The kinetics of intrinsic factor-vitamin B12 binding

The kinetic behaviour of intrinsic factor-vitamin B₁₂ binding has been examined under varying conditions using an albuminised charcoal separation technique. The overall reaction obeys second order rate laws. The intrinsic factor considered alone obeys first order laws; the velocity of reaction of vitamin B₁₂ is too fast for measurement by the technique described but by deduction obeys first order laws. Rate constants as three temperatures, (k₂ at 25°C=1.56·10⁸·mole^?1·s^?1) the activation energy (E=12.7 kJ·mole^?1) and Arrhenius constant (A=2.7·10¹⁰ 1·mole^?1·s^?1 have been calculated. There is the possibility of diffusion control of the reaction in which case the E and A values are invalid. The effect of pH on the reaction has been studied and the results discussed in relation to the pH studies of other workers whose results show disagreement. Albumin coated charcoal was shown to discriminate between intrinsic factor-vitamin B₁₂ and free vitamn B₁₂ over a wide pH range. The apparent under-estimation of intrinsic factor in dilute solution was shown to be due to adsorption of the intrinsic factor to plastic tubes.     

Interaction between insulin receptors and glucose transport: Effect of prostaglandin E2

The effect of prostaglandin E₂ on the adipocyte glucose transport system was measured. Prostaglandin E₂ enhanced insulin's effects to accelerate adipocyte glucose transport without increasing insulin binding. However, prostaglandin E₂ did not increase transport in the absence of insulin, and is thus not an insulin-like agent. This augmenting effect appeared specific for the insulin stimulatory process since prostaglandin E₂ did not enhance the ability of “insulin-like” agents to increase glucose transport. These results suggest that prostaglandin E₂ may play a role in the interaction between insulin receptors and the glucose transport system.     

Characterization and localization of prostaglandin E1 receptors in rat liver plasma membranes

Methodology for measurement and characterization of prostaglandin binding to membranes has been developed. The binding assay was used to study the presence of prostaglandin receptors in high purified cell fractions derived from rat liver. High affinity binding receptors which have a saturation value of 1.0 pmole/mg protein and a dissociation constant of 1.2 nM were found exclusively in the plasma membrane. High affinity receptors were not found in cell fractions containing nuclei, rough microsomes. Golgi complex or mitochondria. The binding by other prostaglandins was competitive with prostaglandin E₁. Competitive binding studies were used to obtain dissociation constants for prostaglandins F_1α, F_2α, B₁, B₂, A₁, A₂, and 15-keto prostaglandin E₂ which were 1100, 100, 300, 180, 16. 16 and 700 nM, respectively. Eicosa-5.8.11.19-tetraynoic acid, an inhibitor of prostaglandin synthesis did not bind appreciably to the prostaglandin E receptor, whereas two prostaglandin analogues, which have high physiological activity compete effectively with prostaglandin E₁ for the receptor. Thus, the binding receptor for the E-type prostaglandins is highly specific both with respect to cell localization as well as the type of substrate. Numerical routines for the fitting of the data and a procedure for the determination of the specific activity of the labelled prostaglandin are provided.     

Changes in prostaglandin E1 stimulation of glucose oxidation in rat adipocytes during maturation and aging

Prostaglandin E₁ stimulates glucose oxidation in isolated rat adipocytes in a time and concentration dependent manner. Maximal stimulation requires 2 hours exposure to prostaglandin, although effects can be detected by 0.5 hours or earlier. In contrast to prostaglandin E₁, prostaglandin F₂α has essentially no effect on glucose oxidation. Maximal stimulation by prostaglandin E₁ at all ages tested occurs at concentrations of 10^?5 ? 10^?4M. Stimulation is greatest in cells of mature (10–12 month old) animals at 81 ± 9% above basal levels of glucose oxidation. This is to reduced to 48 ± 8% in cells of senescent (23–26 month old) animals, and at 23 ± 18% in cells of young (2–3 month old) rats is not significantly different from basal oxidation in most animals. These results are consistent with data for adipocytes and other cell types indicating that responsiveness to certain hormones is altered during maturation and aging.     

