The role of the anion-binding site of transferrin in its interaction with the reticulocyte

Specific and tight binding of Fe(III) by transferrin does not occur unless a suitable anion is concomitantly bound. Bicarbonate, which normally occupies the anion binding site of the protein, may be replaced by an oxalate ion. The resulting ternary complex of Fe(III), transferrin and oxalate is less than 35% as effective as the bicarbonate complex in serving as an iron donor for heme synthesis by the reticulocyte. However, the binding of transferrin to the reticulocyte is not altered by the substitution of oxalate for bicarbonate. When both the oxalate and bicarbonate forms are incubated with reticulocytes, the uptake of iron from the bicarbonate complex is substantially depressed. The free oxalate ion, at the same concentration as the ternary Fe-transferrin-oxalate complex, does not alter the uptake of iron by reticulocytes from the native form of transferrin. The ternary Fe-transferrin-malonate complex is also less efficient than the bicarbonate complex as an iron donor to the reticulocyte, but the effect is less striking than that observed with the oxalate complex. The hypothesis is advanced that the mechanism of iron uptake from transferrin during the transferrin-reticulocyte interaction first entails an attack upon the anion bound to the protein, following which iron release to the heme-synthesizing apparatus of the cell takes place.     

Ion binding properties of the dehydrin ERD14 are dependent upon phosphorylation

The ERD14 protein (early response to dehydration) is a member of the dehydrin family of proteins which accumulate in response to dehydration-related environmental stresses. Here we show the Arabidopsis dehydrin, ERD14, possesses ion binding properties. ERD14 is an in vitro substrate of casein kinase II; the phosphorylation resulting both in a shift in apparent molecular mass on SDS-PAGE gels and increased calcium binding activity. The phosphorylated protein bound significantly more calcium than the nonphosphorylated protein, with a dissociation constant of 120 microm and 2.86 mol of calcium bound per mol of protein. ERD14 is phosphorylated by extracts of cold-treated tissues, suggesting that the phosphorylation status of this protein might be modulated by cold-regulated kinases or phosphatases. Calcium binding properties of ERD14 purified from Arabidopsis extracts were comparable with phosphorylated Escherichia coli-expressed ERD14. Approximately 2 mol of phosphate were incorporated per mol of ERD14, indicating a minimum of two phosphorylation sites. Western blot analyses confirmed that threonine and serine are possible phosphorylation sites on ERD14. Utilizing matrix assisted laser desorption ionization-time of flight/mass spectrometry we identified five phosphorylated peptides that were present in both in vivo and in vitro phosphorylated ERD14. Our results suggest that the polyserine (S) domain is most likely the site of phosphorylation in ERD14 responsible for the activation of calcium binding.     

High resolution structure of an alternate form of the ferric ion binding protein from Haemophilus influenzae

The periplasmic iron binding protein of pathogenic Gram-negative bacteria performs an essential role in iron acquisition from transferrin and other iron sources. Structural analysis of this protein from Haemophilus influenzae identified four amino acids that ligand the bound iron: His(9), Glu(57), Tyr(195), and Tyr(196). A phosphate provides an additional ligand, and the presence of a water molecule is required to complete the octahedral geometry for stable iron binding. We report the 1.14-A resolution crystal structure of the iron-loaded form of the H. influenzae periplasmic ferric ion binding protein (FbpA) mutant H9Q. This protein was produced in the periplasm of Escherichia coli and, after purification and conversion to the apo form, was iron-loaded. H9Q is able to bind ferric iron in an open conformation. A surprising finding in the present high resolution structure is the presence of EDTA located at the previously determined anion ternary binding site, where phosphate is located in the wild type holo and apo structures. EDTA contributes four of the six coordinating ligands for iron, with two Tyr residues, 195 and 196, completing the coordination. This is the first example of a metal binding protein with a bound metal.EDTA complex. The results suggest that FbpA may have the ability to bind and transport iron bound to biological chelators, in addition to bare ferric iron.     

