Effect of phenylmercury derivatives on human oxyhemoglobin

By the dynamics of human oxyhemoglobin coagulation in the presence of phenyl mercury acetate in tris-AcOH buffer, pH 7.2 the number of moles of PhHg+ stechiometrically bound with protein at different temperatures was estimated. Within the temperature range 15-30 degrees C this value is constant--32-34 mole per 1 mole of HBO2-tetramer. Within the range 30-40 degrees C it rises to approximately 40. Coagulation of oxyhemoglobin modified with PhHg+ cation is reversible in contrast to HBO2 coagulation modified with uncharged PhHgCl.     

The aggregative stability of human oxyhemoglobin in aqueous media in the presence of mercury(II) compounds]

It was shown that the mechanism regulating the oxyhemoglobin coagulation in presence of a mercury reagent in large amount differs from that in presence of the reagent in relatively small concentrations. The significance of a large class of ligands at mercury atom during the oxyhemoglobin coagulation was demonstrated. Several theoretical generalizations are drawn.     

The coagulation characteristics of human oxyhemoglobin in the presence of a mercury (II) ion in a neutral phosphate buffer

The kinetics of human oxyhemoglobin coagulation in neutral phosphate buffer in the presence of mercury acetate at 20 degrees has been studied using turbidimetric methods. The addition of small amounts of concentrated Hg2+ solution leads to rapid local protein coagulation with subsequent dissolution of the formed coagulate. Coagulation can be inhibited by addition of Tris that binds to mercury ions. The pattern of oxyhemoglobin coagulation is determined by molar Hg2+/protein ration rather than by total Hg2+ concentration.     

Compartmentation and turnover of the low density lipoprotein receptor in skin fibroblasts

The low density lipoprotein receptor (LDLR) was immunoprecipitated from [35S]methionine-labeled skin fibroblasts derivatized at 4 or 18 degrees C with an impermeant biotinylating reagent. Separation of derivatized and underivatized receptor from immunoprecipitates by selective binding to streptavidin-agarose allowed assessment of receptor protein cellular compartmentation and rates of intercompartmental transfer. At both 4 and 18 degrees C the amount of LDLR that is derivatized in cells labeled to near steady state saturates after 1-2 h of reaction at, respectively, 47 and 70% of total immunoprecipitable receptor protein. On the basis of temperature titration experiments, protein exposed only to the cell surface reacts at 4 degrees C; raising the temperature of biotinylation to 18 degrees C provides access to an additional pool of receptor protein. Remaining LDLR is derivatized at 37 degrees C. LDLR unreactive at 18 degrees C largely resides in membrane compartment(s) devoid of plasma membrane on the basis of its fractionation on Percoll gradients. While total cellular LDLR and 4 degrees C-derivatized LDLR labeled to steady state turn over in a first order manner (t1/2 = 12-13 h), the specific activity of pulse-labeled, 4 degrees C-accessible protein peaks after 1-2 h of chase and reaches a reduced level by 3 h of chase. These latter results show that the newly synthesized LDLR is transiently enriched at the cell surface prior to achieving equilibrium distribution between the cell surface and intracellular pools.     

Temperature-induced changes in the flexibility of the loop between SH1 (Cys-707) and SH3 (Cys-522) in myosin subfragment 1 detected by cross-linking

The ability of dibromobimane to cross-link SH1 (Cys-707) in the 21-kDa C-terminal segment to SH3 (Cys-522) in the 50-kDa middle segment of the myosin S1 heavy chain has been examined as a function of nucleotide binding and temperature. The results obtained indicate that, while the reagent rapidly reacts with SH1 at both 25 and 4 degrees C, its ability to cross-link to SH3 is highly dependent on temperature. At 25 degrees C, substantial cross-linking from monofunctionally labeled SH1 to SH3 occurs, in agreement with recent work of Mornet, Ue, and Morales (1985, Proc. Natl. Acad. Sci, USA 82, 1658-1662) and of Ue (1987, Biochemistry 26, 1889-1894) and with their conclusion that a loop, allowing SH1 and SH3 to reside at the cross-linking span of dibromobimane, preexists in the protein. At 4 degrees C, however, negligible amounts of cross-linking are observed whether or not a nucleotide is present, despite indications that SH1 is labeled rapidly by the reagent at this temperature. The inability to form this cross-link is not due to an alternate cross-link between monofunctionally labeled SH1 and another thiol in the 21-kDa segment. These results indicate that this loop exists at 25 degrees C and does not exist (or exists only transiently) at the lower temperature.     

