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1.
Active transport of D-alanine and related amino acids by whole cells of Bacillus subtilis 总被引:4,自引:2,他引:2
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Whole cells of Bacillus subtilis transported d-alanine and l-alanine by two different systems. The high-affinity system (K(m) of 1 muM and V(max) of 0.6 to 0.8 nmol/min per mg of protein) was specific for the two stereoisomers of alanine. The low-affinity system (K(m) of 10 muM for l-alanine and 20 muM for d-alanine and glycine) had a V(max) of 5 to 12 nmol/min per mg of protein. This system transported glycine, d-cycloserine, and d-serine, in addition to d- and l-alanine. Azide inhibited the uptake of these amino acids and caused the efflux of d-alanine from preloaded cells. These data suggest that transport of these amino acids is energized by the electron transport chain. 相似文献
2.
d-Glucose decreases phosphate reabsorption in rat proximal tubule. It is also postulated that some amino acids interact with phosphate reabsorption. To investigate the mechanism of these interactions, phosphate, d-glucose and l-alanine transport kinetics were measured in brush border membrane vesicles isolated from superficial rat kidney cortex by the calcium precipitation technique. At pH 7.4, Na+-dependent phosphate transport was inhibited in the presence of either d-glucose (39 mM) or l-alanine (2.4 mM). In this model, with d-glucose or with l-alanine the value of the phosphate uptake was decreased, whereas the apparent for the phosphate uptake was not affected. However, some inhibition of phosphate transport was observed in the presence of l-glucose, d-alanine or d-glucose after phlorizin preincubation. A 30% Na+-dependent l-alanine (0.1 mM) transport inhibition was observed in the presence of 5 mM phosphate. d-Glucose (1 mM) was also inhibited by 20% when 5 mM phosphate was added to incubation medium. According to several authors, in our model, d-glucose decreased the l-alanine transport and vice versa. Moreover, when the membrane potential was abolished, a clear inhibition of d-glucose by l-alanine persisted. These multiple interactions could be explained by the accelerated dissipation of the Na+ gradient insofar as the rate of the Na+ uptake was increased with d-glucose, l-alanine or phosphate and since the absence of variations in membrane potential did not suppress these inhibitions. 相似文献
3.
The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium. 相似文献
4.
Summary Chloroquine is an antimalarial and antirheumatic lysosomotropic drug which inhibits taurine uptake into and increases efflux from cultured human lymphoblastoid cells. It inhibits taurine uptake by rat lung slices and affects the uptake and release of cystine from cystinotic fibroblasts. Speculations on its mode of action include a proton gradient effect, a non-specific alteration in membrane integrity, and membrane stabilization. In this study, the effect of chloroquine on the uptake of several amino acids by rat renal brush border membrane vesicles (BBMV) was examined. Chloroquine significantly inhibited the secondary active, NaCl-dependent component of 10µM taurine uptake at all concentrations tested, but did not change equilibrium values. Analysis of these data indicated that the inhibition was non-competitive. Taurine uptake was reduced at all osmolarities tested, but inhibition was greatest at the lowest osmolarity. Taurine efflux was not affected by chloroquine, nor was the NaCl-independent diffusional component of taurine transport. Chloroquine (1 mM) inhibited uptake of the imino acids L-proline and glycine, and the dibasic amino acid L-lysine. It inhibited the uptake of D-glucose, but not the neutral-amino acids L-alanine or L-methionine. Uptake of the dicarboxylic amino acids, L-glutamic acid and L-aspartic acid, was slightly enhanced. With regard to amino acid uptake by BBMV, these findings may support some of the currently proposed mechanisms of the action of chloroquine but further studies are indicated to determine why it affects the initial rate of active amino acid transport. 相似文献
5.
