The hydrolysis of α-amino-acid esters in mixed ligand complexes with copper(II)-iminodiacetate

α-Amino-acid esters (EH⁺) interact with [Cu(IMDA)]° to give mixed ligand complexes according to the equilibrium,

where EH⁺ represents the protonated ester ⁺NH₃CH(R)CO₂R′ and IMDA^2? is HN(CH₂CO_2^?)₂. The mixed ligand complexes are only formed over a rather narrow pH range (ca. pH 5.8–6.5). At higher pH there is kinetic evidence for the competing equilibrium,

Rate constants k_OH have been obtained by pH-stat for the hydrolyses [where A^? = NH₂CH(R)CO_2^?]

The complexed α-amino-acid esters undergo base hydrolysis ca. 10⁴ times faster than the free esters E. Values of k_OH show little dependence on the nature of the alkyl substituent R but the normal leaving group effect of methyl esters hydrolyzing at ca. twice the rate of ethyl esters is observed. Activation parameters have been determined for base hydrolysis of [Cu(IMDA)(glyOMe)]°, and possible mechanisms for the reaction are considered.     

Nonenzymic hydrolysis of ATP–infrared investigations of intermolecular interactions

The nonenzymic hydrolysis of

and

were studied by infrared (IR) spectroscopy. Protons resulting from hydrolysis of ATP are not bound to the N₁ atoms of the adenine residues. With hydrolysis of

, these protons are partially bound to the terminal phosphate group of ADP, namely,

,

,

, and

, present after hydrolysis. With decreasing pH or when Mg²⁺ ions are present, all hydrolysis protons are attached to the orthophosphate molecules.With hydrolysis of

the pH decreases up to 40% degree of hydrolysis. Then the system becomes self-buffered in the physiological pH region. A similar pH decrease is found with hydrolysis of

. With these systems, however, the pH decreases slightly also at degrees of hydrolysis larger than 40%. No other systems show pronounced pH changes during hydrolysis; in other words, they are buffer systems.The IR bands demonstrate that mesomeric bond resonance in the phosphate groups strongly depends on whether protons are present at these groups. Regarding the equilibria of proton attachment mentioned above, mesomeric bond resonance in these groups strongly depends on pH and on the presence of

ions.With hydrolysis of ATP, two POH groups are formed that bind H₂O molecules via strong hydrogen bonds, changing the solvate structure. Finally, easily polarizable hydrogen bonds are formed, for instance,

bonds with the hydrolysis of

, and

bonds with the hydrolysis of

. These bonds strongly interact with their environment. The formation of these hydrogen bonds strongly depends on pH and the presence of

ions.All these effects, especially the intermolecular ones, contribute to the change of free energy during ATP hydrolysis.     

The effect of tin chloride on the structure and function of dna in human white blood cells

Tin compounds are being used increasingly in

in industry and in medicine. There have been relatively few studi

ng term biological effects of this metal, although acute effects h

cumented. In this report we describe experiments which show tha

stannous chloride, is readily taken up by human white blood cells

an cause damage to DNA. Damage was detected in WBC after e

-50 μM tin(II) for 30 min at either 0°C or 37°C. The amount o

served was more extensive than that produced by exposure of

nolar amounts of chromium(VI), a known carcinogen and DNA

agent. Additional indication of cellular damage is that exposm

n lymphocytes or mouse splenocytes to tin(II) interfered with thei

o be stimulated by the polyvalent mitogen concanavalin A (Con A).

rast, tin(IV) was not taken up by cells, did not cause DNA damage nor d

inhibit stimulation of DNA synthesis in cells that were exposed to Con A.     

Guanine complexes with dysprosium(III), thorium(IV) and uranium(IV) chlorides

By refluxing mixtures of guanine (guH) and DyCl₃, ThCl₄ or UCl₄ in ethanol-triethyl orthoformate, solid complexes of the Dy(guH)₂(gu)Cl₂ and M(gu)₂Cl₂ (M = Th, U) types were isolated. The insolubility of the new complexes in organic media, combined with the coordination number six suggested by the spectral evidence, favors polymeric configurations. Most likely structures involve a linear, chainlike, single-bridged polymeric backbone

The Dy³⁺ complex is probably a linear polymer, also containing terminal unidentate guanine ligands, whilst for M = Th⁴⁺, U⁴⁺ highly polymeric structures arising from cross-linking between linear polymeric

units seems most likely. IR evidence rules out participation of the O(6) oxygen of guanine in coordination, despite the hard acid character of the metal ions under study. Guanine apparently coordinates exclusively through ring nitrogens in the new metal complexes; N(9) and N(7). N(9) are, respectively, the most likely binding sites of terminal unidentate and bridging bidentate guanine. The chloro ligands present in the complexes seem to be exclusively terminal.     

