 3.8 mM whereas L4 enzyme shows both positive and negative homotropic interactions towards phosphoenolpyruvate and has [S] 0.5 of 1.2 mM. The characteristics of L2L2' are roughly intermediate between those of L4' and of L4. Fructose 1,6-biphosphate decreases [S]0.5 for these three pyruvate kinase forms without suppressing the differences in the apparent affinity for phosphoenolpyruvate of these enzymes. 5. L4 pyruvate kinase is more inhibited by Mg-ATP than L4', with L2L2' in the intermediate range. 6. Tryptic treatment of each enzyme form studied transforms its kinetic behaviour into that observed for L4.
Erythrocyte pyruvate kinase (PK) deficiency was detected in a boy of dutch origin. Immunologic, electrophoretic, and kinetic studies of the enzymes of propositus and the members of his family demonstrated that the boy was heterozygote for two different mutant PK alleles. The mutant enzyme, for which his mother was heterozygote, was characterized by a lower immunologic specific activity, a decreased affinity for the substrate phospho-enol-pyruvate, and a loss of homotropic interactions toward this substrate, an increased affinity toward the allosteric inhibitor MgATP2-, a decreased affinity for the activator fructose-1,6-diphosphate, and a lowered pH optimum. Electrophoresis, Km app. for MgADP, and the reactivity toward the purine nucleotide substrate analogues were normal. The mutant enzyme for which his father was heterozygote was characterized by a decreased affinity for the substrate phospho-enol-pyruvate and a loss of homotropic interactions toward this substrate. All other parameters mentioned above were normal. The use of a kinetic study of mutant enzymes for the detection of heterozygotes is discussed.
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Erythrocyte pyruvate kinase (ATP:pyruvate 2-0-phosphotransferase, EC 2.7.1.40) has been purified 40 000 times from human erythrocytes, according to an original method. The whole purification procedure included toluene extraction, ammonium sulphate fractionation, DEAE-Sephadex batchwise chromatography and affinity chromatography on a Dextran Blue-Sepharose column with specific elution by fructose 1,6-diphosphate. The final preparation had specific activity of 290 I.U./mg of proteins and the overall yield was about 30%. Pyruvate kinase showed only one protein band as judged by sodium dodecyl sulphate acrylamide gel electrophoresis. Pure enzyme was injected into rabbits and monospecific antiserum was obtained able to neutralize, per ml, 150 I.U. of erythocyte-type pyruvate kinase as well as of L-type enzyme. L-type and erythrocyte-type pyruvate kinases showed reactions of complete identity when tested in immunodiffusion against anti-erythrocyte type pyruvate kinase sera; in all cases a single precipitation line could be detected. L-type pyruvate kinase when mixed with anti-erythocyte pyruvate kinase serum suppressed all ability of that antiserum to react immunological with erythocyte enzyme. Finally the microcomplement fixation curves using anti-erythrocyte pyruvate kinase serum were identical for erythrocyte and L-type enzymes. From these results it appeared that no antigenic difference between L-type and erythocyte enzyme could be detected. Consequently the most likely hypothesis is that both these enzymes are coded by the same single gene, the slight electrophoretic differences between them being due to post-synthetic tissue-specific changes.
Erythrocyte PK deficiency was detected in a 38-year-old man from Catalonia, in Spain. His father and his three children were proven to be heterozygous for the same mutant PK variant. This variant was characterized by low immunologic specific activity, normal (or slightly increased) stability to heat and to urea; normal isoelectric point, increased K0.5 for phosphoenolpyruvate, increased inhibition by ATP and normal activation by 0.35 mM fructose 1,6-diphosphate. The mutant PK variant was antigenically identical with wild enzyme as tested against anti wild erythrocyte PK serum by double immunodiffusion and micro complement fixation. The utility and the significance of the immunologic methods to be used in the study of mutant PK variants are discussed.
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The M type isozymes of Pyruvate-kinase have been studied by isoelectrofocusing in thin layer acrylamide ampholine gel, during the ontogeny of rat muscle in vivo and during the differentiation in vitro of myoblasts of a line established by Yaffe. In both cases, multiple subbands have been seen; the most acid (pHi 5.2) was the predominant band in myoblasts and in fetal muscle at the 15th day. Several more cathodic bands appear sequentially in vitro as in vivo, one of them corresponding to the "M2" or "K" band (predominant in kidney). The most cathodic band M1 (pHi 7.3), characteristic of the adult muscle, appears at the 9th day of culture in vitro in multinucleated myotubes and at the 20th day of fetal life in vivo. Kinetic results confirm these electrofocusing results, showing in fetal muscle and in myoblasts a sigmoid saturation curve of pyruvate kinase activity with phosphoenolpyruvate as substrate. This allosteric kinetic is progressively replaced by a Michaëlian kinetics in vitro as in vivo. Consequently, the studies in vitro may serve as a model for myogenesis in vivo, and may contribute to the understanding of the significance of the multiple forms of pyruvate-kinase during this myogenesis.
