Abstract 049: Phosphodiesterase-3a Catalytic-domain Mutation and Hypertension

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Sylvia Bähring ◽  
Carolin Schächterle ◽  
Atakan Aydin ◽  
Enno Klussmann ◽  
Friedrich C Luft
Keyword(s):  
Blood Pressure ◽  
Turnover Rate ◽  
Catalytic Domain ◽  
Hydrolytic Activity ◽  
Hek293 Cells ◽  
Michaelis Constant ◽  
Salt Form ◽  
Cysteine Substitution ◽  
Camp Hydrolysis ◽  
Kinetics Of

We recently discovered phosphodiesterase-3A (PDE3A) mutations causing a 50 mm Hg increase in blood pressure and stroke >50 years, as the first non-salt form of Mendelian genetic hypertension, autosomal-dominant hypertension with brachydactyly (HTNB). The mutations cause increased PDE3A phosphorylation and higher cAMP affinity. We now have found a completely different PDE3A mutation causing a similar syndrome in a New Zealand pedigree. The mutation resides in the enzyme’s catalytic domain, results in an arginine-to-cysteine substitution, and represents a more direct mechanism of PDE3A activation. For Michaelis-Menten kinetics of cAMP hydrolysis, we transfected HEK293 cells transiently expressing Flag-tagged versions of PDE3A1, PDE3A2, or PDE3A3 mutant vs. wildtype and stimulated with forskolin and phorbol-12-myristate-13-acetate (PMA) to enhance intrinsic phosphorylation. Vmax and Km (Michaelis constant) were calculated using GraphPad Prism software to reveal the maximum cAMP turnover rate at saturated substrate concentration and the affinity of cAMP to wildtype and mutated PDE3A1, PDE3A2 and PDE3A3. For PDE3A1 hydrolytic activity (triplicate), we observed: Vmax Km Wildtype 7.5 340 Wildtype+forskolin/PMA 7.2 203 Mutant 6.6 116 Mutant+forskolin/PMA 6.3 81 The dramatically lower Km of mutant PDE3A indicates a substantially greater affinity for cAMP consistent with gain-of-function. These data underscore the importance of PDE3A to high blood pressure by means of a different, novel genetic mechanism directly implicating the catalytic domain.

scholarly journals PfPDE1, a novel cGMP-specific phosphodiesterase from the human malaria parasite Plasmodium falciparum

2005 ◽  
Vol 392 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Keizo Yuasa ◽  
Fumika Mi-Ichi ◽  
Tamaki Kobayashi ◽  
Masaya Yamanouchi ◽  
Jun Kotera ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Cyclic Nucleotide ◽  
Malaria Parasite ◽  
Catalytic Domain ◽  
Hydrolytic Activity ◽  
Human Malaria Parasite ◽  
Pde Inhibitors ◽  
Ic50 Value ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum

This is the first report of molecular characterization of a novel cyclic nucleotide PDE (phosphodiesterase), isolated from the human malaria parasite Plasmodium falciparum and designated PfPDE1. PfPDE1 cDNA encodes an 884-amino-acid protein, including six putative transmembrane domains in the N-terminus followed by a catalytic domain. The PfPDE1 gene is a single-copy gene consisting of two exons and a 170 bp intron. PfPDE1 transcripts were abundant in the ring form of the asexual blood stages of the parasite. The C-terminal catalytic domain of PfPDE1, produced in Escherichia coli, specifically hydrolysed cGMP with a Km value of 0.65 μM. Among the PDE inhibitors tested, a PDE5 inhibitor, zaprinast, was the most effective, having an IC50 value of 3.8 μM. The non-specific PDE inhibitors IBMX (3-isobutyl-1-methylxanthine), theophylline and the antimalarial chloroquine had IC50 values of over 100 μM. Membrane fractions prepared from P. falciparum at mixed asexual blood stages showed potent cGMP hydrolytic activity compared with cytosolic fractions. This hydrolytic activity was sensitive to zaprinast with an IC50 value of 4.1 μM, but insensitive to IBMX and theophylline. Furthermore, an in vitro antimalarial activity assay demonstrated that zaprinast inhibited the growth of the asexual blood parasites, with an ED50 value of 35 μM. The impact of cyclic nucleotide signalling on the cellular development of this parasite has previously been discussed. Thus this enzyme is suggested to be a novel potential target for the treatment of the disease malaria.