Marc R. Hammerman 《生物化学与生物物理学报:生物膜》1982,685(1):71-77
Na+-independent l-arginine uptake was studied in rabbit renal brush border membrane vesicles. The finding that steady-state uptake of l-arginine decreased with increasing extravesicular osmolality and the demonstration of accelerative exchange diffusion after preincubation of vesicles with l-arginine, but not d-arginine, indicated that the uptake of l-arginine in brush border vesicles was reflective of carrier-mediated transport into an intravesicular space. Accelerative exchange diffusion of l-arginine was demonstrated in vesicles preincubated with l-lysine and l-ornithine, but not l-alanine or l-proline, suggesting the presence of a dibasic amino acid transporter in the renal brush border membrane. Partial saturation of initial rates of l-arginine transport was found with extravesicular [arginine] varied from 0.005 to 1.0 mM. l-Arginine uptake was inhibited by extravesicular dibasic amino acids unlike the Na+-independent uptake of l-alanine, l-glutamate, glycine or l-proline in the presence of extravesicular amino acids of similar structure. l-Arginine uptake was increased by the imposition of an H+ gradient (intravesicular pH<extravesicular pH) and H+ gradient stimulated uptake was further increased by FCCP. These findings demonstrate membrane-potential-sensitive, Na+-independent transport of l-arginine in brush border membrane vesicles which differs from Na+-independent uptake of neutral and acidic amino acids. Na+-independent dibasic amino acid transport in membrane vesicles is likely reflective of Na+-independent transport of dibasic amino acids across the renal brush border membrane. 相似文献
6.
Labeled ((3)H or (14)C) l-alanine was perfused through the xylem vessels of isolated tomato internodes (Lycopersicon esculentum cv. Moneymaker) at various concentrations (10(-6) molar to 10(-2) molar). At each concentration the escape of l-alanine from the xylem vessels was apparently a first order process, which is in agreement with Horwitz' (1958, Plant Physiology 33:81-93) model for irreversible escape from the xylem vessels. The escape constant (K) decreased at higher concentrations of l-alanine, which implies that Horwitz' model is inappropriate to describe the kinetics of l-alanine escape, and that the escape at least partly is a saturable process. To obtain data that relate the concentration of l-alanine in the xylem vessels and the escape rate of the amino acid, average escape rates per internode were measured and the corresponding concentrations were calculated from the integrated form of the Michaelis-Menten equation.AS THE CONCENTRATION DEPENDENCE OF THE ESCAPE RATE WAS BIPHASIC, THREE POSSIBLE MECHANISMS WERE CONSIDERED, ESCAPE BEING CAUSED BY: (a) saturable amino acid uptake of cells around the xylem vessels and diffusion into the free space; (b) saturable uptake of the cells around the xylem vessels, but at higher amino acid concentrations in the xylem vessels the number of cells, that participate in the uptake, increases; (c) two, simultaneously operating, saturable uptake systems in the cells around the xylem vessels. 相似文献
7.
The transport of l-leucine by two human breast cancer cell lines has been examined. l-Leucine uptake by MDA-MB-231 and MCF-7 cells was via a BCH-sensitive, Na+-independent pathway. l-Leucine uptake by both cell lines was inhibited by l-alanine, d-leucine and to a lesser extent by l-lysine but not by l-proline. Estrogen (17β-estradiol) stimulated l-leucine uptake by MCF-7 but not by MDA-MB-231 cells. l-Leucine efflux from MDA-MB-231 and MCF-7 cells was trans-stimulated by BCH in a dose-dependent fashion. The effect of external BCH on l-leucine efflux from both cell types was almost abolished by reducing the temperature from 37 to 4 °C. There was, however, a significant efflux of l-leucine under zero-trans conditions which was also temperature-sensitive. l-Glutamine, l-leucine, d-leucine, l-alanine, AIB and l-lysine all trans-stimulated l-leucine release from MDA-MB-231 and MCF-7 cells. In contrast, d-alanine and l-proline had little or no effect. The anti-cancer agent melphalan inhibited l-leucine uptake by MDA-MB-231 cells but had no effect on l-leucine efflux. Quantitative real-time PCR revealed that LAT1 mRNA was approximately 200 times more abundant than LAT2 mRNA in MCF-7 cells and confirmed that MDA-MB-231 cells express LAT1 but not LAT2 mRNA. LAT1 mRNA levels were higher in MCF-7 cells than in MDA-MB-231 cells. Furthermore, LAT1 mRNA was more abundant than CD98hc mRNA in both MDA-MB-231 and MCF-7 cells. The results suggest that system L is the major transporter for l-leucine in both MDA-MB-231 and MCF-7 cells. It is possible that LAT1 may be the major molecular correlate of system L in both cell types. However, not all of the properties of system L reflected those of LAT1/LAT2/CD98hc. 相似文献
8.