Interleukin-1 inhibits PGE2 binding to macrophage-like P388D1 cells by a cyclic AMP-independent process

The interaction between interleukin IL-1α and PGE₂ on P388D₂ on cells has been investigated. Preincubation of murine macrophage-like cells, P388D₁, with IL-1α (0–73 pM) reduced the binding of PGE₂ to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1α decreased the PGE₂ binding by lowering both the high and low affinity receptor binding capacities (from 0.31 ± 0.02 to 0.12 ± 0.01 fmol/10⁶ cells for the high affinity receptor binding sites and from 2.41 ± 0.12 to 1.51 ± 0.21 fmol/10⁶ cells for the low affinity receptor binding sites). However, the dissociation constants of the receptor of the IL-1α-treated cells remained unchanged. Inhibition of PGE₂ binding IL-1α did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1α inhibits the binding of PGE₂ to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE₂.     

Comparison of the effects of prostaglandin I2 and prostaglandin E2 stimulation of the rat kidney adenylate cyclase-cyclic AMP systems

Prostacyclin (Prostaglandin I₂) effects on the rat kidney adenylate cyclase-cyclic AMP system were examined. Prostaglandin I₂ and prostaglandin E₂, from 8 · 10^?4 to 8 · ^?7 M stimulated adenylate cyclase to a similar extent in cortex and outer medulla. In inner medulla, prostaglandin I₂ was more effective than prostaglandin E₂ at all concentrations tested. Both prostaglandin I₂ and prostaglandin E₂ were additive with antidiuretic hormone in outer and inner medulla. Prostaglandin I₂ and prostaglandin E₂ were not additive in any area of the kidney, indicating both were working by similar mechanisms. Prostaglandin I₂ stimulation of adenylate cyclase correlated with its ability to increase renal slice cyclic AMP content. Prostaglandin I₂ and prostaglandin E₂ (1.5 · 10^?4 M) elevated cyclic AMP content in cortex and outer medulla slices. In inner medulla, with Santoquin® (0.1 mM) present to suppress endogenous prostaglandin synthesis, prostaglandin I₂ and prostaglandin E₂ increased cyclic AMP content. 6-Ketoprostaglandin F_1α, the stable metabolite of prostaglandin I₂, did not increase adenylate cyclase activity or tissue cyclic AMP content. Thus, prostaglandin I₂ activates renal adenylate cyclase. This suggests that the physiological actions of prostaglandin I₂ may be mediated through the adenylate cyclase-cyclic AMP system.     

Prostaglandin E1 binding and adenylate cyclase activation in normal and transformed fibroblasts

The binding of [³H]prostaglandin E₁ to membranes of clones of normal rat kidney fibroblasts (NRK cells) has been measured. Cell lines that responded to prostaglandin E₁, such as NRK and NRK transformed with Schmitt-Ruppin strain of Rous sarcoma virus (SR-NRK cells), have a high affinity prostaglandin E₁ binding site. Murine-sarcoma-virus-transformed lines of NRK cells are unresponsive to prostaglandin E₁ and have reduced prostaglandin E₁ binding. Exposure of cells to prostaglandin E₁ results both in decreases prostaglandin E₁ responsiveness and reduced prostaglandin E₁ binding.Activation of adenylate cyclase is correlated to binding of prostaglandin E₁ to receptors in both NRK and SR-NRK cell membranes. Mathematical models suggest that GTP decreases the affinity of hormone for its receptor while increasing the catalytic efficiency of adenylate cyclase, and that aggregates of occupied receptors may play an important role in the activation of adenylate cyclase.     

The interaction of norepinephrine and prostaglandin E1 on the adenyl cyclase system of human and rabbit blood platelets

Prostaglandin E₁ markedly increased the formation of cyclic [³H]AMP from labeled adenine in human and rabbit blood platelets. Norepinephrine alone had no stimulatory effect, but it reduced cyclic AMP levels elevated by prostaglandin E₁. Phentolamine overcame the inhibitory effect of norepinephrine, whereas propranolol did not. Homogenization of platelets reduced, but did not abolish, the inhibitory effect of norepinephrine on adenyl cyclase activity induced by prostaglandin E₁.     