The aggregative stability of human oxyhemoglobin in aqueous media in the presence of mercury(II) compounds]

An analytical review of studies on human oxyhemoglobin coagulation has been performed by the author jointly with V. S. Koniaeva and L. D. Bogdanova within a period from 1985 to 1990. It was shown that the oxyhemoglobin coagulation modified by mercurials proceeded without any essential alteration of native protein conformation. A hypothesis is discussed that the oxyhemoglobin coagulation results from the primary polyaggregation of dimer fragments and that hydrophobic sites which provide for dimer-to-dimer contacts in native tetrameric oxyhemoglobin, participate in this process.     

Calorimetry of apolipoprotein-A1 binding to phosphatidylcholine-triolein-cholesterol emulsions.

The thermotropic properties of triolein-rich, low-cholesterol dipalmitoyl phosphatidylcholine (DPPC) emulsion particles with well-defined chemical compositions (approximately 88% triolein, 1% cholesterol, 11% diacyl phosphatidylcholine) and particle size distributions (mean diameter, approximately 1000-1100 A) were studied in the absence and presence of apolipoprotein-A1 by a combination of differential scanning and titration calorimetry. The results are compared to egg yolk PC emulsions of similar composition and size. Isothermal titration calorimetry at 30 degrees C was used to saturate the emulsion surface with apo-A1 and rapidly quantitate the binding constants (affinity Ka = 11.1 +/- 3.5 x 10(6) M-1 and capacity N = 1.0 +/- 0.09 apo-A1 per 1000 DPPC) and heats of binding (enthalpy H = -940 +/- 35 kcal mol-1 apo-A1 or -0.92 +/- 0.12 kcal mol-1 DPPC). The entropy of association is -3070 cal deg-1 mol-1 protein or -3 cal deg-1 mol-1 DPPC. Without protein on the surface, the differential scanning calorimetry heating curve of the emulsion showed three endothermic transitions at 24.3 degrees C, 33.0 degrees C, and 40.0 degrees C with a combined enthalpy of 1.53 +/- 0.2 kcal mol-1 DPPC. With apo-A1 on the surface, the heating curve showed the three transitions more clearly, in particular, the second transition became more prominent by significant increases in both the calorimetric and Van't Hoff enthalpies. The combined enthalpy was 2.70 +/- 0.12 kcal mol-1 DPPC and remained constant upon repeated heating and cooling. Indicating that the newly formed DPPC emulsion-Apo-A1 complex is thermally reversible during calorimetry. Thus there is an increase in delta H of 1.17 kcal mol-1 DPPC after apo-A1 is bound, which is roughly balanced by the heat released during binding (-0.92 kcal) of apo-A1. The melting entropy increase, +3.8 cal deg-1 mol-1 DPPC of the three transitions after apo-A1 binds, also roughly balances the entropy (-3 cal deg-1 mol-1 DPPC) of association of apo-A1. These changes indicate that apo-A1 increases the amount of ordered gel-like phase on the surface of DPPC emulsions when added at 30 degrees C. From the stoichiometry of the emulsions we calculate that the mean area of DPPC at the triolein/DPPC interface is 54.5 A2 at 41 degrees C and 54.2 A2 at 30 degrees C. The binding of apo-A1 at 30 degrees C to the emulsion reduces the surface area per DPPC molecule from 54.2 A2 to 50.8 A2. At 30 degrees apo-A1 binds with high affinity and low capacity to the surface of DPPC emulsions and increases the packing density of the lipid domain to which it binds. Apo-A1 was also titrated onto DPPC emulsions at 45 degrees C. This temperature is above the gel liquid crystal transition. No heat was released or adsorbed. Furthermore, egg yolk phosphatidylcholine emulsions of nearly identical composition were also titrated at 30 degrees C with apo-A1 and were euthermic. Association constants were previously measured using a classical centrifugation assay and were used to calculate the entropy of apo-A1 binding (+28 cal deg-1 mol-1 apo-A1). This value indicates that apo-A1 binding to a fluid surface like egg yolk phosphatidylcholine or probably DPPC at 45 degrees C is hydrophobic and is consistent with hydrocarbon lipid or protein moities coming together and excluding water. Thus the binding of apo-A1 to partly crystalline surfaces is entropically negative and increases the order of the already partly ordered phases, whereas binding to liquid surfaces is mainly an entropically driven hydrophobic process.     

