Irreversible inhibition of folate transport in Lactobacillus casei by covalent modification of the binding protein with carbodiimide-activated folate

Activated folate formed by reaction of folic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide irreversibly inhibits the folate transport system of Lactobacillus casei. Complete inhibition of both folate binding to the carrier protein and folate transport was achieved by pretreatment of the cells at low temperature (4 °C) and at neutral pH with 200 nm activated folate. Fifty percent inhibition of binding and transport occurred at 35 and 40 nm activated folate, respectively. Specificity was demonstrated by the fact that excess nonactivated folate added during the pretreatment step afforded complete protection of the binding protein against inhibition, and that activated folate had no effect on the binding or transport of thiamine. Rapid measurements at 4 °C were employed to show that, prior to the appearance of irreversible inhibition, activated folate (K_i = 15 nM) interacted reversibly with the binding site for folate (K_d = 0.8 nM). Cells treated with activated [³H]folate incorporated 1 mol of folate per mole of binding protein. Purification of the labeled protein followed by digestion with Pronase led to the isolation of a compound identified as ?-N-folyl lysine. The ?-amino group of a lysyl residue thus appears to be the nucleophilic group at the binding site that reacts with activated folate.     

Peroxidation of lysozyme treated with Cu(II) and hydrogen peroxide

When lysozyme was treated with Cu(II) and H₂O₂ at pH 7.4, the protein underwent polymerization as well as changes in its fluorescent characteristics. Upon prolonged incubation, most of the protein aggregates were degraded into smaller peptides. Amino acid analysis indicated that the basic amino acid residues were most susceptible to the oxidation. Tryptophan residues were converted to N-formylkynurenine and kynurenine, and lysine residues were deaminated to form α-aminoadipic acid δ-semi- aldehyde. During Cu(II)H₂O₂ treatment, the formation of carbonyl groups was accompanied by the loss of free amino groups in the protein. Succinylation of free amino groups protected lysine residues from oxidation by Cu(II)H₂O₂, but failed to prevent polymerization. The studies with the modified lysozyme suggest that Cu(II)H₂O₂ can oxidize various amino acid residues in addition to lysine to generate different types of carbonyl compounds and these carbonyl compounds may be responsible for the formation of crosslinks in the polymerization process.     

A fluorometric-HPLC assay for quantitating the binding of benzo[a]pyrene metabolites to DNA

A nonradiometric method is presented for quantitating low levels of benzo[a]pyrene (BP) derivatives that are covalently bound to the DNA of BP-treated mice. This method consists of hydrolyzing the DNA with acid to liberate the BP-adducts in the form of the isomeric tetrols of BP. These tetrols have fluorescence quantum yields of ～0.7 in deoxygenated solution at 298 K. Hence they are easily quantitated, following HPLC separation, by means of fluorescence detection. The sensitivity of the method is such that one bound BP residue per 10⁷ bases can be detected in 100 μg of DNA.     

A direct fluorometric assay of benzo[a]pyrene hydroxylase.

A technique to measure the activity of pyruvate carboxylase spectrophotometrically in crude liver homogenates is described. The assay is based on the transformation of oxaloacetate, which is formed during the carboxylation reaction, into citrate in the presence of excess acetyl CoA and citrate synthase. After removal of pyruvate with KBH₄ and of protein with HClO₄, citrate is cleaved with citrate lyase into oxaloacetate and acetate, and oxaloacetate then is measured spectrophotometrically. Optimal concentrations of pyruvate, Mg²⁺, ATP, and KHCO₃ for the carboxylation reaction and the V_max were in good correlation with the data found by others using [¹⁴C]pyruvate.     

Synthesis of a new class of polydentate ligands: [bis(2-imidazolyl)methyl] amino-thioether-thiols

A new class of polydentate bis(imidazole)-thioether-thiol polydentate ligands has been synthesized by the reactions of functionalized primary amines with bis(2-imidazolyl)nitromethane. The molecules contain a bis(2-imidazolyl)methylamino group attached to chains of varying length with thiol (3, 23) and thioether/thiol (7,11,15,19) binding sites.     