The authors have looked for altered proteins in senescent cultured cells from adult liver. Four enzymes (phosphoglycerate kinase, M2 type pyruvate kinase, glucose phosphate isomerase and glucose-6-phosphate dehydrogenase) have been studied by immunological and enzymatic titration and electrofocusing. In addition, heat stability of glucose-6-phosphate dehydrogenase (G6PD) was appraised in cell crude extracts and in partially purified preparations. Enzymatic aactivity as well as immunological reactivity of the four enzymes studied were identical 16 lines in phase II and in 13 lines in phase III. Electrofocusing pattern of the enzymes from 'young cells' was identical to the ones from 'old cells'. Finally, G6PD from old cells seemed to be more unstable than G6PD from young cells when studied in crude extracts. These differences, however, disappeared as G6PD was partially purified from old or young cultured cells. Consequently, no evidence of altered protein, either missynthesized or posttranslationally modified, was found in the senescent cultured cells studied. Moreover, this work indicated that the modification of heat stability of G6PD from old cells was not due to the enzyme molecule itself but rather to the cell medium.
While the error theory of ageing has attracted most interest in recent times it cannot yet be regarded as being demonstrated. Posttranslational modifications of proteins genetic theory appears loical but has little in vivo evidence to prove it. Basic mechanisms of ageing probably involve the interaction of several processes.
L-type pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase; EC 2.7.1.40) was purified from human liver by an original method. This purification included toluene extraction, a-monium sulphate fractionation, DEAE-Sephadex bactchwise, CM-Sephadex batchwise with elective elution by ATP and affinity chromatography on a Blud Dextran-Sepharose column with specific elution by fructose 1, 6-diphosphate. This purification procedure allowed us to obtain 6 mg protein with a specific activity of 420 IU/mg protein, i.e. 2,690-fold purification with an overall yield of 34%. This preparation was homogeneous as judged by immuno-diffusion, acrylamide and sodium dodecyl sulphate acrylamide-gel electrophoresis.
Blood from 10 normal healthy adults and cord blood from 8 healthy full term infants were infiltrated through a mixture sulfoethylethycellulose-Sephadex G 25 in order to eliminate the platelets and the leukocytes. Then the erythrocytes were fractionated into young and old cells by centrifugation in microhematocrit tubes. The enzyme activity and the immunologic reactivity of glucose phosphate isomerase (EC.5.3.1.9), phosphoglycerate kinase (EC.2.7.2.3), pyruvate kinase (ec.2.7.1.40), glucose 6-phosphate dehydrogenase (EC. 1.1.1.49), and 6-phosphogluconate dehydrogenase (EC.1.1.1.44) were measured in every fraction. As previously reported, the enzyme activities were far higher in cord blood than in adult blood red cells; nevertheless, the age-related loss of enzyme activity was similar in both cord and adult blood. The decrease of the enzyme activity of glucose phosphate isomerase and phosphoglycerate kinase in old cells was singly associated with a lowered concentration of the enzyme-related antigen; by contrast, the age-related decrease of the enzyme activity of pyruvate kinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase was associated with both a lowered concentration of the enzyme-related antigen and a lowered "molecular specific activity" (i.e., a lowered ratio of enzyme activity to enzyme-related antigen concentration). This phenomenon was especially marked for pyruvate kinase, which had a molecular specific activity in old cells that was 68% of that in young cells. Phosphofructokinase had a lower enzyme activity in cord blood erythrocytes than in adult blood erythrocytes; the difference was especially important in old cells from infants in which phosphofructokinase activity was 53% of that in old cells from adults. Phosphofructokinase from old cells of full term infants and from unfractionated cells from two premature infants (21 and 32 weeks of gestation) was less neutralized by anti-muscle phosphofructokinase serum and more inhibited by ATP than the enzyme from adult blood erythrocytes.
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Acquired enzymatic activity defects of erythrocyte pyruvate kinase, glucose phosphate isomerase and phosphofructokinase have been studied in patients with acute myeloid leukemias, sideroblastic refractory anemias and unclassified acquired dyserythropoiesis. 6 patients with acute myeloid leukemia had a lowered erythrocyte pyruvate kinase activity; in 5 of them the concentration of the "pyruvate kinase"-antigen was parallely decreased, in such a manner that the ratio enzyme activity/immunologic reactivity (i.e. the molecular specific activity) was normal. In 1 patient with acute leukemia, 4 with refractory anemia and 1 with acquired dyserythropoiesis the defect of the pyruvate kinase activity was associated with a normal antigen concentration (and, therefore, the molecular specific activity in whole hemolysate was lowered). The enzyme activity was restored by incubation with SH reagents in two cases and by partial purification as often as it was performed. The electrofocusing pattern of erythrocyte pyruvate kinase was normal in both these types of defects. In two patients with so-called "acquired dyserythropoiesis" an erythrocyte glucose phosphate isomerase deficiency has been detected; in both the cases it was associated with a parallel decrease of the antigen concentration. The residual enzyme had a normal electrofocusing and electrophoretic pattern and a normal heat stability; the enzyme activity could not be restored by any treatment. In 1 patient with erythroleukemia and in 1 other with acquired dyserythropoiesis the erythrocyte phosphofructokinase activity was lowered. The enzyme activity was not restored by cross incubation in isologous plasma or by the SH reagents. In one case immunologic study could be performed, indicating that the enzyme defect was mainly due to the decreased ratio of the muscle type subunit of the erythrocyte phosphofructokinase. The electrofocusing pattern of deficient phosphofructokinases was normal. Finally, we point out the probable existence of several direct mechanisms, genetic and post translational, accounting for the acquired enzyme defects of red blood cells in various blood disorders.