Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Kinetic studies on the two common inherited forms of human erythrocyte adenylate kinase.

ABSTRACT: 1. The kinetic properties of two genetic variants of human erythrocyte adenylate kinase were studied at limiting concentrations of both ADP and MgADP(-) in the forward direction and at limiting concentrations of both AMP and MgATP(2-) in the reverse direction. 2. Primary reciprocal plots rule out the possibility of a Ping Pong mechanism for both forms of the enzyme. 3. Analysis of the kinetic data by an appropriate computer program gave the following K(m) values for the type 1 enzyme: AMP, 0.33mm+/-0.1; MgATP(2-), 0.95mm+/-0.13; ADP, 0.12mm+/-0.03; MgADP(-), 0.22mm+/-0.04. Values for the type 2 enzyme were: AMP, 0.27mm+/-0.03; MgATP(2-), 0.40mm+/-0.05; ADP, 0.08mm+/-0.07; MgADP(-), 0.20mm+/-0.04. 4. Product inhibition studies were done by studying the reverse reaction. With ADP as product inhibitor competitive inhibition patterns were obtained with AMP and/or MgATP(2-) as variable substrate. Similar results were obtained for product inhibition by MgADP(-) with AMP as variable substrate. The results are consistent with a Rapid Equilibrium Random mechanism. 5. Secondary plots of slope versus product concentration were linear. The data were fitted to the appropriate equation and analysed by computer to give values for the product inhibition constants. 6. Differences between the values of certain kinetic constants for the two forms of the enzyme were observed.

SUBMITTER: Brownson C

PROVIDER: S-EPMC1174519 | biostudies-other | 1972 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Partial purification and properties of the two common inherited forms of human erythrocyte adenylate kinase.

Project description:1. The partial purification of adenylate kinase, types 1 and 2, from human erythrocytes is described. 2. Gel chromatography of both forms of the enzyme gave estimates of the molecular weights in the range 20000-23000. 3. Studies on crude haemolysates at various pH values indicated that the type 2 enzyme was less stable than the type 1. Heat denaturation studies on the partially purified enzymes confirmed these findings. 4. Measurements of rates of inhibition by iodoacetate and iodoacetamide showed that the type 2 enzyme reacts more readily than the type 1 enzyme with both reagents. 5. The effect of temperature on the initial velocity of ADP formation was measured at a single concentration of both AMP and MgATP(2-). The two forms of the enzyme responded differently to increasing temperature.

| S-EPMC1174518 | biostudies-other

Assessing the Interactions of Statins with Human Adenylate Kinase Isoenzyme 1: Fluorescence and Enzyme Kinetic Studies.

Project description:Statins are the most effective cholesterol-lowering drugs. They also exert many pleiotropic effects, including anti-cancer and cardio- and neuro-protective. Numerous nano-sized drug delivery systems were developed to enhance the therapeutic potential of statins. Studies on possible interactions between statins and human proteins could provide a deeper insight into the pleiotropic and adverse effects of these drugs. Adenylate kinase (AK) was found to regulate HDL endocytosis, cellular metabolism, cardiovascular function and neurodegeneration. In this work, we investigated interactions between human adenylate kinase isoenzyme 1 (hAK1) and atorvastatin (AVS), fluvastatin (FVS), pravastatin (PVS), rosuvastatin (RVS) and simvastatin (SVS) with fluorescence spectroscopy. The tested statins quenched the intrinsic fluorescence of hAK1 by creating stable hAK1-statin complexes with the binding constants of the order of 104 M-1. The enzyme kinetic studies revealed that statins inhibited hAK1 with significantly different efficiencies, in a noncompetitive manner. Simvastatin inhibited hAK1 with the highest yield comparable to that reported for diadenosine pentaphosphate, the only known hAK1 inhibitor. The determined AK sensitivity to statins differed markedly between short and long type AKs, suggesting an essential role of the LID domain in the AK inhibition. Our studies might open new horizons for the development of new modulators of short type AKs.

| S-EPMC8197361 | biostudies-literature

Two polymorphic forms of human histamine methyltransferase: structural, thermal, and kinetic comparisons.