β2-Adrenergic regulation of prostaglandin D2 receptor in rabbit platelets

[³H]Prostaglandin D₂ binding to rabbit platelets was increased by about 150% in the presence of β-adrenoceptor agonist, isoproterenol. The isoproterenol-induced potentiation of the [³H]prostaglandin D₂ binding gave a bell-shaped dose-response relationship (maximum response at 3·10⁻⁸ M) in a stereospecific manner. Similar and moderate potentiation was obtained with terbutaline. On the other hand, β-adrenoceptor antagonists such as alprenolol, propranolol and butoxamine (β₂-specific) had no potentiating effect on [³H]prostaglandin D₂ binding; rather, they abolished the isoproterenol-induced increase of [³H]prostaglandin D₂ binding. The β₁-specific antagonist, metoprolol, did not have any effect. Rabbit platelets were found to possess one [³H]prostaglandin D₂ binding site (K_d = 6·10⁻⁷ M, B_max = 787 fmol/mg protein). In the presence of isoproterenol at 3·10⁻⁸ M, B_max was increased with unaltering K_d value. Isoproterenol did not increase [³H]prostaglandin E₁, [³H]prostaglandin E₂ and [³H]prostaglandin F_2α bindings to platelets. The potential effect of isoproterenol was mimicked by forskolin, theophylline, dibutyryl cyclic AMP, prostaglandin E₁ and prostaglandin I₂, but it was abolished by 2′, 5′-dideoxyadenosine, an inhibitor of adenylate cyclase, indicating that elevated level of cyclic AMP may be available for the induction of the increase of [³H]prostaglandin D₂ binding. Prostaglandin D₂-induced cyclic AMP synthesis and antiaggregation activity were also augmented in the presence of isoproterenol. These results suggest a β₂-adrenoceptor-mediated cyclic AMP-dependent mechanism for the regulation of prostaglandin D₂ receptor binding in rabbit platelets.     

The treatment of the hepatorenal syndrome with intra-renal administration of prostaglandin E1

Three patients with the hepatorenal syndrome were treated with prostaglandin E₁ administered through a selective renal arterial catheter. Prostaglandin E₁ was given in progressively increasing doses (2 to 100 ng/kg/min) over a 60-minute period. Control plasma prostaglandin E levels were elevated in all three patients, 0.98, 0.91, and 0.83 ng/ml, respectively. At the end of the infusion, plasma prostaglandin E levels had risen to 10.4, 2.63, and 10.3 ng/ml in the three patients respectively. Plasma renin activity increased during the course of the infusion in two of the patients. The plasma aldosterone concentration did not change during the prostaglandin E₁ infusion. Intrarenal prostaglandin E₁ failed to increase urine volume or urinary sodium concentration in three patients with the hepatorenal syndrome.     

Prostaglandins and renin release: III. Effects of PGE1, E2 F2α and D2 on renin release from rabbit renal cortical slices

We have investigated the direct effects of prostaglandins E₁, E₂, F_2α and D₂ on renin release from rabbit renal cortical slices. Prostaglandin E₁ (PGE₁) was the most potent stimulant of renin release, while PGE₂ was 20–30 fold less active. PGF_2α was found not to be an inhibitor of renin release as reported by others, but rather a weak agonist. PGD₂ up to a concentration of 10 μg/ml had no activity in this system. That the stimulation of renin release by PGE₁ is a direct effect is supported by the finding that PGE₁-induced release is not blocked by L-propranolol or by Δ^5,8,11,14-eicosatetraynoic acid (ETYA), a prostaglandin synthesis is inhibitor. The fatty acid precursor of PGE₁, Δ^8,11,14-eicosatrienoic acid, also stimulated renin release, an effect which was blocked by ETYA. In addition to the above findings, ethanol, a compound frequently used to dissolve prostaglandins, was shown to inhibit renin release.     

