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1.
Transport of folates at maternal and fetal sides of the placenta: lack of inhibition by methotrexate 总被引:1,自引:0,他引:1
Folate (pteroylglutamate) and methotrexate rapid (seconds) uptake by the trophoblast was investigated from either the maternal or fetal circulations of the isolated dually-perfused guinea-pig placenta. Tissue uptake was measured by using a single-circulation paired-tracer (3H-test and 14C-extracellular marker) technique. [3H]Folate uptakes were 80 and 52% (mean) in perfusates without unlabelled folate, on maternal and fetal sides, respectively. There was negligible 3H-tracer backflux into the circulation up to 6 min probably due to metabolic sequestration. [3H]Methotrexate uptakes were about 85 and 22% on maternal and fetal sides, respectively; however these uptakes were followed by rapid and complete backflux of the label. Specific transplacental transfer of [3H]folate or [3H]methotrexate in either direction was not detectable within 5-6 min. At the brush-border side (maternal) uptake of [3H]folate was highly inhibited by 100 nM unlabelled folate or its reduced form, methyltetrahydrofolate (the main form in plasma); however, equimolar methotrexate (an antifolate chemotherapeutic agent) failed to produce any inhibition of folate uptake. Our findings demonstrate that on both sides of the placenta a high-affinity transport system exists for trophoblast uptake of folate compounds. For methotrexate, either a separate transport system may exist or methotrexate may have a very low affinity for the folate system. These results are distinct from the findings reported in mouse L1210 leukemia cells. 相似文献
2.
Methionine S-adenosyltransferase (MAT) catalyzes the only reaction that produces the major methyl donor in mammals. Low-dose methotrexate is the most commonly used disease-modifying antirheumatic drug in human rheumatic conditions. The present study was conducted to test the hypothesis that methotrexate inhibits MAT expression and activity in vitro and in vivo. HepG2 cells were cultured under folate restriction or in low-dose methotrexate with and without folate or methionine supplementation. Male C57BL/6J mice received methotrexate regimens that reflected low-dose clinical use in humans. S-adenosylmethionine and MAT genes, proteins and enzyme activity levels were determined. We found that methionine or folate supplementation greatly improved S-adenosylmethionine in folate-depleted cells but not in cells preexposed to methotrexate. Methotrexate but not folate depletion suppressed MAT genes, proteins and activity in vitro. Low-dose methotrexate inhibited MAT1A and MAT2A genes, MATI/II/III proteins and MAT enzyme activities in mouse tissues. Concurrent folinate supplementation with methotrexate ameliorated MAT2A reduction and restored S-adenosylmethionine in HepG2 cells. However, posttreatment folinate rescue failed to restore MAT2A reduction or S-adenosylmethionine level in cells preexposed to methotrexate. Our results provide both in vitro and in vivo evidence that low-dose methotrexate inhibits MAT genes, proteins, and enzyme activity independent of folate depletion. Because polyglutamated methotrexate stays in the hepatocytes, if methotrexate inhibits MAT in the liver, then the efficacy of clinical folinate rescue with respect to maintaining hepatic S-adenosylmethionine synthesis and normalizing the methylation reactions would be limited. These findings raise concerns on perturbed methylation reactions in humans on low-dose methotrexate. Future studies on the clinical physiological consequences of MAT inhibition by methotrexate and the potential benefits of S-adenosylmethionine supplementation on methyl group homeostasis in clinical methotrexate therapies are warranted. 相似文献
3.
Transport of folates at maternal and fetal sides of the placenta: lack of inhibition by methotrexate
Folate (pteroylglutamate) and methotrexate rapid (seconds) uptake by the trophoblast was investigated from either the maternal or fetal circulations of the isolated dually-perfused guinea-pig placenta. Tissue uptake was measured by using a single-circulation paired-tracer (3H-test and 14C-extracellular marker) technique. [3H]Folate uptakes were 80 and 52% (mean) in perfusates without unlabelled folate, on maternal and fetal sides, respectively. There was negligible 3H-tracer backflux into the circulation up to 6 min probably due to metabolic sequestration. [3H]Methotrexate uptakes were about 85 and 22% on maternal and fetal sides, respectively; however these uptakes were followed by rapid and complete backflux of the label. Specific transplacental transfer of [3H]folate or [3H]methotrexate in either direction was not detectable within 5–6 min. At the brush-border side (maternal) uptake of [3H]folate was highly inhibited by 100 nM unlabelled folate or its reduced form, methyltetrahydrofolate (the main form in plasma); however, equimolar methotrexate (an antifolate chemotherapeutic agent) failed to produce any inhibition of folate uptake. Our findings demonstrate that on both sides of the placenta a high-affinity transport system exists for trophoblast uptake of folate compounds. For methotrexate, either a separate transport system may exist or methotrexate may have a very low affinity for the folate system. These results are distinct from the findings reported in mouse L1210 leukemia cells. 相似文献
4.
