Pressure-adaptive differences in the binding and catalytic properties of muscle-type (M4) lactate dehydrogenases of shallow- and deep-living marine fishes

Summary The pressure sensitivities of substrate (pyruvate) and cofactor (NADH) binding and catalytic rate of purified muscle-type (M₄) lactate dehydrogenases (LDH, EC 1.1.1.27; NAD⁺: lactate oxidoreductase) from shallow- and deep-living teleost fishes were compared. The LDH's of the shallow species are significantly more pressure-sensitive than the LDH's of the deep-living fishes. The apparent Michaelis constant (K _m)¹ of pyruvate of the deep-living species' LDH's is pressure-insensitive over the entire pressure range used in these studies, 1 to 476 atmospheres (Fig. 1). For the LDH's of the shallow species, theK _m of pyruvate increases significantly between 1 and 68 atmospheres, and then remains stable up to 476 atmospheres. TheK _m of NADH displays a much higher pressure sensitivity. For the LDH's of the deep species, theK _m of NADH increases slightly (approximately 32%) between 1 and 68 atmospheres, and then remains stable up to 476 atmospheres (Fig. 1). TheK _m of the shallow species' LDH's rises sharply (approximately 113%) between 1 and 68 atmospheres, and then continues to increase at a slower rate up to 476 atmospheres. This marked inhibition of cofactor binding by pressure for the shallow species' LDH's may be of sufficient magnitude to seriously impair the function of these LDH's at pressures typical of those encountered by the deeper-living species.Pressure effects on optimal velocity, measured under high (optimal) concentrations of pyruvate and NADH, were generally lower for the LDH's of the deep species (Table 1).These results indicate that M₄-LDH's of shallow water fishes are not pre-adapted for function at deepsea pressures, and that the reduction of pressure sensitivities ofK _m's and catalysis may be a ubiquitous feature of adaptation to life at depth. The virtually identical pressure responses of M₄-LDH's from deepliving teleosts belonging to four different families represents a striking example of convergent evolution at the molecular level.     

Differences and similarities in binding of pyruvate and l-lactate in the active site of M4 and H4 isoforms of human lactate dehydrogenase

We present QM/MM calculations that show differences in geometries of active sites of M₄ and H₄ isoforms of human LDH ligated with oxamate, pyruvate or l-lactate. As the consequence of these differences, binding isotope effects of the methyl hydrogen atoms of pyruvate and l-lactate may be used to experimentally distinguish these isoforms. Based on the FEP calculations we argue that l-lactate is a better candidate for the experimental studies. Our calculations of energies of interactions of ligands with the active site residues provide explanation for the observed experimentally sensitivity to inhibition of the M₄ isoenzyme isoform and pinpoint the differences to interactions of the ligand with the histidine residue. We conclude that pyruvate interacts much stronger in the active site of H₄ than M₄ isoform and that the latter interactions are weaker than with water molecules in the aqueous solution.     

Substrate channeling of NADH and binding of dehydrogenases to complex I

The binding of porcine heart mitochondrial malate dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase to bovine heart NADH:ubiquinone oxidoreductase (complex I), but not that of bovine heart alpha-ketoglutarate dehydrogenase complex, is virtually abolished by 0.1 mM NADH. The malate dehydrogenase and beta-hydroxyacyl-CoA enzymes compete in part for the same binding site(s) on complex I as do the malate dehydrogenase and alpha-ketoglutarate dehydrogenase complex enzymes. Associations between mitochondrial malate dehydrogenase and bovine serum albumin were observed. Subtle convection artifacts in short-time centrifugation tests of enzyme association with the Beckman Airfuge are described. Substrate channeling of NADH from both the mitochondrial and cytoplasmic malate dehydrogenase isozymes to complex I and reduction of ubiquinone-1 were shown to occur in vitro by transient enzyme-enzyme complex formation. Excess apoenzyme causes little inhibition of the substrate channeling reaction with both malate dehydrogenase isozymes in spite of tighter equilibrium binding than the holoenzyme to complex I. This substrate channeling could, in principle, provide a dynamic microcompartmentation of mitochondrial NADH.     

