Localization of chicken muscle FBPase in cardiomyocyte nuclei.

Fructose 1,6-bisphosphatase (FBPase; EC 3.1.3.11) localization in cardiomyocyte nuclei has recently been investigated in mammals [FEBS Lett. 539 (2003) 51]. In this study, nuclear localization of FBPase in the cardiac muscle of the chicken was studied by immunohistochemistry and other methods. A result of the electron microscopic investigation was confirmed by immunoblotting analysis. Using MALDI Q-TOF mass spectrometry and Mascot program, the nuclear FBPase was identified as muscle chicken FBPase. FBPase activity in isolated cardiomyocyte nuclei was 5.9 mU/g. Nuclear FBPase was strongly inhibited by allosteric inhibitor AMP. I(0.5) for AMP was 0.16 microM and was the same as for the purified chicken muscle enzyme.

The interaction of FBPase with aldolase: a kinetic and fluorescence investigation on chicken muscle enzymes.

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) is strongly inhibited by AMP in vitro and, therefore, at physiological concentrations of substrate and AMP, FBPase should be completely inhibited. Desensitization of rabbit muscle FBPase against AMP inhibition was previously observed in the presence of rabbit muscle aldolase. In this study, we analysed the kinetics of an FBPase catalyzed reaction and interaction between chicken muscle FBPase and chicken muscle aldolase. The initial rate of FBPase reaction vs. substrate concentration shows a maximum activity at a concentration of 20 microM Fru-1,6P2 and then decreases. Assuming rapid equilibrium kinetics, the enzyme-catalyzed reaction was described by the substrate inhibition model, with Ks approximately 5 microM and Ksi approximately 39 microM and factor beta approximately 0.2, describing change in the rate constant (k) of product formation from the ES and ESSi complexes. Based on ultracentrifugation studies, aldolase and FBPase form a hetero-complex with approximately 1:1 stoichiometry with a dissociation constant (Kd) of 3.8 microM. The FBPase-aldolase interaction was confirmed via fluorescence investigation. The aldolase-FBPase interaction results in aldolase fluorescence quenching and its maximum emission spectrum shifting from 344 to 356 nm. The Kd of the FBPase-aldolase complex, determined on the basis of fluorescence changes, is 0.4 microM at 25 degrees C with almost 1:1 stoichiometry. This interaction increases the I(0.5) for the AMP inhibition of FBPase threefold, and slightly affects FBPase affinity to magnesium ions, increasing the Ka and Hill coefficient (n). No effect of aldolase on the FBPase pH optimum was observed. Thus, the decrease in FBPase sensitivity to AMP inhibition enables FBPase to function in vivo thanks to aldolase.

The effect of high dose of cortisol on glucose-6-phosphatase and fructose-1,6-bisphosphatase activity, and glucose and fructose-2,6-bisphosphate concentration in carp tissues (Cyprinus carpio L.).

The effect of a high dose of cortisol (200 mg kg(-1) body mass) on juvenile carp was investigated. The activity of glucose-6-phosphatase in liver and of fructose-1,6-bisphosphatase in liver, kidney and muscle, the serum glucose and fructose-2,6-bisphosphate concentration as well as the serum concentration of the injected hormone were measured after 24, 72 and 216 h after intraperitoneal cortisol injection. The activities of fructose-1,6-bisphosphatase in liver and kidney and glucose-6-phosphatase in liver were elevated in comparison with the control, while the fructose-1,6-bisphosphatase activity in the muscle tissue was unchanged. After cortisol injection, the serum glucose level was nearly two times higher after 24 and 72 h and was still 50% higher after 216 h compared with controls. In contrast, the liver fructose-2,6-bisphosphate concentration was unchanged after 24 h. More than two times higher fructose-2,6-bisphosphate concentration was observed in liver after 72 h and it was still elevated after 216 h after the cortisol injection.

Mitochondrial genome organization and divergence in hybridizing central European waterfrogs of the Pelophylax esculentus complex (Anura, Ranidae).

Natural transfer of mitochondrial DNA has occurred between three western Palaearctic waterfrog taxa: Pelophylax lessonae, Pelophylax ridibundus and their hybridogenetic hybrid, Pelophylax kl. esculentus. The transfer is asymmetric with most P. kl. esculentus and approximately one third of all central European P. ridibundus having mtDNA derived from P. lessonae (L-mtDNA). We obtained complete nucleotide sequences of multiple mitochondrial genomes (15,376-78 bp without control regions) from all 3 taxa, including a P. ridibundus frog with introgressed L-mtDNA. The gene content and organization of the mitogenomes correspond to those typical of neobatrachians. Divergence between the mtDNAs of P. lessonae and P. ridibundus is high with an uncorrected p-distance of 11.9% across the entire mitogenome. However, the rate of nucleotide substitution depends on the degree of functional constraint with up to 30-fold differences in levels of divergence. In general, mitochondrial genes encoding the translational machinery evolve very slowly, whereas genes encoding polypeptides of the electron transport system, especially the ND genes, evolve rapidly. Only 25 of 211-213 observed amino acid replacements could be classified as radical and are therefore more likely to be exposed to selection. A disproportionately high number of amino acid substitutions has occurred in the ND4, ND4L and cytb genes of the P. lessonae lineage (including 36% of all radical changes). In contrast to the interspecific divergence, nucleotide polymorphism within L- and R-mtDNA is very low: L-mtDNA haplotypes differed on average by only 19 nucleotides, while there was no variation within two mtDNAs derived from P. ridibundus. This is an expected finding considering that we have sampled a post-glacial expansion area. Moreover, the introgressed L-mtDNA on a P. ridibundus background differed from other L-mtDNAs by only a few substitutions, indicative of a very recent introgression event. We discuss our findings in the context of natural selection acting on L-mtDNA and its potential significance in cytonuclear epistasis.

