Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Pyruvate kinase from Trypanosoma brucei is a labile enzyme, losing its activity within several hours. In mixtures containing 50 mM triethanolamine buffer, pH 7.2, 25% glycerol and 0.5 mM inorganic phosphate the enzyme remained active and could be purified to homogeneity with a specific activity of 417 units mg-1 and a yield of 65%. The enzyme has an activation energy of 31.9 kJ mol-1. Magnesium and potassium ions are essential for activity. Cobalt or manganese ions replace Mg2+ but this leads to a decrease in maximal velocity. Potassium ions can be substituted by ammonium ions, while sodium ions behave as a competitive inhibitor with respect to both K+ and NH4+. All metal ions studied displayed sigmoidal kinetics. The enzyme is activated, with decreasing efficiency by fructose 2-phosphorothioate 6-phosphate, fructose 2,6-bisphosphate, fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. They all display hyperbolic kinetics. Glycerate 2,3-bisphosphate, glyceraldehyde 3-phosphate, CoASAc, oxalate, AMP, ADP, and ATP inhibit the enzyme. At substrate saturation PK was activated by Pi up to a concentration of 0.8 mM. At higher Pi concentrations the enzyme is inhibited. The enzyme is unaffected by most amino acids, only phenylalanine stimulates and tyrosine inhibits.
Mol Biochem Parasitol 1991 Jul
PMID:Characterization of pyruvate kinase of Trypanosoma brucei and its role in the regulation of carbohydrate metabolism. 185 83

Epicardial adenosine levels and venous adenosine release were measured in isovolumically contracting (ISO) and empty non-isovolumic (non-ISO) guinea-pig hearts subjected to graded perfusion (approximately 7.5, 5.5, 4.0, 2.0, and 1.0 ml/min/g). Myocardial metabolism was monitored using 31P-NMR spectroscopy. At flows of 5.5 ml/min/g or higher epicardial adenosine levels were stable and comparable in ISO and non-ISO hearts (approximately 160 nM). At flows of 4.0 ml/min/g or higher venous adenosine release was stable and comparable in ISO and non-ISO hearts (approximately 30 pmol/min/g). At lower flows, epicardial adenosine and venous adenosine release both increased and were significantly higher in ISO hearts, compared to non-ISO hearts, at each flow rate. Whereas epicardial adenosine increased linearly in ISO and non-ISO hearts at low flows, venous adenosine release stabilized in ISO hearts perfused at 1.0 ml/min/g. Epicardial adenosine, venous adenosine release, and log [ATP]/[ADP] [Pi] all displayed significant correlations with the O2 supply:demand ratio which were comparable in ISO and non-ISO hearts. Elevated levels of epicardial adenosine were linearly related to log [ATP]/[ADP] [Pi] and cytosolic [AMP] and these relationships were comparable in ISO and non-ISO hearts. Alternatively, changes in venous adenosine release did not display simple relationships with log [ATP]/[ADP] [Pi] and cytosolic [AMP] and they were not comparable in ISO and non-ISO hearts. The data indicate that: (i) myocardial adenosine formation increases only below a metabolic threshold corresponding to log [ATP]/[ADP] [Pi] = 5.0 and O2 supply:demand = 1.5 in ISO and non-ISO guinea-pig hearts; (ii) stimulated epicardial adenosine levels appear to be consistently related to changes in cytosolic metabolism below this threshold in ISO and non-ISO hearts; (iii) more complex relationships exist between venous adenosine release and myocardial metabolism during graded perfusion, possibly reflecting the variety of factors modulating venous adenosine release.
J Mol Cell Cardiol 1991 Mar
PMID:Effects of graded perfusion and isovolumic work on epicardial and venous adenosine and cytosolic metabolism. 188 Aug 15