Abstract P501: Editing of Myosin Phosphatase Gene as a Novel Approach for Vasodilator Sensitization and Lowering of Blood Pressure

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Mariam Meddeb ◽  
Jeanine Ursitti ◽  
John Reho ◽  
Steven A Fisher
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Leucine Zipper ◽  
Splice Variants ◽  
Species Conservation ◽  
Mesenteric Arteries ◽  
Hek293 Cells ◽  
Myosin Phosphatase ◽  
Novel Approach

Myosin Phosphatase (MP) is the primary effector of vascular smooth muscle (VSM) relaxation and a key end target of signaling pathways that regulate vessel tone. Regulated splicing of alternative Exon24 (E24) of Myosin Phosphatase Regulatory/ Targeting subunit (MYPT1) sets vasodilator sensitivity. Skipping E24 codes for a Mypt1 isoform that contains a C-terminal leucine zipper (LZ) motif required for cGK1α binding and NO/cGMP activation of MP resulting in vasodilation. Inclusion of 31 nt E24 shifts the reading frame coding for a Mypt1 isoform with a distinct C-terminus (LZ-) that is unresponsive to NO/cGMP. We are using two editing approaches to test the function of Mypt1 E24 splice variants in the control of BP in vivo. First, LoxP sites were inserted in introns flanking E24, crossed with smMHCCre ER , and treated with Tamoxifen to achieve smooth muscle-specific cKO of E24 (SMcKO E24), thereby converting Mypt1 to the LZ+ isoform. E24 cKO mice had mean BP that was 15 + 3 mmHg lower than control (n=3-5; p<0.05). Mesenteric arteries from these mice were significantly more sensitive to DEA/NO mediated relaxation (EC 50 : 2.1+0.5 nM vs 18.2+5.6 μM; n=5-6, p<0.05). We now are developing CRISPR/CAS9 editing of Mypt1 for translation into humans with hypertension. Guide(g)RNAs targeting E24 were designed using Benchling.com and selected for further study based on predicted efficacy, specificity (>10%,>60%) and cross-species conservation. Plasmids were generated by sub-cloning of oligonucleotides into the parent pX601 plasmid for the purpose of co-expression of gRNA and saCas9. These plasmids were transfected into HEK293 cells singly and in combinations and Mypt1 gene editing assayed by PCR, Surveyor nuclease assays and sequencing of genomic DNA. Single gRNAs yielded deletions of 1-3 nt. Combinations yielded deletions of 104-334 nt that removed >80% of E24 with an efficiency of editing that varied from 10% (gRNAs 6+9 and 5+9) to 40% (gRNAs 6+11 and 5+11). We have now generated AAVgE24 and are testing their efficiency of editing of VSM in vivo. These studies support that AAV mediated CRISPR/Cas9 editing of Mypt1 E24 could be a novel strategy for vasodilator sensitization and effective lowering of blood pressure in humans.

Charakterisierung und Kinetik des in der Rattenplacenta vorkommenden mikrosomalen Enzyms, das den Wasserstoff-Transfer von Oestradiol auf Androstendion katalysiert / Characterization and Kinetics of the Microsomal Enzyme of Rat Placenta which Functions as a “Transhydrogenase” between Estradiol-17β and Androstenedione

1971 ◽  
Vol 26 (7) ◽  
pp. 710-719 ◽  
Author(s):  
Kunhard Pollow ◽  
Barbara Pollow
Keyword(s):  
Hydrogen Transfer ◽  
Microsomal Fraction ◽  
Close Relation ◽  
Inhibitory Effect ◽  
Magnesium Ions ◽  
Michaelis Constant ◽  
Temperature Optimum ◽  
Optimum Ph ◽  
Kinetics Of ◽  
Estradiol 17Β