Reaction kinetics of P-700, cytochrome c-553 and cytochrome f in the cyanobacterium, Synechococcus sp

Absorption changes invoked by short flashes in the Soret band region were measured in the thermophilic cyanobacterium Synechococcus sp. and photoresponses of P-700, cytochrome c-553 and cytochrome f were resolved with the aid of a microcomputer. Cytochrome c-553 was oxidized very rapidly with a half-time of less than 20 μs, while the half oxidation time of cytochrome f was 35–45 μs. The two cytochromes were reduced monophasically with half-time of 2 ms after a lag lasting a few milliseconds. The reduction kinetics of P-700 showed three exponential phases with half-times of 40 μs, 200 μs and 2 ms, which are ascribed to electron donation from cytochrome f, the Rieske iron-sulfur protein and plastoquinone, respectively. The results support the following sequence and rates of linear electron transport at the physiological temperature of the cyanobacterium: P-700

cytochrome c-553

cytochrome f

Rieske protein

plastoquinone.     

The amino acid sequences of homologous hydroxyproline-containing myotoxins from the marine snail Conus geographus venom

Two homologous toxic peptides containing hydroxyproline from the venom of the marine snail Conus geographus have been sequenced.Geographutoxin I:

Geographutoxin II: Arg-Asp-Cys-Cys-Thr-Hyp-Hyp-Arg-Lys-Cys-Lys-Asp-Arg-Arg-Cys-Lys-Hyp-Met-Lys-Cys-Cys-Ala-NH₂These peptides inhibit the contractile response of directly stimulated mouse diaphragm.     

Pyruvate carboxylase: Mechanism of the second partial reaction

The stoichiometry of the products of the pyruvate carboxylase reaction has been investigated and shown to vary as the concentration of pyruvate was altered. At high concentrations of pyruvate, the ratio of orthophosphate liberated to oxaloacetate produced approached one, but as the pyruvate concentration was decreased, the ratio increased. On the basis of this evidence, a model for the reaction mechanism was proposed in which the

complex could react either with pyruvate to form oxaloacetate, or water to produce

and HCO₃^?. The nonproductive breakdown of the enzyme-substrate complex and the resulting lack of stoichiometry provides an explanation for the nonlinear double reciprocal plots obtained for both the overall reaction and the pyruvate:oxaloacetate exchange reaction. Since neither the rate of breakdown of the isolated

complex nor the rate of decarboxylation of oxaloacetate in the absence of pyruvate could account for the difference in the amount of the two products formed during the overall reaction, it was postulated that the presence of pyruvate was necessary for hydrolysis to occur. Rate equations were derived describing the dependence of the initial velocity of the release of oxaloacetate in the overall reaction and the rate of the pyruvate:oxaloacetate exchange reaction, on pyruvate concentration. By assigning appropriate values to the various rate constants, theoretical curves were generated and fitted to the experimental data.     

Cytochrome f and plastocyanin kinetics in Chlorella pyrenoidosa. I. Oxidation kinetics after a flash

P-700, plastocyanin and cytochrome f redox kinetics were measured after one flash, using dark-adapted Chlorella in the presence of hydroxylamine and 3(3,4-dichlorophenyl)-1,1-dimethylurea. Plastocyanin becomes increasingly oxidized with a half-time of 70 μs, then undergoes reduction with a half-time of 7 ms. Cytochrome f oxidation has a sigmoidal time-course and a half-time of 100 μs. Its reduction exhibits a half-time of 4 ms. These results are interpreted in a linear scheme:

An equilibrium constant of 2 between cytochrome f and plastocyanin (PC), which contrasts with the large equilibrium constant between PC and P-700 is computed.The presence of cytochrome b₆ in a cyclic path around Photosystem I is confirmed under these conditions.     