Synthesis and thermal stability of Zr(BH4)4 and Zr(BD4)4 produced by mechanochemical processing

Zirconium tetrahydroborate Zr(BH₄)₄ and its deuteride compound Zr(BD₄)₄ were successfully synthesized by mechanochemical reaction between NaBH₄ or NaBD₄ and ZrCl₄, reaching yields of 55% and 46%, respectively. The influence of the synthesis parameters on the yield of Zr(BH₄)₄ was analyzed. The composition of the ZrCl₄:NaBH₄ starting mixture and the use of LiBH₄ instead of NaBH₄ as reactive show a clear effect on the Zr(BH₄)₄ yield. Instead, milling atmosphere does not affect the amount of the obtained product. FTIR analysis of atmosphere inside of milling vial allows to determine the formation of diborane during milling from Zr(BH₄)₄ decomposition. Thermal stability of pure Zr(BH₄)₄ and its deuterated compound was studied by combined gas-phase FTIR and DSC measurements under flowing Ar. We found that Zr(BH₄)₄/Zr(BD₄)₄ melt at about 305/303 K, decompose at about 430 K from the gas-phase and show evolution of B₂H₆/B₂D₆ under heating.     

Heterobimetallic aggregates of copper(I) with thiotungstates and thiomolybdates. Synthesis and characterization of polymeric (NEt4)3[Cu4(NCS)5WS4], (NEt4)2[(CuNCS)3WS4] and (NEt4)2[(CuNCS)4WS4], M = Mo, W

Reaction of (NEt₄)₂MS₄ (M = Mo, W) with CuCl and KSCN (or NH₄SCN) in acetone or acetonitrile affords a new set of mixed metal–sulfur compounds: infinite anionic chains Cu₄(NCS)₅MS_4³⁻ (1,2), (CuNCS)₃WS_4²⁻ (3) and two dimensional polymeric dianions (CuNCS)₄MS_4²⁻ (4,5). Crystal of 1 (M = W) and 3 are triclinic, space group P1(1:a = 10.356(2),b = 15.039(1),c = 17.356(2)Å, = 78.27(1)°, β = 88.89(2)° and γ = 88.60(1)°,Z = 2,R = 0.04 for 3915 independent data;3:a = 8.449(2),b = 14.622(4),c = 15.809(8)Å, = 61.84(3)°, β = 73.67(3)° and γ = 78.23(2)°,Z = 2,R = 0.029 for 6585 independent data). Crystals of 4 (M = W) and 5 (M = Mo) are monoclinic, space group P2₁/m,Z = 2 (4:a = 12.296(4),b = 14.794(4),c = 10.260(3)Åand β = 101.88(3)°,R = 0.034 for 4450 independent data;5:a = 12.306(2),b = 14.809(3),c = 10.257(2)Åand β = 101.99(3)°,R = 0.043 for 3078 independent data). The crystal structure determinations of 4 and 5 show that four edges of the tetrahedral MS_4²⁻ core are coordinated by copper atoms forming WS₄Cu₄ aggregates linked by eight-membered Cu(NCS)₂Cu rings. A two-dimensional network is thus formed in the diagonal (101) plane. The space between the anionic two-dimensional networks is filled with the NEt_4⁺ cations. Additional NCS groups lead to the [Cu₄(NCS)₅WS₄]³⁻ (1) trianion connected by NCS bridges forming pseudo-dimers. These latter are held together by weak CuS(NCS) interactions giving rise to infinite chains along a direction parallel to [100]. In contrast complex3 develops infinite chains from WS₄Cu₃ aggregates with the same Cu(NCS)₂Cu bridges as in 4 and 5. These chains are running along a direction parallel to [010]. The structural data of the different types of polymeric compounds containing MS_4²⁻ and CuNCS have been used to interpret vibrational spectroscopic data of the thiocyanate groups.     