The kinetics of the influx and efflux of radioactive l-glycine was studied in slices of rat cerebral cortex. The influx showed saturation kinetics and was inhibited by l-alanine. Influx was dependent on the presence of Na(+) ions and a metabolizable substrate. The efflux of glycine was accelerated by alanine. It was concluded that carrier-mediated facilitated diffusion was the mechanism of glycine uptake by, and efflux from, cerebral slices. 相似文献
9.
Diamide directly added to renal cortical slices inhibits the uptake of amino acids. Steady-state kinetic analysis indicates an inhibition of α-amino acid influx without effect on efflux. The effect could be reversed by addition of pyruvate to the incubation medium. Although there was a good correlation of the transport effect of diamide with its ability to decrease cellular reduced glutathione concentration, there did not appear to be a necessary connection between them. This was shown by the fact that renal cortical slices stored at 4°C have no alteration in amino acid uptake despite the fact that GSH concentration is as low as that seen with diamide. Diamide was shown to have a direct effect on the uptake of glycine by isolated renal brush border membrane vesicles. 相似文献
10.
The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na+ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K+, Li+ or choline+ gradient and was enhanced as extravesicular Na+ was increased from 10 mM to 100 mM. Dissipation of the Na+ gradient by the ionophore gramicidin D resulted in diminished Na+-stimulated glycine uptake. Na+-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na+-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent Km = 996 μM and Vmax = 348 pmol glycine/mg protein per min. Inhibition observed when the Na+-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [3H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids. 相似文献
11.
Kei Higuchi Koki Sugiyama Ryuto Tomabechi Hisanao Kishimoto Katsuhisa Inoue 《The Journal of biological chemistry》2022,298(4)
Monocarboxylate transporter 7 (MCT7) is an orphan transporter expressed in the liver, brain, and in several types of cancer cells. It has also been reported to be a survival factor in melanoma and breast cancers. However, this survival mechanism is not yet fully understood due to MCT7’s unidentified substrate(s). Therefore, here we sought to identify MCT7 substrate(s) and characterize the transport mechanisms by analyzing amino acid transport in HEK293T cells and polarized Caco-2 cells. Analysis of amino acids revealed significant rapid reduction in taurine from cells transfected with enhanced green fluorescent protein-tagged MCT7. We found that taurine uptake and efflux by MCT7 was pH-independent and that the uptake was not saturated in the presence of taurine excess of 200 mM. Furthermore, we found that monocarboxylates and acidic amino acids inhibited MCT7-mediated taurine uptake. These results imply that MCT7 may be a low-affinity facilitative taurine transporter. We also found that MCT7 was localized at the basolateral membrane in polarized Caco-2 cells and that the induction of MCT7 expression in polarized Caco-2 cells enhanced taurine permeation. Finally, we demonstrated that interactions of MCT7 with ancillary proteins basigin/CD147 and embigin/GP70 enhanced MCT7-mediated taurine transport. In summary, these findings reveal that taurine is a novel substrate of MCT7 and that MCT7-mediated taurine transport might contribute to the efflux of taurine from cells. 相似文献
12.
13.