Effects of growth rate and methotrexate on folate polyglutamates and folylpolyglutamate hydrolase activity in Krebs ascites cells 总被引:1,自引:0,他引:1
The activity of folylpolyglutamate hydrolase was measured throughout intraperitoneal growth of Krebs ascites cells in mice and after exposure to methotrexate. Hydrolase activity was lowest during the log phase of growth. Methotrexate administered intraperitoneally during log growth caused a dose- and time-dependent increase in hydrolase activity. Modest changes were observed in endogenous folate polyglutamate chain length distributions throughout growth and upon exposure to methotrexate, but these changes could not be correlated with hydrolase activity. 相似文献
5.
The Clostridium botulinum C2 toxin is the prototype of the family of binary actin-ADP-ribosylating toxins. C2 toxin is composed of two separated nonlinked proteins. The enzyme component C2I ADP-ribosylates actin in the cytosol of target cells. The binding/translocation component C2II mediates cell binding of the enzyme component and its translocation from acidic endosomes into the cytosol. After proteolytic activation, C2II forms heptameric pores in endosomal membranes, and most likely, C2I translocates through these pores into the cytosol. For this step, the cellular heat shock protein Hsp90 is essential. We analyzed the effect of methotrexate on the cellular uptake of a fusion toxin in which the enzyme dihydrofolate reductase (DHFR) was fused to the C-terminus of C2I. Here, we report that unfolding of C2I-DHFR is required for cellular uptake of the toxin via the C2IIa component. The C2I-DHFR fusion toxin catalyzed ADP-ribosylation of actin in vitro and was able to intoxicate cultured cells when applied together with C2IIa. Binding of the folate analogue methotrexate favors a stable three-dimensional structure of the dihydrofolate reductase domain. Pretreatment of C2I-DHFR with methotrexate prevented cleavage of C2I-DHFR by trypsin. In the presence of methotrexate, intoxication of cells with C2I-DHFR/C2II was inhibited. The presence of methotrexate diminished the translocation of the C2I-DHFR fusion toxin from endosomal compartments into the cytosol and the direct C2IIa-mediated translocation of C2I-DHFR across cell membranes. Methotrexate had no influence on the intoxication of cells with C2I/C2IIa and did not alter the C2IIa-mediated binding of C2I-DHFR to cells. The data indicate that methotrexate prevented unfolding of the C2I-DHFR fusion toxin, and thereby the translocation of methotrexate-bound C2I-DHFR from endosomes into the cytosol of target cells is inhibited. 相似文献
6.
M A Kane R M Portillo P C Elwood A C Antony J F Kolhouse 《The Journal of biological chemistry》1986,261(1):44-49
Methotrexate accumulation, subcellular distribution, metabolism, and cytotoxicity were studied in human epidermoid carcinoma (KB) cells that were exposed to a low extracellular concentration of methotrexate (25 nM) following culture in widely differing concentrations of folic acid. KB cells cultured in standard medium with a high folic acid concentration (2.3 microM) had high levels of cellular folate (21.4 pmol/10(6) cells). Five passages through low folate (2.7 nM) medium reduced the level of cellular folate to near physiologic levels (0.4-1.0 pmol/10(6) cells). In contrast to KB cells cultured in standard medium, in KB cells cultured in low folate medium, 1) methotrexate inhibited growth; 2) methotrexate uptake was markedly increased; 3) methotrexate polyglutamation was almost complete; 4) methotrexate binding to dihydrofolate reductase was markedly enhanced; and 5) significant methotrexate binding to a previously undescribed membrane-associated protein occurred. The amount of methotrexate bound to the membrane-associated protein from KB cells cultured in low folate medium equaled the quantities bound by dihydrofolate reductase. Further characterization of this membrane-associated protein indicated that it was soluble in solutions containing Triton X-100, was capable of binding folic acid as well as methotrexate, had an apparent Mr of 160,000 by gel filtration in the presence of Triton X-100, and was precipitated by antiserum to human placental folate receptor. This membrane-associated protein may play an important role in the uptake and metabolism of methotrexate under physiologic conditions. 相似文献
7.