THE DIFFERENTIATION OF PHOSPHOLIPASE A1 AND A2 IN RAT AND HUMAN NERVOUS TISSUES

—Lipid-free extracts of rat and human brain have been prepared and shown to contain phospholipase A₁ and A₂ activities and a lysophospholipase. The phospholipase Aj activity has pH optima of 4·2 and 4·6 in rat and human brain, respectively; it can be partially purified and isolated in high yields by dialysing the extracts at low pH. The purified preparations hydrolyse the ester bond at the 1-position in lecithin, phosphatidyl-ethanolamine and phosphatidylserine, but have little or no action on triglyceride or cholesterol ester. An assay system for the enzyme is described. Phospholipase A₂ activity is optimal at pH 5·5 in rat brain extracts and at pH 5·0 in extracts of human brain. The phospholipase A₂ activity of human cerebral cortex is largely unaffected by heating extracts at 70°C for 5 min, whereas this treatment substantially inactivates phospholipase A₁ and completely destroys lysophospholipase. Phospholipase A₁ is widely distributed in both grey and white matter of human brain and is also present in peripheral nerve. Phospholipase A₂ activity is lower than A₁ in all regions of the CNS examined so far, and is absent from peripheral nerve. Neither enzyme appears to require Ca²⁺ but both are inhibited by di-isopropylfluorophosphate (DFP, 2 × 10^?6 m) and thus differ from phospholipase A of pancreas. These studies confirm that the phospholipase A₁ and A₂ activities in brain are due to separate enzymes.     

Desensitization of PGE1 receptors in neuroblastoma-glioma hybrid cells

Prostaglandin E₁ receptor sites were measured in homogenates of NG108-15 neuroblastoma-glioma hybrid cells after exposure of intact cells to PGE₁. Scatchard analysis of competitive binding studies showed that incubation of NG108-15 cells in the presence of 2.5 μM PGE₁ for 16 h resulted in a loss of PGE₁ receptors and an increase in the dissociation constant of the remaining receptors. Thus, cells challenged with PGE₁ not only lose adenylate cyclase activity, but also lose PGE₁ receptors and decreased the affinity of the remaining receptors for PGE₁.     

MOUSE NEUROBLASTOMA CELL ADENYLATE CYCLASE: REGULATION BY 2-CHLOROADENOSINE, PROSTAGLANDIN E1 AND THE CATIONS Mg2+, Ca2+ AND Mn

—Some basic kinetic properties of adenylate cyclase in cell free preparations of mouse neuroblastoma were investigated. Production of cAMP from ATP by the enzyme requires the presence of either Mg²⁺ or Mn²⁺ in addition to ATP. In the presence of Mg²⁺, the K_m for ATP is 120 ± 15 μM and the interaction of ATP and adenylate cyclase appears to be non-cooperative (Hill coefficient of 1). Magnesium ion concentrations in excess of the ATP concentration cause stimulation although similar excess concentrations of Mn²⁺ cause inhibition. Prostaglandin E₁ and 2-chloroadenosine activate the enzyme. The K_m of the cyclase for 2-chloroadenosine is 6 μm . Activation by 2-chloroadenosine leads to an increase in V_max but does not effect the K_m for ATP. At a fixed ATP concentration, the extent of activation caused by prostaglandin E₁ and 2-chloroadenosine is inversely related to the Mg²⁺ concentration. Calcium ion causes inhibition of adenylate cyclase from 0.1 to 4mM with a K_i of 5 ± 10^?4m . Ca²⁺ interaction with the enzyme in the absence or presence of either 2-chloroadenosine or prostaglandin E₁ appears cooperative (i.e. Hill coefficients of ?2). Ca²⁺ inhibition is non-competitive with respect to either ATP or 2-chloroadenosine but is progressively diminished by increasing Mn²⁺ concentrations. Divalent cation effects and activation by 2-chloroadenosine and prostaglandin E₁ of the neuroblastoma adenylate cyclase are compared with ion effects and hormone activation of the enzyme obtained from non-neuronal tissue.     