The uptake of glutamate and other acidic amino acids into barnacle single muscle fibres has been characterized. The uptake of glutamate consists of two components, one Na-independent and one Na-dependent. The Na-dependent uptake is saturable (half-maximal at 250 microM external glutamate) and is inhibited by a variety of analogues of which L-cysteate and D- and L-aspartate are the most potent. These amino acids are also transported into the muscle in a Na-dependent manner. The excitatory agonists kainate, quisqualate, and N-methyl-D-aspartate do not inhibit or affect uptake in any way. Progressive replacement of external Na by choline reduces uptake with very little effect on the apparent affinity for glutamate, suggesting that Na and glutamate bind to the transporter independently. The kinetics of activation are consistent with a requirement for at least two Na ions. Na activation of glutamate uptake can be inhibited by guanidinium with kinetics that are consistent with competitive inhibition at the Na binding site. Studies on the efflux of L-glutamate and other analogues have shown that efflux rates are only slightly increased by the removal of Na and do not seem to be affected in any clear manner by external levels of acidic amino acids. 相似文献
14.
Aspartic acid (Asp) undergoes l-isomer-selective efflux transport across the blood-brain barrier (BBB). This transport system appears to play an important role in regulating l- and d-Asp levels in the brain. The purpose of this study was to identify the responsible transporters and elucidate the mechanism for l-isomer-selective Asp transport at the BBB. The l-isomer-selective uptake of Asp by conditionally immortalized mouse brain capillary endothelial cells used as an in vitro model of the BBB took place in an Na+- and pH-dependent manner. This process was inhibited by system ASC substrates such as l-alanine and l-serine, suggesting that system ASC transporters, ASCT1 and ASCT2, are involved in the l-isomer selective transport. Indeed, l-Asp uptake by oocytes injected with either ASCT1 or ASCT2 cRNA took place in a similar manner to that in cultured BBB cells, whereas no significant d-Asp uptake occurred. Although both ASCT1 and ASCT2 mRNA were expressed in the cultured BBB cells, the expression of ASCT2 mRNA was 6.7-fold greater than that of ASCT1. Moreover, immunohistochemical analysis suggests that ASCT2 is localized at the abluminal side of the mouse BBB. These results suggest that ASCT2 plays a key role in l-isomer-selective Asp efflux transport at the BBB. 相似文献
15.
The renal clearance of amino acids was measured in canine pups between 5 days and 12 weeks of age. The reabsorption of glycine was incomplete at 5 and 21 days, indicating a physiologic aminoaciduria of immaturity. An adult pattern of 97–100% reabsorption appeared by 8 weeks of age. The uptake of glycine by isolated renal tubules from 5-day-old, 3-month-old and adult dogs was examined towards an understanding of the events underlying this aminoaciduria. The initial uptake of 0.042 mM glycine by isolated tubules from the newborn was lower than that of the adult, but after 30 min of incubation the newborn surpassed the adult. A steady state of uptake was not achieved by the newborn even after 90 min of incubation, while it was achieved in the adult after 30 min. The uptake by the 3-month-old tubules resembled the adult at the early time points and the newborn at later points. With 1.032 mM glycine, a similar relationship of uptake between adult and newborn tubules was found, except with this concentration, the uptake by both the newborn and adult tubules reached a steady state. The concentration dependence of glycine uptake showed two saturable transport systems with similar apparent and values after 30 min of incubation for all three age groups. Determination of glycine flux by compartmental analysis revealed decreased influx and efflux in the newborn, but with a greater decrease in efflux, compared to adult. These changes of influx and efflux which accompany renal tubule maturation could contribute to the increased intracellular amino acid levels and decreased reabsorption of amino acids seen in the immature dog. 相似文献
16.
Amino acid and glucose uptake by rat brown adipose tissue. Effect of cold-exposure and acclimation.