A periplasmic iron-binding protein contributes toward inward copper supply

Periplasmic substrate binding proteins are known for iron, zinc, manganese, nickel, and molybdenum but not copper. Synechocystis PCC 6803 requires copper for thylakoid-localized plastocyanin and cytochrome oxidase. Here we show that mutants deficient in a periplasmic substrate binding protein FutA2 have low cytochrome oxidase activity and produce cytochrome c6 when grown under copper conditions (150 nm) in which wild-type cells use plastocyanin rather than cytochrome c6. Anaerobic separation of extracts by two-dimensional native liquid chromatography followed by metal analysis and peptide mass-fingerprinting establish that accumulation of copper-plastocyanin is impaired, but iron-ferredoxin is unaffected in DeltafutA2 grown in 150 nm copper. However, recombinant FutA2 binds iron in preference to copper in vitro with an apparent Fe(III) affinity similar to that of its paralog FutA1, the principal substrate binding protein for iron import. FutA2 is also associated with iron and not copper in periplasm extracts, and this Fe(III)-protein complex is absent in DeltafutA2. There are differences in the soluble protein and small-molecule complexes of copper and iron, and the total amount of both elements increases in periplasm extracts of DeltafutA2 relative to wild type. Changes in periplasm protein and small-molecule complexes for other metals are also observed in DeltafutA2. It is proposed that FutA2 contributes to metal partitioning in the periplasm by sequestering Fe(III), which limits aberrant Fe(III) associations with vital binding sites for other metals, including copper.     

ELECTRON STAINS : I. Chemical Studies on the Interaction of DNA with Uranyl Salts

Chemical studies have been carried out on the interaction of DNA with uranyl salts. The effect of variations in pH, salt concentration, and structural integrity of the DNA on the stoichiometry of the salt-substrate complex have been investigated. At pH 3.5 DNA interacts with uranyl ions in low concentration yielding a substrate metal ion complex with a UO₂⁺⁺/P mole ratio of about ½ and having a large association constant. At low pH's (about 2.3) the mole ratio decreases to about ⅓. Destruction of the structural integrity of the DNA by heating in HCHO solutions leads to a similar drop in the amount of metal ion bound. Raising the pH above 3.5 leads to an apparent increase in binding as does increasing the concentration of the salt solution. This additional binding has a lower association constant. Under similar conditions DNA binds about seven times more uranyl ion than bovine serum albumin, indicating useful selectivity in staining for electron microscopy.     

High pressure enzyme kinetics of dextransucrase.

The pressure dependence of enzymatic dextran formation has been observed up to 1000 at for several substrate concentrations. First order denaturation effects could be separated from the thermodynamic effects, which lead to a volume of 30.4 to 44.0 ccm per mole for the formation and -13.6ccm per mole for the activation of the enzyme-substrate complex. Denaturation depends on the substrate concentration. This leads to the conslusion that only the free enzyme is denatured, wheras the ES complex is stable.     

Trinitrophenyl-ATP binding to the ArsA protein: the catalytic subunit of an anion pump.

The ars operon of the conjugative R-factor R773 confers resistance to arsenicals by coding for an anion pump for extrusion of arsenicals from cells of Escherichia coli. Extrusion of arsenite requires only two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein exhibits oxyanion-stimulated ATPase activity and has been shown to bind ATP by photoaffinity labeling with [alpha-32P]ATP. From sequence analysis the ArsA protein is predicted to have two nucleotide binding folds, one in the N-terminal half and one in the C-terminal half of the protein. Purified ArsA protein bound a fluorescent ATP analogue, 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidene)adenosine- 5'-triphosphate, with an apparent stoichiometry of 2 mol of nucleotide per mole of ArsA. Strains expressing plasmids with mutations in the N-terminal consensus nucleotide sequence bound only 1 mol of nucleotide per mole of protein.     