Effect of chronic streptozotocin-induced diabetes on [3H]ouabain binding in the rat left ventricle

Earlier results from our laboratory have revealed that the inotropic response to ouabain was depressed in chronically diabetic rat heart (1). In this study we examined the effect of chronic streptozotocin induced diabetes (3 months) on [3H]ouabain binding in the rat heart. Scatchard analysis of [3H]ouabain binding to membrane preparations of rat left ventricle revealed two classes of binding sites; a high affinity/low capacity and a low affinity/high capacity binding site. The maximum number of binding sites of the high and low affinity binding sites in membrane preparations obtained from the chronically diabetic rats was significantly (p less than 0.05) reduced to 60.4 and 48.8% of controls, respectively. The dissociation constant of the high and the low affinity binding/sites in the chronically diabetic rat heart, compared to controls, was significantly (p less than 0.05) increased and decreased, respectively. These results suggest that the decreased inotropic response of ouabain in the intact tissue obtained from chronically diabetic rats (1) may be related to a decreased number of ouabain binding sites.     

The metabolism of benzo[a]pyrene phenols by rat liver microsomal fractions

The oxidative metabolism of benzo[a]pryrene (B[a]P) phenols catalyzed by liver microsomes in vitro leads to multiple products. High-pressure liquid chromatography analysis of the organic-soluble products formed indicates that regardless of the animal pretreatment regime, 3-hydroxy-B[a]P is metabolized to the 3,6-quinone and to a hydroxylated derivative tentatively identified as 3,9-dihyroxy-B[a]P. However, the distribution of products obtained with 9-hydroxy-B[a]P varied with animal pretreatment. A maximum of three distinct metabolites was obtained when the 9-phenol was metabolized in vitro with microsomes from phenobarbital-pretreated rats and the tentative 3,9-dihydroxy derivative was a common metabolite for all pretreatment regimes. Physical characterization, including mass spectrometry, indicates that all three products have an extra oxygen atom incorporated into their molecular structure from molecular oxygen. Studies utilizing specific inhibitors of the cytochrome P-450-dependent monooxygenase clearly suggest that the formation of dihydroxy or phenol-oxide derivatives is catalyzed by the hemoprotein, cytochrome P-450. These metabolites of the benzo[a]pyrene phenols are most likely related to the putative phenol-oxides of benzo[a]pyrene which have been demonstrated to alkylate DNA and protein. Repetitive scan difference spectrophotometric analysis of incubation mixtures containing rat liver microsomes, 3- or 9-hydroxy-B[a]P, NADPH, and oxygen shows the conversion of the phenols into products which absorb in the region from 400 to 500 nm. During and after the steady state of the reaction, it can be seen that certain of the hydroxy compounds produced are in equilibrium with their respective quinone form and may be involved in an oxygen-coupled redox cycle.     

Synthesis and X-ray crystal structure determination of two pseudopolymorphic forms of μ-[1,2-bis(diphenylphosphino)ethane] bis [chlorogold(I)]: a digold(I) DNA binder

An improved synthetic route to the linearly coordinated digold(I) complex, μ-[1,2-bis(diphenylphosphino)ethane]bis[chlorogold(I)], is reported. This complex crystallizes in two pseudopolymorphic forms from a chloroform/methylene chloride solution; the crystal and molecular structures of both are discussed and compared. In both crystal forms the potentially chelating diphenylphosphinoethane (dppe) ligand instead coordinates to two separate gold atoms. The coordination environment of each gold atom is linear in both pseudopolymorphs and the structures display normal goldchloride and goldphosphorus bond distances. On the molecular level, the pseudopolymorphs differ fundamentally by a twist about one of the gold phosphorous bonds with the phosphorous atoms of the dppe ligand adopting a transoid orientation relative to one another in both polymorphs. These conformations thus place the intramolecular gold atoms 6 Å apart and preclude intramolecular AuAu bonding interactions. As has been observed for related gold(I) complexes there are short intermolecular AuAu contacts of the order of 3.2 Å present in both structures. The conformational flexibility of the gold complex relative to its observed biological activity as a DNA binder is discussed.     