Manganese(II), iron(II) and nickel(II) chloride complexes with monodeprotonated anionic guanine

Upon refluxing 2:1 mixtures of guanine (guH) and MnCl₂, FeCl₂ or NiCl₂ in a 7:3 (v/v) mixture of ethanol and triethyl orthoformate for 1–2 weeks, partial substitution of gu^? for Cl^? groups occurs, and solid complexes of the M(gu)Cl·2ROH (R = C₂H₅ for M = Mn; R = H for M = Fe, Ni) type are obtained. The new complexes are pentacoordinated and appear to be linear chainlike polymeric species, involving a single-bridged

_n backbone. Coordination number five is attained by the presence of one terminal chloro and two terminal ROH ligands per metal ion. Most probable binding sites of bidentate bridging gu^? are the N(7) and N(9) imidazole ring nitrogens. IR evidence rules out the possibility of coordination of gu^? through any of the exocyclic potential ligand sites (O(6) oxygen or N(2) nitrogen) [1].     

Studies of the self-association of bacteriophage T4 gene 32 protein by equilibrium sedimentation.

The self-association of the bacteriophage T4 gene 32 protein has been examined in the analytical ultracentrifuge under varying conditions to determine the nature of the process. The process is not a simple indefinite association with one association constant (monomer

dimer

trimer

etc.). The complexity of the process is shown by (1) peculiarities in the molecular weight versus concentration curves, in the region of the dimer (observed with increasing ionic strength, at pH 10, in 0.04 m-MgCl₂, with aged preparations, at 19 °C and in the presence of the oligonucleotide d(pT)₁₀), (2) the increased sigmoidicity of the association curve in the presence of glycerol or oligo[d(pT)₄], and (3) the discontinuity in the association curve at the tetramer at a pH value of approximately 9.4. A model with two association constants which could vary independently (monomer

dimer

tetramer

etc.) explained many of the findings. However, a more complex model was required to explain curves which had a plateau at the dimer with increased association at higher protein concentrations. Thus, under all conditions examined there is evidence for more than one type of protein-protein interaction. These different interactions may be involved in a physiological function such as recombination.     

An arabinoglucuronomannan from suspension-cultured cells of Nicotiana tabacum

From the soluble polysaccharides of suspension-cultured, tobacco cells (Nicotiana tabacum), an arabinoglucuronomannan (AGM) was isolated from the extract with phosphate buffer. Application of partial hydrolysis with acid, carboxyl reduction, methylation analysis, and ¹H- and ¹³C-n.m.r. spectroscopy indicated AGM to consist of the tetrasaccharide repeating-unit

.     

Genetic and sequence analysis of frameshift mutations induced by ICR-191

A genetic and sequence analysis of 373 ICR-191-induced mutations in the lacI gene of Escherichia coli reveals that 365 of the mutations (97·9%) are frameshifts involving the addition or deletion of a single

base-pair from a

sequence including, in one case, a

sequence. Some of the remaining eight mutations (2·1%) represent the loss or gain of a

base-pair from a

sequence. Certain mutational sites are relative hotspots for ICR-191-induced mutations, and we have analyzed the role of surrounding sequences on relative mutation rates. The preference for +1 frameshifts or ?1 frameshifts is site-specific, so that at some sites +1 frameshifts predominate by a 10:1 ratio, whereas at other sites ?1 frameshifts are favored by an approximately 2:1 ratio. The characterized frameshift mutations in lacI described here are useful for constructing systems to detect other frameshift and deletion mutations.     

Activation of phosphates to substitution(II). A comparison of catalyses initiated directly by VO2+ and by oxidation of VO2+ by MnO4−

Vanadium(V)-induced hydrolyses of triphosphates in aqueous solutions were initiated in two ways: (1) oxidizing vanadium(IV)-polyphosphate complexes to produce metastable vanadium(V) complexes; (2) forming VO₂⁺-polyphosphate complexes by acidification of solutions of VO₄^3? and polyphosphate to yield equilibrium mixtures of V(V), polyphosphate, and their complexes. Hydrolysis rates for the complexes formed at 40°C ? T ? 25°C follow the order V₂PPP_i = 2(VPPP_i) ≌ (VO₂) ATP ? V(PPP_i)₂ ≌ PPP_i. The hydrolysis of (V(V))₂(PPP_i) was not very temperature sensitive; the activation enthalpy appears small and the activation entropy large and negative. Mechanistic studies reveal that requirements for the activated state in metal-ion-catalyzed hydrolysis of polyphosphates include monodentate polyphosphate ligated cis to H₂O or OH^? in the coordination sphere of the metal ion:

     

A new site-specific endonuclease BbeI from Bifidobacterium breve: Restriction endonucleases; ; 3′-cohesive termini; DNA sequence analysis

A new site-specific endonuclease, BbeI, has been partially purified from the anaerobic bacterium, Bifidobac-terium breve. BbeI recognizes the hexanucleotide sequence

and cleaves it at the sites indicated by the arrows, producing 3′-cohesive termini four bases long.     

