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Query: UNIPROT:P06889 (Mol)
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Hexokinase (ATP: hexose 6-phosphotransferase, E.C.2.7.1.1) and phosphofructokinase (ATP:fructose-6-phosphate 1-phosphotransferase, E.C.2.7.1.11), two key regulatory enzymes of the glycolytic pathway in vertebrate cells, have been isolated and partially purified from Trypanosoma (Schizotrypanum) cruzi epimastigotes. Both enzymes are associated with particles sedimentable at 105 000 X gav for 1 h and have a high degree of latency; they can be solubilized by sonication. Hexokinase catalyses the phosphorylation of a series of monosaccharides at the following relative rates: D-glucose (100) congruent to D-fructose (97) greater than 2-deoxy-D-glucose (72) congruent to mannose (69) greater than 2-amino-D-glucose (63) greater than 3-O-methyl-D-glucose (21). Very little or no phosphorylating activity was found for D-galactose, N-acetyl-2-amino-D-glucose or 1-alpha-methyl-D-glucose. D-Glucose phosphorylation at fixed ATP concentration follows simple Michaelis-Menten kinetics with Km = 40 microM and Vmax = 440 nmol min-1 mg-1 protein. D-Mannose, 2-deoxy-D-glucose and N-acetyl-2-amino-D-glucose act as competitive inhibitors of glucose phosphorylation, suggesting a single kinase. Mg2+-ATP is the preferred phosphoryl donor, ITP and GTP being much less effective. T. cruzi hexokinase is not inhibited by D-glucose 6-phosphate, or by any of the following compounds (2 mM):D-fructose 6-phosphate, D-fructose 1,6-diphosphate, D-glucose 1,6-diphosphate, phosphoenol pyruvate, L-malate and citrate. Phosphofructokinase displays simple Michaelis-Menten kinetics with no evidence of sigmoidicity with respect to D-fructose 6-phosphate at all ATP concentrations tested, giving a Km of 1.31 mM and Vmax = 400 nmol min-1 mg-1 protein at optimal ATP levels. With respect to ATP, the enzyme exhibits Michaelis-Menten kinetics at low concentration (less than 1 mM) of the substrate (Km = 40 microM at 5 mM MgCl2, pH 7.4). A moderate inhibition is observed at high ATP levels (70% of maximal activity at 2 mM). GTP can substitute for ATP as the phosphoryl donor (Km = 79 microM under the same conditions), but produces only very small inhibitory effects at high concentrations. 5'-AMP activates the enzyme by decreasing its Km with respect to D-fructose 6-phosphate without affecting Vm. Other well-known regulators of the activity of this enzyme in procaryote and vertebrate systems such as citrate, phosphoenol pyruvate, ammonium and phosphate ions have no effect in T. cruzi.
Mol Biochem Parasitol 1984 Apr
PMID:Regulation of energy metabolism in Trypanosoma (Schizotrypanum) cruzi epimastigotes. I. Hexokinase and phosphofructokinase. 623 52

Pause location along primary structure of two RNA fragments each 200 nucleotide residues in the length synthesized from A1 promoters of T7 phage DNA and delta D111 T7 phage DNA was analyzed. No correlation between the location of pauses and GC-rich or self complementary regions of RNA were found. The location of pauses does not change upon the variation of the temperature or ionic strength. Concurrent variation of all four NTP concentrations also did not influence pausing pattern. However the distribution of pauses depends highly on the ratio of the individual substrate concentrations. Substitution of GTP by ITP changes the pausing pattern completely. Inorganic pyrophosphate (PPi) of inhibits RNA elongation preferentially in the regions: NAUN, CGUAG. The study of PPi action on RNA terminated with 3' OCH3-NMP suggest that the sequence-specific inhibition of RNA elongation may be a result of pyrophosphorolysis of terminal nucleotide residues of RNA. It was proposed that the pulse character of RNA elongation stems rather from differences in the kinetic constants of nucleotides attachment and pyrophosphorolysis from the 3'-termini of RNA than by termination signals encoded in the primary structure of DNA. The stable location of pauses in certain short oligonucleotides: AUG, AUU, AAU and some others is in favour of the hypothesis.
Mol Biol (Mosk)
PMID:[Factors influencing the pulse character of RNA elongation in vitro by E. coli RNA polymerase]. 626 62