A highly specific assay for folate reductase mRNA activity from Sarcoma 180 cells was developed using the rabbit reticulocyte lysate protein synthesizing system. Quantitation of in vitro folate reductase synthesis was accomplished by direct immunoprecipitation from lysate reactions. The in vitro labeled folate reductase was synthesized in a linear response to a wide range of RNA concentrations, migrated as a single prominent radioactive species upon polyacrylamide gel electrophoresis, and was indistinguishable from authentic 14C-labeled folate reductase on the basis of molecular weight and immunotitration with anti-folate reductase gamma-globulin. The assay was used to quantitate folate reductase mRNA activity in various cell lines and under several conditions known to affect folate reductase synthesis. These included (a) sensitive and methotrexate-resistant Sarcoma 180 cells, (b) two lines of resistant cells having different relative rates of folate reductase synthesis, (c) growth of methotrexate-resistant cells in the absence of methotrexate, and (d) growth phase. The results indicate that the relative rate of folate reductase synthesis in each case can be explained solely by the level of translatable folate reductase mRNA. The use of poly(U)-Sepharose and sucrose gradient fractionation procedures indicated that folate reductase mRNA contains poly(A) and has a sedimentation coefficient of approximately 14 S. These two fractionation steps were combined to achieve an approximately 90-fold purification of folate reductase mRNA over total cytoplasmic RNA. 相似文献
8.
Wang Y Rajgopal A Goldman ID Zhao R 《American journal of physiology. Cell physiology》2005,288(1):C65-C71
Intestinal folate transport has been well characterized, and rat small intestinal epithelial (IEC-6) cells have been used as a model system for the study of this process on the cellular level. The major intestinal folate transport activity has a low-pH optimum, and the current paradigm is that this process is mediated by the reduced folate carrier (RFC), despite the fact that this carrier has a neutral pH optimum in leukemia cells. The current study addressed the question of whether constitutive low-pH folate transport activity in IEC-6 cells is mediated by RFC. Two independent IEC-6 sublines, IEC-6/A4 and IEC-6/PT1, were generated by chemical mutagenesis followed by selective pressure with antifolates. In IEC-6/A4 cells, a premature stop resulted in truncation of RFC at Gln420. A green fluorescent protein (GFP) fusion with the truncated protein was not stable. In IEC-6/PT1 cells, Ser135 was deleted, and this alteration resulted in the failure of localization of the GFP fusion protein in the plasma membrane. In both cell lines, methotrexate (MTX) influx at neutral pH was markedly decreased compared with wild-type IEC-6 cells, but MTX influx at pH 5.5 was not depressed. Transient transfection of the GFP-mutated RFC constructs into RFC-null HeLa cells confirmed their lack of transport function. These results indicate that in IEC-6 cells, folate transport at neutral pH is mediated predominantly by RFC; however, the folate transport activity at pH 5.5 is RFC independent. Hence, constitutive folate transport activity with a low-pH optimum in this intestinal cell model is mediated by a process entirely distinct from that of RFC. folic acid; folate absorption; methotrexate 相似文献
9.
Methotrexate (MTX) is widely used for the treatment of childhood acute lymphoblastic leukemia (ALL). The accumulation of MTX and its active metabolites, methotrexate polyglutamates (MTXPG), in ALL cells is an important determinant of its antileukemic effects. We studied 194 of 356 patients enrolled on St. Jude Total XV protocol for newly diagnosed ALL with the goal of characterizing the intracellular pharmacokinetics of MTXPG in leukemia cells; relating these pharmacokinetics to ALL lineage, ploidy and molecular subtype; and using a folate pathway model to simulate optimal treatment strategies. Serial MTX concentrations were measured in plasma and intracellular MTXPG concentrations were measured in circulating leukemia cells. A pharmacokinetic model was developed which accounted for the plasma disposition of MTX along with the transport and metabolism of MTXPG. In addition, a folate pathway model was adapted to simulate the effects of treatment strategies on the inhibition of de novo purine synthesis (DNPS). The intracellular MTXPG pharmacokinetic model parameters differed significantly by lineage, ploidy, and molecular subtypes of ALL. Folylpolyglutamate synthetase (FPGS) activity was higher in B vs T lineage ALL (p<0.005), MTX influx and FPGS activity were higher in hyperdiploid vs non-hyperdiploid ALL (p<0.03), MTX influx and FPGS activity were lower in the t(12;21) (ETV6-RUNX1) subtype (p<0.05), and the ratio of FPGS to γ-glutamyl hydrolase (GGH) activity was lower in the t(1;19) (TCF3-PBX1) subtype (p<0.03) than other genetic subtypes. In addition, the folate pathway model showed differential inhibition of DNPS relative to MTXPG accumulation, MTX dose, and schedule. This study has provided new insights into the intracellular disposition of MTX in leukemia cells and how it affects treatment efficacy. 相似文献
10.
