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G B Henderson

Publications and source records attributed to G B Henderson.

At least 37 records · Page 2Linked to original sources
PubMed1990

Preliminary crystallographic analysis of trypanothione reductase from Crithidia fasciculata.

Trypanothione reductase, a flavoprotein disulfide reductase specific to trypanosomatid parasites, has been crystallized by vapor diffusion of a protein solution (10 mg/ml) against 22% polyethylene glycol (average Mr 8000) containing 100 mM-ammonium sulfate. Crystals of a size suitable for structure determination by X-ray diffraction have been obtained by seeding protein solutions with smaller crystals. The space-group is P21 (a = 60.9 A, b = 161.8 A, c = 58.4 A, beta = 99.1 degrees). The molecular mass and volume of the unit cell suggest that there is a dimer of the enzyme in the asymmetric unit, and this is confirmed by self-rotation functions calculated using data to 4.5 A resolution. The crystals diffract to beyond 3 A resolution. Crystals of another P21 form (a = 91.3 A, b = 114.4 A, c = 92.0 A, beta = 141.3 degrees) are observed to grow under similar conditions.

Animals↗
PubMed1990

Pyrimidine biosynthesis in parasitic protozoa: purification of a monofunctional dihydroorotase from Plasmodium berghei and Crithidia fasciculata.

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl-L-aspartate (L-CA) to L-5,6-dihydroorotate (L-DHO), which is the third enzyme in de novo pyrimidine biosynthesis. The enzyme was purified from two parasitic protozoa, Crithidia fasciculata (about 16,000-fold) and Plasmodium berghei (about 790-fold). The C. fasciculata enzyme had a native molecular weight (Mr) of 42,000 +/- 5000, determined by gel filtration chromatography, and showed a single detectable protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Mr 44,000 +/- 3000. The DHOase from P. berghei had a native molecular weight of 40,000 +/- 4000 and a subunit molecular weight on SDS-PAGE of 38,000 +/- 3000. The DHOase from both parasites, in contrast to the mammalian enzyme which resides on a trifunctional protein of the first two enzymes of the pathway, carbamoyl-phosphate synthase and aspartate transcarbamylase, is monomeric and has no oligomeric structure as studied by chemical cross-linking with dimethyl suberimidate. The rate of cyclization of L-CA by the C. fasciculata enzyme was relatively high at acidic pH, decreasing to a very low rate at alkaline pH. In contrast, the rate of ring cleavage of L-DHO was very low at acidic pH and increased to a higher rate at alkaline pH. These pH-activity profiles gave an intersection at pH 6.6. The Km and kcat for L-CA were 0.846 +/- 0.017 mM and 39.2 +/- 6.4 min-1, respectively; for L-DHO, they were 25.85 +/- 2.67 microM and 258.6 +/- 28.5 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Growth inhibition by homofolate in tumor cells utilizing a high-affinity folate binding protein as a means for folate internalization.

A subline (JT-1) of L1210 mouse leukemia cells that contains elevated levels of a high-affinity folate binding protein is sensitive to growth inhibition by homofolate. Inhibition was observed at nanomolar concentrations of folate or 5-formyltetrahydrofolate where the high-affinity binding protein is the predominant uptake route for folate compounds. At 1.0 nM folate, inhibition of growth by 50% occurred at 0.7 nM homofolate, and maximal inhibition exceeded 90% at homofolate concentrations above 10 nM. Homofolate also inhibited the uptake of 1.0 nM [3H]folate by L1210/JT-1 cells in 72-hr cultures, and the extent of uptake inhibition by 1.0 and 20 nM homofolate was comparable to the inhibition of cell growth by the same concentrations of homofolate. At a growth-limiting concentration of 5-formyltetrahydrofolate (0.5 nM), half-maximal inhibition of L1210/JT-1 cell growth occurred at 1.0 nM homofolate. When excess concentrations of folate (5 microM) or 5-formyltetrahydrofolate (0.5 microM) were added to the medium, no growth inhibition was observed for homofolate at concentrations up to 100 microM. Parental cells lacking the folate binding protein did not respond to homofolate either at growth-limiting (0.5 microM) or excess (5.0 microM) levels of folate. Binding measurements showed that homofolate has a high affinity for the folate-binding protein (Ki = 0.03 nM) but interacts poorly with the reduced-folate transport system (Ki = 203 microM). These results indicate that homofolate inhibits the growth of L1210 cells when intracellular folates are acquired via the high-affinity folate binding protein. The basis for this inhibition appears to be competition by homofolate for substrate binding and internalization.