Characterization of an adenosine 3'':5''-cyclic monophosphate phosphodiesterase from baker''s yeast. Its binding to subcellular particles, catalytic properties and gel-filtration behaviour.

1. The 3':5'-cyclic AMP phosphodiesterase in the microsomal fraction of baker's yeast is highly specific for cyclic AMP, and not inhibited by cyclic GMP, cyclic IMP or cyclic UMP. Catalytic activity is abolished by 30 micrometer-EDTA. At 30 degrees C and pH8.1, the Km is 0.17 micrometer, and theophylline is a simple competitive inhibitor with Ki 0.7 micrometer. The pH optimum is about 7.8 at 0.25 micrometer-cyclic AMP, so that over the physiological range of pH in yeast the activity changes in the opposite direction to that of adenylate cyclase [PH optimum about 6.2; Londesborough & Nurminen (1972) Acta Chem. Scand. 26, 3396-3398].2. At pH 7.2, dissociation of the enzyme from dilute microsomal suspensions increased with ionic strength and was almost complete at 0.3 M-KCl. MgCl2 caused more dissociation than did KCl or NaCl at the same ionic strength, but at low KCl concentrations binding required small amounts of free bivalent metal ions. In 0.1 M-KCl the binding decreased between pH 4.7 and 9.3. At pH 7.2 the binding was independent of temperature between 5 and 20 degrees C. These observations suggest that the binding is electrostatic rather than hydrophobic. 3. The proportion of bound activity increased with the concentration of the microsomal fraction, and at 22 mg of protein/ml and pH 7.2 was 70% at I0.18, and 35% at I0.26. Presumably a substantial amount of the enzyme is particle-bound in vivo. 4. At 5 degrees C in 10 mM-potassium phosphate, pH 7.2, the apparent molecular weight of KCl-solubilized enzyme decreased with enzyme concentration from about 200 000 to 40 000. In the presence of 0.5M-KCl, a constant mol.wt. of about 55 000 was observed over a 20-fold range of enzyme concentrations.     

Temperature-dependent switching between "wild-type" and "mutant" forms of p53-Val135

The p53 gene is a suppressor of abnormal cell growth but is also subject to oncogenic activation by mutation. The mutant allele p53-Val135, has recently been discovered to be temperature-sensitive and functions as an oncogene at 37 degrees C and as a tumor suppressor at 32.5 degrees C. In order to investigate the molecular mechanism underlying the temperature sensitivity of p53-Val135 rabbit reticulocyte lysate was used to translate the p53 mRNAs in vitro at 37 degrees C and at 30 degrees C. The immunoreactivity and T antigen binding of wild-type protein p53-Ala135 were unaffected by temperature and were similar to wild-type p53 expressed in vivo. In contrast, the mutant p53-Val135 protein was markedly affected by temperature. At 37 degrees C p53-Val135 showed reduced T antigen binding and did not react with monoclonal antibodies PAb246 and PAb1620. At 30 degrees C, p53-Val135 behaved as the wild-type p53. Temperature also exerted a post-translational effect on p53-Val135 with complete conversion from wild-type to mutant phenotype within two minutes of temperature shift from 30 degrees C to 37 degrees C. There was incomplete conversion from mutant to wild-type phenotype when the temperature was shifted down from 37 degrees C to 30 degrees C. We propose that the temperature dependent forms of p53-Val135 represent conformational variants of the p53 protein with opposing functions in cell growth control.     

