PK Variant Research Articles

Nitrogen and Phosphorus Availability Effect on Activity of Cellulolytic Microorganisms in Meadows

Meadows occupy more than 23 % of agricultural land in the Czech Republic and also represent the largest pool of carbon in soil organic matter. The organic material is an essential component of healthy soil. Decomposition of organic matter is a biological process, affected by high amount of N and P fertilizer applied in the second half of the twentieth century. This work presents the analysis of the effect of available nutrients nitrogen and phosphorus on the activity of cellulolytic microorganisms in permanent grassland of Sanguisorba-Festucetum comutatae association in the soil surface in the Protected Landscape Area of Žďárské Hills. Contents of available nutrients established by the Mehlich III method were measured ex situ. Data referring cellulose decomposition rates were obtained in situ using the standard mesh-bag method. In the field experiment, the highest loss of cellulose was recorded in variants with the addition of nutrients (PK, 90N + PK and 180N + PK) with result 90.38 % on average. The difference was statistically significant, compared with the control variant without added nutrients 27.87 % (P < 0.05). The added amount of nitrogen in the variant 90 kg of N + PK per ha had no significant effect on loss of cellulose compared to PK variant, as well as variant 180 kg of N + PK per ha. From the results obtained, it is evident that the highest decomposition of cellulose was observed at variant with added phosphorus, compared to variant with nitrogen. Moreover, the effect of different intensity of grassland utilization was found: the amount of decomposed cellulose was higher in two variants (control and 90N + PK) of two cut system in comparison with the same variant in three cut system.

The efficiency of nutrient utilization by permanent grassland in the Kameničky locality

This work presents the analysis of the effect of grassland management on leaching of mineral nitrogen from rhizosphere and plant biomass production in area of our interest. The determined values show the influence of fertilization and species composition of permanent grassland on leaching of mineral nitrogen and biomass production. The values were determined during field and laboratory experiments. The highest availability of Nmin in the field experiment was detected in the variant N90+PK; the lowest availability of Nmin was recorded in the PK variant. The difference was statistically significant (P < 0.05). In the pot experiment, the highest loss of Nmin was recorded in the variant with N180+PK (16.88 mg.dm−3) with the difference being statistically significant as compared with the other variants (P < 0.05). Measured values of the availability of mineral nitrogen and plant biomass production indicate the importance of species composition at the establishment of new grassland. For example, species composition of permanent grassland has a great importance at the establishment of new grassland on erosion-affected soils in protection areas of drinking water. Because selected species of grasses can immobilize nitrogen in their bodies, thus preventing it’s leaching from the soil.

Molecular Characterization of Hemoglobinopathies and Red Cell Enzymopathies in the Czech and Slovak Populations: An Update

Abstract 5307 Background:Thalassemias are rare disorders in Middle Europe. However, as a result of historical and recent migration, thalassemias became common cause of congenital anemia in the Czech and Slovak populations. Abnormal hemoglobin variants and red-cell enzymopathies are rare cause of congenital anemia in this region.The aim of this work was to update the original reports of this research published almost two decades ago (Indrak et al., Hum Genet 1992; 88:399–404, Xu et al., Blood 1995; 85:257–63, Lenzner et al., Blood 1997; 89:1793–9). We assessed the frequency and spectrum of β-globin gene mutations in the patients with clinical symptoms of β-thalassemia or δ,β-thalassemia, the α-globin gene status in the patients with clinical symptoms of α-thalassemia, and we characterized red cell enzymopathies on molecular level in the Czech and Slovak populations. Patients and methods:Nearly 390 cases with clinical symptoms of thalassemia or hereditary nonspherocytic hemolytic anemia from several centers of Czech and Slovak Republic were analyzed. Hematological parameters, hemoglobin electrophoresis and enzyme activities were measured by standard procedures. Genomic DNA was used for PCR-sequencing analysis. Results:We identified 22 β-thalassemia mutations in more than 260 heterozygotes; most of the mutations were of Mediterranean origin. The newly discovered insertion of transposable element L1 into the HBB gene represents a novel etiology of β-thalassemia due to a silencing effect of repressive chromatin associated with retrotransposon insertion. The list of abnormal hemoglobins now contains 14 β-globin variants, involving Heinz body hemolytic anemia variant Hb Hana (β63(E7) His-Asn), phenotype of which was worsened by concomitant partial glutathione reductase deficiency (Mojzikova et al., Blood Cells Mol Dis 2010; 45:219–22).Several G6PD and PK variants were described in the Czech and Slovak populations; the G6PD variants include G6PD Olomouc, G6PD Varnsdorf and G6PD Praha. Recently, we identified a new frameshift mutation c. 1553delG (p. Arg518fs) at the homozygous state in exon 11 of the PKLR gene of the pediatric patient who suffered from transfusion dependent hemolytic anemia with Hb=9.4 g/dL, Ret=4.5%. His red cells PK activity was 4.52 IU/gHb (normal range 13–17 IU/gHb). The mutation occurs in C domain of PK-R subunit containing the binding site for fructose-1,6-bisphosphate. The patient’s extremely elevated level of growth differentiation factor 15 (GDF15, 3577 pg/mL, healthy controls 231–345 pg/mL) could explain hereditary hemochromatosis and signs of iron overload in this patient. Conclusions:In the Czech and Slovak populations, hemoglobinopathies and red-cell enzymopathies appear to be an uncommon disorder, which, however, must be considered as the prevailing cause of congenital anemia. Most of the thalassemia patients were heterozygous, manifesting thalassemia minor. Most of the hemoglobin variants were described in single families, some of them originated locally. Among hemolytic anemias due to red-cell enzymopathies is the most frequent PK deficiency.This work was supported by grants NT11208, NS10281 (Ministry of Health Czech Republic), MSM6198959205 (Ministry of Education, Youth and Sports) and student projects LF_2011_006 and LF_2011_011 of the Palacky University. Disclosures:No relevant conflicts of interest to declare.