Project description:BackgroundHistamine plays important biological roles in cell-to-cell communication; it is a mediator in allergic responses, a regulator of gastric acid secretion, a messenger in bronchial asthma, and a neurotransmitter in the central nervous system. Histamine acts by binding to histamine receptors, and its local action is terminated primarily by methylation. Human histamine N-methyltransferase (HNMT) has a common polymorphism at residue 105 that correlates with the high- (Thr) and low- (Ile) activity phenotypes.ResultsTwo ternary structures of human HNMT have been determined: the Thr105 variant complexed with its substrate histamine and reaction product AdoHcy and the Ile105 variant complexed with an inhibitor (quinacrine) and AdoHcy. Our steady-state kinetic data indicate that the recombinant Ile105 variant shows 1.8- and 1.3-fold increases in the apparent K(M) for AdoMet and histamine, respectively, and slightly (16%) but consistently lower specific activity as compared to that of the Thr105 variant. These differences hold over a temperature range of 25 degrees C-45 degrees C in vitro. Only at a temperature of 50 degrees C or higher is the Ile105 variant more thermolabile than the Thr105 enzyme.ConclusionsHNMT has a 2 domain structure including a consensus AdoMet binding domain, where the residue 105 is located on the surface, consistent with the kinetic data that the polymorphism does not affect overall protein stability at physiological temperatures but lowers K(M) values for AdoMet and histamine. The interactions between HNMT and quinacrine provide the first structural insights into a large group of pharmacologic HNMT inhibitors and their mechanisms of inhibition.

| S-EPMC4030376 | biostudies-literature

The Immunome in Two Inherited Forms of Pulmonary Fibrosis

Project description:We analyzed via microarray gene expression profiles from patients with familial pulmonary fibrosis and HPS-1 pulmonary fibrosis collected under IRB-approved protocols.

2018-01-10 | GSE107797 | GEO

The Immunome in Two Inherited Forms of Pulmonary Fibrosis.

Project description:The immunome (immune cell phenotype, gene expression, and serum cytokines profiling) in pulmonary fibrosis is incompletely defined. Studies focusing on inherited forms of pulmonary fibrosis provide insights into mechanisms of fibrotic lung disease in general. To define the cellular and molecular immunologic phenotype in peripheral blood, high-dimensional flow cytometry and large-scale gene expression of peripheral blood mononuclear cells and serum proteomic multiplex analyses were performed and compared in a cohort with familial pulmonary fibrosis (FPF), an autosomal dominant disorder with incomplete penetrance; Hermansky-Pudlak syndrome pulmonary fibrosis (HPSPF), a rare autosomal recessive disorder; and their unaffected relatives. Our results showed high peripheral blood concentrations of activated central memory helper cells in patients with FPF. Proportions of CD38+ memory CD27- B-cells, IgA+ memory CD27+ B-cells, IgM+ and IgD+ B-cells, and CD39+ T helper cells were increased whereas those of CD39- T helper cells were reduced in patients affected with either familial or HPSPF. Gene expression and serum proteomic analyses revealed enrichment of upregulated genes associated with mitosis and cell cycle control in circulating mononuclear cells as well as altered levels of several analytes, including leptin, cytokines, and growth factors. In conclusion, dysregulation of the extra-pulmonary immunome is a phenotypic feature of FPF or HPSPF. Further studies investigating the blood immunome are indicated to determine the role of immune system dysregulation in the pathogenesis of pulmonary fibrosis. Clinical Trial Registration:www.ClinicalTrials.gov, identifiers NCT00968084, NCT01200823, NCT00001456, and NCT00084305.

| S-EPMC5797737 | biostudies-literature

Partial purification and properties of the common inherited forms of adenosine deaminase from human erythrocytes.

Project description:1. The partial purification of adenosine deaminase, types 1, 2 and 2-1, from human erythrocytes is described. 2. The isoenzyme components characteristic of the three forms of the enzyme were partially resolved by chromatography on DEAE-Sephadex. 3. Gel chromatography of the various forms of the enzyme gave estimates of the molecular weights in the range 30000-35000. 4. Electrophoresis in starch gels containing increasing percentages of starch did not reveal any differences in molecular weight between the genetic variants or their isoenzyme components. 5. Analytical isoelectric-focusing experiments in polyacrylamide gels gave the following pI values for the four isoenzyme components present in type 2-1 erythrocytes: 4.70, 4.83, 4.94 and 5.06. 6. All forms of the enzyme gave K(m) values for adenosine of about 30mum and K(i) values of about 8mum for the competitive inhibitor purine riboside. 7. Reaction rates of the type 1 and 2 enzymes were measured at different temperatures. Both enzymes gave values for the energy of activation for hydrolysis of adenosine of about 33.4kJ/mol (8kcal/mol). 8. Heat inactivation of all forms of the enzyme was markedly dependent on ionic strength, the rate of inactivation increasing with increasing ionic strength. The type 1 and type 2 forms of the enzyme differed significantly in their susceptibility to heat inactivation. From the variation of rates of inactivation with temperature, values were obtained for the energies of activation for the heat inactivation of both enzymes as follows: type 1 enzyme 275.5kJ/mol (65.9kcal/mol) and type 2 enzyme 241.6kJ/mol (57.8kcal/mol.).

| S-EPMC1177676 | biostudies-other

Study into the kinetic properties and surface attachment of a thermostable adenylate kinase.