Animals↗
PubMed1990

Biosynthesis of the trypanosomatid metabolite trypanothione: purification and characterization of trypanothione synthetase from Crithidia fasciculata.

Trypanothione synthetase from Crithidia fasciculata has been purified ca. 14,500-fold to homogeneity in an overall yield of 40%. The pure enzyme catalyzed the synthesis of N1- and N8-glutathionylspermidine and N1,N8-bis(glutathionyl)spermidine (trypanothione) from ATP/magnesium, glutathione (GSH), and spermidine, N1- and N8-glutathionylspermidines being intermediates of trypanothione synthesis. The enzyme showed a sharp pH optimum of 7.5-7.75 for the synthesis of both mono- and diglutathionylspermidine conjugates. It was highly specific for its physiological substrates ATP/Mg2+, GSH, spermidine, and N1- and N8-glutathionylspermidine with Km values of 400 microM, 914 microM, 1.07 mM, 20 microM, and 7 microM, respectively. Trypanothione synthetase was active in the monomeric form with Mr = 87,000 and absorption maxima lambda max = 225 and 280 nm (A280/A260 = 1.85). Trypanothione synthetase is a new member of the ATP-dependent class of ligases which form amide linkage with concomitant production of ADP and orthophosphate.

Amide Synthases↗
PubMed1990

Characteristics of a novel transport system for folate compounds in wild-type and methotrexate-resistant L1210 cells.

The growth requirements and transport characteristics of folate compounds in a methotrexate-resistant L1210/R81 cell line were compared with parental cells. Concentrations for half-maximal growth of the resistant cells with folate (350 nM) and 5-formyltetrahydrofolate (20 nM) were found to be higher by 2.7-fold and 20-fold, respectively, relative to the parent, suggesting that changes had occurred in the transport of these folate compounds. Transport measurements revealed that the resistant cells have lost the capacity to transport methotrexate and other folate compounds via the reduced-folate transport system but that a second previously undescribed transport system is present. Uptake of folate via this second route is energy dependent, exhibits saturation kinetics, can be inhibited by substrate analogues, and is activated by a reduction in pH. The pH effect is substantial since uptake at 5.0 microM folate can be increased 10-fold by decreasing the pH from 7.4 to 6.2. The observed Kt for half-maximal influx of folate at pH 6.8 was 5.2 microM, and the Vmax was 0.55 pmol/min/mg of protein. Ki values for methotrexate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate were 10.2, 3.2, and 2.7 microM, respectively. Transport activity was not affected by depleting internal stores of folate by growth in folate-free medium, and this same transport system was also present in comparable amounts in wild-type cells. In the latter case, interfering uptake via the reduced-folate transport system was blocked by bromosulfophthalein and thiamine pyrophosphate. Methotrexate and 5-formyltetrahydrofolate were also transported via this alternative, pH-dependent route, whereas little or no uptake of 5-methyltetrahydrofolate could be detected. The results indicate that antifolate resistance in L1210/R81 cells was induced by inactivating the reduced-folate transport system and by utilizating a secondary route which has a low capacity for transporting methotrexate but whose activity is sufficient to permit cell growth in medium supplemented with folate or 5-formyltetrahydrofolate.

Animals↗
PubMed1990

Identification of cholate as a shared substrate for the unidirectional efflux systems for methotrexate in L1210 mouse cells.

The bidirectional transport properties of cholate have been examined in leukemic L1210 mouse cells and compared with the transport of methotrexate. The cell entry of [3H]cholate was Na(+)-independent, linear with increasing concentrations of substrate, enhanced by decreasing pH, and uneffected by excess unlabeled cholate or by various anion-transport inhibitors and hence had the characteristics of passive diffusion or a pH-dependent mediated process with a high Kt for cholate. The efflux of [3H]cholate, however, could be attributed to carrier-mediated and energy-dependent transport. Efflux was rapid (t1/2 = 1.5 min) and could be increased with glucose and decreased with metabolic inhibitors, and it was inhibited by various compounds including bromosulfophthalein, probenecid, prostaglandin A1, reserpine, verapamil, quinidine, diamide, 1-methyl-3-isobutylxanthine and vincristine. The most potent inhibitor was prostaglandin A1, which reduced efflux by 50% at a concentration of 0.10 microM. Half-maximal inhibition by vincristine occurred at 4.8 microM. The maximum extent of inhibition with most of the inhibitors was 95%, although a lower value was observed with bromosulfophthalein (85%). When cholate efflux was compared with the efflux of methotrexate, both processes responded similarly to changes in the metabolic state of the cell. Moreover, the various inhibitors of cholate efflux also inhibited the efflux of methotrexate and the same concentration of each inhibitor was required for half-maximal inhibition of both processes. The efflux of folate and urate also proceeded via outwardly directed, unidirectional processes which were sensitive to bromosulfophthalein and probenecid. The results suggest that L1210 cells have the capacity for the unidirectional extrusion of cholate, methotrexate and probably other large, structurally dissimilar organic anions and that this efflux occurs via two or more very similar transport systems with a broad anion specificity. The function of an organic anion efflux system in vivo may be to facilitate the extrusion of cytotoxic metabolic anions which are too large to exit via the general anion-exchange carrier of these cells. Similarities in inhibitor specificity were also apparent between unidirectional anion efflux in L1210 cells and the drug efflux pump which is over-produced in cells with multidrug resistance.

