Zinc inhibition of pyruvate kinase of M-type isozyme

Inhibitory effect of Zn on the pyruvate kinase of M (muscle)-type isozyme was analyzed for the purpose of elucidating the cytotoxicity of Zn. Zn inhibited pyruvate kinase uncompetitively with respect to the substrate PEP, and competitively with respect to ADP. Quotient velocity plot calculated from the Zn-inhibition curves showed that Zn²⁺ as a ZnADP complex acted as competitive and uncompetitive inhibitors of the enzyme with respect to the substrate ADP and PEP, respectively: Zn²⁺ forms a ZnADP complex, which may bind to the ADP-binding site of the free enzyme with the K_i value of 1.4 μM causing competitive inhibition, or to the ADP-site of the enzyme-PEP complex with 2.6 μM resulting in uncompetitive inhibition. The inhibition of pyruvate kinase by Zn²⁺ may be responsible for the cytotoxicity of this metal by decreasing glycolytic flux.     

Immunohistochemical localization of pyruvate kinase isoenzymes in chicken tissues

Summary A method for the localization of pyruvate kinase isoenzymes type L, M₂ and M₁ in tissue sections is described. Mono-specific antibodies directed against isoenzymes of pyruvate kinase from chicken and the peroxidase antiperoxidase method were used. The following preferential localizations of the isoenzymes in chicken tissues were observed: Pyruvate kinase M₁ was found in skeletal muscle. The white muscle fibers were more intensely stained than the red. Some dark muscles (e.g., anterior latissimus dorsi) and the heart muscle showed no reaction with antiserum against pyruvate kinase M₁. Pyruvate kinase type L was found in the hepatocytes and in kidney cortex. Pyruvate kinase type M₂ was seen in the distal tubules of kidney, in hepatocytes and sinusoidal cells in liver, in lung, adipose tissue, and in the spleen mainly in the bursa dependent areas. Pyruvate kinase type M₂ was detected in high concentrations in the granulation tissue of type M₂ was detected in high concentrations in the granulation tissue of regenerating liver after partial hepatectomy. Liver sections of a hen bearing a pancreatic tumor showed an unusually high content of pyruvate kinase type M₂ in some hepatocytes, which were each clustered to spots in the liver parenchyma. Thus, contrary to previous reports, the tissue distribution of isoenzymes in chicken is similar to that of other vertebrates.     

Immunohistochemical localization of pyruvate kinase isoenzymes in chicken tissues.

A method for the localization of pyruvate kinase isoenzymes type L, M2 and M1 in tissue sections is described. Mono-specific antibodies directed against isoenzymes of pyruvate kinase from chicken and the peroxidase antiperoxidase method were used. The following preferential localizations of the isoenzymes in chicken tissues were observed: Pyruvate kinase M1 was found in skeletal muscle. The white muscle fibers were more intensely stained than the red. Some dark muscles (e.g., anterior latissimus dorsi) and the heart muscle showed no reaction with antiserum against pyruvate kinase M1. Pyruvate kinase type L was found in the hepatocytes and in kidney cortex. Pyruvate kinase type M2 was seen in the distal tubules of kidney, in hepatocytes and sinusoidal cells in liver, in lung, adipose tissue, and in the spleen mainly in the bursa dependent areas. Pyruvate kinase type M2 was detected in high concentrations in the granulation tissue of regenerating liver after partial hepatectomy. Liver sections of a hen bearing a pancreatic tumor showed an unusually high content of pyruvate kinase type M2 in some hepatocytes, which were each clustered to spots in the liver parenchyma. Thus, contrary to previous reports, the tissue distribution of isoenzymes in chicken is similar to that of other vertebrates.     

Kinetic properties of human muscle pyruvate kinase

Summary The steady-state kinetics of human skeletal muscle pyruvate kinase (M_A) and its RNA-complex (M_B) has been examined and compared. Kinetic studies revealed significant differences in kinetic properties with respect to free and complex form of pyruvate kinase.The M_A form follows a simple Michaelis-Menten kinetics in contrast with the M_B form, which displays a negative cooperativity with respect to ADP. V_max for the complex is 40–60% that for free enzyme. Heterologous RNA is a noncompetitive inhibitor of free enzyme but the kinetics of the complex (M_B) is not affected.In presence of 1.0 mM ATP in an assay mixture the kinetic constants of the complex were unchanged except for V_max, which increased by nearly 60%. Aged preparations of free enzyme (M_A) were activated by 100% and more, but the native enzyme was inhibited by 22%.Inorganic phosphate is a potent activator of both forms of pyruvate kinase. In presence of 50 mM K-phosphate the apparent Michaelis constant and interaction coefficient are unchanged, but V_max for free enzyme increases by 35% and for the complex by 70%, respectively. The specific activity of aged M_A form can be restored to the original value after incubation of the enzyme in 50 mM K-phosphate, pH 7.6, or by addition of ATP (1.0 mM) to the assay mixture.     

Ontogenesis of pyruvate kinase in the brain and liver tissues of the rat

The ontogenetic study of pyruvate kinase in the brain and liver tissues was performed in different batches of rats, from the fœtus at the 13th day of gestation to the adult subject.