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F J López-Soriano J A Fernández-López T Mampel F Villarroya R Iglesias M Alemany 《The Biochemical journal》1988,252(3):843-849
The net uptake/release of glucose, lactate and amino acids from the bloodstream by the interscapular brown adipose tissue of control, cold-exposed and cold-acclimated rats was estimated by measurement of arteriovenous differences in their concentrations. In the control animals amino acids contributed little to the overall energetic needs of the tissue; glucose uptake was more than compensated by lactate efflux. Cold-exposure resulted in an enhancement of amino acid utilization and of glucose uptake, with high lactate efflux. There was a net glycine and proline efflux that partly compensated the positive nitrogen balance of the tissue; amino acids accounted for about one-third of the energy supplied by glucose to the tissue. Cold-acclimation resulted in a very high increase in glucose uptake, with a parallel decrease in lactate efflux and amino acid consumption. Branched-chain amino acids, however, were more actively utilized. This was related with a much higher alanine efflux, in addition to that of glycine and proline. It is suggested that most of the glucose used during cold-exposure is returned to the bloodstream as lactate under conditions of active lipid utilization, amino acids contributing their skeletons largely in anaplerotic pathways. On the other hand, cold-acclimation resulted in an important enhancement of glucose utilization, with lowered amino acid oxidation. Amino acids are thus used as metabolic substrates by the brown adipose tissue of rats under conditions of relatively scarce substrate availability, but mainly as anaplerotic substrates, in parallel to glucose. Cold-acclimation results in a shift of the main substrates used in thermogenesis from lipid to glucose, with a much lower need for amino acids. 相似文献
17.
Sandhya Sankaranarayanan Margarita de la Llera-Moya Denise Drazul-Schrader Bela F. Asztalos Ginny L. Weibel George H. Rothblat 《Journal of lipid research》2010,51(11):3243-3249
Net flux of cholesterol represents the difference between efflux and influx and can result in net cell-cholesterol accumulation, net cell-cholesterol depletion, or no change in cellular cholesterol content. We measured radiolabeled cell-cholesterol efflux and cell-cholesterol mass using cholesterol-normal and -enriched J774 and elicited mouse peritoneal macrophage cells. Net cell-cholesterol effluxes were observed when cholesterol-enriched J774 cells were incubated with 3.5% apolipoprotein (apo) B depleted human serum, HDL3, and apo A-I. Net cell-cholesterol influxes were observed when cholesterol-normal J774 cells were incubated with the same acceptors except apo A-I. When incubated with 2.5% individual sera, cholesterol mass efflux in free cholesterol (FC)-enriched J774 cells correlated with the HDL-cholesterol (HDL-C) concentrations (r2 = 0.4; P=0.003), whereas cholesterol mass influx in cholesterol-normal J774 cells correlated with the LDL cholesterol (LDL-C) concentrations (r2 = 0.6; P<0.0001) of the individual sera. A positive correlation was observed between measurements of [3H]cholesterol efflux and reductions in cholesterol mass (r2 = 0.4; P=0.001) in FC-enriched J774 cells. In conclusion, isotopic efflux measurements from cholesterol-normal or cholesterol-enriched cells provide an accurate measurement of relative ability of an acceptor to remove labeled cholesterol under a specific set of experimental conditions, i.e., efflux potential. Moreover, isotopic efflux measurements can reflect changes in cellular cholesterol mass if the donor cells are enriched with cholesterol. 相似文献
18.
