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1.
Enhancement of Stimulation-Evoked Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells by Forskolin 总被引:7,自引:9,他引:7
Katsuya Morita Toshihiro Dohi Shigeo Kitayama Yutaka Koyama Akira Tsujimoto 《Journal of neurochemistry》1987,48(1):243-247
Acetylcholine (ACh) increased cyclic AMP levels in cultured bovine chromaffin cells with a peak effect at 1 min after the addition. Pretreatment with forskolin (0.3 microM) enhanced the ACh-evoked cyclic AMP increase. The catecholamine (CA) release induced by ACh was enhanced by forskolin, but forskolin alone did not enhance the CA release. The effect of forskolin increased dose-dependently up to 1 microM, but decreased at higher concentrations. Dibutyryl cyclic AMP (DBcAMP) also enhanced ACh-evoked CA release, but the effect was less potent than that of forskolin. Forskolin enhanced both [3H]norepinephrine ([3H]NE) and endogenous CA release evoked by 30 mM K+ from cells that were preloaded with [3H]NE. The effects of forskolin were substantial when CA release was evoked with low concentrations of ACh or excess K+, but decreased with higher concentrations of the stimulants. Forskolin also enhanced the CA release induced by ionomycin and veratrine, or by caffeine in Ca2+-free medium. The potentiation by forskolin of the ACh-evoked CA release was manifest in low Ca2+ concentrations in the medium, but decreased when Ca2+ concentration was increased. These results suggest that cyclic AMP may play a role in the modulation of CA release from chromaffin cells. 相似文献
2.
A system to discriminate the real-time dynamics of the secretory function in cultured adrenal chromaffin cells, using a cell bed perfusion technique and an amperometric detector, was established. Examination of basal conditions revealed that the electrode potential and flow rate are crucial factors for monitoring precise dynamics of the secretory process. Stimulation of the cells either with acetylcholine (ACh) or with high K+ concentration caused a transient current response. The current responses showed concentration dependence for both stimuli, and also showed a high correlation with the amount of catecholamines (CA) in the respective peak fraction of perfusate. Either prolonged cholinergic stimulation or maintained depolarization produced a transient response, which is not attributable to a depletion of releasable storage of CA as indicated by double-stimulation experiments. Stimulation with high K+ concentration evoked an additional release of CA even after the cellular response to prolonged ACh was inactivated, whereas maintained depolarization with high K+ produced both facilitatory and inhibitory effects on the cell responsiveness to ACh. Most probably the transient natures of the secretory responses to ACh and to high K+ are mediated by different mechanisms. All the results suggest that the direct monitoring is profitable for studies on the regulatory mechanisms of the secretory function. 相似文献
3.
Ca2+ binding to chromaffin vesicle matrix proteins: effect of pH, Mg2+, and ionic strength 总被引:5,自引:0,他引:5
Recently we found that Ca2+ within chromaffin vesicles is largely bound [Bulenda, D., & Gratzl, M. (1985) Biochemistry 24, 7760-7765]. In order to explore the nature of these bonds, we analyzed the binding of Ca2+ to the vesicle matrix proteins as well as to ATP, the main nucleotide present in these vesicles. The dissociation constant at pH 7 is 50 microM (number of binding sites, n = 180 nmol/mg of protein) for Ca2+-protein bonds and 15 microM (n = 0.8 mumol/mumol) for Ca2+-ATP bonds. When the pH is decreased to more physiological values (pH 6), the number of binding sites remains the same. However, the affinity of Ca2+ for the proteins decreases much less than its affinity for ATP (dissociation constant of 90 vs. 70 microM). At pH 6 monovalent cations (30-50 mM) as well as Mg2+ (0.1-0.5 mM), which are also present within chromaffin vesicles, do not affect the number of binding sites for Ca2+ but cause a decrease in the affinity of Ca2+ for both proteins and ATP. For Ca2+ binding to ATP in the presence of 0.5 mM Mg2+ we found a dissociation constant of 340 microM and after addition of 35 mM K+ a dissociation constant of 170 microM. Ca2+ binding to the chromaffin vesicle matrix proteins in the presence of 0.5 mM Mg2+ is characterized by a Kd of 240 microM and after addition of 15 mM Na+ by a Kd of 340 microM.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
