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21.
Giorgio M. Hanozet Barbara Giordana Paolo Parenti Andrea Guerritore 《The Journal of membrane biology》1984,81(3):233-240
Summary In brush border membrane vesicles from the midgut ofPhilosamia cynthia larvae (Lepidoptera) thel- andd-alanine uptake is dependent on a potassium gradient and on transmembrane electrical potential difference. Each isomer inhibits the uptake of the other form: inhibition ofl-alanine uptake byd-alanine is competitive, whereas inhibition ofd-alanine uptake byl-alanine is noncompetitive. Transstimulation experiments as well as the different pattern of specificity to cations suggest the existence of two transport systems. Kinetic parameters for the two transporters have been calculated both when Kout>Kin and Kout=Kin.d-alanine is actively transported also by the whole midgut, but it is not metabolized by the intestinal tissue. 相似文献
22.
Summary DNA sequences homologous to the T-DNA region of the octopine Ti plasmid from Agrobacterium tumefaciens are found in various fast-growing Rhizobium fredii strains. The largest fragment (BamHI fragment 2) at the right-boundary region of the core T-DNA hybridizes to more than one plasmid present in R. fredii. However, one smaller fragment (EcoRI fragment 19a) adjacent to the core T-DNA shows homology only with the plasmid carrying the symbiotic nitrogen-fixation genes (pSym). Hybridization data obtained with digested R. fredii USDA193 pSym DNA suggests that the homology is mainly with two HindIII fragments, 1.7 kb and 8.8 kb in size, of the plasmid. The 1.7 kb HindIII fragment also hybridizes to two regions of the virulence plasmid of A. tumefaciens, pAL1819, a deletion plasmid derived from the octopine Ti plasmid, pTiAch5. Hybridization studies with an insertion element IS66 from A. tumefaciens indicate that the 1.7 kb HindIII fragment of R. fredii plasmid, homologous to the T-DNA and the virulence region of Ti plasmid, is itself an IS66 homologue. 相似文献
23.
Intestinal brush border vesicles of a Mediterranean sea fish (Dicentrarchus labrax) were prepared using the Ca2+-sedimentation method. The transport of glucose, glycine and 2-aminoisobutyric acid is energized by an Na+ gradient (). In addition, amino acid uptake requires Cl? in the extravesicular medium (2-aminoisobutyric acid more than glycine). This Na+- and Cl?-dependent uptake is electrogenic, since it can be stimulated by negative charges inside the vesicles. The specific Cl? requirement of glycine and 2-aminoisobutyric acid transport is markedly influenced by pH, a change from 6.5 to 8.4 reducing the role played by Cl?. In the presence of Cl?, the of 2-aminoisobutyric acid uptake is reduced and its is enhanced. Cl? affects also a non-saturable Na+-dependent component of this amino acid uptake. Amino acid transport is also increased by intravesicular Cl? (2-aminoisobutyric acid less than glycine). This effect is more concerned with glucose uptake, which can be then multiplied by 2.3. A concentration gradient () as well as the presence of Na+ in the incubation medium seems to enter into this requirement. This intravesicular Cl? effect is not influenced by pH between 6.5 and 8.4. 相似文献
24.
Summary We first present two simple dimeric models of cotransport that may account for all of the kinetics of Na++-d-glucose cotransport published so far in the small intestine. Both the sigmoidicity in the Na++ activation of transport (positive cooperativity) and the upward deviations from linearity in the Eadie-Hofstee plots relative to glucose concentrations (negative cooperativity) can be rationalized within the concept of allosteric kinetic mechanisms corresponding to either of two models involving sequential or mixed concerted and sequential conformational changes. Such models also allow for 2 Na++ 1 S and 1 Na++ 1 S stoichiometries of cotransport at low and high substrate concentrations, respectively, and for partial inhibition by inhibitors or substrate analogues. Moreover, it is shown that the dimeric models may present physiological advantages over the seemingly admitted hypothesis of two different cotransporters in that tissue. We next address the reevaluation of Na++-d-glucose cotransport kinetics in rabbit intestinal brush border membrane vesicles using stable membrane preparations, a dynamic approach with the Fast Sampling Rapid Filtration Apparatus (FSRFA), and both nonlinear regression and statistical analyses. Under different conditions of temperatures, Na++ concentrations, and membrane potentials clamped using two different techniques, we demonstrate that our data can be fully accounted for by the presence of only one carrier in rabbit jejunal brush border membranes since transport kinetics relative to glucose concentrations satisfy simple Michaelis-Menten kinetics. Although supporting a monomeric structure of the cotransporter, such a conclusion would conflict with previous kinetic data and more recent studies implying a polymeric structure of the carrier protein. We thus consider a number of alternatives trying to reconcile the observation of Michaelis-Menten kinetics with allosteric mechanisms of cotransport associated with both positive and negative cooperativities for Na++ and glucose binding, respectively. Such models, implying energy storage and release steps through conformational changes associated with ligand binding to an allosteric protein, provide a rational hypothesis to understand the long-time debated question of energy transduction from the Na++ electrochemical gradient to the transporter.This research was supported by grant MT-7607 from the Medical Research Council of Canada. One of the authors (A.B.) was supported by a scholarship from the Fonds de la Recherche en Santé du Québec and C. C. was supported by a fellowship from the GRTM. The technical assistance of Mrs. C. Leroy has been greatly appreciated. The authors also thank D.D. Maenz and C. Malo for insightful discussions and C. Gauthier for the art work. 相似文献
25.