Animals↗
PubMed1990

Hemoglobin degradation in the malaria parasite Plasmodium falciparum: an ordered process in a unique organelle.

The malaria parasite Plasmodium falciparum uses host erythrocyte hemoglobin as a major nutrient source. We report the purification of P. falciparum digestive vacuoles and characterization of the degradative process therein. Vacuoles were isolated by a combination of differential centrifugation and density gradient separation. The pure vacuoles were capable of degrading hemoglobin to small fragments with a pH optimum of 5-5.5. Proteolysis in the vacuoles appears to be an ordered process, requiring an aspartic protease to clip intact hemoglobin before other proteolytic activities can function efficiently. The vacuoles do not contain other hydrolases commonly found in lysosomes and therefore appear to be unique proteolytic organelles designed specifically to degrade hemoglobin.

Animals↗
PubMed1990

Folate-binding proteins.

Folate-binding proteins of three major classes have been observed in various bodily fluids and in the plasma membrane and cytoplasm of normal and neoplastic cells. A major class, the high-affinity folate-binding proteins, show a preferential and tight binding of folic acid relative to reduced folates and methotrexate and consist of water-soluble and membrane-associated forms. Soluble forms of the high-affinity binders are present in serum and milk and in the growth medium of certain cultured cell lines, whereas membrane-associated forms are observed on the surface of various cells and tissues. The binders in serum have no clearly defined function, whereas the milk binders serve to accumulate and stabilize reduced-folate compounds in milk and they may also facilitate the absorption of folates by the intestinal mucosa of neonates. Membrane-bound forms of high-affinity folate-binding proteins mediate the transport of folate compounds across plasma membranes and appear to utilize endocytosis as the transport mechanism. Membrane-associated high-affinity binding proteins contain covalently bound phospholipids and hydrophobic C-terminal amino acid sequences that are absent in the soluble forms. The remaining protein portions of these binders show considerable sequence homology. The second class is composed of folate-binding proteins that reside solely in the plasma membrane and are structurally and mechanistically distinct from the high-affinity binders. These proteins function in transport, exhibit varied substrate specificities that accommodate reduced-folate compounds with equal or higher affinity than folate, and do not utilize endocytosis as the mechanism for substrate internalization. The third class of folate-binding proteins consists of enzymes that reside in the cytoplasm of cells.

Animals↗
PubMed1989

Mediation of cellular anion detoxification in leukemic cells by unidirectional efflux pumps.