Kinetics of methyl viologen reduction by hydrogen catalyzed by hydrogenase from Desulfovibrio vulgaris

The reduction of methyl viologen by hydrogen with hydrogenase was studied kinetically. The initial rate of the reduction was expressed as

where k′, K₂, and K₃ are constants and [S′] is the concentration of methyl viologen.According to this equation, a sequential mechanism was proposed. By combining the mechanism of hydrogen production from the reduced methyl viologen, a reaction mechanism for the reduction and oxidation of methyl viologen was proposed.     

Structure of the extracellular polysaccharide from xanthomonas campestris

Xanthan gum, the extracellular polysaccharide from Xanthomonas campestris, has been reinvestigated by methylation analysis, and by uronic acid degradation followed by oxidation and elimination of the oxidized residue. The polysaccharide is composed of pentasaccharide repeating-units with the following structure:

     

Nascent α2-macroglobulin-trypsin complex: Incorporation of amines and water at the thiol esterified GLX-residues of α2-macroglobulin during activation with trypsin

During complex formation between α₂-macroglobulin and trypsin the internal thiol esters (one in each of the four M_r 180,000 subunits) are activated. In this activated state (nascent α₂-macroglobulin-trypsin complex) addition of low M_r amines lead to their covalent incorporation into α₂M. Evidence is presented showing that covalent binding of added amines occurs at the γ-carbonyl group of the Glx-residue in the thiol ester sequence:

     

Evidence for a convalent intermediate between alpha-glucosidase and glucose

A stable enzyme-glucose intermediate has been obtained in the short-term reaction between α-methyl-

-glucosidase and α-methyl-

-¹⁴C-glucopyranoside. A rapid-flow technique was employed in which phenol was used to terminate the reaction and to trap the product. It is believed that a covalent linkage is involved because (a) continued washing of the denatured protein failed to remove the radioactivity and (b) the radioactivity was retained by a tryptic peptide isolated by gel filtration. Treatment of the labeled protein with 2 $\text{N}$ HCl at room temperature released over 80% of the radioactivity as a compound with the same chromatographic mobility as glucose. No radioactive product was formed when bovine serum albumin replaced the enzyme, nor when glucosylamine, a potent glucosidase inhibitor, was present with the enzyme.     

Conformations of the d-glucarolactones and d-glucaric acid in solution

The conformations of d-glucaric acid (1), d-glucaro-1,4-lactone (2), d-glucaro-6,3-lactone (3), and d-glucaro-1,4:6,3-dilactone (4) in solution were investigated by ¹H-n.m.r. and ¹³C-p.F.t., n.m.r. spectroscopy. The solvents used were deuterium oxide, methanol-d₄, and dimethyl sulfoxide-d₆, and praseodymium chloride was employed as a lanthanide shift-reagent. For 2, it was found that the conformational equilibrium ³E(d)

E₃(d) exists in solution, and that the OH-5 group tends to occupy the position over the lactone ring in the favored E₃(d),gg conformation. The n.m.r. data for 3 indicated that the conformational equilibrium is shifted in favor of the ⁴E(d)

E₄(d),gt conformation in solution. The dienvelope conformation ³E:E₄(d) was found to be the favored conformation of 4. For 1, a conformational equilibrium between one planar, zigzag form and two sickle forms was indicated by the n.m.r. data observed. ¹³C-N.m.r. spectroscopy proved to be a convenient method for monitoring the lactonization of 1, and the hydrolysis of its lactones. Lactones other than 2–4 were not found in solutions prepared from 1–4, either during their mutarotation or after equilibration at 30°.     

Stimuli-responsive polymeric materials for separation of biomolecules

Different stages in the SRP Virtuous Cycle for the development of temperature responsive polymers.