scholarly journals Kinetic properties and tissular distribution of mammalian phosphomannomutase isozymes

1999 ◽  
Vol 339 (1) ◽  
pp. 201-207 ◽  
Author(s):  
Michel PIRARD ◽  
Younes ACHOURI ◽  
Jean-François COLLET ◽  
Els SCHOLLEN ◽  
Gert MATTHIJS ◽  
...  
Keyword(s):  
Kinetic Studies ◽  
Time Dependent ◽  
Chemical Characteristics ◽  
Kinetic Properties ◽  
Rat Tissues ◽  
Northern Blots ◽  
Carbohydrate Deficient Glycoprotein Syndrome ◽  
Fructose 1,6 Bisphosphate ◽  
Acyl Phosphate ◽  
Stimulation Of

Human tissues contain two types of phosphomannomutase, PMM1 and PMM2. Mutations in the PMM2 gene are responsible for the most common form of carbohydrate-deficient glycoprotein syndrome [Matthijs, Schollen, Pardon, Veiga-da-Cunha, Jaeken, Cassiman and Van Schaftingen (1997) Nat. Genet.19, 88–92]. The protein encoded by this gene has now been produced in Escherichia coli and purified to homogeneity, and its properties have been compared with those of recombinant human PMM1. PMM2 converts mannose 1-phosphate into mannose 6-phosphate about 20 times more rapidly than glucose 1-phosphate to glucose 6-phosphate, whereas PMM1 displays identical Vmax values with both substrates. The Ka values for both mannose 1,6-bisphosphate and glucose 1,6-bisphosphate are significantly lower in the case of PMM2 than in the case of PMM1. Like PMM1, PMM2 forms a phosphoenzyme with the chemical characteristics of an acyl-phosphate. PMM1 and PMM2 hydrolyse different hexose bisphosphates (glucose 1,6-bisphosphate, mannose 1,6-bisphosphate, fructose 1,6-bisphosphate) at maximal rates of ≈ 3.5 and 0.3% of their PMM activity, respectively. Fructose 1,6-bisphosphate does not activate PMM2 but causes a time-dependent stimulation of PMM1 due to the progressive formation of mannose 1,6-bisphosphate from fructose 1,6-bisphosphate and mannose 1-phosphate. Experiments with specific antibodies, kinetic studies and Northern blots indicated that PMM2 is the only detectable isozyme in most rat tissues except brain and lung, where PMM1 accounts for about 66 and 13% of the total activities, respectively.

scholarly journals Difference in kinetic properties between hexokinase type I isoenzymes from various rat tissues with reference to the effect of a thiol inhibitor (Short Communication)

1974 ◽  
Vol 137 (1) ◽  
pp. 139-142 ◽  
Author(s):  
T. Kamikashi ◽  
H. Kizaki ◽  
K. Murakami ◽  
S. Ishibashi
Keyword(s):  
Short Communication ◽  
Kinetic Studies ◽  
Kinetic Properties ◽  
Rat Tissues ◽  
Type I ◽  
Different Tissues ◽  
Hexokinase Type I

Hexokinase isoenzyme type I was purified from various rat tissues, and was subjected to kinetic studies in the presence or the absence of p-chloromercuribenzenesulphonate. The mode of the inhibition by the thiol inhibitor was different for the type I isoenzymes obtained from different tissues, suggesting that the type I isoenzymes from different tissues were not identical proteins.

scholarly journals pH and kinetic studies of chloroplast sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea)

1988 ◽  
Vol 253 (1) ◽  
pp. 249-254 ◽  
Author(s):  
F Cadet ◽  
J C Meunier
Keyword(s):  
Spinacia Oleracea ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Kinetic Properties ◽  
Enzyme Specificity ◽  
Active Centre ◽  
Specificity Constant ◽  
The One ◽  
Fructose 1,6 Bisphosphate