1. Dihydrofolate reductase was purified from Lactobacillus casei MTX/R, and studied on affinity columns containing folic acid and methotrexate. Two forms of the enzyme were interconverted by incubation with substrates. 2. Affinity columns were prepared from agarose activated with cyanogen bromide and coupled with 1,6-diaminohexane. Stable folate derivatives were covalently attached by using a carbodi-imide condensation. 3. Columns containing folic acid retarded but did not retain the enzyme. 4. Methotrexate at pH 6.0 was particularly effective for retention of the enzyme. 5. There is selective loss of one form of the enzyme during affinity chromatography in the absence of added NADPH. This loss is due to conversion into a single enzyme form on the column. 6. NADPH has a dual effect in stabilizing the enzyme and in sensitizing it to inactivation by methotrexate, particularly in the presence of glycine. 7. Protein with affinity for methotrexate, but without dihydrofolate reductase activity, may also be eluted from the columns. 8. In a single-step procedure the enzyme was purified nearly 4000-fold from mammalian skin. 相似文献
11.
Inoue K Nakai Y Ueda S Kamigaso S Ohta KY Hatakeyama M Hayashi Y Otagiri M Yuasa H 《American journal of physiology. Gastrointestinal and liver physiology》2008,294(3):G660-G668
Proton-coupled folate transporter/heme carrier protein 1 (PCFT/HCP1) has recently been identified as a transporter that mediates the translocation of folates across the cellular membrane by a proton-coupled mechanism and suggested to be the possible molecular entity of the carrier-mediated intestinal folate transport system. To further clarify its role in intestinal folate transport, we examined the functional characteristics of rat PCFT/HCP1 (rPCFT/HCP1) expressed in Xenopus laevis oocytes and compared with those of the carrier-mediated folate transport system in the rat small intestine evaluated by using the everted tissue sacs. rPCFT/HCP1 was demonstrated to transport folate and methotrexate more efficiently at lower acidic pH and, as evaluated at pH 5.5, with smaller Michaelis constant (K(m)) for the former (2.4 microM) than for the latter (5.7 microM), indicating its characteristic as a proton-coupled folate transporter that favors folate than methotrexate as substrate. rPCFT/HCP1-mediated folate transport was found to be inhibited by several but limited anionic compounds, such as sulfobromophthalein and sulfasalazine. All these characteristics of rPCFT/HCP1 were in agreement with those of carrier-mediated intestinal folate transport system, of which the K(m) values were 1.2 and 5.8 microM for folate and methotrexate, respectively, in the rat small intestine. Furthermore, the distribution profile of the folate transport system activity along the intestinal tract was in agreement with that of rPCFT/HCP1 mRNA. This study is the first to clone rPCFT/HCP1, and we successfully provided several lines of evidence that indicate its role as the molecular entity of the intestinal folate transport system. 相似文献
12.
We have isolated stable variants of the L1210 cell exhibiting increased transport inward of the folate analog, methotrexate. These variants show 3- to 14-fold increases in [3H]methotrexate influx compared to parental cells but are unaltered for [3H]methotrexate efflux. This increased influx in each variant is quantitatively reflected in corresponding elevations in intracellular exchangeable levels of drug at steady state, but there is no alteration in membrane potential. The increases in influx are associated with increased values for influx Vmax for a system normally transporting reduced folates and the same increase in the amount of a specific binding component at the cell surface. Otherwise, values for influx Km and specificity for various folate structures are unchanged. This alteration in [3H]methotrexate influx is biochemically and genetically stable, since it is expressed in isolated plasma membrane vesicles and is retained during growth in non-selective medium. Following addition of cycloheximide, the same rate of decay of this transport activity (t 1/2 = 126 +/- 24 to 137 +/- 26 min) was shown for parental and variant cells. From these results we conclude that turnover of this transport property occurs in these cells which is genetically regulated. Also, the elevated transport activity inward for this folate analog in these variant cells is probably the result of a genetic alteration up-regulating the rate of synthesis of the "putative" carrier protein itself. The absence of any effect on efflux of [3H]methotrexate in these variants in the face of evidence for increased synthesis of the carrier protein for the system mediating influx of this folate analog is construed as further evidence for the nonidentity of systems mediating each flux that we proposed on the basis of earlier kinetic studies. 相似文献
13.