Desensitization to Nicotinic Cholinergic Agonists and K+ , Agents That Stimulate Catecholamine Secretion, in Isolated Adrenal Chromaffin Cells 总被引:4,自引:0,他引:4
Desensitization of catecholamine (CA) release from cultured bovine adrenal chromaffin cells was studied to characterize the phenomenon of desensitization and to attempt an elucidation of the mechanism(s) involved in this phenomenon at the level of the isolated chromaffin cell. Prior exposure of chromaffin cells to nicotinic cholinergic agonists [acetylcholine (ACh) or nicotine] caused a subsequent depression or desensitization of CA release during restimulation of the cells with the same agonists. Rates of development of and recovery from nicotinic desensitization were in the minute time range and the magnitude of nicotinic desensitization of CA release was greater at 37 degrees C than at 23 degrees C. ACh- (or nicotine)-induced desensitization was shown to be the result of two processes: (1) a Ca2+-dependent component of desensitization, possibly due to a depletion of intracellular CA stores and (2) a Ca2+-independent, depletion-independent component of desensitization. Prior exposure of cultured chromaffin cells to an elevated concentration of K+ also resulted in desensitization of K+-induced CA release in these cells. K+-induced desensitization was completely Ca2+-dependent and was shown to be the result, at least in part, of a mechanism that is independent of depletion of CA stores. 相似文献
5.
Adrenal chromaffin cells secrete catecholamines (CA) and ATP in response to acetylcholine (ACh) and high [K+]o. The release process is relatively fast making it difficult to measure the early phase of the secretory response. Recently we were able to resolve the time course of the secretory response by measuring the release of ATP using luciferin-luciferase included in the extracellular medium. For the three secretagogues studied, ACh, nicotine and high [K+]o, the early phase of release followed a complex kinetics. Allowing for an initial delay of the secretory response, the kinetics could be described as the sum of two power exponential processes. Increasing the temperature from 23 to 37 degrees C induced a marked decrease in the two time constants needed to fit the early time course of the ATP secretion. The activation energies, estimated from Arrhenius plots, were approx. 20 and 16 kcal/mol for both phases of ATP release induced by either cholinergic agonists or high [K+]o. These results suggest that cholinergic receptor activation and membrane depolarization induce ATP (and CA) secretion through a common pathway. The initial delay in the onset of the secretory response decreased with increasing doses of secretagogue and with temperature. We propose that the delay preceding the actual onset of ATP release represents the time required for generation of intracellular second messengers. The effective concentration attained by these messengers depend apparently on both receptor occupancy by the agonist and the ensuing Ca2+ channel activation. 相似文献
6.
The effect of 55 mM K+ and nicotine on intracellular free calcium was monitored in bovine adrenal chromaffin cells microinjected with aequorin. In contrast to results with quin 2, which suggested that stimulation of chromaffin cells resulted in sustained rises in free calcium, aequorin measurements showed that 55 mM K+ and nicotine resulted in a transient (60-90 s) elevation of free calcium. The peak free calcium and duration of the transient elicited by nicotine were dose-dependent. The concentration of nicotine (10 microM) giving a maximal secretory response gave a peak rise in free calcium of up to 1 microM. 55 mM K+ which only releases 30% of the catecholamine released by 10 microM nicotine generated a calcium transient indistinguishable from that due to 10 microM nicotine. These results support the idea that nicotine agonists generate an alternative second messenger in addition to the rise in free calcium. 相似文献
7.