Binding of human factor VIII to phospholipid vesicles

Factor VIII, a protein cofactor involved in blood coagulation, functions in vitro on a phospholipid membrane surface to greatly increase the rate of factor X activation by factor IXa. Using gel filtration, rapid sedimentation, and resonance energy transfer we have studied the interaction of recombinant-derived human factor VIII with small and large unilamellar phospholipid vesicles composed of phosphatidylserine and phosphatidylcholine. Resonance energy transfer, from intrinsic fluorophores in factor VIII to dansyl-phosphatidylethanolamine incorporated into vesicles, has been adapted for quantitative equilibrium measurements. Factor VIII binds rapidly and reversibly to small and large vesicles. At 8 degrees C the interaction of factor VIII with small vesicles fits a simple bimolecular model with a KD of 2 nM and a phospholipid binding site defined by 180 phospholipid monomers. At 25 degrees C the binding of factor VIII to small vesicles containing 20% phosphatidylserine can be described by an apparent KD of 4 nM; the phospholipid/protein ratio at saturation was 170. Binding to large vesicles was demonstrated with a KD of 2 nM and a phospholipid/protein ratio at saturation of 385. Binding was dependent upon the phosphatidylserine mole fraction and was nonlinear from 0 to 30% phosphatidylserine content. A direct comparison of factor VIII and factor V binding indicated that the affinity of factor V to phospholipid vesicles was equivalent to that of factor VIII and that the phosphatidylserine requirement was lower. A model is proposed to explain the nonlinear phosphatidylserine dependence of binding for factor VIII.     

Unmasking of GDP binding sites on hamster brown adipose tissue mitochondria and uncoupling protein.

1. A rapid unmasking of GDP binding sites on brown adipose tissue (BAT) mitochondria was observed when hamsters acclimatized to 28 degrees C were exposed to a temperature of 4 degrees C for 2 hr. 2. No rapid unmasking of GDP binding sites was observed when hamsters housed at 22 degrees C were briefly exposed to 4 degrees C. 3. The amount of GDP bound to BAT mitochondria from hamsters increased during 2 weeks of exposure to 4 degrees C, but did not change between 2 weeks and 30 days of cold exposure. 4. Incubation of mitochondria with 10 mM Mg2+ prior to the GDP binding assay increased the subsequent GDP binding to BAT mitochondria from hamsters housed at 28, 22 or 4 degrees C, albeit to different degrees. 5. The amount of GDP bound to uncoupling proteins isolated from untreated and Mg(2+)-treated mitochondria of hamsters and rats was measured. Scatchard analyses of the binding of GDP to purified uncoupling protein indicate that increases in the number of binding sites due to Mg2+ treatment of mitochondria do not change the affinity of the protein for GDP.     

Abscisic acid-induced heat tolerance in Bromus inermis Leyss cell-suspension cultures. Heat-stable, abscisic acid-responsive polypeptides in combination with sucrose confer enhanced thermostability.

Increased heat tolerance is most often associated with the synthesis of heat-shock proteins following pre-exposure to a nonlethal heat treatment. In this study, a bromegrass (Bromus inermis Leyss cv Manchar) cell suspension cultured in a medium containing 75 microM abscisic acid (ABA) without prior heat treatment had a 87% survival rate, as determined by regrowth analysis, following exposure to 42.5 degrees C for 120 min. In contrast, less than 1% of the control cells survived this heat treatment. The heat tolerance provided by treatment with 75 microM ABA was first evidenced after 4 d of culture and reached a maximum tolerance after 11 d of culture. Preincubation with sucrose partially increased the heat tolerance of control cells and rendered ABA-treated cells tolerant to 45 degrees C for 120 min (a completely lethal heat treatment for control cells). Comparative two-dimensional polyacrylamide gel electrophoresis of cellular protein isolated from heat-tolerant cells identified 43 ABA-responsive proteins of which 26 were heat stable (did not coagulate and remained soluble after 30 min at 90 degrees C). Eight heat-stable, ABA-responsive proteins ranging from 23 to 45 kD had similar N-terminal sequences. The ABA-responsive (43-20 kD), but none of the control heat-stable, proteins cross-reacted to varying degrees with a polyclonal antibody directed against a conserved, lysine-rich dehydrin sequence. A group of 20- to 30-kD heat-stable, ABA-responsive proteins cross-reacted with both the anti-dehydrin antibody and an antibody directed against a cold-responsive winter wheat protein (Wcs 120). In ABA-treated cells, there was a positive correlation between heat- and pH-induced coagulation of a cell-free homogenate and the heat tolerance of these cells. At 50 degrees C, control homogenates coagulated after 8 min, whereas cellular fractions from ABA-treated cells showed only marginal coagulation after 15 min. In protection assays, addition of heat-stable, ABA-responsive polypeptides to control fractions reduced the heat-induced coagulation of cell-free homogenates. Sucrose (8%) alone and control, heat-stable fractions enhanced the thermostability of control fractions, but the most protection was conferred by ABA-responsive, heat-stable proteins in combination with sucrose. These data suggest that stress-tolerance mechanisms may develop as a result of cooperative interactions between stress proteins and cell osmolytes, e.g. sucrose. Hypotheses are discussed implicating the role of these proteins and osmolytes in preventing coagulation and denaturation of cellular proteins and membranes.     