Reductive thiocyanolysis of tetraoxorhenate (VII): Synthesis, crystal structure, catalytic oxidation and kinetic studies of (PPh4)2[Re(NCS)6] and (PPh4)2[ReO(NCS)5]

Thiocyanate ions reduce perrhenate in aqueous acidic solution, and on addition of a suitable countercation (PPh₄Cl) afford (PPh₄)₂[Re(NCS)₆] (1) and (PPh₄)₂[ReO(NCS)₅] (2), which have been confirmed by X-ray crystallography. The kinetics of the above reaction has been studied. Both the complexes exhibit efficient and highly selective catalytic epoxidation ability in the presence of NaHCO₃ as a co-catalyst and competent catalytic properties in the selective oxidation of alcohols to the corresponding aldehydes or ketones in the presence of pyrazole as an additive and sulfides to sulfoxides and sulfones. H₂O₂ was used as the terminal oxidant in all the above-mentioned oxidation reactions.     

DNA binding and cleavage activity of [Ru(NH3)4(diimine)]Cl2 complexes

The interaction of a series of mixed ligand complexes of the type [Ru(NH₃)₄(diimine)]Cl₂, where diimine=2,2^′-bipyridine (bipy), 1,10-phenanthroline (phen), 5,6-dimethyl-1,10-phenanthroline (5,6-dmp), 4,7-dimethyl-1,10-phenanthroline (4,7-dmp), 2,9-dimethyl-1,10-phenanthroline (2,9-dmp), 3,4,7,8-tetra-methyl-1,10-phenanthroline (Me₄phen), with calf thymus DNA has been studied using absorption, emission and circular dichroic spectral measurements and viscometry and electrochemical techniques. On interaction with DNA the complexes show hypochromism and red-shift in their MLCT band suggesting that the complexes bind to DNA. The magnitude of the binding constant (K_b) obtained from absorption spectral titration varies depending upon the nature of the diimine ligand: Me₄phen > 5,6-dmp > 4,7-dmp > phen suggesting the use of diimine ‘face’ of the octahedral complexes in binding to DNA. The interaction of phen complex possibly involves phen ring partially inserted into the DNA base pairs. In contrast, the methyl-substituted phen complexes would involve hydrophobic interaction of the phen ring in the grooves of DNA, which is supported by hydrogen bonding interactions of the ammonia ligands with the intrastrand nucleobases. Also the shape and size of the phen ligand as modified by the methyl substituents determine the DNA binding site sizes (0.12-0.45 base pairs). The relative emission intensities (I/I₀) of the DNA-bound complexes parallel the variation in K_b values. Almost all the metal complexes exhibit induced CD bands on binding to B DNA, with the 4,7-dmp and Me₄phen complexes inducing certain structural modifications on the biopolymer. DNA melting curves obtained in the presence of metal complexes reveal a monophasic melting of the DNA strands, the Me₄phen complex exhibiting a slightly enhanced tendency to stabilize the double-stranded DNA. There were slight to appreciable changes in the relative viscosities of DNA, which are consistent with enhanced hydrophobic interaction of the methyl-substituted phen rings. Upon interaction with CT DNA, the Me₄phen, 4,7-dmp and 5,6-dmp complexes, in contrast to bipy, phen and 2,9-dmp complexes, show a decrease in anodic peak current in their cyclic voltammograms suggesting that they exhibit enhanced DNA binding. DNA cleavage experiments show that all the complexes induce cleavage of pBR322 plasmid DNA, the Me₄phen and 5,6-dmp complexes being remarkably more efficient than other complexes.     

In vivo covalent binding of aflatoxin B1 and aflatoxin M1 to liver DNA of rat,mouse and pig

[¹⁴C]Aflatoxin B₁ (AFB₁) was isolated from cultures of Aspergillus parasiticus grown on [1-¹⁴C]sodium acetate. Covalent binding of AFB₁ to liver DNA of rat and mouse was determined 6–8 h after oral administration. The effectiveness of covalent binding, expressed as DNA binding per dose in the units of a ‘Covalent Binding Index’ (CBI), (μmol aflatoxin/mol DNA nucleotides)/(mmol aflatoxin/kg animal), was found to be 10 400 for rats and 240 for mice. These CBI partly explain the different susceptibility of the two species for the incidence of hepatic tumors.The corresponding values for pig liver DNA, 24 and 48 h after oral administration, were found to be as high as 19 100 and 13 300. DNA-binding has not so far been reported for this species although it could represent an appropriate animal model for studies where a human-like gastrointestinal tract physiology is desirable.Aflatoxin M₁ (AFM₁) is a metabolite found in the milk of cows that have been fed AFB₁-contaminated diet. [¹⁴C]AFM₁ was also found to be produced by cultures of A. parasiticus giving a yield of about 0.3% of the total aflatoxins. A test for covalent binding to rat liver DNA revealed a CBI of 2100 showing that AFM₁ must also be regarded as a strong hepatocarcinogen. It is concluded that AFB₁ contaminations should be avoided in dairy feed.     