Ribonucleosides of some pyrazolo [3,4-d] pyrimidines have been shown to be potent anticoccidial agents. To investigate their interactions with adenosine kinase, this enzyme was purified by affinity chromatography from the sporulated oocysts of 3 avian coccidia, Eimeria tenella, E. acervulina and E. brunetti as well as from chicken liver. Comparative studies revealed several differences among the enzymes. Magnesium appeared not to be inhibitor of the E. tenella enzyme but did inhibit the enzymes from the other three sources. ATP in excess of the magnesium concentration strongly inhibited the E. brunetti enzyme but had only a small effect on the other enzymes. The chicken liver enzyme utilized a broader variety of triphosphate donors than did any of the enzymes from Eimeria species. ATP, dATP, GTP, dGTP and ITP was the best substrates. Studies with pyrazolo [3,4-d] pyrimidine nucleosides revealed two groups of enzymes with similar inhibitor specificities, the chicken liver and E. Acervulina vs. the E. tenella and E. brunetti enzyme. This grouping roughly correlates with the in vivo anticoccidial specificity of these compounds. Substrate specificity studies using two 4-substituted pyrazolo [3,4-d] pyrimidine ribonucleosides (ethylthio- and cinnamylthio-), which have shown potent anticoccidial activity in vivo, revealed that each served as a substrate for the enzymes from E. tenella and E. acervulina. The E. tenella enzyme was the more efficient at the phosphorylation of those compounds. However, only the ethylthio- compound was detectably phosphorylated by the enzyme from E. brunetti. In contrast to the inhibitor specificity, the substrate activities of these nucleosides do not correlate well with their in vivo anticoccidial activity.
Mol Biochem Parasitol 1982 Oct
PMID:Purification, characterization, substrate and inhibitor specificity of adenosine kinase from several Eimeria species. 629 13

There are 3 loci in the phosphoglycerate kinase (PGK) gene complex of Trypanosoma brucei. The PGK-A gene product, which we term 56PGK, is targeted to glycosomal microbodies and is highly homologous to the parasite's 2 known PGKs (one cytoplasmic and one glycosomal). However, 56PGK contains an 80 amino acid insertion as well as numerous substitutions compared to the other PGKs. The complementation and kinetic analyses described here demonstrate that 56PGK is an authentic phosphoglycerate kinase--the largest yet described. When expressed in Escherichia coli, 56PGK complements the pgk- phenotype. 56PGK was expressed as a fusion protein and purified to near homogeneity. The Michaelis constants are similar to those of other PGKs, being 0.12 and 2.4 mM for Mg-ATP and 3-phosphoglycerate, respectively. As with other T. brucei PGKs, ATP but not GTP or ITP can serve as a phosphate donor during catalysis. No evidence was obtained for phosphate transfer to atypical substrates. 56PGK shows sulfate inhibition at all concentrations tested, rather than the sulfate activation observed with yeast PGK.
Mol Biochem Parasitol 1993 Aug
PMID:Characterization of a divergent glycosomal microbody phosphoglycerate kinase from Trypanosoma brucei. 823 17

A 23-kD pathogenesis-related protein (P23) is induced in tomato (Lycopersicon esculentum Mill, cv Rutgers) plants when infected with citrus exocortis viroid. This protein is homologous to the salt-induced tomato NP24 protein (I. Rodrigo, P. Vera, R. Frank, V. Conejero [1991] Plant Mol Biol 16: 931-934). Further characterization of P23 has shown that this protein accumulates in vacuoles in association with dense inclusion bodies. In vitro assays indicated that the purified P23 protein inhibits the growth of several phytopathogenic fungi. P23-coding cDNA clones were isolated from viroid-induced and ethylene-induced libraries. Southern analysis showed that at least two genes could encode P23 or P23-related products. The accumulation of P23 protein correlated with the accumulation of its mRNA. Sequence analysis revealed significant differences in both coding and downstream untranslated regions between the cDNA sequences corresponding to the viroid-induced P23 and the salt stress-induced NP24 proteins.
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PMID:cDNA cloning of viroid-induced tomato pathogenesis-related protein P23. Characterization as a vacuolar antifungal factor. 827 38