The aim of this paper is to study some steady-state kinetic properties of sedoheptulose-1,7-bisphosphatase, its pH-dependence and the effect of a substrate analogue, fructose 2,6-bisphosphate. Studies were carried out with sedoheptulose 1,7-bisphosphate and with fructose 1,6-bisphosphate, an alternative substrate. The pK values are identical for both substrates, and fructose 2,6-bisphosphate behaves like a competitive inhibitor. These results suggest that there exists a unique active site for either sedoheptulose 1,7-bisphosphate or fructose 1,6-bisphosphate on the enzyme molecule. Increasing Mg2+ concentrations shifted the optimum pH. As for fructose-1,6-bisphosphatase, we believe that this shift is due to the neutralization of negative charges near the active centre [Cadet, Meunier & Ferté (1987) Eur. J. Biochem. 162, 393-398]. The free species of sedoheptulose 1,7-bisphosphate and fructose 1,6-bisphosphate are not the usual substrates of enzyme, nor is Mg2+. But the kinetics relative to the (Mg2+-substrate4-)2- complex is not consistent with this complex being the substrate. An explanation of this discrepancy is proposed, involving both the negative charges near the active centre and the positive charges of Mg2+. The observed Vmax. of the reduced enzyme is 65% of the theoretical Vmax. for both substrates, but the observed Vmax. relative to sedoheptulose 1,7-bisphosphate is 3 times the one relative to fructose 1,6-bisphosphate. The specificity constant (kcat./Km), 1.62 × 10(6) M-1.s-1 with respect to sedoheptulose 1,7-bisphosphate compared with 5.5 × 10(4) M-1.s-1 with respect to fructose 1,6-bisphosphate, indicates that the enzyme specificity towards sedoheptulose 1,7-bisphosphate is high but not absolute.

scholarly journals On the mechanism of stimulation of the Na/K pump of LK sheep erythrocytes by anti-L antibody.

1987 ◽  
Vol 90 (1) ◽  
pp. 3-25 ◽  
Author(s):  
P Dunham ◽  
C Anderson
Keyword(s):  
Maximum Velocity ◽  
Kinetic Studies ◽  
Cell Types ◽  
Kinetic Properties ◽  
Sheep Erythrocytes ◽  
Noncompetitive Inhibition ◽  
L Cells ◽  
Significant Stimulation ◽  
K Concentration ◽  
Stimulation Of

Studies were undertaken to explore the mechanism of stimulation of the Na/K pump in LK sheep erythrocytes by anti-L antibody. First, the numbers of functioning pump sites were determined by correlating [3H]ouabain binding with levels of inhibition of the pump. Untreated (control) cells had approximately 41 pumps per cell, and anti-L treatment caused an increase in the number of functioning pumps to approximately 85 per cell. Reducing the intracellular K concentration, [K]c, to near zero caused an increase in the number of pumps in control cells, but not in anti-L cells, such that the numbers of pumps per cell were about the same in the two cell types. These results led to the prediction that Kc is a noncompetitive inhibitor of the pump in control cells, and that anti-L stimulates the pump and increases number of functioning pumps by reducing noncompetitive inhibition by Kc. Kinetic studies were undertaken to test this prediction: activation of the pump by increasing [Na]c was measured at three fixed levels of [K]c. In control cells, the apparent maximum velocity of the pump (J'max) was reduced approximately threefold by raising [K]c from 0.2 to 9 mmol/liter cells, demonstrating noncompetitive inhibition by Kc. In anti-L cells, J'max did not vary with [K]c, which shows that, as predicted, anti-L abolishes the noncompetitive inhibition by Kc. The modification of the kinetic properties of the pumps by the antibody is highly specific in that affinities for Nac and Ko as substrates are unaffected. However, the effect of the antibody on noncompetitive inhibition by Kc does not explain the stimulation of the pump fully since there is significant stimulation at near-zero [K]c.