Two different species of messenger RNAs specify synthesis of M1 and M2 pyruvate kinase subunits

A study was performed to determine whether M₁ and M₂ pyruvate kinases were synthesized under the direction of one or two messenger RNAs. We compared M₁ and M₂ pyruvate kinases purified from fresh tissues with those neosynthesized under the direction of messenger RNAs from tissues synthesizing either M₁ or M₂. RNA was isolated from rat muscle, lung, spleen and kidney by ethanol precipitation in 7 M guanidium chloride, translated in rabbit reticulocyte system and newly-synthesized pyruvate kinase subunits were purified by microimmunoaffinity chromatography. Pyruvate kinase from fresh muscle and spleen was purified in one step by a similar process. Muscle and spleen RNA directed the synthesis of M subunits with molecular weights of approx. 61000 and 62000, respectively, the same as those of the corresponding fresh tissue monomers. In addition, peptide maps obtained by partial digestion of neosynthesized M₁ and M₂ with V₈ protease from Staphylococcus aureus confirmed that these polypeptides were clearly different.     

Pressure-tuning infrared spectra of the three Magnus’ Green salts, [Pt(NH3)4][PtCl4], [Pt(ND3)4][PtCl4] and [Pt(NH3)4][PtBr4]

Pressure-tuning infrared spectra (up to ca. 40 kbar) are reported for Magnus’ Green salt, [Pt(NH₃)₄][PtCl₄] and two of its derivatives, [Pt(ND₃)₄][PtCl₄] and [Pt(NH₃)₄][PtBr₄]. The spectroscopic data indicate that there is restricted rotation of the coordinated ammonia groups about the Pt-N bonds in the complexes. It is possible that this restricted rotation is due to the presence of weak hydrogen bonding to the halogens, i.e., N-H?X (X = Cl, Br) interactions.     

Leukotriene B4 binding to human neutrophils

[³H] Leukotriene B₄ (LTB₄) binds concentration dependency to intact human polymorophonuclear leukocytes (PMN's). The binding is saturable, reaches equilibrium in 10 min at 4°C, and is readily reversible. Mathematical modeling analysis reveals biphasic binding of [³H] LTB₄ indicating two discrete populations of binding sites. The high affinity binding sites have a dissociation constant of 0.46 × 10⁻⁹M and B_max of 1.96 × 10⁴ sites per neutrophil; the low affinity binding sites have a dissociation constant of 541 × 10⁻⁹M and a B_max of 45.6 × 10⁴ sites per neutrophil. Competitive binding experiments with structural analogues of LTB₄ demonstrate that the interaction between LTB₄ and the binding site is stereospecific, and correlates with the relative biological activity of the analogs. At 25°C[³H] LTB₄ is rapidly dissociated from the binding site and metabolized to 20-OH and 20-COOH-LTB₄. Purification of neutrophils in the presence of 5-lipoxygenase inhibitors significantly increases specific [³H] LTB₄ binding, suggesting that LTB₄ is biosynthesized during the purification procedure. These data suggest that stereospecific binding and metabolism of LTB₄ in neutrophils are tightly coupled processes.     

Synthesis and crystal structure of tetranuclear copper(I) and silver(I) complexes bridged by 2-amino-1,3,4-thiadiazole (atdz): [Cu4(atdz)6](ClO4)4·2CH3OH and [Ag4(atdz)6](ClO4)4

Two novel tetracopper(I) and tetrasilver(I) complexes [Cu₄(atdz)₆](ClO₄)₄·2CH₃OH (1) and [Ag₄(atdz)₆](ClO₄)₄ (2), have been prepared using 2-amino-1,3,4-thiadiazole (atdz), and their crystal structures and properties have been determined. On each tetranuclear complex, two Cu or Ag atoms (M) are bridged by two atdz ligands to form a six-membered N₂M₂N₂ framework. The two N₂M₂N₂ frameworks are in parallel linked by another atdz ligand to provide the tetranuclear structure with a rectangular M₄ core. The four Cu or Ag atoms possess a trigonal-square geometry. The two adjacent MM separations are (3.096(1) and 3.412(1) Å) and (3.316(2) and 3.658(2) Å) for 1 and 2, respectively. On both tetranuclear complexes there are two species of hydrogen bonds between the ClO₄ ⁻ anions and the NH₂ group of atdz ligands. It is proposed that the hydrogen bonds are related to the stabilization of the tetranuclear structure during the crystallization process.     