Project description:A thermostable adenylate kinase (tAK) has been used as model protein contaminant on surfaces, so used because residual protein after high temperature wash steps can be detected at extremely low concentrations. This gives the potential for accurate, quantitative measurement of the effectiveness of different wash processes in removing protein contamination. Current methods utilise non-covalent (physisorbtion) of tAK to surfaces, but this can be relatively easily removed. In this study, the covalent binding of tAK to surfaces was studied to provide an alternative model for surface contamination. Kinetic analysis showed that the efficiency of the enzyme expressed as the catalytic rate over the Michaelis constant (kcat/KM) increased from 8.45±3.04 mM-1 s-1 in solution to 32.23±3.20 or 24.46±4.41 mM-1 s-1 when the enzyme was immobilised onto polypropylene or plasma activated polypropylene respectively. Maleic anhydride plasma activated polypropylene showed potential to provide a more robust challenge for washing processes as it retained significantly higher amounts of tAK enzyme than polypropylene in simple washing experiments. Inhibition of the coupled enzyme (luciferase/luciferin) system used for the detection of adenylate kinase activity, was observed for a secondary product of the reaction. This needs to be taken into consideration when using the assay to estimate cleaning efficacy.

| S-EPMC4547157 | biostudies-literature

The crystal structure of human adenylate kinase 6: An adenylate kinase localized to the cell nucleus.

Project description:Adenylate kinases (AKs) play important roles in nucleotide metabolism in all organisms and in cellular energetics by means of phosphotransfer networks in eukaryotes. The crystal structure of a human AK named AK6 was determined by in-house sulfur single-wavelength anomalous dispersion phasing methods and refined to 2.0-A resolution with a free R factor of 21.8%. Sequence analyses revealed that human AK6 belongs to a distinct subfamily of AKs present in all eukaryotic organisms sequenced so far. Enzymatic assays show that human AK6 has properties similar with other AKs, particularly with AK5. Fluorescence microscopy showed that human AK6 is localized predominantly to the nucleus of HeLa cells. The identification of a nuclear-localized AK sheds light on nucleotide metabolism in the nucleus and the energetic communication between mitochondria and nucleus by means of phosphotransfer networks.

| S-EPMC544302 | biostudies-literature

Comparative kinetic studies on the two interconvertible forms of Streptococcus faecalis inorganic pyrophosphatase.

Project description:In this work the two interconvertible forms of inorganic pyrophosphatase (EC 3.6.1.1) of Streptococcus faecalis were shown to differ in kinetics. The highly active form of the enzyme was more sensitive to the changes in the Mg2+ concentration, and thus also more sensitive to the inhibition caused by ATP, which competes with PPi for the chelation of Mg2+ ions. We have previously described a kinetic model for the less-active form of S. faecalis inorganic pyrophosphatase [Lahti & Jokinen (1985) Biochemistry 24, 3526-3530]. The kinetic model of the highly active enzyme form is proposed to be a modification of the model of the less-active form in which enzyme activation by free Mg2+ is necessary for the reaction to occur. In this model the enzyme exists in two states, referred to as R- and T-states. In the absence of ligands the enzyme is in the T-state. R-state, i.e. the catalytically active state, exists only in the presence of free Mg2+. Mg1PPi2- is the primary substrate, and free pyrophosphate is a weak inhibitor that cannot serve as a substrate for the highly active form of S. faecalis inorganic pyrophosphatase. This model closely resembles that previously presented for yeast inorganic pyrophosphatase.

| S-EPMC1152772 | biostudies-other

Life-long sports engagement enhances adult erythrocyte adenylate energetics.

Project description:Regular physical activity reduces age-related metabolic and functional decline. The energy stored in adenine nucleotides (ATP, ADP, and AMP) is essential to enable multiple vital functions of erythrocytes and body tissues. Our study aimed to predict the rate of age-related changes in erythrocyte adenylate energetics in athletes and untrained controls. The erythrocyte concentration of adenylates was measured in 68 elite endurance runners (EN, 20-81 years), 58 elite sprinters (SP, 21-90 years), and 62 untrained individuals (CO, 20-68 years). Resting concentrations of ATP, total adenine nucleotide pool, and ADP/AMP ratio were lowest in the CO group and highest in the SP group. The concentration of erythrocyte ADP and AMP was lowest in the EN group and highest in the CO group. In all studied groups, we found a significant increase in the concentration of most erythrocyte adenylate metabolites with age. For ADP and AMP, the trend was also significant but decreasing. Our study strongly suggests that lifelong sports and physical activity participation supports erythrocyte energetics preservation. Although the direction and the predicted rates of change are similar regardless of the training status, the concentrations of particular metabolites are more advantageous in highly trained athletes than in less active controls. Of the two analyzed types of physical training, sprint-oriented training seems to be more efficient in enhancing erythrocyte metabolism throughout adulthood and old age than endurance training.

| S-EPMC8660807 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data