The microsomal fraction of rat placenta contains a 17β-hydroxysteroid-oxidoreductase which transfers hydrogen from position 17 of estradiol to androstenedione. This hydrogen transfer is dependent on NAD, NADP as cofactor is without effect. The optimum pH is at 6,9. In the presence of NAD the Michaelis constant for estradiol is 4,17 · 10-5м at pH 7,4. In the presence of androstenedione in the incubation medium the Km-value for estradiol is decreased, which indicates an increased affinity for the enzyme. The temperature optimum of the enzyme is 38 °C. Addition of SH-blocking agents inhibited the enzyme activity. Zinc and magnesium ions had an inhibitory effect on the “transhydrogenase” and B-NADPT specifically labelled from [1-T]-glucose showed that the non-effect of NADP on transhydrogenation from estradiol to androstenedione resulting in reduction of position 17 is not due to different stereospecifity.The results show a close relation between the oxidative metabolism of estradiol and the reduction of androstenedione, indicating that estradiol-17β, as the preferred hydrogen-donating substrate, is an essential component of the androstenedione-hydrogenating system in the microsomal fraction of rat placenta.

scholarly journals Graphical Approach to Compare Concentration Constants of Hill and Michaelis-Menten Equations

2018 ◽  
Vol 1 (3) ◽  
pp. 94-99 ◽  
Author(s):  
Elena V. Emelyanova
Keyword(s):  
Catalytic Activity ◽  
Substrate Concentration ◽  
Hill Equation ◽  
Enzymatic Process ◽  
Michaelis Constant ◽  
Graphical Approach ◽  
Graphical Technique ◽  
Kinetics Of ◽  
Permissible Rate ◽  
Concentration Constants

The aim of present study was to describe the graphical technique how to go from Hill concentration constant to Michaelis constant. To compare enzymatic processes, the kinetics of which is subjected to different regularities, it is possible to use constants that characterize catalytic activity (Vmax) and concentration constants that are the substrate concentration at which the rate of the enzymatic process is equal to a half of maximum permissible rate. Concentration constants are S0.5 for Hill equation and Km for Michaelis-Menton equation. The graphical approach was proposed in order to go from concentration constant of Hill equation to Michaelis concentration of the process that could be characterized by the same catalytic activity (the same values of minimum and maximum rates) similar to that observed in the process described by Hill equation.

scholarly journals The third chitinase gene (chiC) of Serratia marcescens 2170 and the relationship of its product to other bacterial chitinases

1999 ◽  
Vol 343 (3) ◽  
pp. 587-596 ◽  
Author(s):  
Kazushi SUZUKI ◽  
Mayumi TAIYOJI ◽  
Noriko SUGAWARA ◽  
Naoki NIKAIDOU ◽  
Bernard HENRISSAT ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Catalytic Domain ◽  
Signal Sequence ◽  
Early Stage ◽  
Hydrolytic Activity ◽  
Chitinase Gene ◽  
Glycoside Hydrolase Family ◽  
Chitin Binding Domain ◽  
The Third ◽  
Fn3 Domain

The third chitinase gene (chiC) of Serratia marcescens 2170, specifying chitinases C1 and C2, was identified. Chitinase C1 lacks a signal sequence and consists of a catalytic domain belonging to glycoside hydrolase family 18, a fibronectin type III-like domain (Fn3 domain) and a C-terminal chitin-binding domain (ChBD). Chitinase C2 corresponds to the catalytic domain of C1 and is probably generated by proteolytic removal of the Fn3 and ChBDs. The loss of the C-terminal portion reduced the hydrolytic activity towards powdered chitin and regenerated chitin, but not towards colloidal chitin and glycol chitin, illustrating the importance of the ChBD for the efficient hydrolysis of crystalline chitin. Phylogenetic analysis showed that bacterial family 18 chitinases can be clustered in three subfamilies which have diverged at an early stage of bacterial chitinase evolution. Ser. marcescens chitinase C1 is found in one subfamily, whereas chitinases A and B of the same bacterium belong to another subfamily. Chitinase C1 is the only Ser. marcescens chitinase that has an Fn3 domain. The presence of multiple, divergent, chitinases in a single chitinolytic bacterium is perhaps necessary for efficient synergistic degradation of chitin.