Structural requirements for chemotactic activity of leukotriene B4 (LTB4)

LTB₄ (5s, 12R dihdroxy-6, 14-CIS-8, 10-trans-eicosatetraenoic acid) formed in activated neutrophils by lipoxygenation of arachidonic acid is an extremely potent chemotaxin. We examined structural requirements for chemotactic and aggregatory activity of the ligand using synthetic LTB₄ and several of its isomers. Additionally we examined the potency of two analogs, nor- and homo- LTB₄. Dose response curves for neutrophil chemotaxis to these compounds were obtained using a modified Boyden chamber. The mean distance cells moved into the filter was determined after 30 minutes. Peak chemotactic activity of LTB₄ was at 10⁻⁷M. At higher concentrations, chemotactic activity was decreased. The shape of the dose response curve was similar to that of FMLP except that maximum chemotaxis to LTB₄ was consistently greater than chemotaxis to FMLP. A mixture of the two epimers at C-5 and c-12 shifted the response curve to the right but did not lower maximum activity. Increasing or decreasing the chain by one carbon between the first hydroxyl group and the carboxyl group also shifted the response curve to the right without lowering maximal activity. Changing the 6 double bond from cis to trans has a greater effect. Activity was only detectable at high concentrations and maximum activity achieved was less than 50% that of LTB₄. Thus the chain length between the carboxyl and C-5 hydroxyl groups, the c-5 and c-12 absolute stereochemistry and the stereochemistry of the delta⁶ double bond are all important structural features for chemotactic activity with delta⁶ stereochemistry apparently having the greatest contribution. The relative potencies of these compounds in inducing aggregation were comparable to their chemotactic potencies. The data suggested that they acted at the same receptor since even the less active isomers were able to desensitive the neutrophils to LTB₄.     

Synthesis and characterization of isopropylamine complexes of lanthanide(II) diiodides: Molecular structure of TmI2(PrNH2)4 and EuI2(PrNH2)4

It was found that the lanthanide diiodides LnI₂ (1) (Ln = Nd, Sm, Eu, Dy, Tm, Yb) are dissolved in isopropylamine (IPA) without redox transformations. Stability of the formed solutions decreases in a row Eu ≈ Yb > Sm > Tm > Dy > Nd. Removing of a solvent in vacuum leaves complexes LnI₂(IPA)_x (2) (Nd, x = 5; Sm, Eu, Dy, Tm, Yb, x = 4) as crystalline colored solids. Stability of 2-Nd,Dy,Tm is higher than that of known THF or DME coordinated salts. Divalent state of metal in the products is confirmed by data of UV-Vis spectroscopy, magnetic measurements and their chemical behavior. Structure of 2-Eu and 2-Tm was established by X-ray diffraction analysis. Oxidation of 2-Nd,Dy in IPA affords amine-amides (PrⁱNH)Ln(IPA)_y (3) (Nd, y = 4; Dy, x = 3). n-Propylamine also dissolves the iodides 1-Sm,Eu,Dy,Tm,Yb but stability of the solutions is significantly lower. 1-Nd vigorously reacts with PrⁿNH₂ even at −30 °C which hampers the formation of the solution.     