Binding of RNA by the alfalfa mosaic virus movement protein is biphasic.

The movement protein of alfalfa mosaic virus was expressed in Escherichia coli and purified by cation exchange chromatography. The purified protein bound single-stranded RNA cooperatively in a biphasic manner. At protein saturation, RNA/protein complexes (designated 'primary complexes') were detected by a nitrocellulose-retention assay within 1 min of mixing, both at 4 and 22 degrees C. In contrast, an incubation of 30 min at 22 degrees C was necessary to obtain electrophoretically retarded complexes ('stabilized complexes'), containing a large number of protein molecules bound stably to each molecule of RNA. Stabilization did not take place at 4 degrees C. The rate of formation of the primary complexes was strongly dependent on protein concentration, and thus appeared limited by a bimolecular interaction. In contrast, the rate of stabilization was independent of protein concentration, suggesting that this process consisted of a rearrangement of the primary complexes without binding of additional protein molecules. In agreement with this suggestion, the amount of complexed RNA at equilibrium was the same when assayed by nitrocellulose retention and by electrophoretic retardation. The possibility that these peculiar kinetics could be caused by the presence of Tween 20 in the incubation media is discussed.     

Metabolism of ochratoxin A by primary cultures of rat hepatocytes.

Association of ochratoxin A with cultured rat hepatocytes occurs at 4 degrees C, and the saturation level in the medium is 0.3 mM ochratoxin A, with maximal binding after 60 min. At 37 degrees C the level of cell-associated ochratoxin A increased up to 6 h and remained at 2 nmol of toxin per mg of cell protein for 30 h. With increasing concentrations of ochratoxin A, increasing amounts of the toxin accumulated in the cells; saturation occurred at a concentration of 0.3 mM. Ochratoxin A was metabolized by hepatocytes at 37 degrees. (4R)-4-Hydroxyochratoxin A appeared in the medium at a maximal level (about 30 nmol/mg of cell protein) at an ochratoxin A concentration of 0.25 mM after 48 h of incubation. Small amounts of (4S)-4-hydroxyochratoxin A were detected only after incubation for 22 h or longer.     

Enthalpic barriers to the hydrophobic binding of oligosaccharides to phage P22 tailspike protein

The structural thermodynamics of the recognition of complex carbohydrates by proteins are not well understood. The recognition of O-antigen polysaccharide by phage P22 tailspike protein is a highly suitable model for advancing knowledge in this field. The binding to octa- and dodecasaccharides derived from Salmonella enteritidis O-antigen was studied by isothermal titration calorimetry and stopped-flow spectrofluorimetry. At room temperature, the binding reaction is enthalpically driven with an unfavorable change in entropy. A large change of -1.8 +/- 0.2 kJ mol(-1) K(-1) in heat capacity suggests that the hydrophobic effect and water reorganization contribute substantially to complex formation. As expected from the large heat-capacity change, we found enthalpy-entropy compensation. The calorimetrically measured binding enthalpies were identical within error to van't Hoff enthalpies determined from fluorescence titrations. Binding kinetics were determined at temperatures ranging from 10 to 30 degrees C. The second-order association rate constant varied from 1 x 10(5) M(-1) s(-1) for dodecasaccharide at 10 degrees C to 7 x 10(5) M(-1) s(-1) for octasaccharide at 30 degrees C. The first-order dissociation rate constants ranged from 0.2 to 3.8 s(-1). The Arrhenius activation energies were close to 50 and 100 kJ mol(-1) for the association and dissociation reactions, respectively, indicating mainly enthalpic barriers. Despite the fact that this system is quite complex due to the flexibility of the saccharide, both the thermodynamic and kinetic data are compatible with a simple one-step binding model.     