Desensitization of substrate inhibition of acto-H-meromyosin ATPase by treatment of H-meromyosin with rho-chloromercuribenzoate. Relation between the extent of desensitization and the amount of bound rho-chloromercuribenzoate1.

H-Meromyosin (CMB leads to betaME-H-meromyosin) was prepared by tryptic digestion of myosin, which had been treated with CMB bound to H-meromyosin and the extent of desensitization of the substrate inhibition of acto-H-meromyosin ATPase [EC 3.6.1.3.] was investigated. Both the dissociation of acto-H-meromyosin induced by ATP and substrate inhibition decreased with increase in the amount of bound CMB to a minimum value at about 1 mole of CMB bound per mole of H-meromyosin. The substrate inhibition of acto-H-meromyosin ATPase was restored to the original level by complete removal of the bound CMB by further treatment of CMB leads to beta ME-H-meromyosin with a large excess of beta-mercaptoethanol. The dissociation constant of acto-H-meromyosin in the presence of ATP decreased markedly on modification with CMB, while the maximum ATPase activity ar a sufficiently high concentration of F-actin remained essentially unchanged. Acto-H-meromyosin was reconstituted from F-actin and CMB LEADS TO beta ME-H-meromyosin, containing less than the stoichiometric amount of bound CMB. Its ATPase activity and the extent of dissociation of acto-H-meromyosin induced by ATP were explained as those of a mixture of unmodified H-meromyosin and CMB leads to beta ME-H-meromyosin containing 1 mole of CMB per mole of H-meromyosin. Half of the light chains (g2), with a molecular weight of 18,000, were removed from myosin by treatment with CMB and beta-mercaptoethanol. After this treatment, on further incubation of the myosin with a large excess of beta-mercaptoethanol, the myosin contained only half of the g2, but the substrate inhibition of acto-H-meromyosin ATPase was restored completely. The initial burst of P1 liberation and the EDTA-ATPase activity decreased to almost zero on specific modification of the SH1-groups with NEM, while the initial burst decreased to some extent and the EDTA-ATPase activity to 50% of the original value on binding of 1 mole CMB per mole of H-meromyosin. The actomyosin-type of ATPase activity was strongly inhibited by modification with CMB. The extent of the dissociation of acto-H-meromyosin induced by ATP was unaffected by modification with NEM, while it decreased on further treatment of NEM-myosin with CMB FOLLOWED BY BETA-MERCAPTOETHANOL.     

Kinetic and physical properties of Co2+ enolase

The activation of yeast enolase by cobaltous ion in 0.1 M KCl is characterized by an activation constant of 1 microM and an inhibition constant of 18 microM. Measurements of binding of Co2+ to the apoenzyme show that a maximum of four Co2+ ions are bound per dimer in the presence or absence of substrate although binding is far tighter in the presence of substrate. Ultraviolet spectral titrations show evidence for a conformational change due exclusively to the binding of the first two ions of Co2+. Both visible and EPR spectra confirm that the environment of the first pair of cobalt ions ("conformational sites") is markedly different from that of the second pair in the "catalytic" sites. Cobalt at the conformational site appears to be a tetragonally distorted octahedral complex while the second pair of metal ions appears to be in a more regular tetrahedral symmetry. Addition of either Mg2+ or substrate to the enzyme with only one pair of cobalt ions per dimer causes striking changes in the metal ion environment. The conformational metal sites appear sufficiently shielded from solvent to be inaccessible to oxidation by H2O2, in contrast to the second pair of cobaltous ions whose ready oxidation by H2O2 inactivates the enzyme. Comparison of kinetic and binding data suggests that only one site of the dimeric enzyme can be active, since activity requires more than two metals bound per dimer and inactivation results from the binding of the fourth ion per dimer.     

Hydrogen ion uptake upon tobacco mosaic virus protein polymerization.

