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1.
Evidence for histidyl and carboxy groups at the active site of the human placental Na+-H+ exchanger. 总被引:1,自引:0,他引:1 下载免费PDF全文
V Ganapathy D F Balkovetz M E Ganapathy V B Mahesh L D Devoe F H Leibach 《The Biochemical journal》1987,245(2):473-477
The Na+-H+ exchanger of the human placental brush-border membrane was inhibited by pretreatment of the membrane vesicles with a histidyl-group-specific reagent, diethyl pyrocarbonate and with a carboxy-group-specific reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. In both cases the inhibition was irreversible and non-competitive in nature. But, if the membrane vesicles were treated with these reagents in the presence of amiloride, cimetidine or clonidine, there was no inhibition. Since amiloride, cimetidine and clonidine all interact with the active site of the exchanger in a mutually exclusive manner, the findings provide evidence for the presence of essential histidyl and carboxy groups at or near the active site of the human placental Na+-H+ exchanger. This conclusion was further substantiated by the findings that Rose Bengal-catalysed photo-oxidation of histidine residues as well as covalent modification of carboxy residues with NN'-dicyclohexylcarbodi-imide irreversibly inhibited the Na+-H+ exchanger and that amiloride protected the exchanger from inhibition caused by NN'-dicyclohexylcarbodi-imide. 相似文献
2.
A proton gradient is the driving force for uphill transport of lactate in human placental brush-border membrane vesicles 总被引:2,自引:0,他引:2
D F Balkovetz F H Leibach V B Mahesh V Ganapathy 《The Journal of biological chemistry》1988,263(27):13823-13830
The characteristics of lactate transport in brush-border membrane vesicles isolated from normal human full-term placentas were investigated. Lactate transport in these vesicles was Na+-independent, but was greatly stimulated when the extravesicular pH was made acidic. In the presence of an inwardly directed H+ gradient ([H+]o greater than [H+]i), transient uphill transport of lactate could be demonstrated. This H+ gradient-dependent stimulation was not a result of a H+ diffusion potential. Transport of lactate in the presence of the H+ gradient was not inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or by furosemide, ruling out the participation of an anion exchanger in placental lactate transport. Many monocarboxylates strongly interacted with the lactate transport system, whereas, with the single exception of succinate, dicarboxylates did not. The monocarboxylates pyruvate and lactate, but not the dicarboxylate succinate, when present inside the vesicles, were able to exert a trans-stimulatory effect on the uptake of radiolabeled lactate. Kinetic analyses provided evidence for a single transport system with a Kt of 4.1 +/- 0.4 mM for lactate and a Vmax of 54.2 +/- 9.9 nmol/mg of protein/30 s. Pyruvate inhibited lactate transport competitively, by reducing the affinity of the system for lactate without altering the maximal velocity. It is concluded that human placental brush-border membranes possess a transport system specific for lactate and other monocarboxylates and that this transport system is Na+-independent and is energized by an inwardly directed H+ gradient. Lactate-H+ symport rather than lactate-OH- antiport appears to be the mechanism of the H+ gradient-dependent lactate transport in these membranes. 相似文献
3.
P. S. Ganapathy 《Biologia Plantarum》1969,11(2):165-174
Floral buds ofBrowallia demissa, at three stages of development, were cultured on Nitsch and Nitsch basal medium. The supplements used include IAA; several cytokinins— benzyladenine, kinetin and 6-benzyl-9 tetrahydropyran-adenine (SD 8339); gibberellic acid (GA3); 2, 3, 5-triiodobenzoic acid (TIBA); arginine and cysteine. All three stages of floral buds failed to complete development. In some treatments stages II and III produced callus and/or roots from the morphological basal end. Cytokinins promoted bud formation whereas both IAA and GA3 depressed bud formation The shoots differentiatedin vitro were capable of setting flowers, fruits and seeds in all the treatments. The seeds were viable. Comparative studies of development of flowersin vivo andin vitro were made. In some treatments the flowers exhibited abnormal corolla, and roecium and gynoecium. Factors affecting normal bud initiation, organization and development are discussed. 相似文献
4.
5.
Evidence for an imipramine-sensitive serotonin transporter in human placental brush-border membranes 总被引:6,自引:0,他引:6
D F Balkovetz C Tiruppathi F H Leibach V B Mahesh V Ganapathy 《The Journal of biological chemistry》1989,264(4):2195-2198
Serotonin is actively transported into brush-border membrane vesicles isolated from normal human term placentas and an inward-directed NaCl gradient provides the driving force for this process. Uptake is negligible if Na+ is replaced by Li+, K+, Rb+, Cs+ or choline. The presence of Cl- seems necessary for the maximal activity of this Na+-dependent uptake system. Intravesicular K+ (20-40 mM) stimulates serotonin uptake, the stimulation being considerably greater at pH 7.5 than at pH 6.5. But, in the absence of K+, uptake at pH 6.5 was twice the uptake at pH 7.5. Unlabeled serotonin and dopamine inhibit the uptake of radiolabeled serotonin and the IC50 values are 70 nM and 20 microM, respectively. Histamine and 5-hydroxytryptophan do not significantly interact with the system (IC50 greater than 1 mM). Kinetic analysis reveals that serotonin uptake in these vesicles occurs via a single, saturable, high affinity system (Kt = 51 +/- 2 nM; Vmax = 6.4 +/- 0.1 pmol/mg of protein/15 s). The transporter is highly sensitive to inhibition by imipramine (IC50 = 32 nM) and desipramine (IC50 = 160 nM) but relatively insensitive to reserpine and hydralazine. 相似文献
6.