The effect of methotrexate on the free intracellular pools of thymidylate triphosphate (dTTP) and deoxyadenosine triphosphate (dATP) in normal human phytohaemagglutinin-transformed lymphocytes has been studied. Methotrexate caused a fall in the dTTP pool ranging from 38% to 88% and a rise in the dATP pool ranging from 24% to 185%.A rise in the free intracellular pool of dATP is thought to inhibit both rubonucleotide reduction and polynucleotide ligase, an enzyme concerned in DNA synthesis and repair. The hypothesis is suggested here that folate deficiency per se, as well as a functional folate deficiency induced by methotrexate may cause reduced DNA synthesis, megaloblastic changes, and chromosome abnormalities by producing a rise in the free intracellular pool of dATP as well as by causing a fall in free intracellular dTTP. 相似文献
14.
Alfredo Guéra Twan America Marieke van Waas Peter J. Weibeek 《Plant molecular biology》1993,23(2):309-324
Protein conformational changes related to transport into chloroplasts have been studied. Two chimaeric proteins carrying the transit peptide of either ferredoxin or plastocyanin linked to the mouse cytosolic enzyme dihydrofolate reductase (EC 1.5.1.3.) were employed. In contrast to observations in mitochondria, we found in chloroplasts that transport of a purified ferredoxin-dihydrofolate reductase fusion protein is not blocked by the presence of methotrexate, a folate analogue that stabilizes the structural conformation of dihydrofolate reductase. It is shown that transport competence of this protein in the presence of methotrexate is not a consequence of alteration of the folding characteristics or methotrexate binding properties of dihydrofolate reductase by fusion to the ferredoxin transit peptide. Binding of dihydrofolate reductase fusion proteins to chloroplast envelopes is not inhibited by low temperature and it is only partially diminished by methotrexate. It is demonstrated that the dihydrofolate reductase fusion proteins unfold, despite the presence of methotrexate, on binding to the chloroplast envelopes. We propose the existence of a strong protein unfolding activity associated to the chloroplast envelopes. 相似文献
15.
Richard D Leprohon P Drummelsmith J Ouellette M 《The Journal of biological chemistry》2004,279(52):54494-54501
The protozoan parasite Leishmania relies on the uptake of folate and pterin from the environment to meet its nutritional requirements. We show here that a novel gene (folate transporter 1 (FT1)) deleted in a Leishmania infantum methotrexate-resistant mutant corresponds to the main folate transporter (K(m), 410 nM). FT1 was established as the main folate transporter by both gene transfection and by targeted gene deletion. Modulation of the expression of FT1 by these manipulations altered the susceptibility of Leishmania cells to methotrexate. Folate transport was stage-regulated with higher activity in the logarithmic phase and less in the stationary phase. FT1 fused to green fluorescent protein led to the observation that FT1 was located in the plasma membrane in the logarithmic phase but was retargeted to an intracellular organelle followed by a degradation of the protein in stationary phase. Leishmania has several folate transporters with different characteristics, and the growth stage-related activity of at least one transporter is regulated post-translationally. 相似文献
16.
《Journal of liposome research》2013,23(2):217-235
AbstractAn in vitro liposome-cell association system has been developed that will allow the study of uptake and metabolism of liposomes by cultured cells at nanomolar lipid concentrations. The fate of cell associated liposomes is followed through the liposome encapsulated marker, methotrexate. Detection is based on the inhibition of dihydrofolate reductase by methotrexate, after its release from cells through boiling. Methotrexate in phospha-tidylglycerol (PG) liposomes is taken up by cells and then subsequently lost from the cells. Uptake is partially blocked by monensin. Loss from the cells is blocked by metabolic inhibitors, monensin, ammonium chloride, and chloroquine. Methotrexate in distearoylphosphatidylglycerol (DSPG) liposomes is taken up by cells slowly, and there is minimal lost of methotrexate after uptake. Pulse studies show that metabolism of PG liposomes after endocytosis is occurring at a much higher rate than that of DSPG liposomes, and substantial retention of encapsulated methotrexate occurs for both liposome compositions. 相似文献
17.