The energy status of mammalian cells is a finely regulated phenomenon. This is especially true in cardiac muscle cells in which energy requirements are high and the system must provide rapid turnover of the adenine nucleotides and instant response to changes in energetic demands. We have examined the acute response of the rat myocardium to ventricular pacing up to 2.5 times the resting heart rate. The purpose of this study was to determine at what level of pacing the normal energy status could be maintained and at what point it was compromised. Myocardial energy charge (EC = (ATP + 0.5 ADP)/(ATP + ADP + AMP] was maintained at 1, 1.5 and 2 times the resting heart rate but declined significantly at 2.5 times. In contrast, phosphorylation potential (PP = ATP/ADPf x Pi) was drastically altered in hearts paced at 1.5, 2 and 2.5 times the resting rate. Tissue lactate increased and glycogen decreased in a linear fashion as pacing rate increased, indicating that the metabolic challenge was proportional to the pacing rate. EC seems to reflect the overall status of the cell and its ability to maintain a dynamic equilibrium. PP may reflect the immediate and necessary driving force for mitochondrial respiration in times of increased demand. These data suggest that the myocardium may meet the increased energy demands of acute ventricular pacing by shifting the molar ratio of ATP to ADP times Pi in favour of driving phosphorylation.
Mol Cell Biochem 1991 Apr 10
PMID:Effects of different rates of cardiac pacing on rat myocardial energy status. 188 88

The effects on sarcoplasmic reticulum (SR) Ca2+ transport of solutions mimicking the important intracellular milieu changes associated with short-term hypoxia (hypoxic solutions, as described by Kammermeier et al. J. Mol. Cell. Cardiol. 14: 267, 1982) were examined. SR Ca2+ content was estimated by measuring the magnitude of the caffeine-induced contracture in saponin-skinned rat papillary muscle. SR Ca2+ uptake was inhibited by hypoxic solutions only at loading times less than or equal to 30 s. This inhibition was primarily due to the increase in Pi. The hypoxic solutions had no effect on Ca(2+)-induced Ca2+ release from the SR. We also tested the effects of ATP-free (rigor) solutions that mimic the intracellular environment during late hypoxia and ischemia. Elevating Pi or ADP alone in rigor solution had no effect on SR Ca2+ content. However, elevating Pi and ADP (+/-Mg2+) produced a 44-48% reduction in SR Ca2+ content. This reduction is most likely due to reversal of the SR Ca2+ pump. We conclude that the changes in milieu with short-term hypoxia can depress contractility in intact cardiac muscle by inhibiting SR Ca2+ uptake. During long-term hypoxia or ischemia, these milieu changes can elevate intracellular Ca2+ by reversing the SR Ca2+ pump.
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PMID:Intracellular milieu changes associated with hypoxia impair sarcoplasmic reticulum Ca2+ transport in cardiac muscle. 188 12

Five ADP-ribosylating bacterial toxins, pertussis toxin, cholera toxin, diphtheria toxin, Escherichia LT toxin and Pseudomonas exotoxin A, show significant homology in selected segments of their sequence. Site-directed mutagenesis and chemical modification of residues within these regions cause loss of catalytic activity and of NAD binding. On the basis of these results and of molecular modelling based on the three-dimensional structure of exotoxin A, the geometry of an NAD binding site common to all the toxins is deduced and described in the paper. For diphtheria toxin, sequence similarity with exotoxin A is such that its preliminary structure can be computed by molecular modelling, whereas for the other toxins similarity appears to be restricted to the NAD binding site. Moreover, an analysis of molecular fitting of the NAD molecule into its binding cavity suggests a new model for the conformation of the bound NAD that better accounts for all available experimental information.
Mol Microbiol 1991 Jan
PMID:Computer modelling of the NAD binding site of ADP-ribosylating toxins: active-site structure and mechanism of NAD binding. 190 17