The phosphoenolpyruvate carboxykinase (PEPCK) from Vibrio costicola catalyzed a 14CO2-oxaloacetate exchange reaction with an unusual nucleotide specificity. ATP gave the higher apparent catalytic efficiency (Vmax/Km, 6.78), followed by GTP (1.30), CTP (0.87) and ITP (0.66). Maximal activity required a divalent cation; CdCl2 and MgCl2 synergistically activated the enzyme, when added in the presence of MnCl2. The sigmoidal saturation curve for MnCl2 (apparent n 2.11) was converted into a hyperbola by 0.01 mM CdCl2 (apparent n 1). The results suggest a double role of the divalent cation in the reaction mechanism, namely as part of the MeATP2- substrate and as free Me2+. Mn2+ would be the best for the first, and Cd2+ for the second role. Preincubation with 0.01 mM CdCl2 increased the activity of the enzyme assayed with MgATP2- through an increase in Vmax; addition of CdCl2 to the reaction mixture elicited further activation, through a 17-fold decrease in the apparent Km for MgATP2-. These results, together with the biphasic curve of activation by CdCl2 when used alone, suggest the existence of two different sites for free Cd2+ on the enzyme.
Biochem Mol Biol Int 1995 Aug
PMID:Effects of divalent cations and nucleotides on the 14CO2-oxaloacetate exchange catalyzed by the phosphoenol pyruvate carboxykinase from the moderate halophile, Vibrio costicola. 853 94

Evaluating cardiac gene therapy in the intact animal requires an index of cardiac function capable of detecting regional differences in contractility in a load-independent fashion. Potentially load-insensitive measures of ventricular performance were therefore evaluated in 10 open- and closed-chested, anesthetized rabbits. LV transmural pressure and myocardial segment length were measured using micromanometry and sonomicrometry during steady-state and transient inferior vena caval occlusion, over a range of inotropic and loading conditions. For each intervention, segmental stroke work was calculated as the area within the left ventricular transmural pressure-length loops at a given end-diastolic segment length during inferior vena caval occlusion; regression analysis was applied to obtain the linear Frank-Starling relationship. In both open- and closed-chested states, these relationships were highly linear (r = 0.97 +/- 0.1) and reproducible. The slope of the linear relationship between segmental stroke work and end-diastolic segment length increased significantly with calcium and epinephrine infusions (P < 0.05 v control) but was not significantly altered by decreased afterload or increased afterload (P > 0.4). The x-intercept was not significantly altered by changes in intropy or afterload (P > 0.4). These data validate the linear Frank-Starling relationship and the slope, MW, as a load-insensitive index of contractility in the intact rabbit. This study presents a novel approach to the quantification of regional cardiac function in smaller animals.
J Mol Cell Cardiol 1996 May
PMID:The in vivo quantification of myocardial performance in rabbits: a model for evaluation of cardiac gene therapy. 876 21

During differentiation, megakaryocytes undergo nuclear endoreplication, an increase in cell size, cytoplasmic granulation, and release of platelets. The changes in highly lobulated nuclei with varying degree of polyploidy and increasing cell size are easily recognized morphologically. However, the actual cytoplasmic changes are more difficult to perceive morphologically. With the peroxidase-antiperoxidase (PAP) method using UEA-1 as the binding protein to the alpha-L-fucose of glycoprotein synthesized by megakaryocytes, we observed significant variation in cytoplasmic staining of megakaryocytes in routinely processed bone marrow biopsy sections. A total of 3344 megakaryocytes in bone marrow sections from 10 patients with nonhematologic diseases and from 10 patients with idiopathic thrombocytopenic purpura (ITP) was studied. According to the intensity and pattern of cytoplasmic staining, we divided megakaryocytes into at least six groups: (1) low granular (LG), (2) diffuse granular (DG), (3) diffuse dense granular (DDG), (4) marginal granular (MG), (5) denuded (DMK), and (6) endomitotic (EndoM). Most of the megakaryocytes were DG (mean, 42.75% +/- 19.21%) and DDG (mean, 50.25% +/- 21.23%). In correlation with nuclear morphology and cell size, it appears that substances binding to UEA-1 are located in the paranuclear region in early megakaryocytes and produce a low granular focal staining pattern (LG cells). Next, the granules spread throughout the cytoplasm (DG cells) and increase in quantity (DDG). This is followed by migration of granules to the periphery of the cytoplasm (MG cells) and is associated with the liberation of platelets and eventual formation of DMK megakaryocytes. Endomitosis, regulated by unknown factors, occurred in the MG stage. In comparing the group with nonhematologic disease (mean DG, 35.4% +/- 18.48%; DDG, 58.4% +/- 21.8%) and the group with ITP (mean DG, 50.1% +/- 17.82%; DDG, 42.1% +/- 18.12%), we found an increasing proportion of DG megakaryocytes in ITP, which suggests a left-shifted maturation of megakaryocytes. By understanding the staining pattern seen in the different stages of megakaryocytic differentiation, UEA-1 staining may be a practical method for studying megakaryocytopoiesis in routinely processed paraffin sections of bone marrow biopsy samples.
Hematopathol Mol Hematol 1996
PMID:Immunohistochemical study of Ulex europaeus agglutinin 1 (UEA-1) binding of megakaryocytes in bone marrow biopsy specimens: demonstration of heterogeneity in staining pattern reflecting the stages of differentiation. 879 51