Methotrexate influx in L1210 cells is mediated almost entirely by a single system, whereas efflux occurs via three routes, the influx carrier functioning in reverse and two additional systems which are operationally unidirectional. The two unidirectional routes show considerable similarities in energy-dependence and inhibitor sensitivities but can be separated by their differential inhibition by bromosulfophthalein (BSP), probenecid, and vincristine. The predominant route is inhibited by BSP and vincristine, whereas the other route is sensitive to probenecid. A search for additional anion substrates for the unidirectional efflux systems for methotrexate led to the finding that cholate exits L1210 cells via the same two unidirectional efflux systems as methotrexate. Cholate efflux is carrier-mediated, energy-dependent, and unidirectional, and it can be inhibited by several compounds which inhibit the efflux of methotrexate. Moreover, the same concentration of each compound which produced a half-maximal inhibition of methotrexate efflux also inhibited the efflux of cholate by 50%. Cholate efflux occurred predominantly (85%) via the BSP-sensitive route. The observation that methotrexate and cholate share the same efflux systems in L1210 cells suggest that perhaps other large anions are also accommodated by these systems since methotrexate and cholate differ in net negative charge and have few common structural features. The hypothesis advanced from these studies has been that the function of the unidirectional efflux systems is to extrude various organic anion catabolites which might otherwise become toxic if allowed to accumulate within cells. Possible intracellular anions in this latter category include sulfonated or carboxylated steroids, bilirubin, and products of vitamin D and prostaglandin catabolism. Unidirectional efflux pumps have been identified for other anionic compounds including cyclic AMP and oxidized glutathione, and in both cases similarities were apparent with the efflux of methotrexate and cholate. The most compelling comparison was with prostaglandin A1 which inhibited the efflux of cyclic AMP, methotrexate, and cholate half-maximally at the same low concentration of 0.1 microM. Energy-dependent unidirectional efflux pumps for large neutral or cationic drugs have also been identified in cells with acquired multidrug resistance. The latter efflux activity is thought to be mediated by a membrane glycoprotein (p170) which is also sensitive to several of the various inhibitors (reserpine, verapamil, and quinidine) which reduce the efflux of methotrexate and cholate.

Animals↗
PubMed1989

Trypanothione is the primary target for arsenical drugs against African trypanosomes.

The trypanosomatid metabolite N1,N8-bis-(glutathionyl)spermidine (trypanothione) has been demonstrated to form a stable adduct with the aromatic arsenical drug melarsen oxide [p-(4,6-diamino-s-triazinyl-2-yl)aminophenyl arsenoxide]. The stability constant of the melarsen-trypanothione adduct (Mel T) has been determined to be 1.05 x 10(7) M-1. When bloodstream Trypanosoma brucei are incubated with either melarsen oxide or the 2,3-dimercaptopropanol adduct of melarsen oxide (melarsoprol), Mel T is the only arsenical derivative detectable in acid-soluble extracts of the cells. Trypanothione may therefore be regarded as a primary target for aromatic arsenical derivatives against African trypanosomes. The selective toxic action of these compounds might arise through sequestration of intracellular trypanothione in the form of Mel T, or Mel T itself may be toxic within the cell. The latter possibility is illustrated by the finding that Mel T is an inhibitor of trypanothione reductase from T. brucei (Ki = 9.0 microM)--an enzyme that is central to the regulation of the thiol/disulfide redox balance in the parasite and absent from the host.

Animals↗
PubMed1988

Identification of the bromosulfophthalein-sensitive efflux route for methotrexate as the site of action of vincristine in the vincristine-dependent enhancement of methotrexate uptake in L1210 cells.

The mechanism by which vincristine enhances the uptake of methotrexate in leukemic L1210 mouse cells has been investigated. Methotrexate uptake after 30 min at 37 degrees C increased 44% relative to untreated controls in cells suspended in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid/bicarbonate-buffered saline medium containing 20 microM vincristine. This stimulation was half-maximal at a vincristine concentration of 4 microM. An enhancement of methotrexate uptake by vincristine was also observed in the presence of glucose but a reversal could be achieved by prior treatment of the cells with taxol, a drug which prevents microtubule disassembly by vincristine. When the effect of vincristine was determined on the individual influx and efflux components for methotrexate, the increased uptake of methotrexate correlated with the inhibition of the unidirectional efflux route for methotrexate that is sensitive to bromosulfophthalein. Inhibition of this route was half-maximal at 3 microM vincristine and it exceeded 90% at high concentrations of the inhibitor. Transport via the bidirectional exchange carrier for methotrexate was not affected by vincristine, while the unidirectional efflux route sensitive to probenecid was inhibited by vincristine but only at concentrations 10-fold higher than required to inhibit the bromosulfophthalein-sensitive route. Vincristine did not increase methotrexate uptake in CCRF-CEM lymphoblasts, a human cell line which contains much lower levels of bromosulfophthalein-sensitive efflux route for methotrexate. Concentrations of vincristine which inhibited the bromosulfophthalein-sensitive efflux route of L1210 cells by 80-90% had little or no effect on the intracellular pH or on intracellular levels of ATP, GTP, cyclic AMP, K+, or oxidized glutathione. A significant increase was observed in the cellular uptake of tetraphenylphosphonium ions, suggesting that vincristine causes a hyperpolarization of the plasma membrane.

Adenosine Triphosphate↗
PubMed1988

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate.