Phosphorylation of Nck in response to a variety of receptors, phorbol myristate acetate, and cyclic AMP

1992 ◽  
Vol 12 (12) ◽  
pp. 5816-5823
Author(s):  
D Park ◽  
S G Rhee
Keyword(s):  
Monoclonal Antibodies ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Immunoglobulin M ◽  
Rat Tissues ◽  
A431 Cells ◽  
Sh3 Domains ◽  
Epidermal Growth ◽  
Stimulation Of

The 47-kDa protein coimmunoprecipitated with phospholipase C (PLC)-gamma 1 by anti-PLC-gamma 1 monoclonal antibodies is proved to be Nck, a protein composed almost exclusively of one SH2 and three SH3 domains. Nck and PLC-gamma 1 are recognized by certain anti-PLC-gamma 1 monoclonal antibodies because Nck and PLC-gamma 1 share an epitope that likely is located in their SH3 domains. Nck is widely distributed in rat tissues, with an especially high level of expression in testes. The expression levels of Nck remains unchanged during the development of rat brain, whereas PLC-gamma 1 decreases during the same developmental period. Stimulation of A431 cells with epidermal growth factor elicits the tight association of Nck with the epidermal growth factor receptor and phosphorylation of Nck on both serine and tyrosine residues. The phosphorylation of Nck is also enhanced in response to stimulation of the nerve growth factor receptor in PC12 cells, the T-cell receptor complex in Jurkat cells, the membrane immunoglobulin M in Daudi cells, and the low-affinity immunoglobulin G receptor (Fc gamma RII) in U937 cells. The phosphorylation of Nck was also enhanced following treatment of A431 cells with phorbol 12-myristate 13-acetate or forskolin. These results suggest that Nck is a target for a variety of protein kinases that might modulate the postulated role of Nck as an adaptor for the physical and functional coordination of signalling proteins.

Tetanic hyperpolarization of single medullated nerve fibers in sodium and lithium

1976 ◽  
Vol 231 (4) ◽  
pp. 1033-1038 ◽  
Author(s):  
GM Schoepfle
Keyword(s):  
Steady State ◽  
Interspike Interval ◽  
Nerve Fibers ◽  
Repetitive Stimulation ◽  
Time Dependent ◽  
Potassium Ions ◽  
Current Densities ◽  
Medullated Nerve Fiber ◽  
Sodium And Potassium ◽  
Stimulation Of

Repetitive stimulation of a single medullated nerve fiber of Xenopus yields a succession of postspike voltage-time curves which are nearly coincident until attainment of a voltage that corresponds to that of the maximum attained by the normal postspike undershoot. Initially the interspike potential returns toward a resting level after this brief phase of hyperpolarization. However, as tetanization proceeds, a pattern of hyperpolarization develops with the result that, in the tetanic steady state, there exists a progressive hyperpolarization throughout each interspike interval. Extent of postspike hyperpolarization in terms of a deviation deltaVm from the resting level of membrane potential is approximated by the variation deltaVm = delta[MNa + MK]/[GNa + GK] where MNa and MK are current densities associated with active pumping of sodium and potassium ions and GNa and GK are corresponding time-dependent leak conductances. Tetanic hyperpolarization is reversibly abolished by cyanide and by exposure to lithium Ringer. Eventual reappearance of tetanic hyperpolarization in the presence of lithium Ringer suggests lithium pumping.

scholarly journals Interacting cell populations in cultures of leukocytes from normal or leukemic peripheral blood

Blood ◽  
1977 ◽  
Vol 49 (2) ◽  
pp. 269-280
Keyword(s):  
Thymidine Incorporation ◽  
Kinetic Studies ◽  
Cell Interaction ◽  
Cell Number ◽  
Cellular Interaction ◽  
Intact Cells ◽  
Quantitative Evidence ◽  
Peripheral Leukocytes ◽  
Limiting Dilution ◽  
Stimulation Of

Kinetic studies in cultures containing 2 X 10(5) peripheral leukocytes from patients with acute myelobastic leukemia revealed extensive, radiation-sensitive increases in thymidine incorporation without parallel increases in cell number. Modest and variable stimulation of 3HTdR incorporation was seen with the addition of either leukocyte- conditioned medium prepared with phytohemagglutin (PHA) or PHA alone. However, using the method of limiting dilution, stimulation was always observed and ranged from 3- to 20-fold in individual patients. By mixing small numbers of intact cells with larger numbers of irradiated autologous cells, quantitative evidence was obtained for a cellular interaction between irradiated, PHA-stimulated populations capable of 3HTdR incorporation. Similar evidence for cell-cell interaction was obtained for normal leukocytes.

