Purification and some of the properties of a novel secondary alcohol dehydrogenase from Alcaligenes eutrophus

Alcaligenes eutrophus utilizing nerolidol, a sesquiterpene alcohol,as the sole source of carbon contains an inducible NAD(P)+-linked secondary alcohol dehydrogenase (SADH). The enzyme was purified to homogeneity by a combination of salt precipitation, ion exchange and affinity matri chromatographies. The apparent molecular mass of the enzyme was estimated to be 139 KDa with four identical subunits of 38.5 KDa. The enzyme carried out both oxidation and reduction reactions. At pH 5.5, enzyme catalyzed the stereospecific reduction of prochiral ketones to secondary alcohols. The pH optimum for the oxidation reaction was 9.5. NADP+ and NADPH were respectively preferred over NAD+ and NADH for oxidation and reduction reactions. Some of the properties of this enzyme were found to be significantly different from those thus far described.

Expression, purification and X-ray analysis of 1,3-propanediol dehydrogenase (Aq_1145) from Aquifex aeolicus VF5

1,3-Propanediol dehydrogenase is an enzyme that catalyzes the oxidation of 1,3-propanediol to 3-hydroxypropanal with the simultaneous reduction of NADP(+) to NADPH. SeMet-labelled 1,3-propanediol dehydrogenase protein from the hyperthermophilic bacterium Aquifex aeolicus VF5 was overexpressed in Escherichia coli and purified to homogeneity. Crystals of this protein were grown from an acidic buffer with ammonium sulfate as the precipitant. Single-wavelength data were collected at the selenium peak to a resolution of 2.4 angstrom. The crystal belonged to space group P3(2), with unit-cell parameters a = b = 142.19, c = 123.34 angstrom. The structure contained two dimers in the asymmetric unit and was solved by the MR-SAD approach.

Stimulation by thyrotropin, long-acting thyroid stimulator, and dibutyryl 3′, 5′-adenosine monophosphate of protein and ribonucleic acid synthesis and ribonucleic acid polymerase activities in porcine thyroid in Vitro

The in vitro incorporation of [3H]uridine into RNA and [3H]leucine into protein in slices of porcine thyroid was studied. Thyrotropin (10-500 mU/ml of medium), when added with [3H]uridine, inhibited incorporation into RNA, but as little as 10 mU of thyrotropin per ml stimulated incorporation of [3H]orotic acid into RNA. Uridine kinase (EC 2.7.1.48) was found to be inhibited in slices incubated with thyrotropin whereas UMP 5′ nucleotidase (EC 2.1.3.5) was not. Preincubation of slices with thyrotropin (5-50 mU/ml) led to enhanced incorporation of subsequently added [3H]uridine and [3H]leucine. When slices were preincubated with long-acting thyroid stimulator-IgG (2.5 or 5 mg per ml of medium) incorporation of [3H]uridine and [3H]leucine was similarly enhanced, with the smaller concentration being more effective. Without preincubation these stimulatory effects were mimicked by 1 mM dibutyryl 3′,5′-AMP and, to a lesser extent, 1 mM 3′,5′-AMP. AMP and ATP also stimulated [3H]uridine incorporation in this system but only after more prolonged periods of incubation than were required for the other nucleotides. RNA polymerase (EC 2.7.7.6) activity measured in isolated thyroid nuclei had two components, one Mg2+-stimulated and the other requ ring Mn2+ and high salt content [0.4 M (NH4)2SO4]. These activities, and particularly the former, were enhanced if thyroid slices were incubated with thyrotropin (5-100 mU/ml of medium), 2.5 mg or 5.0 mg of long-acting thyroid stimulator-IgG per ml, or 1 mM dibutyryl 3′,5′-AMP, before isolatior of the nuclei and measurement of enzyme activities; 1 mM AMP, ADP, or 2′,3′-GMP had no influence. Added directly to the nuclei, thyrotropin, long-acting thyroid stimulator-IgG, and dibutyryl 3′,5′-AMP had no effect on RNA polymerase activities. These data are seen as affording evidence for mediation by 3′,5′-AMP of effects of thyrotropin and long-acting thyroid stimulator on thyroid RNA and protein synthesis, at least in part through an indirect stimulation of nuclear RNA polymerase activities.

Stimulation by adenosine 3′,5′-cyclic monophosphate of protein synthesis by adenohypophyseal polyribosomes

Addition of dibutyryl 3′,5′-cyclic AMP to slices of bovine pituitary stimulated incorporation of [3H]leucine into protein, whether or not actinomycin D was present; therefore the influence of 3′,5′-cyclic AMP on protein synthesis by bovine pituitary polysomes was studied. If the cyclic nucleotide was added to the complete protein-synthesizing system (including pH 5.0 enzyme), stimulation of [3H]leucine incorporation occurred only with pH 5.0 enzyme from rat liver; there was no stimulation when homologous enzyme, i.e., from bovine pituitary, was used. Addition of 3′,5′-cyclic AMP to the polysomes, before addition of pH 5.0 enzyme, resulted in stimulation of protein synthesis with either source of enzyme, but stimulation was facilitated to a greater degree, over the range 0.5-2 mM 3′,5′-cyclic AMP, when rat liver was the source. The stimulation of protein synthesis was prevented by the addition of cycloheximide. With rat liver pH 5.0 enzyme the product of hydrolysis of 3′,5′-cyclic AMP was mainly 5′-AMP whereas with pituitary pH 5.0 enzyme there was also dephosphorylation and deamination resulting in production of hypoxanthine and other bases. However, using either source of pH 5.0 enzyme and the complete protein-synthesizing system (i.e., including an ATP-regenerating mechanism) most of the 3H from hydrolysis of [3H]3′,5′-cyclic AMP was incorporated into ATP. The data are seen as compatible with a stimulation by 3′,5′-cyclic AMP of translation by pituitary polysomes; the significance of the importance of the source of pH 5.0 enzyme used in the system is obscure.