A simplified molecular method for distinguishing among species and ploidy levels in European water frogs (Pelophylax).

Western Palearctic water frogs in the genus Pelophylax are a set of morphologically similar anuran species that form hybridogenetic complexes. Fully reliable identification of species and especially of hybrid ploidy depends on karyological and molecular methods. In central Europe, native water frog populations consist of the Pelophylax esculentus complex, that is, P. lessonae (LL), P. ridibundus (RR) and the hybrid form P. esculentus that can have different karyotypes (RL, LLR and RRL). We developed existing molecular methods further and propose a simple PCR method based on size-differences in the length of the serum albumin intron-1 and the RanaCR1, a non-LTR retrotransposon of the chicken repeat (CR) family. This PCR yields taxon-specific banding patterns that can easily be screened by standard agarose gel electrophoresis and correctly identify species in all of the 160 samples that had been identified to karyotype with other methods. To distinguish ploidy levels in LR, LLR and RRL specimens, we used the ratio of the peak heights of the larger (ridibundus specific) to the smaller (lessonae specific) bands of fluorescently labelled PCR products resolved on a capillary DNA sequencer and obtained a correct assignment of the karyotype in 93% of cases. Our new method will cut down time and expenses drastically for a reliable identification of water frogs of the P. esculentus complex and potentially for identification of other hybridogenetic complexes and/or taxa, and it even serves as a good indicator of the ploidy status of hybrid individuals.

Kinetic properties of Pelophylax esculentus muscle FBPase.

D-Fructose-1,6-bisphosphate 1-phosphohydrolase FBPase; [EC 3.1.3.11] was isolated from Pelophylax esculentus muscle in an electrophoretically homogeneous form with ca 30% yield. Its subunit molecular mass is ca 37 kDa. In this study, we determined the basic kinetic properties of the frog muscle enzyme. FBPase exhibited a maximum activity at pH 7.5. Like other FBPases the frog enzyme requires magnesium ions for its activity (K(a)=263 microM) and is activated by potassium ions (K(a)=63.6 microM). I(0.5) for calcium ion (91 microM) is 100 times higher than the corresponding value of mammalian muscle FBPase. K(s) for the substrate was 1.68 microM. Substrate excess inhibited the enzyme (K(si)=55 microM). AMP and fructose-2,6-bisphosphate (Fru-2,6P(2)) are potent inhibitors of frog muscle FBPase with I(0.5) of 0.2 microM and K(i) of 114 nM, respectively. Both inhibitors act synergistically on the frog muscle FBPase. In the presence of 0.05-0.5 microM of AMP, K(i) for Fru-2,6P(2) is 92 and 28 nM. I(0.5) for AMP for P. esculentus muscle FBPase is 55 times lower than the corresponding value for P. esculentus liver isozyme.

Mononuclear and polymorphonuclear leukocytes show increased fructose-1,6-bisphosphatase activity in patients with type 1 diabetes mellitus.

The activity and localization of fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) in blood leukocytes of patients with type 1 diabetes mellitus and healthy adults were investigated immunocytochemically. The amount of polymorphonuclear (PMN) and mononuclear (MN) cells with positive FBPase immunocytochemical reaction was 57% and 68%, respectively, in pathological, and 38% and 42%, respectively, in healthy donors. Results of light microscopic investigations were confirmed by measurements of FBPase activity following lysis of PMN and MN cells. The enzyme activity of PMN and MN leukocytes was higher in diabetes mellitus than in healthy adults, by 30% and 127%, respectively. Using immunocytochemistry together with electron microscopy, FBPase was detected not only in the cytoplasm but also in the nucleus of leukocytes of both patients with insulin-dependent diabetes mellitus and healthy donors.

F-actin, beta-tubulin, aldolase, and fructose-1,6-bisphosphatase in heteropteran ovarioles--I. Immunocytochemical investigations of whole-mounted ovarioles.

The distribution of F-actin, beta-tubulin, aldolase, and fructose-1,6-bisphosphatase (FBPase) in ovarioles of four heteropteran species (Ilyocoris cimicoides, Coreus marginatus, Lygus pratensis, and Notostira elongata) was investigated biochemically and immunocytochemically. Aldolase was found to be uniformly distributed in the cytoplasm of trophocytes and follicular cells, with the highest concentration in prefollicular cells. Its concentration in follicular cells increased during differentiation and reached a peak in ovarian follicles at the stage of late choriogenesis. FBPase was observed in the cytoplasm (weak reaction) and on cell borders (strong reaction) of both germ line and somatic cells. No FBPase or aldolase signal was observed on the F-actin trophic core mesh or on stress fibers.