Previously, we proposed the following reaction machanism for the transport ATPase (EC 3.6.1.3) reaction in the presence of high concentrations of Mg2+ and Na+:(see article). Some kinetic and thermodynamic properties of steps 3 and 4 were investigated, and the following results were obtained. 1. When the reaction was started by adding ATP to the enzyme in the presence of 50 mM Na+ and 0.5 mM K+ or in the presence of 50mM Na+ and 0.5mM Rb+, the amount of E ADP P increased with time and maintained a constant level after reaching a maximum. We could not observe the initial burst of EP formation, which was observed by Post er al. in the presence of 8 mM Na+ and 0.01 mM Rb+. 2. The existence of quasi-equilibrium between E2ATP and E ADP P in the presence of low concentrations of Na+ was suggested by the fact that the values of the reciprocal of the equilibrium constant, K3 of step 3 obtained by the following three methods were almost the same. a) The value of 1+K3 was estimated from the ratio of vo/[EP] to kd, where vo is the rate of ATP hydrolysis in the steady state, [EP] the concentration of EP, and kd the first-order rate constant of EP disappearance after stopping EP formation. b) This value was also calculated from the ratio of the amount of P1 liberated to that of decrease in EP after stopping EP formation. c) The value of K3 was also calculated from the initial rapid decrease in EP on adding K+ and EDTA, assuming that the rapid decrease was due to a shift of the equilibrium toward E2ATP on adding K+. For example, the value of K3 with 10mM NaCL and 0.5mM KCL was 7--11. Although ATP formation due to a shift of the equilibrium toward E2ATP by a K+ jump in the presence of a low concentration of Na+ was observed at 0 degrees, the amount of ATP formed by a K+ jump at 15 degrees was less than the value expected from the shift of the equilibrium. 3. The values of delta H degrees and delta S degrees of step 3 were estimated in the presence of a sufficient amount of Na+ and in the absence of K+. They were +4--+5 kcal mole minus 1 and +15--+16 entropy units mole minus1, respectively. On the basis of kinetic studies of the elementary steps and the overall reaction of Na+-K+-dependent ATPase [EC 3.6.1.3], we (1--4) showed that a phosphorylated intermediate, EP, is formed via two kinds of enzyme-substrate complex, E1ATP and E2ATP, that the EP is in K+-dependent quasi-equilibrium with E2ATP, and that in the presence of high concentration of Mg2+, EP is in a high-energy state and contains bound ADP, E ADP P.(see article). 相似文献
8.
1. The ATP sites. Homotropic interactions between ATP sites have been studied in a very large range of Na+ and K+ concentrations. The ( Na+, K+)-activated ATPase displays Michaelis-Menten kinetics for ATP under standard concentration conditions of Na+ (100 mM) and K+ (10 mM). The steady-state kinetics behavior changes at very low concentrations of K+ where negative cooperativity is observed. The existence of a high affinity and a low affinity site for ATP was clearly demonstrated from the study of the ATP stimulated hydrolysis of p-nitrophenylphosphate in the presence of Na+ and K+. The ratio of apparent affinities of high and low affinity sites for ATP is 86 at pH 7.5. 2. The Na+ sites. The binding of Na+ to its specific stimulatory sites (internal sites) is characterized by positive cooperativity with a Hill coefficient n(H(Na+))=2.0. Homotropic interactions between Na+ sites are unaffected by variations of the K+ concentration. 3. The K+ sites. (a) Binding of K+ to the (external) stimulatory site of the ATPase has been analyzed by following the (Na+, K+)-ATPase activity as well as the p-nitrophenylphosphatase activity in the presence of Na+ and K+ (with or without ATP). Binding is characterized by a Hill coefficient of 1.0 and a K(0.5(K+))=0.1 to 0.8 mM. The absence of positive or negative cooperativity persists between 5 mM and 100 mM Na+. (b) The analysis of the p-nitrophenylphosphatase or of the 2, 4 dinitrophenylphosphatase activity in the presence of K+ alone indicates the existence of low affinity sites for K+ with positive homotropic interactions. The characteristics of stimulation in that case are, K(0.5)=5 mM, n(H)=1.9. The properties of this family of site(s) are the following: firstly, saturation of the low affinity site(s) by K+ prevents ATP binding to its high affinity internal site. Secondly, saturation of the low affinity sites for K+ prevents binding of Na+ to its internal sites. Thirdly, this family of sites disappears in the presence of ATP, p-nitrophenylphosphate or of both substrates, when Na+ binds to its internal sites. Na+ binding to its specific stimulatory sites provokes the formation of the high affinity type of site for K+. 4. Mg2+ stimulation of the (Na+, K+)-ATPase is characterized by a Hill coefficient n(H(Mg2+))=1.0 and a K(0.5(Mg2+))=1 mM stimulation is essentially a V effect. Heterotropic effects between binding of Mg2+ and substrate to their respective sites are small. Heterotropic interactions between the Ms2+, Na+ and K+ sites are also small. 5. The fluidity of membrane lipids also controls the (Na+, K+)-ATPase activity. Phase transitions or separations in the membrane hardly affect recognition properties of substrates, Na+, K+ and Mg2+ for their respective sites on both sides of the membrane. Only the rate of the catalytic transformation is affected. 相似文献
9.