Properties of Excitatory Synaptic Connections Mediated by the Corpus Callosum in the Developing Rat Neocortex

2001 ◽  
Vol 86 (6) ◽  
pp. 2973-2985 ◽  
Author(s):  
Sanjay S. Kumar ◽  
John R. Huguenard
Keyword(s):  
Corpus Callosum ◽  
Ampa Receptors ◽  
Pyramidal Neurons ◽  
Epileptiform Activity ◽  
Cell Voltage ◽  
Inward Rectification ◽  
Kinetic Properties ◽  
Postsynaptic Receptors ◽  
Minimal Stimulation ◽  
Stimulation Of

Despite the major role of excitatory cortico-cortical connections in mediating neocortical activities, little is known about these synapses at the cellular level. Here we have characterized the synaptic properties of long-range excitatory-to-excitatory contacts between visually identified layer V pyramidal neurons of agranular frontal cortex in callosally connected neocortical slices from postnatal day 13 to 21( P13–21) rats. Midline stimulation of the corpus callosum with a minimal stimulation paradigm evoked inward excitatory postsynaptic currents (EPSCs) with an averaged peak amplitude of 56.5 ± 5 pA under conditions of whole cell voltage clamp at −70 mV. EPSCs had fixed latencies from stimulus onset and could follow stimulus trains (1–20 Hz) without changes in kinetic properties. Bath application of 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) abolished these responses completely, indicating that they were mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs). Evoked responses were isolated in picrotoxin to yield purely excitatory PSCs, and a low concentration of NBQX (0.1 μM) was used to partially block AMPARs and prevent epileptiform activity in the tissue. Depolarization of the recorded pyramidal neurons revealed a late, slowly decaying component that reversed at ∼0 mV and was blocked by d-2-amino-5-phosphonovaleric acid. Thus AMPA and N-methyl-d-aspartate receptors (NMDARs) coexist at callosal synapses and are likely to be activated monosynaptically. The peak amplitudes and decay time constants for EPSCs evoked using minimal stimulation (±40 mV) were similar to spontaneously occurring sEPSCs. Typical conductances associated with AMPA and NMDAR-mediated components, deduced from their respective current-voltage ( I-V) relationships, were 525 ± 168 and 966 ± 281 pS, respectively. AMPAR-mediated responses showed age-dependent changes in the rectification properties of their I-V relationships. While I-Vs from animals > P15 were linear, those in the younger (< P16) age group were inwardly rectifying. Although Ca2+ permeability in AMPARs can be correlated with inward rectification, outside-out somatic patches from younger animals were characterized by Ca2+-impermeable receptors, suggesting that somatic receptors might be functionally different from those located at synapses. While the biophysical properties of AMPAR components of callosally-evoked EPSCs were similar to those evoked by stimulation of local excitatory connections, the NMDA component displayed input-specific differences. NMDAR-mediated responses for local inputs were activated at more hyperpolarized holding potentials in contrast with those evoked by callosal stimulation. Paired stimuli used to assay presynaptic release properties showed paired-pulse depression (PPD) in animals < P16, which converted to facilitation (PPF) in older animals, suggesting a developmental transition from low probability of transmitter release to high P r at these synapses and/or alterations in the properties of the underlying postsynaptic receptors. Physiologic properties of neocortical e-e connections are thus input specific and subject to developmental changes in their postsynaptic receptors.