To determine the stage at which H⁺ ions are bound during the entropy-driven polymerization of tobacco mosaic virus protein, acid-base titrations were carried out at a concentration of 5 mg/ml in 0.1 m-KCl from pH 8 to pH 5.2 and back to pH 8 at 4, 10, 15 and 20 °C. The titration was always completely reversible when the addition of acid or base was so slow that the experiment required seven hours in each direction. When the titration was started at pH 7 and performed down and up twice as rapidly, a hysteresis loop, indistinguishable from one previously published, was obtained at 20 °C.Ultracentrifugation experiments were carried out at selected pH values at the four temperatures. H⁺ ion uptake, as determined from the reversible titration curves, is correlated with the disappearance of the 4 S component and is independent of whether the polymerized species is in a 20 S or higher state of aggregation. At pH 7, approximately 1 mole of H⁺ ion is bound per mole of monomer. At pH values between 6.56 and 6.05, 1.5 moles of H⁺ ion are bound per mole of monomer upon polymerization. At pH 6.05, 0.5 mole of H⁺ ion is bound before any polymerization takes place.Tobacco mosaic virus protein at 20 °C in an unbuffered 0.1 m-KCl solution at pH 7.18 at a concentration of 41 mg/ml, largely in the 20 S state, was depolymerized entirely to the 4 S state by dilution with 0.1 m-KCl adjusted to the same pH. Under these conditions, there was no pH change, indicating that no H ⁺ ions are released.These seemingly contradictory findings can be explained by assuming that the 4 S component polymerizes to form either double discs without binding H⁺ ions, or, alternatively, two-turn helices accompanied by the binding of H⁺ ions. Both double discs and two-turn helices sediment at approximately 20 S. Whether polymerization in the neighborhood of pH 7 leads to helices or discs depends upon the availability of H⁺ ions.     

Dephosphorylation of tubulin-bound guanosine triphosphate during microtubule assembly.

1. Tubulin purified from porcine brain in the presence of GTP contained 0.16 mole of GDP and 0.73 mole of GTP per 60,000 g of protein. 2. Microtubules reconstituted from the purified tubulin contained 0.43 mole of GDP and 0.41 mole of GTP per 60,000 g of protein. Guanine nucleotide bound to the exchangeable site of tubulin was converted to GDP during microtubule assembly, while GTP at the non-exchangeable site remained intact. 3. Guanine nucleotide which had been bound to the exchangeable site of tubulin before microtubule assembly was also exchangeable during disassembly.     

Properties and catalytic function of the two nonequivalent flavins in sarcosine oxidase

Sarcosine oxidase from Corynebacterium sp. U-96 contains 1 mol of noncovalently bound flavin and 1 mol of covalently bound flavin per mole of enzyme. Anaerobic titrations of the enzyme with either sarcosine or dithionite show that both flavins are reducible and that two electrons per flavin are required for complete reduction. Absorption increases in the 510-650-nm region, attributed to the formation of a blue neutral flavin radical, are observed during titration of the enzyme with dithionite or substrate, during photochemical reduction of the enzyme, and during reoxidation of substrate-reduced enzyme. Fifty percent of the enzyme flavin forms a reversible, covalent complex with sulfite (Kd = 1.1 X 10(-4) M), accompanied by a complete loss of catalytic activity. Sulfite does not prevent reduction of the sulfite-unreactive flavin by sarcosine but does interfere with the reoxidation of reduced enzyme by oxygen. The stability of the sulfite complex is unaffected by excess acetate (an inhibitor competitive with sarcosine) or by removal of the noncovalent flavin to form a semiapoprotein preparation where 75% of the flavin reacts with sulfite (Kd = 9.4 X 10(-5) M) while only 3% remains reducible with sarcosine. The results indicate that oxygen and sulfite react with the covalently bound flavin and suggest that sarcosine is oxidized by the noncovalently bound flavin.     