We used an intracellular fluorescent probe, FURA-2M, to examine the responses of isolated rat chromaffin cells to applications of 1 mM acetylcholine (ACh). Our data showed two different populations of the cell responses to such stimulation. Responses of the first type demonstrated fast rise and decay phases of the Ca2+ transients and no significant decrease in their amplitude during repetitive stimulation of the cell with ACh. Cell responses of the second type showed remarkably slower rise and decay phases of the Ca2+ transients and a noticeable drop of the cell responses during repetitive ACh stimulation that could be recovered after KCl depolarization. We find no significant differences in the amplitudes of the transients in these two populations of the cells. We conclude that there is heterogeneity of the chromaffin cells according to their ACh receptors: the first subpopulation predominantly expresses ionotropic (nicotinic) receptors (n cells), whereas the second cell population has mainly metabotropic (muscarinic) ones (m cells), which are associated with Ca2+ release from the intracellular stores. 相似文献
10.
Showdomycin inhibited pig brain (Na+ + K+)-ATPase with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na+, K+, Mg2+ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1) Na+ + Mg2+ + ATP, (2) Mg2+, Mg2+ + K+, K+ and none, (3) Na+ + Mg2+, Na+, K+ + Na+ and Na+ + K+ + Mg2+, (4) Mg2+ + K+ + ATP, K+ + ATP and Mg2+ + ATP, (5) K+ + Na + + ATP, Na+ + ATP, Na+ + K+ + Mg2+ + ATP and ATP. The highest rate was obtained in the presence of Na+, Mg2+ and ATP. The apparent concentrations of Na+, Mg2+ and ATP for half-maximum stimulation of inhibition (KS0.5) were 3 mM, 0.13 mM and 4 MicroM, respectively. The rate was unchanged upon further increase in Na+ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E1, E2, ES, E1-P and E2-P, i. e., E2 has higher reactivity with showdomycin than E1, while E2-P has almost the same reactivity as E1-P. We conclude that the reaction of (Na+ + K+)- ATPase proceeds via at least four kinds of enzyme form (E1, E2, E1 . nucleotide and EP), which all have different conformations. 相似文献
11.
K+-induced alterations in airway muscle responsiveness to electrical field stimulation 总被引:1,自引:0,他引:1
We investigated possible pre- and postsynaptic effects of K+-induced depolarization on ferret tracheal smooth muscle (TSM) responsiveness to cholinergic stimulation. To assess electromechanical activity, cell membrane potential (Em) and tension (Tm) were simultaneously recorded in buffer containing 6, 12, 18, or 24 mM K+ before and after electrical field stimulation (EFS) or exogenous acetylcholine (ACh). In 6 mM K+, Em was -58.1 +/- 1.0 mV (mean +/- SE). In 12 mM K+, Em was depolarized to -52.3 +/- 0.9 mV, basal Tm did not change, and both excitatory junctional potentials and contractile responses to EFS at short stimulus duration were larger than in 6 mM K+. No such potentiation occurred at a higher K+, although resting Em and Tm increased progressively above 12 mM K+. The sensitivity of ferret TSM to exogenous ACh appeared unaffected by K+. To determine whether the hyperresponsiveness in 12 mM K+ was due, in part, to augmented ACh release from intramural airway nerves, experiments were done using TSM preparations incubated with [3H]choline to measure [3H]ACh release at rest and during EFS. Although resting [3H]ACh release increased progressively in higher K+, release evoked by EFS was maximal in 12 mM K+ and declined in higher concentrations. We conclude that small elevations in the extracellular K+ concentration augment responsiveness of the airways, by increasing the release of ACh both at rest and during EFS from intramural cholinergic nerve terminals. Larger increases in K+ appear to be inhibitory, possibly due to voltage-dependent effects that occur both pre- and postsynaptically. 相似文献
12.