Stimulation of P(i) transport in opossum kidney cells by hyposmotic media

1993 ◽  
Vol 264 (4) ◽  
pp. F585-F592
Author(s):  
M. Loghman-Adham ◽  
G. T. Motock
Keyword(s):  
Fluid Phase ◽  
Kinetic Studies ◽  
Uptake Kinetic ◽  
Opossum Kidney ◽  
Pi Transport ◽  
Opossum Kidney Cells ◽  
Fluid Phase Endocytosis ◽  
Peroxidase Uptake ◽  
Stimulation Of

Exposure of various cells to hyposmotic media (Hypo) results in a rapid inhibition of both receptor-mediated and fluid-phase endocytosis. We used this maneuver to investigate the role of endocytosis in regulation of Pi transport in opossum kidney (OK) cells. Following exposure to Hypo, Na(+)-dependent Pi uptake increased rapidly, reaching a maximum within 5 min, and remained elevated up to 30 min. This was associated with a simultaneous reduction of horseradish peroxidase uptake. Kinetic studies showed increased apparent Vmax for Pi (9.38 +/- 0.93 vs. 13.08 +/- 1.04 nmol.mg-1.5 min-1 for control and Hypo, respectively; P < 0.05, n = 6) with no change in apparent Km. The effect was specific for Pi with no change in the Na(+)-dependent or -independent uptake of L-proline, L-glutamine, or methyl-alpha-D-glucopyranoside. Stimulation of Pi transport persisted when control and Hypo had identical ionic compositions. Stimulation of Pi transport was rapidly reversed when cells were returned to an isosmotic medium. Preincubation with Hypo at 4 degrees C had no effect on Pi transport. Addition of cycloheximide or actinomycin D did not prevent the increased Pi uptake after exposure to Hypo. The effect also persisted after protein kinase C downregulation. Stimulation of Pi transport by Hypo is consistent with reduced endocytic retrieval of Na(+)-Pi cotransporters from brush-border membrane (BBM), resulting in an increase in their number on the BBM.

Regulation of Fructose 1,6-Bisphosphatase Activity of Chlorella by Mole Mass Change

1996 ◽  
Vol 51 (9-10) ◽  
pp. 639-645 ◽  
Author(s):  
N. Grotjohann
Keyword(s):  
Enzyme Regulation ◽  
Fold Increase ◽  
Substrate Affinity ◽  
Kinetic Properties ◽  
Enzyme Form ◽  
Phosphorylated Compounds ◽  
Fructose 1,6 Bisphosphatase ◽  
Chloroplast Stroma ◽  
Fructose 1,6 Bisphosphate

Fast protein liquid chromatography on Superose 6 of partially purified FBPase II from Chlorella reveals a 1350 kDa-form at pH 6.0 and a 67 kDa-form at pH 8.5. Treatment of the large enzyme form with 5mᴍ concentrations of Mg2+, F1,6P2, DTT or ATP leads to dissociation into smaller ones of 215 -470 kDa. Aggregation/dissoziation is a reversible process, as has been shown for the effect of F1,6P2 and of pH, by rechromatography. The change in mole mass results in alterations of the activitiy and of the kinetic properties of the enzyme forms, obtained. Dissociation results in a 4 - 6 fold increase in activity, as can be shown for F1,6P2-treated samples. Halfsaturation constants, as well as the degree of cooperativity of the 67- and the 1350- kDa form, are different for substrate affinity, activation by Mg2+ and DTT, and for inhibition by ATP. Both enzyme forms hydrolyse fructose 1,6 bisphosphate and seduheptulose 1,7 bisphosphate better than other phosphorylated compounds. The ratio of F1,6P2- to SDP-cleavage is 100:58 for the small enzyme form and 100: 84 for the large one. Activation of FBPase II in the light and inactivation in the dark is discussed on the basis of different oligomeric forms of the enzyme, generated by changes in the concentration of intermediates and effectors in the chloroplast stroma, leading to dissociation or aggregation. The conclusion is drawn that oligomerization of key enzymes, resulting in enzyme forms with different activities and different kinetic properties, might provide an effective mechanism for enzyme regulation in vivo

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