Studies of human zinc kinetics using the stable isotope 70Zn

1993 ◽  
Vol 84 (1) ◽  
pp. 113-117 ◽  
Author(s):  
N. M. Lowe ◽  
A. Green ◽  
J. M. Rhodes ◽  
M. Lombard ◽  
R. Jalan ◽  
...  
Keyword(s):  
Liver Disease ◽  
Alcoholic Liver Disease ◽  
Turnover Rate ◽  
Kinetic Studies ◽  
Normal Subjects ◽  
Short Term ◽  
Control Subjects ◽  
Two Component ◽  
Kinetics Of ◽  
Fractional Turnover

1. The short-term (120 min) kinetics of Zn turnover has been studied in control subjects and patients with alcoholic liver disease after intravenous injection of 0.5 mg of 96.5% enriched 70ZnCl2. 2. The 70Zn enrichment of plasma was found closely to obey two-compartment kinetics and the derived two-component decay equation has been used to calculate the size and turnover of the initial two rapidly exchanging pools of body Zn. 3. In normal subjects isotopic Zn appears initially to equilibrate with the whole of the plasma Zn which comprises the first metabolic compartment, pool a. This has a size of 0.72 ± 0.1 μmol/kg. 70Zn equilibration then occurs with a second compartment, pool b, consistent with a rapidly exchanging liver Zn pool of size 3.60 ± 0.93 μmol/kg. The fractional turnover rate of pool b was found to be fivefold slower than that of pool a. 4. In the alcoholic group an expansion of pool a was observed (1.63 ± 0.39 μmol/kg), but the size of the second pool was not significantly different from that of control subjects (5.55 ± 1.0 μmol/kg), although its fractional turnover was significantly increased (Kab: control subjects, 0.018 ± 0.002 min−1, alcoholic patients, 0.031 ± 0.006 min−1). 5. These data therefore demonstrate that kinetic studies using stable isotopes of Zn can provide novel information on exchangeable Zn pools in man, but provide no support for the possibility of an underlying Zn depletion in patients with alcoholic liver disease.

scholarly journals Turnover as a control of ribonucleic acid accumulation in bacteria undergoing stepdown

1976 ◽  
Vol 154 (2) ◽  
pp. 541-552
Author(s):  
J E. M. Midgley
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Turnover Rate ◽  
Amino Acid Starvation ◽  
High Rate ◽  
Glucose Medium ◽  
Amino Acid Deprivation ◽  
Acid Accumulation ◽  
Associated Proteins ◽  
Kinetics Of

The synthesis of ribosomes was compared in rel+ and rel- strains of Escherichia coli undergoing “stepdown” in growth from glucose medium to one with lactate as principal carbon source. Two strains (CP78 and CP79), isogenic except for rel, showed similar behaviour with respect to (1) the kinetics of labelling total RNA and ribosomes with exogenous uracil, (2) the proportion of newly formed protein that could be bound with nascent rRNA in mature ribosomes, and (3) the rate of induction of enzymically active β-galactosidase (relative to the rate of ribosome synthesis). It was concluded that, as there was no net accumulation of RNA during stepdown in either strain, rRNA turnover must be occurring at a high rate. The general features of ribosome maturation in rel+ and rel- cells were almost identical with those found in auxotrophic rel+ organisms starved of required amino acids. In both cases, there was a considerable delay in the maturation of new ribosomal particles, owing to a relative shortfall in the rate of synthesis of ribosome-associated proteins. Only about 4-5% of the total protein labelled during stepdown was capable of binding with newly formed rRNA. This compared with 3.5% for rel+ and 0.5% for rel- auxotrophs during amino acid starvation. The turnover rate for newly formed mRNA and rRNA was virtually the same in “stepped-down” rel+ and rel- strains and was similar to that of the same fraction in amino acid-starved rel+ cells. The functional lifetime of mRNA was also identical. It seems that in the rel- strain many of the characteristics typical of the isogenic rel+ strain are displayed under these conditions, at least as regards the speed of ribosome maturation and the induction of β-galactosidase. Studies on the thermolability of the latter enzyme induced during stepdown indicate that inaccurate translation, which occurs in rel- strains starved for only a few amino acids, is less evident in this situation than in straightforward amino acid deprivation.