An L1210 cell line (JT-1), which can grow in medium supplemented with 1 nM folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37 degrees C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23 +/- 0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [3H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37 degrees C remained as unmetabolized folic acid. Binding was also rapid at 0 degrees C but uptake at the plateau was only one-half the value obtained at 37 degrees C. Half-maximal saturation of the binding component (KD) occurred at a folate concentration of 0.065 nM at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (KD = 2.0 nM). 5-Methyltetrahydrofolate was also bound by this component (Ki = 13 nM at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (Ki = 45 nM) and methotrexate (Ki = 325 nM). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500 nM caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Physiological↗
PubMed1988

"Subversive" substrates for the enzyme trypanothione disulfide reductase: alternative approach to chemotherapy of Chagas disease.

The trypanosomatid flavoprotein disulfide reductase, trypanothione reductase, is shown to catalyze one-electron reduction of suitably substituted naphthoquinone and nitrofuran derivatives. A number of such compounds have been chemically synthesized, and a structure-activity relationship has been established; the enzyme is most active with compounds that contain basic functional groups in side-chain residues. The reduced products are readily reoxidized by molecular oxygen and thus undergo classical enzyme-catalyzed redox cycling. In addition to their ability to act as substrates for trypanothione reductase, the compounds are also shown to effectively inhibit enzymatic reduction of the enzyme's physiological substrate, trypanothione disulfide. Under aerobic conditions, trypanothione reductase is not inactivated by these redox-cycling substrates, whereas under anaerobic conditions the nitrofuran compounds cause irreversible inactivation of the enzyme. When tested for biological activity against Trypanosoma cruzi trypomastigotes, many of the test compounds were trypanocidal, and this activity correlated with their relative ability to act as substrates for trypanothione reductase. The activity of the enzyme with these redox-cycling derivatives constitutes a subversion of its normal antioxidant role within the cell. For this reason these compounds may be termed "subversive" substrates for trypanothione reductase.

Aerobiosis↗
PubMed1987

Methotrexate efflux in L1210 cells. Kinetic and specificity properties of the efflux system sensitive to bromosulfophthalein and its possible identity with a system which mediates the efflux of 3',5'-cyclic AMP.

Methotrexate exits L1210 mouse leukemia cells via multiple routes that include a unidirectional efflux component which is sensitive to bromosulfophthalein. This efflux component has been characterized in the present study after eliminating the contribution from the other efflux routes by treatment of the cells with an active ester of methotrexate and by reducing the assay pH to 6.2. The remaining efflux at pH 6.2 was greater than 90% sensitive to bromosulfophthalein. This route was also inhibited by probenecid, prostaglandin A1, diamide, 1-methyl-3-isobutylxanthine, various metabolic inhibitors, and by transfer of the cells to a buffer containing high concentrations of KCl. The inhibition by prostaglandin A1 was exceptionally potent and reached 50% at a concentration of 0.5 microM. An enhancement in efflux occurred upon the addition of glucose or by transfer of the cells to a non-saline buffer. When parameters relating to cellular energetics were measured, a reduction in ATP level was associated with the inhibition of efflux by probenecid, carbonylcyanide m-chlorophenylhydrazone, valinomycin, and antimycin A, whereas the increase in efflux by glucose was accompanied by an increase in intracellular ATP. Changes in ATP, however, were not associated with the inhibition by various other compounds or additions or with the enhancement in efflux by the non-anionic buffer. When the relative sensitivity of methotrexate efflux to bromosulfophthalein, 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and lactic anhydride was compared with other anion transport systems, differences in specificity indicated that methotrexate was not exiting the cells via the bicarbonate/chloride exchange carrier, the lactate/H+ co-transport system, or a system which mediates the efflux of phthalate. However, a correlation was apparent between the sensitivity of methotrexate efflux to inhibition by prostaglandin A1, probenecid, and certain metabolic inhibitors and the ability of these same compounds to inhibit the unidirectional efflux of 3',5'-cyclic AMP in other cell lines, suggesting that methotrexate may share a common efflux route with cyclic nucleotides.

Adenosine Triphosphate↗
PubMed1987

Transport of folate compounds by leukemic cells. Evidence for a single influx carrier for methotrexate, 5-methyltetrahydrofolate, and folate in CCRF-CEM human lymphoblasts.