Temperature sensitivity of catecholamine secretion and ion fluxes in bovine adrenal chromaffin cells
The effects of temperature on ion fluxes and catecholamine secretion that are mediated by nicotinic acetylcholine receptors (nAChRs), voltage-sensitive calcium channels (VSCCs), and voltage-sensitive sodium channels (VSSCs) were investigated using bovine adrenal chromaffin cells. When the chromaffin cells were stimulated with DMPP, a nicotinic cholinergic agonist, or 50 mM K+, the intracellular calcium ([Ca2+]i) elevation reached a peak and decreased more slowly at lower temperatures. The DMPP-induced responses were more sensitive to temperature changes compared to high K+-induced ones. In the measurement of intracellular sodium concentrations ([Na+]i), it was found that nicotinic stimulation required a longer time to attain the maximal level of [Na+]i at lower temperatures. In addition, the VSSCs-mediated [Na+]i increase evoked by veratridine was also reduced as the temperature decreased. The measurement of [3H]norepinephrine (NE) secretion showed that the secretion within the first 3 min evoked by DMPP or high K+ was greatest at 37 degrees C. However, at 25 degrees C, the secretion evoked by DMPP, but not that by the 50 mM K+, was greater after 10 min of stimulation. This data suggest that temperature differentially affects the activity of nAChRs, VSCCs, and VSSCs, resulting in differential [Na+]i and [Ca2+]i elevation, and in the [3H]NE secretion by adrenal chromaffin cells. 相似文献
13.
《Neurochemistry international》1988,13(2):265-270
Effect of benzodiazepines on evoked catecholamine (CA) release from a primary culture of bovine adrenal medullary cells was investigated. Midazolam at high doses (> 10 μ M) inhibited CA release evoked by acetylcholine (ACh), excess K+ and veratridine but not by A23187 or caffeine in Ca2+ -free media. Other benzodiazepines, diazepam, clonazepam, nitrazepam and R05-4864, as well as 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195) and ethyl-β-carboline-3-carboxylate (βCCE) also inhibited ACh-evoked CA release but only at high concentrations. The inhibitory effect of midazolam on ACh-evoked CA release was not affected by R015-1788, a central-type benzodiazepine receptor antagonist which itself had no effect on basal and ACh-evoked CA release. Facilitatory action of Bay K 8644 on CA release evoked by 20 mM K+ was reduced by midazolam, PK11195 and R05-4864. Further, ACh-evoked 45Ca uptake was markedly reduced by midazolam and R05-4864 in association with the inhibition of CA release. These results suggest that benzodiazepines at high doses, inhibit the evoked CA release from adrenal chromaffin cells possibly through the blockade of Ca2+ influx. Possible involvement of receptor subtypes of benzodiazepines in regulating CA secretion is discussed. 相似文献
14.
Partial characterization of the ouabain-insensitive, Na+-stimulated ATPase activity of kidney basal-lateral plasma membranes 总被引:1,自引:0,他引:1
The present paper characterizes the Na+-stimulated ATPase activity present in basal-lateral plasma membranes from guinea-pig kidney proximal tubular cells. These characteristics are compared with those of the (Na+ + K+)-stimulated ATPase activity, and they are: (A) Na+-ATPase activity: (1) requires Mg2+; (2) may be activated by mu molar quantities of Ca2+; (3) optimal ratio Mg:ATP = 5:1-2 and Ka for Mg:ATP = 3:0.60 mM; (4) Ka for Na+:8 mM; (5) does not require K+; (6) is only stimulated by Na+ and Li+ (in a lower extent); (7) is similarly stimulated by the Na+ salt of different anions; (8) hydrolyzes only ATP; (9) optimal temperature: 47 degrees C; (10) optimal pH: 6.9; (11) is ouabain insensitive; (12) is totally inhibited by 1.5 mM ethacrynic acid, 2 mM furosemide and 0.75 mM triflocin. (B) (Na+ + K+)-ATPase activity: (1) also requires Mg2+; (2) is inhibited by Ca2+; (3) optimal ratio Mg:ATP = 1.25:1 and Ka for Mg:ATP = 0.50: 0.40 mM; (4) Ka for Na+: 14 mM (data not shown); (5) needs K+ together with Na+; (6) K+ may be substituted by: Rb+ greater than NH+4 greater than Cs+; (7) is anion insensitive; (8) hydrolyzes mostly ATP and to a lesser extent GTP, ITP, UTP, ADP, CTP; (9) optimal temperature: 52 degrees C; (10) optimal pH: 7.2; (11) 100% inhibited by 1 mM ouabain; (12) 63% inhibited by 1.5 mM ethacrynic acid, 10% inhibited by 2 mM furosemide and insensitive to 0.75 mM triflocin. 相似文献
15.