Chemical modification of the Ca2+ -dependent ATPase of sarcoplasmic reticulum from skeletal muscle. II. Use of 2, 4, 6-trinitrobenzenesulfonate to show functional movements of the ATPase molecule.

1. By changing the concentrations of Mg2+ and Ca2+ ions and ATP, the Ca2+, Mg2+-dependent ATPase [EC 3.6.1.3] of SR was converted to various enzymatic states, E, MgE, MgECa, MgEATP' CaECa, and CaECap at pH 8.0 and 0 degrees (cf. Eq. 1). 2. SR vesicles were allowed to react with 0.5 mM 2,4,6-trinitrobenzenesulfonate (TBS) at pH 8.0 and 0 degrees, keeping the ATPase in one of the enzymatic states listed above. Trinitrophenyl (TNP)-protein and TNP-lipid were separated by gel-filtration, and the amounts of TNP incorporated into protein and lipid were determined. 3. In all the enzymatic states of ATPase tested, the amount of TBS bound with SR protein increased exponentially with time, and reached a maximum level 10 min after starting the reaction. On the other hand, the amount of TBS bound to lipid increased with time, and did not reach a maximum level for at least 20 min. 4. The SR ATPase activity and the amount of EP formed decreased only slightly, even when the amount of TBS bound to SR protein reached the maximum level. 5. The maximum amount of TBS bound to SR protein varied on changing the enzymatic state of SR ATPase. Namely, about 2,3,1,3, or 4, and 3 moles of TNP were incorporated per mole of SR ATPase, when SR was allowed to react with TBS in the enzymatic states MgE, MgECa, MgEATP' CaECa, and CaECap, respectively. 6. When the enzymatic state was changed from MgE to MgECa 10 min after starting the reaction with TBS, about 4 moles of TBS was bound per mole of ATPase within 10 min, while the maximum levels of TBS bound in states MgE and MgECa were about 2 and 3 moles per mole of ATPase, respectively, as mentioned above. 7; When the enzymatic state was changed from MgE to MfEATP 10 min after starting the reaction with TBS, about 3 moles of TBS was bound per mole of ATPase within 10 min, while the maximum levels of TBS bound in states MgE and MgEATP were about 2 and 1 moles per mole of ATPase, respectively, as mentioned above. 8. When the enzymatic state was changed from CaECa to CaECap 10 min after starting the reaction with TBS, about 4 moles of TBS was bound per mole of ATPase, while the maximum levels of TBS bound in states CaECa and CaECap were about 3 or 4 moles per mole of ATPase, respectively. 9. A diagrammatic model of functional movements of the ATPase molecule coupled with elementary steps in the ATPase reaction is proposed on the basis of these results.     

Glucose degradation, molar growth yields, and evidence for oxidative phosphorylation in Streptococcus agalactiae

In a complex medium with the energy source as the limiting nutrient factor and under anaerobic growth conditions, Streptococcus agalactiae fermented 75% of the glucose to lactic acid and the remainder to acetic and formic acids and ethanol. By using the adenosine triphosphate (ATP) yield constant of 10.5, the molar growth yield suggested 2 moles of ATP per mole of glucose from substrate level phosphorylation. Under similar growth conditions, pyruvate was fermented 25% to lactic acid, and the remainder was fermented to acetic and formic acids. The molar growth yield suggested 0.75 mole of ATP per mole of pyruvate from substrate level phosphorylation. Under aerobic growth conditions about 1 mole of oxygen was consumed per mole of glucose; about one-third of the glucose was converted to lactic acid and the remainder to acetic acid, acetoin, and carbon dioxide. Molar growth yields indicated 5 moles of ATP per mole of glucose. Estimates based on products of glucose degradation suggested that about one-half of the ATP was derived from substrate level phosphorylation and one-half from oxidative phosphorylation. Addition of 0.5 m 2,4-dinitrophenol reduced the growth yield to that occurring in the absence of oxygen. Aerobic pyruvate degradation resulted in 30% of the substrate becoming reduced to lactic acid and the remainder being converted to acetic acid and carbon dioxide, with small amounts of formic acid and acetoin. The molar growth yields and products found suggested that 0.70 mole of ATP per mole of pyruvate resulted from substrate level phosphorylation and 0.4 mole per mole of pyruvate resulted from oxidative phosphorylation.     