Molecular dissection of Drosophila Prickle isoforms distinguishes their essential and overlapping roles in planar cell polarity

Prickle-Spiny-Legs (Pk) is an essential component of the planar cell polarity (PCP) pathway, together with Frizzled (Fz) and Dishevelled (Dsh). A role for Pk was proposed to mediate feedback amplification of asymmetric Fz/Dsh activity across cell boundaries, ensuring a single prehair initiates at each distal vertex. Here we show that apical localisation of Pk Pk and Pk Sple isoforms are mutually independent and regulated by the C-terminal domain. The N-terminus of Pk Pk is dispensable for PCP, whereas the unique N-terminal domain of Pk Sple contains an additional localisation function, which confers a qualitatively different activity. Our results suggest that endogenous Pk Pk and Pk Sple can affect each other's function via the C-terminal domain, yet may not form heteromeric complexes. Overexpressing PET domain-deleted Pk variants interferes with a branch of Fz/Dsh signalling that regulates the number of wing hairs, and blocks non-cell-autonomous repolarisation. We infer that Pk Pk is sufficient to mediate the intercellular feedback signalling. Significantly, Pk Pk but not Pk Sple is required for hexagonal cell packing in the pupal wing. We propose that Fz-dependent PCP readout reflects short-range, cell-contact based, interactions between hexagonal cells, rather than a direct response to an as yet unidentified diffusible ligand.

Catalytic Properties of Two Pyruvate Kinase Isoforms in Nordic Krill, Meganyctiphanes norvegica, With Respect to Seasonal Temperature Adaptation

Two isoforms of pyruvate kinase (PK I and PK II) were partly purified and characterized from the Nordic krill Meganyctiphanes norvegica. Both PK variants were present in summer and winter specimens with a tissue specificity in abdominal muscle (PK I) and cephalothorax (PK II). Obvious differences were found in chromatographic and kinetic characteristics. Enzymatic adaptations to low temperatures were found in PK I only, whereas PK II did not contribute to seasonal temperature adaptation. In winter specimens, the activation energy of PK I decreased significantly from 53.2 ± 1.5 to 50.2 ± 1.2 kJ·mol−1. The affinity of PK I to phosphoenol-pyruvate was higher in winter (KM = 0.024 ± 0.002 mmol·l−1) compared to summer (KM = 0.033 ± 0.003 mmol·l−1). Both effects lead to an increased efficiency of this enzyme isoform in the cold. In contrast, KM values of PK II showed no significant differences between summer (KM = 0.181 ± 0.014 mmol·l−1) and winter specimens (KM = 0.193 ± 0.015 mmol·l−1). The effects of cooperativity remained unchanged during the seasons with approximate values of nHill = 1.0 (PK I) and 1.5 (PK II). Fructose-1,6-bis-phosphate affected only PK II by shifting sigmoidal kinetics to hyperbolic curves resulting in a decrease of KM to 0.027 mmol·l−1. Further effectors were tested showing an inhibiting effect of oxalate on both isoforms with a reduction to 20% and 50% in PK I and PK II, respectively. Presumably, the ecophysiological effect of the capacity to regulate muscle PK is related to the necessity to increase motility during vertical migration and phases of feeding activity.

Modification of pyruvate kinase from the foot muscle of Patella caerulea (L) during anaerobiosis

AbstractPyruvate kinase (PK) from the foot muscle of Patella carulea (L) is present as two variants. These variants are eluted by ion‐exchange chromatography from DEAE‐cellulose as peak I and peak II at different concentrations of KCl. Studies of their kinetic properties show that both variants of PK exhibit sigmoidal curves with respect to phosphoenolpyuvate (PEP), but the PK in peak I is more sensitive to the activation by PEP and fructose diphosphate (FDP) and less sensitive to inhibition by alanine, ATP, and H+ than the PK in peak II. The proportion of the two PK variants changes during anaerobiosis. The ratio of the number of units of enzyme in peak I to that in peak II increases up to the second hour and then decreases up to the eight hour of anaerobiosis. Studies on the electrophoretic mobility of these two variants on strips of cellulose acetate show that the less active from or peak II PK migrates more quickly toward the anode (Rf = 0.13) than the more active from or peak I PK (Rf = 0.07). Peak II PK, after treatment with alkaline phosphatase, exhibits kinetic properties and electrophoretic mobility quite similar to those of peak I PK. The above data suggest that the two variants of PK from the foot muscle of P. caerulea are interconvertible and the direction of the interconversion depends on the duration of anaerobiosis.