Effect of prostaglandins A1, A2, B1, B2, E2 and F2α on human umbilical cord vessels

The effect of six naturally occurring prostaglandins on isolated umbilical arteries and veins has been studied. All six prostaglandins had a constricting effect on the umbilical vessels. On the umbilical artery preparations the potencies in decreasing order were A₂>B₂>F_2α>B₁>E₂>A₁. Prostaglandin B₂ was more potent than PGA₂ on the umbilical vein. Polyphloretin phosphate (PPP) antagonised the constricting effect of all six prostaglandins without altering responses to 5-hydroxytryptamine.     

The reaction between cytochrome C1 and cytochrome C

The kinetics of electron transfer between the isolated enzymes of cytochrome c₁ and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c₁ and ferricytochrome c is fast; the second-order rate constant (k₁) is 3.0 · 10⁷ M^?1 · s^?1 at low ionic strength (I = 223 mM, 10°C). The value of this rate constant decreases to 1.8 · 10⁵ M^?1 · s^?1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c₁ and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c₁ and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c₁ and ferricytochrome c and the reverse reaction are k₁ = 2.4 · 10⁵ M^?1 · s^?1 and k_?1 = 3.3 · 10⁵ M^?1 · s^?1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10°C). The ‘equilibrium’ constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c²⁺₁ + cytochrome c³⁺ai cytochrome c³⁺₁ + cytochrome c²⁺.     

Thromboxane A2 and prostaglandin H2: Potent stimulators of the swine coronary artery

Thromboxane A₂ was generated by incubation of arachidonic acid with a suspension of human platelets. The filtrate contained 266 ± 46 ng/ml (n=10) of thromboxane A₂ and 25 ng/ml or less of prostaglandin endoperoxides (prostaglandins G₂+H₂). Thromboxane A₂ was 2–10 times more potent than prostaglandin H₂ and 9–102 times and 26–308 times more potent than prostaglandins E₂ and F₂α, respectively, in causing contractions of the superfused swine coronary artery.     

Kinetics of nucleotide binding to chloroplast coupling factor (CF1)

Studies of the kinetics of association and dissociation of the formycin nucleotides FTP and FDP with CF₁ were carried out using the enhancement of formycin fluorescence. The protein used, derived from lettuce chloroplasts by chloroform induced release, contains only 4 types of subunit and has a molecular weight of 280 000.In the presence of 1.25 mM MgCl₂, 1 mol of ATP or FTP is bound to the latent enzyme, with K_d = 10^?7 or 2 · 10^?7, respectively. The fluorescence emission (λ_max 340 nm) of FTP is enhanced 3-fold upon binding, and polarization of fluorescence is markedly increased. The fluorescence changes have been used to follow FTP binding, which behaves as a bimolecular process with K₁ = 2.4 · 10⁴ M^?1 · s^?1. FTP is displaced by ATP in a process apparently involving unimolecular dissociation of FTP with k_?1 = 3 · 10^?3 s^?1. The ratio of rates is comparable to the equilibrium constant and no additional steps have been observed.The protein has 3 sites for ADP binding. Rates of ADP binding are similar in magnitude to those for FTP. ADP and ATP sites are at least partly competitive with one another.The kinetics of nucleotide binding are strikingly altered upon activation of the protein as an ATPase. The rate of FTP binding increases to at least 10⁶ M^?1 · s^?1. This suggests that activation involves lowering of the kinetic barriers to substrate and product binding-dissociation and has implications for the mechanism of energy transduction in photophosphorylation.     

Studies on the effects of prostaglandins E1, E2, A1, and A2 on the ductus arteriosus of swine in vivo using cineangiography

Prostaglandins E₁, E₂, A₁, and A₂ have been shown by cineaortography to open and dilate the ductus arteriosus in anesthetised piglets 3 to 6 hours of age. The dosage of PGEs required was 1 to 4 μg/kg/min. and of PGAs 20 to 40 μg/kg/min. The effect of PGEs faded within 20 minutes of stopping infusion but the effect of PGAs was still evident up to 45 minutes after stopping the infusion. Little effect was noted from hypoxia or from the addition of indomethacin to prostaglandin infusion. Side effects were not troublesome with the dosage employed but hypotension and apnea sometimes occurred at the onset of PGE infusions.