Influx kinetics and inhibitor specificity have been compared for the transport of methotrexate, 5-methyltetrahydrofolate, and folate in CCRF-CEM human lymphoblastoid cells. Influx of each folate compound proceeded with approximately the same Vmax, fluctuated in the same fashion with the ionic composition of the medium, and was blocked by low concentrations of an N-hydroxysuccinimide ester of methotrexate in both an anion-deficient buffer and in a buffered saline medium containing physiological concentrations of glucose and bicarbonate. Moreover, methotrexate influx was inhibited by 5-methyltetrahydrofolate and folate, and the inhibition constants (Ki) of the latter compounds were equivalent to their Kt values for half-maximal influx. Folate influx was likewise inhibited by methotrexate. The Ki for methotrexate was equivalent to its Kt for influx, and o-phthalate and phosphate each inhibited folate and methotrexate with the same degree of effectiveness. Various reversible and irreversible inhibitors reduced the influx of each folate substrate by greater than 90%, and the progression of inhibition in each case was indicative of a single uptake component. Folate influx exhibited the same high sensitivity to inhibitors of methotrexate influx when measurements were performed at folate concentrations near the Kt for influx (10-50 microM) or at concentrations approximating physiological conditions (5-20 nM). These results indicate that CCRF-CEM cells possess a single shared transport system for the uptake of methotrexate, 5-methyltetrahydrofolate, and folate and that other high- or low-affinity uptake processes are not present in these cells.

Biological Transport↗
PubMed1987

Substrate specificity of the flavoprotein trypanothione disulfide reductase from Crithidia fasciculata.

The substrate specificity of the trypanosomatid enzyme trypanothione reductase has been studied by measuring the ability of the enzyme to reduce a series of chemically synthesized cyclic and acyclic derivatives of N1,N8-bis(glutathionyl)spermidine disulfide (trypanothione). Kinetic analysis of the enzymatic reduction of these synthetic substrates indicates that the mutually exclusive substrate specificity observed by the NADPH-dependent trypanothione disulfide reductase and the related flavoprotein glutathione disulfide reductase is due to the presence of a spermidine binding site in the substrate binding domain of trypanothione reductase. Trypanothione reductase will reduce the disulfide form of N1-monoglutathionylspermidine and also the mixed disulfide of N1-monoglutathionylspermidine and glutathione. The Michaelis constants for these reactions are 149 microM and 379 microM, respectively. Since the disulfide form of N1-monoglutathionylspermidine and the mixed disulfide of N1-monoglutathionylspermidine and glutathione could be formed in trypanosomatids, the binding constants and turnover numbers for the enzymatic reduction of these acyclic disulfides are consistent with these being potential alternative substrates for trypanothione reductase in vivo.

Animals↗
PubMed1987

Folate transport in Lactobacillus salivarius. Characterization of the transport mechanism and purification and properties of the binding component.

Lactobacillus salivarius cells contain an inducible transport system for folate. Influx via this system is time- and temperature-dependent, requires glucose and glutamine for optimum activity, and is half-maximal at folate concentrations in the nanomolar range. The folate internalized after 30 min at 30 degrees C is not released from the cells by excess extracellular folate and is recovered in cell extracts primarily in metabolized forms. A membrane-associated folate-binding protein is also present in cells that have been induced to transport folate. This binding protein constitutes 1% of total cellular protein, exhibits a high affinity for folate (KD = 0.40 nM), and requires divalent cations for optimum binding activity. Folate binds rapidly to this protein, while the exchange of bound substrate with folate added subsequently is relatively slow and dependent on the metabolic state of the cell. The transport rate per binding site is 0.05/min at 30 degrees C. A comparison of substrate specificity showed that folate binding and transport are both inhibited to the same extent by several different folate compounds, and a parallel irreversible inhibition of both processes is observed by prior treatment of the cells with a carbodiimide-activated derivative of folic acid. Binding protein labeled covalently with [3H]folate and solubilized with Triton X-100 was purified by a fractionation procedure involving absorption and elution from microgranular silica and molecular sieve chromatography. The isolated protein appeared homogeneous by gel electrophoresis and had an apparent molecular weight of 21,000. Monoclonal antibodies to the folate transport protein of Lactobacillus casei showed a high degree of cross-reactivity to the isolated binding protein from L. salivarius, indicating that these proteins share common epitopes. These results suggest that folate uptake by L. salivarius proceeds via an abundant membrane-associated binding protein which facilitates the movement of folate across the membrane as an electroneutral complex with cations. The substrate then slowly dissociates from internalized binding sites and is metabolized sequentially to coenzyme forms and then to membrane-impermeable folylpolyglutamates.

Biological Transport↗