The release of endogenous vasoactive intestinal polypeptide (VIP) from enteric nerves of isolated rat ileum and the role of extracellular calcium on the release mechanism have been investigated. Evaluation of simultaneous release of endogenous acetylcholine (ACh) and adenosine 5'-triphosphate (ATP) from enteric nerves was used to establish the reliability of the technique. Electrical field stimulation of the ileal preparation induced an increase in the release of endogenous ACh, ATP and VIP. The evoked, but not the basal, release of these substances was blocked by tetrodotoxin (TTX), indicating that the release was a result of nerve stimulation. However, while increase in release of ACh and ATP during nerve stimulation was suppressed in Ca2+-free medium and by the addition of the Ca2+ channel blocker cadmium, nerve-mediated VIP release was unaffected. Further, while K+-depolarization induced release of ACh and ATP from the ileal preparations, it did not lead to an increase in the release of VIP. These results demonstrate that, unlike ACh and ATP release, release of endogenous VIP from enteric nerves is independent of extracellular calcium. The implications of these results in terms of the mechanism of transmitter release in the gastrointestinal tract are discussed. 相似文献
16.
Manganese (2.2mM) blocked catecholamine (CA) secretion evoked by acetylcholine (ACh) in perfused bovine adrenals. This effect was reverted when the concentration of Mn2+ was increased to 6.6mM. Similar results were observed when higher concentrations (11 and 22mM respectively) were used. Mn2+ substituted for Ca2+ in the ACh evoked CA secretion, and this response was concentration dependent. The removal of Mn2+ from the perfusion medium potentiated the secretory response with respect to the first ACh stimulation. The subcellular distribution of Mn2+ in perfused adrenals showed a poor association with storage granules. It is concluded that Mn2+ inhibits Ca2+ entry during secretion and also substitutes for Ca2+ in the excitation-secretion coupling. 相似文献
17.
Inhibition of (Na+ + K+)-dependent adenosine triphosphatase phosphatase by vanadate is thought to occur through the tight binding of vanadate to the same site from which Pi is released. To see if ATP binds to [48V] vanadate-enzyme complex, just as it does to the phosphoenzyme, the effects of Na+, K+, and ATP on the dissociation rate of the complex at 10 degrees C were studied. The rate constant was increased by Na+, and this increase was blocked by K+, indicating that either Na+ or K+ binds to the complex. ATP alone, or in combination with K+, had no effect on the rate constant. In the presence of Na+, however, ATP caused a further increase in the rate constant. The value of K0.5 of Na+ was the same in the presence or absence of ATP; K0.5 of ATP (0.2 mM) did not seem to change significantly when Na+ concentration was varied, and K0.5 of K+, at a constant Na+ concentration, was the same in the presence or absence of ATP. The data indicate that ATP binds to the enzyme-vanadate complex regardless of the presence or absence of Na+ or K+, but it affects the dissociation rate only when Na+ is bound simultaneously. The value of K0.5 of Na+ decreased as pH was increased in the range of 6.5-7.8, but K0.5 of ATP was independent of pH. Demonstration of ATP binding to the enzyme-vanadate complex provides further support for the suggestion that the oligomeric enzyme contains a low-affinity regulatory site for ATP that is distinct from the interacting high-affinity catalytic sites. 相似文献
18.