Binding of the antibiotic vernamycin in Balpha to Escherichia coli ribosomes

The interaction of the antibiotic vernamycin Bα with Escherichia coli ribosomes has been studied. The antibiotic is bound to 70S ribosomes and 50S subunits but not to the 30S subunit or to polysomes. The binding of the antibiotic requires K⁺ or NH⁺₄ and Mg²⁺. At saturation approximately 0.5 mole of antibiotic is bound per mole of ribosomes. The vernamycin Bα-ribosome complex is unstable. The bound antibiotic is readily displaced by nonradioactive vernamycin Bα and by a number of other antibiotics which are known to interact with the 50S subunit. The dissociation of the vernamycin Bα-ribosome complex is prevented by the simultaneous binding of vernamycin A. The binding sites for A and Bα are distinguishable since both drugs are able to bind simultaneously and neither prevents binding of the other, Ribosomes isolated from an erythromycin-resistant mutant are incapable of binding vernamycin A and Bα, indicating that the mutated protein responsible for resistance to erythromycin distorts the ribosome making it also unreceptive for the vernamycins.     

Structure of putrebactin, a new dihydroxamate siderophore produced by Shewanella putrefaciens

 Shewanella putrefaciens is a bacterium implicated in oil pipeline corrosion and fish spoilage, and is one of very few isolated microorganisms able to use iron(III) as an electron acceptor. S. putrefaciens strain 200 produced a novel cyclic dihydroxamate siderophore, putrebactin, during aerobic growth. Putrebactin was determined to be 1,11-dihydroxy-1,6,11,16-tetraazacycloeicosane-2,5,12,15-tetrone, a cyclic dimer of succinyl-(N-hydroxyputrescine), by ¹H and ¹³C NMR spectroscopy, fast atom bombardment and chemical ionization mass spectrometry, and X-ray crystallography. The protonation constants of putrebactin were determined to be 8.82 and 9.71. Potentiometric titration of the ferric complex revealed a sharp equivalence point at 3.0 equivalents of base per mole of Fe(III), consistent with loss of 3 protons per equivalent of bound ferric ion, while Job's method of continuous variation supported a shift from 1 : 1 to 3 : 2 complex stoichiometry as a function of pH. Putrebactin is similar in structure to two other siderophores, bisucaberin and alcaligin, produced by unrelated bacteria. Received: 21 May 1996 / Accepted: 9 November 1996     

Phenothiazine binding by a homolog of calpactin, the pp60src tyrosine kinase substrate

Microvilli isolated from 13762 mammary ascites tumor cells contain a major calcium-sensitive protein (AMV-p35) that can be isolated with microvillar microfilament cores prepared by Triton X-100 extraction in the presence but not absence of calcium. AMV-p35 can be readily purified from ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid extracts of the microfilament cores by chromatography on an anion exchange column, to which it does not bind. Immunoblot analysis indicates that AMV-p35 is related to calpactin I, the pp60src tyrosine kinase substrate. In the presence of calcium, AMV-p35 binds approximately 4 mol of chlorpromazine per mole of protein in a binding process showing apparent positive cooperativity, similar to calmodulin; however, in contrast to calmodulin, AMV-p35 also binds phenothiazine in the absence of calcium.     