Purification and properties of aerobic and anoxic forms of pyruvate kinase from red muscle tissue of the channelled whelk, Busycotypus canaliculatum.

Aerobic and anoxic variants of radular retractor muscle pyruvate kinase (PK-aerobic and Pk-anoxic) from the gastropod mollusc, Busycotypus canaliculatum, were purified to homogeneity and respective specific activities of 368 and 186 mumol of product min-1 mg protein-1. Both PK variants were apparent homotetramers with native molecular masses of about 235 kDa, but differed in several other physical characteristics including pI (5.81 +/- 0.06 for PK-aerobic, 5.42 +/- 0.03 for PK-anoxic) and chromatographic behavior on several columns used during their respective purifications. The two enzymes differed greatly in several kinetic properties. Affinity for phosphoenolpyruvate was more than tenfold greater for PK-aerobic (K0.5 = 0.067 +/- 0.002 mM; h = 0.99 +/- 0.10), whereas the cooperative effect for phosphoenolpyruvate binding was greatly enhanced for PK-anoxic (K0.5 = 0.85 +/- 0.02 mM, h = 2.57 +/- 0.01). Although the affinities for the second substrate, ADP, were identical for both enzyme forms (apparent Km = 0.25 mM) pK-anoxic showed greater substrate inhibition by high concentrations of ADP. Likewise, affinities for K+ and Mg2+ were similar but PK-anoxic showed a greater degree of cooperativity with Mg2+ (h = 2.50 +/- 0.02) than did PK-aerobic (h = 1.70 +/- 0.06). Saturating concentrations of fructose 1,6-bisphosphate (50 microM) activated PK-anoxic resulting in an enzyme with properties similar to fructose-1,6-bisphosphate-activated PK-aerobic, with K0.5 values for phosphoenolpyruvate of about 0.04 mM and Hill coefficients of 1.1. PK-anoxic showed much stronger regulation by the allosteric inhibitors MgATP, phenylalanine, proline and alanine. Fructose 1,6-bisphosphate partially relieved the inhibitions by ADP, MgATP, alanine, proline and arginine phosphate of both enzyme forms. However, at 0.1 mM phosphoenolpyruvate PK-aerobic was much more sensitive to activation by fructose 1,6-bisphosphate, Ka values being 0.05 +/- 0.01 microM for PK-aerobic and 1.3 +/- 0.1 microM for PK-anoxic. In the presence of 1.0 mM alanine and 1.5 mM MgATP much higher concentrations of fructose 1,6-bisphosphate were required for activation of PK-anoxic (Ka = 5.2 +/- 0.4 microM) than for PK-aerobic (Ka = 0.02 +/- 0.01 microM). Variations in pH over the range likely occurring in vivo during anaerobiosis caused no significant additional kinetic differences between the two enzyme forms. The dissimilarity in kinetic properties of PK-aerobic and PK-anoxic indicate that red muscle PK activity is probably strongly depressed in vivo during anoxia stress.

Identification of an Erythrocyte Pyruvate Kinase Variant in a Family from Latium with Non-Spherocytic Congenital Haemolytic Anaemia

Erythrocyte PK deficiency was detected in a family from Latium in Italy. This PK variant is characterized by normal or increased activity immediately after blood collection, instability to storage, to heat and to urea. Only in the propositus the mutant enzyme exhibited an increased Michaelis constant for PEP, slightly increased inhibition by ATP and an altered optimum pH value. The kinetic anomaly was only partially corrected by activation with F-1, 6-DP and by addition of 2-ME. From these results it can be concluded that in the family observed two distinct erythrocyte PK alterations were demonstrable: instability in the propositus and his father; low affinity for PEP and altered optimum pH value only in the propositus.

Electrophoretic demonstration of heterozygosis in hereditary pyruvate kinase deficiency

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.

A spanish family with erythrocyte pyruvate kinase deficiency: Contribution of various immunologic methods in the study of the mutant enzyme

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 K 0.5 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.

Pyruvate kinase electrophoresis in normal and pyruvate kinase deficient hemolysates

Two bands of pyruvate kinase activity were demonstrated in normal hemolysates with high voltage electrophoresis at pH 5.3 using cellulose acetate strips. Heterozygotes with red cell pyruvate kinase deficiency also exhibited two bands. Six of ten homozygous patients showed two bands, the remaining four only one band. No mutant enzyme with abnormal electrophoretic mobility could be demonstrated among the ten homozygous individuals with pyruvate kinase deficiency. High voltage electrophoresis may be an additional procedure for the characterization of PK variants in patients with congenital hemolytic anemia associated with PK deficiency.