The effects of monovalent ions on endogenous pyruvate dehydrogenase (PDH) kinase activity in purified bovine heart pyruvate dehydrogenase complex were investigated. Activity of PDH kinase was stimulated 1.9-, 1.95-, 1.65-, and 1.4-fold by 10 mM K+, Rb+, NH+4, and Cs+, respectively, whereas Na+ and Li+ had no effect on PDH kinase activity. The crystal radii of stimulatory ions were in the range of 1.33 to 1.69 A while the crystal radii of nonstimulatory ions were in the range of 0.6 to 0.94 A. Stimulation of PDH kinase by monovalent ions was not pH dependent. Protein dilution studies showed that monovalent ion stimulation was measurable within 10 s after protein addition to PDH kinase assays. Furthermore, stimulation occurred at all protein concentrations tested. At ATP concentrations from 12.5 to 25 microM, K+ and NH+4 stimulation was constant from 0 to 110 and 0 to 30 mM, respectively. At higher ATP concentrations, from 50 to 500 microM, K+ and NH+4 stimulation peaked at approximately 30 and 3 mM, respectively, and thereafter declined as the ion concentration increased. Maximal PDH kinase stimulation by K+ or NH+4 also declined as Na+ was increased from 0 to 120 mM, but at a fixed salt concentration of 120 mM, both K+ and NH+4 stimulated PDH kinase activity. Phosphopeptide analysis demonstrated that K+ and NH+4 stimulated phosphorylation at sites 1 and 2, but that site 3 phosphorylation was relatively constant under all conditions. Thiamin pyrophosphate and 5,5'-dithiobis-(2-nitrobenzoate) blocked monovalent ion stimulation half-maximally at 4 and 6 microM, respectively. However, neither thiamin pyrophosphate nor 5,5'-dithiobis-(2-nitrobenzoate) significantly inhibited PDH kinase activity in the absence of monovalent ions. The results indicate that heart PDH kinase stimulation by monovalent ions does not occur by changing the binding equilibrium between PDH and dihydrolipoyl transacetylase core. Instead, monovalent ions bind and exert their regulatory effects at or near the active site of PDH kinase. 相似文献
19.
Cytosolic adenylate kinase synthesis thiamin triphosphate (TTP) from thiamin diphosphate (TDP) in vitro by a reversible reaction: TDP + ADP Mg2+ in equilibrium TTP + AMP. The backward (TTP----TDP) reaction rate was 3-times faster than the forward (TDP----TTP) reaction rate when all the substrate concentrations were 0.1 mM. This property of TTP-synthesizing activity of the enzyme did not explain the fact that the [TTP]/[TDP] molar ratio determined in chicken white skeletal muscle is 5.0 (Miyoshi, K., Egi, Y., Shioda, T. and Kawasaki, T. (1990) J. Biochem. 108, 267-270). To solve this problem, we have studied the properties of TTP-synthesizing activity of the purified recombinant chicken cytosolic adenylate kinase preparation and the effect of adenine nucleotides, especially of ATP. The backward reaction of the TTP synthesis did not proceed in the presence of 8.8 mM ATP, a physiological concentration in chicken white skeletal muscle, while the forward reaction proceeded at a reduced rate. The [TTP]/[TDP] ratio found after a long incubation period was 3.0 and 0.7, respectively, in the presence and absence of 8.8 mM ATP. These results indicate that the high [TTP]/[TDP] molar ratio found in chicken white muscle was demonstrated in vitro by the purified chicken cytosolic adenylate kinase and support in vivo TTP synthesis by this enzyme. 相似文献
20.
Cells of Bacillus acidocaldarius that were grown with 2.7 mM K+ expressed a low-affinity K+ uptake system. The following observations indicate that its properties closely resemble those of the Escherichia coli Trk and Streptococcus faecalis KtrI systems: (i) the B. acidocaldarius system took up K+ with a Km of 1 mM; (ii) it accepted Rb+ (Km of 6 mM; same Vmax as for K+); (iii) it was still active in the presence of low concentrations of sodium; (iv) the observed accumulation ratio of K+ maintained by metabolizing cells was consistent with K+ being taken up via a K+-H+ symporter; and (v) K+ uptake did not occur in cells in which the ATP level was low. Under the latter conditions, the cells still took up methylammonium ions via a system that was derepressed by growth with low levels of ammonium ions, indicating that in the acidophile ammonium (methylammonium) uptake requires a high transmembrane proton motive force rather than ATP. 相似文献