Hydrothermal synthesis and structure of an open framework Co0.7Zn1.3(PO4)2(NH3-CH2CH2NH3) and Co6.2(OH)4(PO4)4Zn1.80, a new adamite type phase

The hydrothermal reaction of cobalt(II)oxalate di-hydrate, zinc oxide, and triethyl-orthophosphate, using 1,2-diaminoethane as structure directing template in water, produced two major crystal phases in almost equal amount: the purple crystals of [NH₃-CH₂CH₂NH₃][Co_0.7Zn_1.3(PO₄)₂] (1) and the red burgundy crystals of Co_6.2(OH)₄(PO₄)₄Zn_1.80 (2), a new adamite type phase. The structure of [NH₃-CH₂CH₂NH₃] [Co_0.7Zn_1.3(PO₄)₂] (1) exhibits a 3D open framework built from PO₄ and (Co/Zn)O₄ tetrahedra, and (Co/Zn)O₅ trigonal bipyramids, forming two major channels, an 8-membered ring channel and a 16-membered ring channel, that host the ethanediammonium ions. The Co_6.2(OH)₄(PO₄)₄Zn_1.80 (2) is isomorphous with adamite-type M₂(OH)XO₄ structure, with a condensed vertex and edge sharing network of (Co/Zn)O₅, and distorted CoO₆, and PO₄ subunits. The cobalt preference for higher coordination numbers is displayed in this structure, where the octahedral sites are wholly occupied by cobalt. Thermal analysis confirmed that these compounds display high thermal stability.     

Rhenium(IV) cyanate complexes: Synthesis, crystal structures and magnetic properties of NBu4[ReBr4(OCN)(DMF)] and (NBu4)2[ReBr(OCN)2(NCO)3]

Two new rhenium(IV) mononuclear compounds of formula NBu₄[ReBr₄(OCN)(DMF)] (1) and (NBu₄)₂[ReBr(OCN)₂(NCO)₃] (2) (NBu₄ = tetrabutylammonium cation, OCN = O-bonded cyanate anion, NCO = N-bonded cyanate anion and DMF = N,N-dimethylformamide) have been synthesized and their crystal structures determined by single-crystal X-ray diffraction. 1 crystallizes in the monoclinic system with the space group P2₁/n, whereas 2 crystallizes in the triclinic one with as space group. In both complexes the rhenium atom is six-coordinated, in 1 by four Br atoms in the equatorial plane, and two trans-oxygen atoms, one of a DMF molecule and another one from a cyanato group, while in 2 by one bromide anion and five cyanate ligands, two of which are O-bonded and three N-bonded, forming a somewhat distorted octahedral surrounding. Magnetic susceptibility measurements on polycrystalline samples of 1 and 2 in the temperature range 1.9-300 K are interpreted in terms of magnetically isolated spin quartets with large values of the zero-field splitting (|2D| is ca. 41.6 and 39.2 cm⁻¹ for 1 and 2, respectively).     

Cyano-bridged cluster-metal coordination compound: synthesis and crystal structure of [Cu(NH3)3][Cu(NH3)4][Cu(NH3)5][W4Te4(CN)12]·5H2O

Dark-brown single crystals of the title compound 1 were obtained in high yield by layering a CuCl₂ solution in 25% aqueous ammonia on a glycerol solution of K₆[W₄Te₄(CN)₁₂]·5H₂O. The complex 1 was characterized by single crystal X-ray diffraction analysis and IR spectroscopy. The X-ray structure of 1 reveals a polymeric chain cyano-bridged cluster-metal coordination compound. The [W₄Te₄(CN)₁₂]⁶⁻ cluster anions are linked one to another by Cu²⁺ cations through coordination by nitrogen atoms of the CN groups.     

葡萄糖氧化酶解除黄曲霉毒素M_1的应用研究

试验旨在研究葡萄糖氧化酶解除黄曲霉毒素M_1毒性的应用效果。选择含有7.2μg/kg(A组)和19.5μg/kg(B组)黄曲霉毒素B_1(AFB_1)的玉米饲料,分别添加5‰固体和液体葡萄糖氧化酶制剂,连续饲喂奶牛10 d,同时和延后测定鲜牛奶和尿中黄曲霉毒素M_1(AFM_1)量。测定数据表明,与AFB_1组相比,固体酶A组产牛奶中AFM_1未检出,B组中AFM_1含量平均减少了76.47%;液体酶A组产牛奶中AFM_1未检出,B组中AFM_1平均减少了82.35%。尿中AFM_1量减少了75%、70.5%和75%、73.1%;与对照组相比,牛奶产量、牛奶中蛋白质及脂肪含量没有变化。