Receptor-mediated endocytosis of a ricin-colloidal gold conjugate in vero cells. Intracellular routing to vacuolar and tubulo-vesicular portions of the endosomal system

We have prepared a conjugate (Ri-Au) of the toxic plant protein ricin and colloidal gold (particle size 5 nm) and used it for internalization studies in monolayer cultures of Vero cells. The Ri-Au conjugate was very stable, with only little release of ricin ([125I]Ri) from the gold particles within a pH range of 4.5-8.0. Within 2 h at 37 degrees C, only very little intracellular degradation of the ricin preparation ([125I]Ri-Au) occurred. The cells bound the same proportion of native ricin ([125I]Ri) and Ri-Au from the medium, and the kinetics of toxicity (decrease in cellular incorporation of [3H]leucine) of [125I]Ri and [125I]Ri-Au were also comparable. At 4 degrees C, the cell-surface binding of Ri-Au was continuous and distinct, as revealed by electron microscopy. This binding was specific, since almost no Ri-Au surface binding occurred at 4 degrees C in the presence of 0.1 M lactose or 1 mg/ml native (unlabelled) ricin. Within the first 30 min of warming prelabelled cells to 37 degrees C, the amount of surface-associated Ri-Au decreased considerably (from 150 to 60 gold particles per micron cell surface in 40 nm sections). Coated pits and vesicles were involved in the internalization of Ri-Au, and within 5-30 min at 37 degrees C Ri-Au had been delivered to vacuolar and tubulo-vesicular portions of the endosomal system, and later also to lysosomes. Analysis of very thin (ca 20 nm) serial sections revealed that most of the tubulo-vesicular elements were separate structures not connected to the membrane of the vacuolar portion. Data here presented indicate that our ricin conjugate, like many "physiological' ligands and viruses, is internalized by receptor-mediated endocytosis via the coated pit-endosomal pathway.     

Fluorescent glucagon derivatives. II. The use of fluorescent glucagon derivatives for the study of receptor disposition in membranes

When monolayer cultured hepatocytes were incubated with 1 nM [125I]glucagon at 30 degrees C, equilibrium was reached after 10 min, whereas at 4 degrees C, equilibrium was reached after 60 min. At the higher temperature, 11.2% of the bound ligand was broken down after 60 min, at the lower temperature, the amount of degradation was negligible. At 30 degrees C, acid-washing did not remove specifically bound ligand; thus, it was assumed that the ligand was internalised at this temperature, since some of the specifically bound ligand could be washed off at lower temperatures. This was confirmed in experiments when monolayer cultures of hepatocytes were incubated with fluorescein-labelled derivatives of glucagon. The distribution of specific binding on the cell surface was studied at both 30 and 4 degrees C using video intensification microscopic techniques. In keeping with studies using radiolabelled glucagon, more fluorescence was detected following incubation at 4 degrees C than at 30 degrees C and it could be removed by washing the cells. Video intensification microscopy indicated that at the lower temperature, the bound ligand was distributed all over the cell surface. At the higher temperature, ligand-derived fluorescence could only be detected in mobile intracellular vesicles.     

Separation of the presynaptic and synaptic phases of homologous pairing promoted by recA protein

Homologous pairing of single strands with duplex DNA promoted by recA protein occurred without a lag only when the protein was preincubated with ATP and single-stranded DNA. The rate-limiting presynaptic interaction of recA protein and single strands showed a high temperature coefficient: it proceeded 30 times more slowly at 30 degrees C than at 37 degrees C, whereas synapsis showed a normal temperature coefficient. Thus, the presynaptic phase could be separated experimentally from the rest of the reaction by preincubation of single strands with recA protein and ATP at 37 degrees C, followed by a shift to 30 degrees C before double-stranded DNA was added. The presynaptic phase was an order of magnitude more sensitive to inhibition by ADP than was subsequent strand exchange. Presynaptic complexes that were formed at 37 degrees C decayed only slowly at 30 degrees C, but Escherichia coli single strand binding protein caused complexes to form rapidly at 30 degrees C which indicates that single strand binding protein accelerated the rate of formation of complexes. Preincubation synchronized the initial pairing reaction, and further revealed the rapid formation of nascent heteroduplex DNA 250-300 base pairs in length.     

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)