— According to the kinetic study, the shape of the curve is transformed from sigmoid to hyperbolic from the 13th day of fœtal life to adulthood in the brain. Hill cœfficient increases with the age of animal in the liver tissue.

— According to polyacrylamide gel isoelectrofocusing, a family of four, transitory or definite bands are detected in the fœtal brain: they are well defined by their pHi: M₄, M₃, M₂, M₁; at the adult stage, M₁ predominates, M₂ is minor. Three principal bands are distinguished in the liver: two are characteristic of fœtal life (Lf and M₂), one of adulthood (L).

— According to the immunochemical tests, there are antigenic determinants common to M₁, M₂, M₃ and M₄.

The confrontation of the first two methods prompts the conclusion that the kinetic of the enzyme (and perhaps its function) varies with the animals age and is linked to its molecular structure. With the third method, it allows to stress the precociousness of the appearance of the common antigenic determinants, simultaneously with immature enzymatic forms.The signification of the kinetic modifications as well as the succession of the isozymes of the M type in a determined order are discussed and the in vivo formation of hybrids is suggested.     

Post-translational modification of human erythrocyte pyruvate kinase

Upon storage, partially purified human erythrocyte pyruvate kinase (ATP: pyruvate-phosphotransferase, E.C. 2.7.1.40) from normal individuals was found to undergo a spontaneous oxidation to a form which displayed markedly reduced activity. This modified form of the enzyme exhibited kinetic patterns similar to those frequently reported for the enzyme in cases of nonspherocytic hemolytic anemia. The data are discussed in relation to the recently proposed theory that post-translational modification of pyruvate kinase is responsible for the abnormal kinetic patterns frequently encountered for this enzyme in the disease state. [Van Berkel, T. J. C., Koster, J. F., Kruyt, J. K. and Staal, G. E. J. 1973 $\text{Biochim. Biophys. Acta 321}$, 496–502].     

Purification and characterization of human muscle pyruvate kinase.

The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240700 and has a sedimentation coefficient (S20,W) of 10.04S. It contains four subunits with identical molecular weights of 61000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross-react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.     

Ontogenesis of pyruvate kinase in the brain and liver tissues of the rat.

The ontogenetic study of pyruvate kinase in the brain and liver tissues was performed in different batches of rats, from the foetus at the 13th day of gestation to the adult subject. -- According to the kinetic study, the shape of the curve is transformed from sigmoid to hyperbolic from the 13th day of foetal life to adulthood in the brain. Hill coefficient increases with the age of animal in the liver tissue. -- According to polyacrylamide gel isoelectrofocusing, a family of four, transitory or definite bands are detected in the foetal brain : they are well defined by their pHi : M4, M3, M2, M1 ; at the adult stage, M1 predominates, M2 is minor. Threee principal bands are distinguished in the liver : two are characteristic of foetal life (Lf and M2), one of adulthood (L). -- According to the immunochemical tests, there are antigenic determinants common to M1, M2, M3 and M4. The confrontation of the first two methods prompts the conclusion that the kinetic of the enzyme (and perhaps its function) varies with the animals age and is linked to its molecular structure. With the third method, it allows to stress the precociousness of the appearance of the common antigenic determinants, simultaneously with immature enzymatic forms. The signification of the kinetic modifications as well as the succession of the isozymes of the M type in a determined order are discussed and the in vivo formation of hydbrids is suggested.     

Spermatogenesis-related change in the synthesis of the creatine kinase B-type and M-type isoforms in human spermatozoa

We have demonstrated earlier that the per sperm creatine-N-phosphotransferase (CK) activity was increased in oligospermic vs. normospermic men. The increased sperm CK activity is related to higher concentrations of cellular CK, which may indicate a defect of cytoplasmic extrusion during spermatogenesis. In the present work, we examined whether in spermatozoa, similar to muscle, there is a change in the synthesis of B-CK and M-CK isoforms during cellular differentiation. In 109 normospermic and 50 oligospermic specimens (sperm concentrations 60.6 +/- 3.7 vs. 8.8 +/- 1.3 million sperm/ml; all values expressed as mean +/- SEM), the relative concentrations of the M-CK isoform (M-CK/M-CK + B-CK) were 27.2% +/- 2.1% vs. 6.7% +/- 0.9% (P less than 0.001). The per sperm CK activities showed comparable differences (0.21 +/- 0.02 vs. 0.89 +/- 0.1 CK IU/100 million sperm; P less than 0.001) in the two groups, and there was a close correlation between per sperm CK activities and M-CK concentrations (R = 0.69, P less than 0.001, N = 159). This indicates that the loss of cytoplasm and the commencement of M-CK isoform synthesis are related events during the last phase of spermatogenesis, also that the incidence of spermatozoa with incomplete cellular maturation is higher in oligospermic specimens. In characterizing the M-CK, we found that sperm (unlike muscle tissue) lack the MB hybrid of CK dimers. However, in the presence of muscle M-CK, the muscle-sperm MB-CK hybrid has formed. Thus in sperm and muscle the M-CK isoforms are structurally different, whereas the B-CKs are apparently homologous.(ABSTRACT TRUNCATED AT 250 WORDS)