A comparative study of the binding of l-tryptophan and bilirubin by plasma proteins

The binding of l-tryptophan and bilirubin by plasma proteins from a variety of animals has been studied. Bilirubin was bound by plasma protein(s) from all animals investigated. l-Tryptophan, on the other hand, was bound only by plasma protein(s) from warm-blooded animals. Our results in vertebrates are consistent with serum albumin being the binding protein concerned.     

Direct demonstration that ferrous ion complexes of di- and triphosphate nucleotides catalyze hydroxyl free radical formation from hydrogen peroxide

Utilizing an electron paramagnetic resonance (EPR) spin-trapping technique it was demonstrated that the di- and triphosphate nucleotides of adenosine, cytidine, thymidine, and guanosine in the presence of Fe(II) catalyze hydroxyl free radical formation from H₂O₂. The triphosphate nucleotides in general were about 20% more effective than the diphosphate nucleotides. The amount of ?H produced from H₂O₂ as a function of nucleotide level tended to increase in a sigmoidal fashion beginning at a nucleotide/Fe(II) ratio of 2 but then rose rapidly up to a ratio of 5 at which point the increase became more gradual. The monophosphate nucleotides did not cause an increase in the amount of hydroxyl free radical produced from H₂O₂ over the low level obtained in the buffer system only. The cations, Mg²⁺ and Ca²⁺, even at much higher than physiological levels and much higher than the level of added Fe(II), did not cause a substantial diminution of the Fe(II)-nucleotide-catalyzed breakdown of H₂O₂ to yield ?H. A study of the time course of the effectiveness of Fe(II)-nucleotide-mediated ?H formation from H₂O₂ demonstrated that Fe(II) in the presence of nucleotides remained in an effective catalytic state with a halftime of about 160 s whereas in the absence of the nucleotides the halftime was 7.5 s. All observations indicate that Fe(II) ligates with di- and triphosphate nucleotides and remains in the ferrous state which is then capable of catalyzing ?H formation from H₂O₂; but with time, oxidation of the metal ion to the ferric state occurs, which either ligated to the nucleotide or to buffer ions, is ineffective in H₂O₂ catalysis to yield ?H. Iron-nucleotide complexes may be of importance in mediating oxygen free radical damage to biological systems. The observations presented here indicate that hydroxyl free radicals will be produced when H₂O₂ is present with ferrous-nucleotide complexes.     

Stoichiometry and structure of a complex of acidic ribosomal proteins

The acidic proteins B-L13 (homologous to Escherichia coli protein L7/L12) and B-L8, from the 50 S subunit of Bacillus stearothermophilus ribosomes, form a stable complex. Trypsin digestion of ribosomes generates an N-terminal fragment of B-L13 (approximately residues 1 to 47) which can associate with B-L8, displacing intact B-L13, and bind to B-L13-deficient ribosomes. Displacement of B-L13 from the B-L8 · B-L13 complex by the B-L13 N-terminal fragment causes a change in gel electrophoretic mobility of the complex, and titration of the complex with fragment indicates unambiguously that it contains four molecules of B-L13. Evidence is presented that B-L13 forms a dimer in solution, and that the dimer associates intact with B-L8. Reconstituted 50 S subunits in which B-L13 is replaced by its N-terminal fragment have the same functional properties as 50 S subunits missing B-L13 altogether: polypeptide synthesis is reduced but not abolished; ability to bind elongation factor EF-G and GTP is severely reduced; and peptidyl transferase activity and ability to associate with a 30 S subunit · Phe-tRNA · poly(U) complex are unaffected (relative to intact 50 S subunits).     