Effects of linoleic acid supplementation on blood pressure and kinetics of red cell sodium transport

1991 ◽  
Vol 9 (6) ◽  
pp. S312 ◽  
Author(s):  
Andrea Semplicini ◽  
Edoardo Casiglia ◽  
Mauro Marzola ◽  
Giulio Ceolotto ◽  
Roberto Businaro ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Linoleic Acid ◽  
Sodium Transport ◽  
Red Cell ◽  
Acid Supplementation ◽  
Kinetics Of

Interactions of transmembrane carbonic anhydrase, CAIX, with bicarbonate transporters

2007 ◽  
Vol 293 (2) ◽  
pp. C738-C748 ◽  
Author(s):  
Patricio E. Morgan ◽  
Silvia Pastoreková ◽  
Alan K. Stuart-Tilley ◽  
Seth L. Alper ◽  
Joseph R. Casey
Keyword(s):  
Gastric Mucosa ◽  
Carbonic Anhydrase ◽  
Catalytic Domain ◽  
Hek293 Cells ◽  
Bicarbonate Transport ◽  
Physical Interaction ◽  
Human Gastric Mucosa ◽  
Bicarbonate Transporter ◽  
Bicarbonate Transporters ◽  
Physical Interactions

Association of some plasma membrane bicarbonate transporters with carbonic anhydrase enzymes forms a bicarbonate transport metabolon to facilitate metabolic CO2-HCO3−conversions and coupled HCO3−transport. The transmembrane carbonic anhydrase, CAIX, with its extracellular catalytic site, is highly expressed in parietal and other cells of gastric mucosa, suggesting a role in acid secretion. We examined in transfected HEK293 cells the functional and physical interactions between CAIX and the parietal cell Cl−/HCO3−exchanger AE2 or the putative Cl−/HCO3−exchanger SLC26A7. Coexpression of CAIX increased AE2 transport activity by 28 ± 7% and also activated transport mediated by AE1 and AE3 (32 ± 10 and 37 ± 9%, respectively). In contrast, despite a transport rate comparable to that of AE3, coexpressed CAIX did not alter transport associated with SLC26A7. The CAIX-associated increase of AE2 activity did not result from altered AE2 expression or cell surface processing. CAIX was coimmunoprecipitated with the coexpressed SLC4 polypeptides AE1, AE2, and AE3, but not with SLC26A7. GST pull-down assays with a series of domain-deleted forms of CAIX revealed that the catalytic domain of CAIX mediated interaction with AE2. AE2 and CAIX colocalized in human gastric mucosa, as indicated by coimmunofluorescence. This is the first example of a functional and physical interaction between a bicarbonate transporter and a transmembrane carbonic anhydrase. We conclude that CAIX can bind to some Cl−/HCO3−exchangers to form a bicarbonate transport metabolon.

scholarly journals Activation and Deactivation Kinetics of α2A- and α2C-Adrenergic Receptor-activated G Protein-activated Inwardly Rectifying K+Channel Currents

2001 ◽  
Vol 276 (50) ◽  
pp. 47512-47517 ◽  
Author(s):  
Moritz Bünemann ◽  
Markus M. Bücheler ◽  
Melanie Philipp ◽  
Martin J. Lohse ◽  
Lutz Hein
Keyword(s):  
Action Potential ◽  
G Protein ◽  
Adrenergic Receptor ◽  
Hek293 Cells ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Intact Cells ◽  
Deactivation Kinetics ◽  
Inwardly Rectifying ◽  
Kinetics Of

Although G protein-coupled receptor-mediated signaling is one of the best studied biological events, little is known about the kinetics of these processes in intact cells. Experiments with neurons from α2A-adrenergic receptor knockout mice suggested that the α2A-receptor subtype inhibits neurotransmitter release with higher speed and at higher action potential frequencies than the α2C-adrenergic receptor. Here we investigated whether these functional differences between presynaptic α2-adrenergic receptor subtypes are the result of distinct signal transduction kinetics of these two receptors and their coupling to G proteins. α2A- and α2C-receptors were stably expressed in HEK293 cells at moderate (∼2 pmol/mg) or high (17–24 pmol/mg) levels. Activation of G protein-activated inwardly rectifying K+(GIRK) channels was similar in extent and kinetics for α2A- and α2C-receptors at both expression levels. However, the two receptors differed significantly in their deactivation kinetics after removal of the agonist norepinephrine. α2C-Receptor-activated GIRK currents returned much more slowly to base line than did α2A-stimulated currents. This observation correlated with a higher affinity of norepinephrine at the murine α2C- than at the α2A-receptor subtype and may explain why α2C-adrenergic receptors are especially suited to control sympathetic neurotransmission at low action potential frequencies in contrast to the α2A-receptor subtype.

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