F. Norbis M. Boll G. Stange D. Markovich F. Verrey J. Biber H. Murer 《The Journal of membrane biology》1997,156(1):19-24
In a previous report we documented an increased Na+-dependent transport of inorganic phosphate (P
i
) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch.
422:211–216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved
in this effect. The identified cDNA (provisionally named PiUS; for P
i
-uptake stimulator) lead to a 3-4-fold stimulation of Na+-dependent P
i
-uptake (10ng cRNA injected, 3–5 days of expression). Na+-independent uptake of P
i
was also affected but transport of sulphate and l-arginine (in the presence or absence of sodium) remained unchanged. The apparent K
m
-values for the induced Na+-dependent uptake were 0.26 ± 0.04 mm for P
i
and 14.8 ± 3.0 mm for Na+. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments.
In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of
∼2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and
heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we
have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P
i
-uptake into Xenopus laevis oocytes, but which is not a P
i
-transporter itself.
Received: 31 July 1996/Revised: 16 October 1996 相似文献
26.
L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral
membrane vesicles l-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective.
The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is
saturable with respect to external lactate with a K
m
of 39.2 ± 4.8 mm and a J
max of 8.9 ± 0.7 nmoles mg protein−1 sec−1. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains
a H+-lactate cotransporter, whereas in the apical membrane both H+-lactate and Na+-lactate cotransporters are present, even if they exhibit a low transport rate.
Received: 22 October 1996/Revised: 11 March 1997 相似文献
27.
P W Pappas 《Journal of cellular biochemistry》1984,25(3):131-137
The specific activities of the alkaline phosphatase (APase), type I phosphodiesterase and 5'-nucleotidase activities associated with the brush-border plasma membrane of the tapeworm, Hymenolepis diminuta, decrease significantly as the tapeworm grows and matures. Kinetic analyses of the APase activity associated with membrane preparations from whole 6-, 12-, and 18-d-old H diminuta, and individual pieces of 18-d-old H diminuta cut into ten pieces of equal length, failed to demonstrate qualitative changes in the APase activity. Therefore, the decreased specific activities are apparently due to changes in the ratios of enzymatically active to enzymatically inactive membrane proteins (ie, quantitative changes in the membrane proteins) which occur as the tapeworm grows. 相似文献
28.
Summary The properties of two sodium-dependentd-glucose transporters previously identified in renal proximal tubule brush border membrane (BBM) vesicles are studied. The low-affinity system, found in BBM vesicles from the outer cortex (early proximal tubule), is shown to be associated with the high-affinity phlorizin binding site typically found in renal BBM preparations. The high-affinity system, found in BBM vesicles from the outer medulla (late proximal tubule), is almost two orders of magnitude less sensitive to inhibition by phlorizin and is apparently not associated with high-affinity phlorizin binding. The sodium/g;ucose stoichiometry of the outer medullary transporter is found to be 21 by two independent methods. Previous measurements have established that the stoichiometry of the outer cortical system is 11. It is suggested that this arrangement of transporters in series along the proximal tubule enables the kidney to reabsorb glucose from the urine in an energy-efficient fashion. The bulk of the glucose load is reabsorbed early in the proximal tubule at an energetic cost of one Na+ per glucose molecule. Then in the late proximal tubule a larger coupling ratio and hence a larger driving force is employed to reabsorb the last traces of glucose from the urine. 相似文献
29.
The observed rate of phenylalanine absorption into rat intestinal rings with 0.5 or 5.0 mM phenylalanine is greater than that for absorption of phenylalanine from 0.25 or 2.5 mM Phe-Phe, respectively. With the amino acid phenylalanine, V for absorption is the same whether Na+ is present (149 mM) or absent, but the concentration at which the half-maximal transport rate occurred (Kt) is greater in the absence of Na+. For Phe-Phe, the V decreases in the absence of Na+ whilst Kt is not influenced by the Na+ concentration. The different effect of Na+ on Phe and Phe-Phe transport indicates that the absorptive mechanism for Phe-Phe is different from that for phenylalanine. Absorption of a mixture of [U-14C]Phe-Phe and Phe-[G-3H]Phe showed identical rates of uptake of the carboxyl and amino terminal amino acids.Studies of transport of radioactive maltose showed that the rates of uptake of the reducing and non-reducing glucosyl moieties are identical. Radioactive maltose absorption is not inhibited by glucose oxidase.These results provide evidence that in intestinal epithelium, hydrolysis of Phe-Phe and maltose does not occur on the cell surface with release of the hydrolyzed products to the medium. Rather, hydrolysis and release of the reaction products occur at a point on the cytosol side of a diffusion barrier located in the brush border membrane. 相似文献
30.
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