P2Y-Purinergic receptors were solubilized from turkey erythrocyte plasma membranes with the nonionic detergent digitonin. Adenosine 5'-O-(2-[35S]thiodiphosphate) ([35S]ADP beta S) labeled a single population of soluble high affinity sites (Kd = 12.9 nM; Bmax = 4.5 pmol/mg of protein) in an equilibrium binding assay; adenine nucleotide analogs competitively inhibited [35S]ADP beta S binding with a rank order of potency consistent with that for P2Y-purinergic receptors. Radioligand binding to solubilized P2Y-purinergic receptors was noncompetitively inhibited by guanine nucleotides with a rank order of potency that was in agreement with the potency order observed for guanine nucleotide-mediated inhibition of [35S]ADP beta S binding in purified turkey erythrocyte plasma membranes. The rate constant for dissociation of [35S]ADP beta S from solubilized receptors was increased 2.3-fold by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Plasma membrane P2Y-purinergic receptors were labeled with [35S]ADP beta S or covalently labeled with the photoaffinity probe 3'-O-(4-benzoyl)benzoyl adenosine 5'-[alpha-32P]triphosphate ([alpha-32P]BzATP) before solubilization and gel filtration chromatography on Superose 12. [35S]ADP beta S- or [alpha-32P]BzATP-labeled species eluted as a single peak of radioactivity of apparent Mr greater than or equal to 300,000. Incubation of the Mr greater than or equal to 300,000 protein species with GTP gamma S before rechromatography resulted in loss of labeling of proteins by [35S]ADP beta S and a shift in apparent size of the covalently [alpha-32P]BzATP-labeled species to a single peak of radioactivity of approximate Mr 70,000. These results suggest that a P2Y-purinergic receptor-guanine nucleotide regulatory protein complex is stable to membrane solubilization with digitonin, even in the absence of prebound agonist.
Mol Pharmacol 1991 Jul
PMID:Solubilization of a guanine nucleotide-sensitive form of the P2Y-purinergic receptor. 190 78

Angiotensin II can inhibit hormone-stimulated adenylyl cyclase in intact hepatocytes or in hepatic membrane preparations. Because the response can be blocked by pertussis toxin, the object of the present study was to determine which of the known variants of Gi can couple angiotensin II receptors to inhibition of adenylyl cyclase. The potential candidates were identified by probing RNA isolated from rat hepatocytes with cDNAs specific for the alpha subunits of known toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins). Hepatocytes contained no detectable RNA for the Go or Gi1 alpha subunits and similar levels of RNA coding for the Gi2 and Gi3 alpha subunits. To determine whether Gi3 could couple angiotensin receptors to inhibition of cyclase, membranes were prepared from hepatocytes whose G proteins were fully ADP-ribosylated with pertussis toxin, and the Gi3 holoprotein purified from rabbit liver was reconstituted into the membranes. The nature of the Gi3 reconstituted into the membrane was assessed by immunoblotting with antibodies specific for the Gi alpha subunits. Reconstitution of 6-10 pmol of Gi3/mg of membrane protein into the toxin-treated membranes restored the ability of 10 nM angiotensin II to inhibit adenylyl cyclase. Because pertussis toxin has nonspecific effects, an assay was developed to measure the interaction of the angiotensin receptor with reconstituted G proteins in normal membranes. In the presence of Mg2+, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused a reduction of the affinity of the angiotensin II receptor for 125I-angiotensin II that was stable to washing and the detergents used to reconstitute G proteins into the membranes. Using this protocol to activate G proteins and "uncouple" receptors, the ability of the GDP-liganded form of Gi to restore high affinity binding was examined. Reconstitution of about 10-15 pmol of oligomeric Gi3/mg of membrane protein restored both the high affinity state of the angiotensin II receptor and the ability of GTP gamma S to shift the affinity to a lower state. The same shift in receptor affinity could be accomplished by reconstituting the Gi3 alpha subunit, resolved free of beta gamma subunits, into the membranes. Reconstitution of up to 50 pmol of Gs/mg of membrane protein had no effect on angiotensin II receptor affinity. The results suggest that a major form of Gi in hepatocytes is Gi3 and that it can couple angiotensin receptors to inhibition of adenylyl cyclase.
Mol Pharmacol 1991 Aug
PMID:Inhibitory GTP-binding regulatory protein Gi3 can couple angiotensin II receptors to inhibition of adenylyl cyclase in hepatocytes. 190 48