7.
A NaCl-tolerant callus line of Cicer arietinum has been isolated, as a spontaneous variant, on agar-solidified MS-medium supplemented with 100 mM NaCl. The growth of this line, in the presence of 100 mM NaCl, was comparable to that of the NaCl-sensitive callus line growing in the absence of NaCl. Regarding relative tolerance of the two callus lines towards NaCl (0 to 200 mM), the tolerant line performed poorly in the absence of NaCl and exhibited optimal growth at 50 mM NaCl. The tolerance persisted even after three passages of 4-wk each, tested so far, away from the selective agent. 相似文献
8.
Regulation of maize catalase by changing rates of synthesis and degradation. 总被引:3,自引:0,他引:3 下载免费PDF全文
Rates of catalase synthesis and degradation were measured in the scutellum of the germinating maize seedling by the technique of Price, Sterling, Tarantola, Hartley & Rechcigl [J. Biol. Chem. (1963) 237, 3468-3475] by using the porphyrinogenic drug 2-allyl-2-isopropylacetamide to inhibit catalase synthesis. Results indicate that developmental changes in catalase activity are determined by changes in the rate of enzyme degradation as well as by changes in the rates of its synthesis. 相似文献
9.
Methionine sulfoxide is transported into purified intestinal and renal brush border membrane vesicles from rabbit by an Na+-dependent mechanism and is accumulated inside the vesicles against the concentration gradient. Both in intestine and kidney, the rate of transport is enhanced with increasing concentrations of Na+ in the external medium. Increasing the Na+ gradient reduces the apparent Kt for methionine sulfoxide without causing any change in Vmax. With an outward K+ gradient (vesicle > medium), valinomycin stimulates the Na+-gradient-dependent transport of methionine sulfoxide in the kidney, showing the electrogenicity of the transport process. A number of amino acids inhibit methionine sulfoxide transport in both the intestine and kidney. An enzymatic activity capable of reducing methionine sulfoxide to methionine is present in the intestinal mucosa, renal cortex and liver. The activity is highest in renal cortex and lowest in intestine. The methionine sulfoxide-reducing activity is stimulated by NADH, NADPH, glutathione and dithiothreitol and the potency of the stimulation is in the order: dithiothreitol > NADPH > glutathione > NADH. 相似文献
10.
P D Prasad F H Leibach V B Mahesh V Ganapathy 《The Journal of biological chemistry》1992,267(33):23632-23639
The interaction of 5-(N-methyl-N-isobutyl)amiloride (MIBA) with brush-border membrane vesicles isolated from normal human term placentas was investigated using two parameters: binding and transport. The binding of MIBA to placental membranes was specific and temperature- and pH-dependent, and the apparent dissociation constant (Kd) for the process was 58 +/- 2 microM. The binding was inhibited by other amiloride analogs and also by clonidine and cimetidine with a rank order potency: MIBA > benzamil > dimethylamiloride > amiloride > clonidine > cimetidine. These compounds also inhibited Na(+)-H+ exchanger activity in these membrane vesicles, but with a different order of potency: dimethylamiloride > MIBA > amiloride > benzamil > cimetidine > clonidine. The membrane vesicles were also able to transport MIBA into the intravesicular space, and the transport was stimulated many-fold by the presence of an outwardly directed H+ gradient across the membrane. The H+ gradient was the driving force for uphill accumulation of MIBA inside the vesicles. The transport process was electrically silent. The transport of MIBA was inhibited by other amiloride analogs and by clonidine and cimetidine, and the order of potency was the same as the order with which these compounds inhibited the binding of MIBA. The Michaelis-Menten constant (Kt) for the transport process was 46 +/- 2 microM. The binding as well as the transport were also inhibited by Na+ and Li+. Interestingly, tetraethylammonium and N1-methylnicotinamide, two of the commonly used substrates in organic cation transport studies, failed to inhibit the binding and transport of MIBA. Furthermore, although the outwardly directed H+ gradient-dependent uphill transport of tetraethylammonium could be demonstrated in renal brush-border membrane vesicles, there was no evidence for the presence of a transport system for this prototypical organic cation in placental brush-border membrane vesicles. It is concluded that the human placental brush-border membranes possess an organic cation-proton antiporter which accepts MIBA as a substrate, the low affinity binding site for MIBA observed in these membranes represents this antiporter, and that the placental organic cation-proton antiporter is distinct from the widely studied renal organic cation-proton antiporter. 相似文献