Crystal structure of [chloro(diethyldithiocarbamato)butyl phenyl]tin(IV)

The crystal structure of the title compound, SnCl(C₆H₅)(C₄H₉)[S₂CN(C₂H₅)₂], was determined and refined to an R factor of 3.2% for 4876 reflections. The molecule contains five-coordinate tin in a distorted trigonal bipyramidal arrangement with the tin atom lying 0.20 Å below the equatorial plane formed by one of the sulphur atoms, S(1), and the donor carbons of the butyl and phenyl groups. The chlorine and the other sulphur atom, S(2), occupy axial sites, making a S(2)SnCl angle of 156.85(1)°. The SnS(2) bond is markedly elongated (2.764(1) Å) compared to the SnCl bond (2.449(1) Å) and the SnS(1) bond (2.454(1) Å). The structure resembles those of analogues such as (C₆H₅)₂Sn(glygly) in having both hydrocarbon ligands located in the equatorial plane. Crystal data: space group P$\text{1}$: a = 8.291(2) Å, b = 14.726(3) Å, c = 9.509(2) Å, α = 96.24(2)°, β = 107.02(3)°, γ = 116.70(2)°, Z = 2, R = 3.2% for 4876 independent reflections.     

Chemical identity of the glucose transporter with the [3H]cytochalasin B-photolabelled component of human erythrocyte membranes. Equal sensitivity to trypsin and endoglycosidase F

The protein photolabelled by [3H]cytochalasin B and band 4.5, which contains the human erythrocyte hexose transporter, were compared by electrophoretically monitoring the effect of digestion with endoglycosidase F and trypsin. Band 4.5 was found to consist of two minor components, Mr 58,000 and 52,000, and one main component, Mr 60,000-50,000. Deglycosylation by endoglycosidase F converted both the [3H]-labelled species and the main polypeptide of band 4.5 from a mixture of polypeptides of Mr 50,000-60,000 to a sharp component of Mr 46,000. Tryptic cleavage of the photolabelled protein produced a [3H]-labelled peptide of 19,000 daltons, which corresponded to an analogous tryptic fragment of the main component of band 4.5. Endoglycosidase F treatment of trypsin-treated samples had no effect on the 19,000 dalton fragment or the labelled 19,000 component, indicating that both species lack the carbohydrate moiety of the parent protein. This parallel chemical behaviour indicates that the photolabelled polypeptide is representative of the main constituent of band 4.5. Photolabelling may be used with confidence to quantitate glucose transporters in other cells.     

Tetrakis-[1-methylimidazoline-2(3H)-thione]-μ2-[1-methylimidazoline- 2(3H)-thione]-Di-Copper(I) sulphate trihydrate: Preparation, thermal analysis and crystal structure

1-emthylimidazoline-2(3H)-thione (mimtH) reacts with copper(II) sulphate pentahydrate in aqueous acetone to produce the dinuclear complex, Cu₂(mimtH)₅SO₄ · 3H₂O; the formula has been established by a combination of chemical and thermal analysis. The monoclinic crystals, (space group P_c, Z = 2), contain dinuclear cations, sulphate ions and water molecules. The dinuclear cation, Cu₂(mimtH)₅²⁺, consists of two trigonal copper(I) atoms, four terminal, monodentate, S-donating mimtH molecules and one S-bridging (μ₂) mimtH molecule. Some average dimensions are:Cu---S, 2.258 Å and S---Cu---S, 120.0°; the Cu---S---Cu bridging angle is 94.8° and the Cu---Cu separation distance is 3.308 Å.     

On the protein crystal chemistry of chloroplatinite ions: general principles and interactions with triose phosphate isomerase

K₂[PtCl₄] has been used to prepare isomorphous derivatives of the majority of proteins for which detailed structure determinations have been published. As the derivatives were prepared under a wide variety of solvent conditions they include a number of chemically different platinum-protein complexes.New observations on the binding of K₂[PtCl₄] to crystalline chicken muscle triose phosphate isomerase are reported which, together with a review of its binding to other proteins and chemical considerations, provide a rational framework for understanding the reactions of this complex salt with crystalline proteins in the presence of various mother liquors. Means by which the reaction may be controlled are also discussed.