Characterization and Distribution of Ferritin Binding Sites in the Adult Mouse Brain

Abstract : Studies on iron uptake into the brain have traditionally focused on transport by transferrin. However, transferrin receptors are not found in all brain regions and are especially low in white matter tracts where high iron concentrations have been reported. Several lines of research suggest that a receptor for ferritin, the intracellular storage protein for iron, may exist. We present, herein, evidence for ferritin binding sites in the brains of adult mice. Autoradiographic studies using ¹²⁵I-recombinant human ferritin demonstrate that ferritin binding sites in brain are predominantly in white matter. Saturation binding analyses revealed a single class of binding sites with a dissociation constant ( K _D) of 4.65 × 10^-9 M and a binding site density ( B _max) of 17.9 fmol bound/μg of protein. Binding of radiolabeled ferritin can be competitively displaced by an excess of ferritin but not transferrin. Ferritin has previously been shown to affect cellular proliferation, protect cells from oxidative damage, and deliver iron. The significance of a cellular ferritin receptor is that ferritin is capable of delivering 2,000 times more iron per mole of protein than transferrin. The distribution of ferritin binding sites in brain vis-à-vis transferrin receptor distribution suggests distinct methods for iron delivery between gray and whi     

Characterization of a novel protein that specifically binds to DNA modified by N-acetoxy-acetylaminofluorene and cis-diamminedichloroplatinum

Proteins recognizing DNA damaged by the chemical carcinogen N-acetoxy-acetylaminofluorene (AAAF) were analyzed in nuclear extracts from rat tissues, using a 36 bp oligonucleotide as a substrate and electrophoretic mobility shift and Southwestern blot assays. One major damage-recognizing protein was detected, whose amount was estimated as at least 10(5) copies per cell. Levels of this protein were similar in extracts from brain, kidney and liver, but much lower in extracts from testis. The affinity of the detected protein for DNA damaged by AAAF was about 70-fold higher than for undamaged DNA. DNA damaged by cis-diamminedichloroplatinum (cis-DDP), benzo(a)pyrene diolepoxide (BPDE) or UV-radiation also bound this protein with an increased affinity, the former more strongly and the latter two more weakly as compared to AAAF-damaged DNA. The detected AAAF/DDP-damaged-DNA-binding (AAAF/DDP-DDB) protein had a molecular mass of about 25 kDa and was distinct from histone H1 or HMGB proteins, which are known to have a high affinity for cis-DDP-damaged DNA. The level of this damage-recognizing protein was not affected in rats treated with the carcinogen 2-acetylaminofluorene. The activity of an AAAF/DDP-DDB protein could also be detected in extracts from mouse liver cells but not from the Hep2G human hepatocellular carcinoma.     

Purification and characterization of the bifunctional thymidylate synthetase-dihydrofolate reductase from methotrexate-resistant Leishmania tropica

Thymidylate synthetase (TS) and dihydrofolate reductase (DHFR) in Leishmania tropica exist as a bifunctional protein. By use of a methotrexate-resistant strain, which overproduces the bifunctional enzyme, the protein was purified 80-fold to apparent homogeneity in two steps. The native protein has an apparent molecular weight of 110 000 and consists of two subunits with identical size and charge. Available data indicate that each of the subunits possesses TS and DHFR. The TS of the bifunctional protein forms a covalent 5-fluoro-2'-deoxyuridylate (FdUMP)-(+/-)-5,10-methylenetetrahydrofolate-enzyme complex in which 2 mol of FdUMP is bound per mole of enzyme. In contrast, titration of DHFR with methotrexate indicated that only 1 mol of the inhibitor is bound per mole of dimeric enzyme. Both TS and DHFR activities of the bifunctional enzyme were inactivated by the sulfhydryl reagent N-ethylmaleimide. Substrates of the individual enzymes afforded protection against inactivation, indicating that each enzyme requires at least one cysteine for catalytic activity. Kinetic evidence indicates that most, if not all, of the 7,8-dihydrofolate produced by TS is channeled to DHFR faster than it is released into the medium. Although the mechanism of channeling is unknown, the possibility that the two enzymes share a common folate binding site has been ruled out.