Transport of a large neutral amino acid in a human intestinal epithelial cell line (Caco-2): uptake and efflux of phenylalanine. 总被引:3,自引:0,他引:3
The processes of L-phenylalanine (Phe) uptake and efflux from the apical (AP) and basolateral (BL) sides of an intestinal epithelial cell line (Caco-2) were investigated to further characterize the mechanism of transcellular transport of this amino acid. The results indicated that the initial uptake rates of Phe were saturable with a Km of 2.7 mM for AP uptake and 0.18 mM for BL uptake. Unlike the uptake, the initial efflux rates were shown to be proportional to the intracellular concentrations of Phe. Based on these kinetic studies and determination of other characteristics (e.g., Na+ dependency) of the uptake and efflux processes, it was concluded that AP uptake, BL uptake and BL efflux were distinctly different. This suggests that either different carriers or a different combination of carriers are responsible for the transmembrane transport of this amino acid. When the results of kinetic studies of Phe uptake and efflux were used to determine the rate-limiting step in the AP-to-BL transcellular transport of this amino acid, it was concluded that the BL efflux is the rate-limiting step in the transcellular transport of Phe in the Caco-2 cell monolayers. 相似文献
19.
Selective uptake of HDL cholesteryl esters and cholesterol efflux from mouse peritoneal macrophages independent of SR-BI 总被引:2,自引:0,他引:2
Brundert M Heeren J Bahar-Bayansar M Ewert A Moore KJ Rinninger F 《Journal of lipid research》2006,47(11):2408-2421
Scavenger receptor class B type I (SR-BI) mediates the selective uptake of HDL cholesteryl esters (CEs) and facilitates the efflux of unesterified cholesterol. SR-BI expression in macrophages presumably plays a role in atherosclerosis. The role of SR-BI for selective CE uptake and cholesterol efflux in macrophages was explored. Macrophages and HDL originated from wild-type (WT) or SR-BI knockout (KO; homozygous) mice. For uptake, macrophages were incubated in medium containing 125I-/3H-labeled HDL. For lipid removal, [3H]cholesterol efflux was analyzed using HDL as acceptor. Selective uptake of HDL CE ([3H]cholesteryl oleyl ether - 125I-tyramine cellobiose) was similar in WT and SR-BI KO macrophages. Radiolabeled SR-BI KO-HDL yielded a lower rate of selective uptake compared with WT-HDL in WT and SR-BI KO macrophages. Cholesterol efflux was similar in WT and SR-BI KO cells using HDL as acceptor. SR-BI KO-HDL more efficiently promoted cholesterol removal compared with WT-HDL from both types of macrophages. Macrophages selectively take up HDL CE independently of SR-BI. Additionally, in macrophages, there is substantial cholesterol efflux that is not mediated by SR-BI. Therefore, SR-BI-independent mechanisms mediate selective CE uptake and cholesterol removal. SR-BI KO-HDL is an inferior donor for selective CE uptake compared with WT-HDL, whereas SR-BI KO-HDL more efficiently promotes cholesterol efflux. 相似文献
20.
Klaus Ring Wolfgang Langheinrich Hella Ehle Beate Foit 《Archives of microbiology》1977,115(2):199-205
The active uptake of 2-aminoisobutyric acid (AIB) and several other amino acids in resting cells of Streptomyces hydrogenans was found to be stimulated by exogenously added adenosine cyclic monophosphate (cAMP). The uptake of glycerol, sorbose, and pyrimidine nucleosides remained unaffected. Among the various cAMP derivatives tested, the dibutyryl derivative was found to be most effective, followed by monobutyryl cAMP, and cAMP. Dibutyryl cGMP was also found to stimulate AIB transport, and its effectivity was as good as that of dibutyryl cAMP. The effect of dibutyryl cAMP is time dependent and attains its maximum after 40–60 min of incubation at 30°C in K-Na-phosphate buffer. Dibutyryl cAMP-dependent transport stimulation has a high temperature coefficient and is prevented by rifamycin SV or chloramphenicol. The rate of leucine incorporation into protein was rapidly increased upon addition of dibutyryl cAMP. Kinetic studies reveal that the stimulation of AIB transport is characterized by an increase in maximum uptake rate and an unaltered apparent Michaelis constant. Analysis of the unidirectional fluxes show that both influx and efflux are enhanced by dibutyryl cAMP. It is concluded that exogenous dibutyryl cAMP stimulates de novo synthesis of certain protein including the transport catalysts for various amino acids. 相似文献