Shin DS Zhao R Yap EH Fiser A Goldman ID 《American journal of physiology. Cell physiology》2012,302(9):C1405-C1412
Proton-coupled folate transporter (PCFT) mediates folate intestinal absorption and transport across the choroid plexus, processes defective in subjects with hereditary folate malabsorption (HFM). PCFT is also widely expressed in human solid tumors where it contributes to the transport of pemetrexed and other antifolates. This study defines the basis for the functional changes due to a P425R mutation detected in a subject with HFM. Among various substitutions, only positively charged mutants (P425R and P425K) lost function but in a highly selective manner. Transport of reduced folates mediated by P425R-PCFT was virtually abolished; the methotrexate influx K(t) was increased fivefold (from 2 to 10 μM). In contrast, the pemetrexed influx K(t) mediated by P425R-PCFT was decreased 30% compared with wild-type (WT)-PCFT. Methotrexate inhibition of pemetrexed influx was competitive with a K(i) for WT-PCFT comparable to its influx K(t). However, the methotrexate influx K(i) for P425R-PCFT was ~15-fold higher than the WT-PCFT influx K(t) and threefold higher than the methotrexate influx K(t) for the P425R-PCFT mutant. The confirmed secondary structure and homology modeling place the P425 residue at the junction of the 6th external loop and 12th transmembrane domain, remote from the aqueous translocation pathway, a prediction confirmed by the failure to label P425C-PCFT with N-biotinylaminoethyl methanethiosulfonate-biotin and the absence of inhibition of P425C-PCFT function by water-soluble sulfhydryl reagents. Hence, despite its location, the P425R-PCFT mutation produces a conformational change that fully preserves pemetrexed binding but markedly impairs binding of methotrexate and other folates to the carrier. 相似文献
18.
S V Klinov N A Chebotareva B M She?man E M Birinberg B I Kurganov 《Bioorganicheskaia khimiia》1987,13(7):908-914
The interaction of rabbit skeletal muscle glycogen phosphorylase b with methotrexate, folic and folinic acids has been studied. Microscopic dissociation constant for the glycogen phosphorylase b--methotrexate complex determined by analytical ultracentrifugation is 0.43 mM. A subunit of glycogen phosphorylase b is shown to have two sites for methotrexate binding. AMP and FMN diminish the affinity of glycogen phosphorylase b to methotrexate, whereas glycogen does not influence the methotrexate binding to the enzyme. Methotrexate, folic and folinic acids are found to be inhibitors of the muscle glycogen phosphorylase b. The inhibition is reversible and characterized by positive kinetic cooperativity (the Hill coefficient exceeds one unity). The value of the pterin concentration causing two-fold diminishing of the enzymatic reaction rate increased in the order: folic acid (0.65 mM), methotrexate (1.01 mM), folinic acid (3.7 mM). The antagonism between methotrexate, folic and folinic acids, on the one hand, and AMP and FMN, on the other, is revealed for their combined action. 相似文献
19.
M Balińska 《Acta biochimica Polonica》1986,33(1):31-37
The conversion of methotrexate to poly-gamma-glutamyl derivatives in Ehrlich ascites carcinoma cells which are characterized by different pools of endogenous folates is described. The cells in which folate pool was high (the 5-fluorodeoxy-uridine-resistant cell line) the ability to convert methotrexate to its polyglutamate derivatives was much lower than in the cells in which folate pool was smaller (the parental cell line). When the cellular folate pool was reduced by treatment of the cells with lysolecithin, a similar methotrexate polyglutamate concentration in both cell lines was observed. These data suggest that cellular folate pool has a regulatory effect on methotrexate polyglutamate synthesis. 相似文献
20.
G Schlenstedt G H Gudmundsson H G Boman R Zimmermann 《The Journal of biological chemistry》1990,265(23):13960-13968
Translocation of large presecretory proteins into the mammalian endoplasmic reticulum requires the ribonucleoparticles, signal recognition particle, and ribosome and is tightly coupled to ongoing protein synthesis. We have shown previously that small presecretory proteins can translocate post-translationally in a reaction that does not require these ribonucleoparticles. We now report that one large protein, a synthetic hybrid between preprocecropin A and dihydrofolate reductase, translocates both cotranslationally (with the aid of signal recognition particle and ribosome) and post-translationally (without the involvement of these ribonucleoparticles) during its in vitro synthesis in the presence of dog pancreas microsomes. The distinction between these two modes of translocation was made possible by adding methotrexate to the translocation reaction. Methotrexate can only form a tight complex with those preprocecropin A-dihydrofolate reductase hybrid chains that have completed their synthesis and folded, but in forming this tight complex, this drug prevents translocation of the dihydrofolate reductase domain across the membrane. 相似文献