The Ca2+ sensitivity of skinned cardiac muscle can be increased by either an increase in sarcomere length or osmotic compression of the myofilament lattice. The length-dependent change in Ca2+ sensitivity is considered to be an important component of the steep force-length relation along the ascending limb of the force-length curve (Frank-Starling relation). Since an increase in sarcomere length is accompanied by a reduction in the spacing between myosin and actin filaments it is not clear whether length-dependent changes in Ca2+ sensitivity are related to changes in sarcomere length, interfilament spacing or some combination of both. To obtain quantitative information on the relative contributions of these two parameters to the determination of Ca2+ sensitivity skinned bovine cardiac muscle bundles of varying sarcomere lengths (1.7-2.3 microns) were exposed to varying concentrations (0-5%) of Dextran T-500. Measurements were made of changes in muscle width in response to Dextran T-500 addition and both force-pCa curves and bound Ca(2+)-pCa curves were obtained as a function of sarcomere length and lattice compression. From the data obtained it was possible to compare Ca2+ sensitivity and Ca(2+)-troponin C affinity at different sarcomere lengths under conditions of changing interfilament spacing and constant interfilament spacing. Both Ca2+ sensitivity and Ca2+ binding correlated more closely with change in interfilament spacing than with change in sarcomere length. These results suggest that length-dependent force generation in cardiac muscle is based primarily on length-dependent changes in the separation between myosin and actin filaments.
J Mol Cell Cardiol 1996 Jul
PMID:Sarcomere length versus interfilament spacing as determinants of cardiac myofilament Ca2+ sensitivity and Ca2+ binding. 884 26

The lifetimes of the first excited singlet states (2(1)A(g)) of diadinoxanthin and diatoxanthin, carotenoids involved in the xanthophyll cycle in some genera of algae, have been measured by femtosecond time-resolved optical spectroscopy to be 22.8 +/- 0.1 ps and 13.3 +/- 0.1 ps, respectively. Using the energy gap law for radiationless transitions set forth by Englman and Jortner (Mol. Phys. 18 (1970) 145-164), these lifetimes correspond to S1 excited state energies of 15210 cm-1 for diadinoxanthin and 14620 cm-1 for diatoxanthin. The lowest excited singlet state energy of Chl a has an energy of 14700 cm-1. The fact that the S1 state energy of diadinoxanthin lies above that of Chl a, whereas the S1 state energy of diatoxanthin lies below that of Chl a, suggests that the xanthophyll cycle involving the enzymatic interconversion of diadinoxanthin and diatoxanthin may play a role in regulating energy flow between these molecules and Chl a in many species of algae, essentially fulfilling a role identical to that proposed for violaxanthin and zeaxanthin in higher plants and green algae (Frank et al. (1994) Photosyn. Res. 41, 389-395).
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PMID:The lifetimes and energies of the first excited singlet states of diadinoxanthin and diatoxanthin: the role of these molecules in excess energy dissipation in algae. 898 90


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