We have investigated the possible role of guanine nucleotide-binding proteins in the process of antigen-induced exocytosis in a cultured rat mast cell line, RBL-2H3 cells. The mRNAs for the alpha subunits of the guanine nucleotide-binding proteins G alpha S (short and long forms), G alpha i-2, G alpha i-3, and G alpha Z were detected by hybridization with G alpha-specific oligonucleotide probes. The corresponding proteins were identified in membranes of RBL-2H3 cells on the basis of size, immunoreactivity with specific antibodies, and their ability to serve as substrates for ADP-ribosylation by cholera toxin or pertussis toxin. Treatment of cells with as little as 10(-9) to 10(-7) M dexamethasone markedly decreased the amount of G alpha Z mRNA and membrane G alpha Z, as well as the responsiveness of the cells to antigen stimulation. In the same cells, the exposure to dexamethasone caused an increase in the amounts of certain other G alpha subunits, particularly G alpha i-3, and in the responsiveness of the cells to an adenosine analog, N(ethylcarboxamido)-adenosine. Because of the apparent decrease in G alpha Z mRNA and protein in dexamethasone-treated cells and the fact that neither cholera toxin nor pertussis toxin inhibits the stimulatory signals to antigen [J. Biol. Chem. 265:745-753 (1990)], we suggest that G alpha Z is a potential candidate for regulating the early signals in antigen-stimulated RBL-2H3 cells.
Mol Pharmacol 1991 Oct
PMID:GTP-binding protein G alpha Z: its down-regulation by dexamethasone and its credentials as a mediator of antigen-induced responses in RBL-2H3 cells. 192 83

The urinary bladder depends on intracellular ATP to support a number of essential intracellular processes including contraction. The concentration of ATP is maintained by mitochondrial oxidative phosphorylation, cytosolic glycolysis and the cytosolic activity of creatine kinase, the enzyme that catalysis the rapid transfer of a phosphate from creatine phosphate (CP) to ADP resulting in the formation of ATP. Prior studies in this lab and others have demonstrated that mitochondrial respiration is significantly lower in hypertrophied bladder tissue (induced by partial outlet obstruction of the white New Zealand Rabbit). In addition to decreased mitochondrial respiration, there are significant increases in glycolysis and lactic acid formation in the hypertrophied tissue. In view of the increased glycolysis and decreased mitochondrial function in the hypertrophied tissue, and the importance in creatine kinase in maintaining cytosolic levels of ATP, the current study was designed to determine if outlet obstruction induces any changes in the activity of creatine kinase. The following is a summary of the results: 1) The bladder mass increased from 2.2 +/- 0.2 gm to 11.5 +/- 1.6 gm at 7 days following outlet obstruction. 2) The intracellular concentrations of both ATP and CP were significantly reduced in the bladder tissue following 7 days of obstruction. 3) The percent of protein (per tissue mass) was significantly lower in the obstructed bladders, although the percent of soluble protein was similar. 4) Creatine kinase activity of control bladders showed linear kinetics with a Vmax = 1120 nmoles/mg protein/4 min and Km = 147 microM CP.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1991 Aug 14
PMID:Creatine kinase activity in normal and hypertrophied rabbit urinary bladder tissue (following partial outlet obstruction). 192 18

Human hearts with idiopathic dilated cardiomyopathy have diminished adenylate cyclase activity and increased amounts of the alpha-subunit of the inhibitory guanine nucleotide-binding regulatory protein (alpha Gi) as measured by pertussis toxin catalyzed ADP-ribosylation. We utilized specific antisera against synthetic peptides corresponding to amino sequences deduced from cDNA's encoding the three alpha Gi subspecies to compare the immunologic and bioactivity levels of Gi in failing and non-failing human hearts. The various antisera detected three peptides with Mr 42,000, 38,000, and 37,000. Only the Mr 42,000 peptide co-migrated with the pertussis toxin substrate. Although functional activity of alpha Gi was increased in the particulate fractions of the failing heart as measured by inhibition of guanine nucleotide-stimulated adenylate cyclase activity and the quantity of pertussis toxin substrate was also increased, there were not associated changes in the levels of immunodetectable Gi. Therefore, the increased functional activity of Gi in the failing human heart as assessed by adenylate cyclase measurements cannot be explained by a relative increase in the among of Gi protein.
J Mol Cell Cardiol 1991 Apr
PMID:Immunodetectable levels of the inhibitory guanine nucleotide-binding regulatory proteins in failing human heart: discordance with measurements of adenylate cyclase activity and levels of pertussis toxin substrate. 194 80


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