Activation of succinate dehydrogenase by 2,4-dinitrophenol-a competitive inhibitor

1.The reported inhibition of the succinate oxidase system at high concentrations of dinitrophenol, considered to be at the primary dehydrogenase level, is now confirmed by measuring the activity of succinate dehydrogenase (succinate:(acceptor) oxidoreductase, EC 1.3.99.1) in the presence of dinitrophenol, using the dye reduction method. 2. 2. The results indicate that the inhibition of substrate-activated succinate dehydrogenase by dinitrophenol is competitive. 3. 3. Low concentrations of dinitrophenol inhibited the basal activity, while at higher concentrations the kinetics were complicated by an apparent activation. 4. 4. Preincubation of mitochondria with dinitrophenol stimulated the enzyme activity, a phenomenon shown by succinate and competitive inhibitors. This activation was very rapid at 37°, compared to that by succinate; activation by dinitrophenol was observed even at 25°, under conditions where succinate had no effect. 5. 5. Repeated washing of the activated mitochondrial samples with the sucrose homogenizing medium reduced the succinate-stimulated activity to the basal level, but only partially reversed the dinitrophenol activation. 6. 6. The relevance of this activation phenomenon to the physiological modulation of this enzyme system is discussed.

On the specificity of activation of mitochondrial succinate dehydrogenase by ubiquinol

Activation of succinate dehydrogenase on preincubation with mitochondria is not specific for ubiquinol-10. Other homologues of ubiquinol, plastoquinol, o-, m- and p-quinols, 2-nitro-, 4-nitro- and 2,4-dinitro- phenols showed different degrees of activation. The lipid quinols, however, showed activation greater than succinate, hitherto considered to give maximum activation.

Structure of 5-methyl-2'-deoxycytidine 5'-monophosphate dihydrate

CloHI6N307P.2H20 , Mr = 357.2, triclinic, P1, a = 4-8520 (8), b = 8"3703 (8), c = 10.0199 (12) A, a= 104.578 (9),/3= 102.332 (13), 7=93.670(11) o, V = 381"75 A 3, Z = 1, Dx = 1"55, Dm = 1"53 Mg m -3, a(Cu Ka) = 1.5418 A,/z = 2.01 mm- l, F(000) = 188, T= 290 K, R = 0-049 for 1568 unique reflections.

X-Ray structure of a ternary complex, copper(II)–lnosine 5-monophosphate–benzimidazole. The first example for the absence of direct metal–nucleotide interaction

The unprecedented absence of direct metal–nucleotide interaction has been observed in the X-ray structure of the ternary metal nucleotide system [Cu(bzim)(H2O)5]2+[IMP]2–·3H2O [IMP = inosine 5-monophosphate(2–), bzim = benzimidazole). The complex crystallizes in the space group P21 with a= 7.013(2), b= 13.179(9), c= 14.565(9)Å, = 94.82(4)°, and Z= 2. The structure was solved by the heavy-atom method and refined by full-matrix least squares on the basis of 1 761 observed (I? 3i) reflections to final R and R values of 0.034 and 0.036 respectively. The CuII has a distorted octahedral co-ordination with a nitrogen of the bzim ligand [Cu–N 1.947(5)Å] and three oxygens of water molecules in the basal plane [mean Cu–O 2.017(3)Å] and two more water oxygens at axial positions [Cu–O 2.194(6) and 2.732(5)Å]. The nucleotide base stacks with the bzim ligand at an average distance of 3.5 Å and an angle of 22°. In the lattice, N(7) of the base is linked to a lattice water through a hydrogen bond, while all the phosphate oxygens are involved in hydrogen bonds with co-ordinated as well as lattice water molecules. The co-ordination behaviour of IMP to CuII is compared in structures containing different -aromatic amines in order to assess the influence of the ternary ligand in complex formation. The present results indicate that, apart from the commonly observed phosphate binding, other modes of co-ordination are possible, these being influenced mainly by the -accepting properties of the ternary ligand.

Increase in alpha-glycerophosphate dehydrogenase and other oxidoreductase activities of hepatic mitochondria on administration of vanadate to the rat

Liver mitochondria isolated from vanadate-administered rats showed increased (20-25%) rates of oxidation of both NAD(+)-linked substrates and succinate. Respiratory control index and ADP/O were unaffected by the treatment. Dormant and uncoupler-stimulated ATPase activity also was not affected by vanadate administration. Membrane-bound, electron-transport-linked dehydrogenase activities (both NAD(+)- and succinate-dependent) increased by 15-20% on vanadate treatment. Mitochondrial alpha-glycerophosphate dehydrogenase activity increased by 50% on vanadate administration. The above effects of vanadate on oxidoreductase activities could be prevented by the prior administration of antagonists to alpha-adrenergic receptors. Substrate-dependent H2O2 generation by mitochondria also showed an increase on vanadate administration.

Crystal and molecular structure of the ammonium salt of the dinucleoside monophosphate d(CpG)

The crystal and molecular structure of the ammonium salt of deoxycytidylyl-(3'-5')-deoxyguanosine has been determined from 0.85 A resolution single crystal X-ray diffraction data. The crystals obtained by acetone diffusion technique at -20 degrees C, are orthorhombic, P212121, a = 12.880(2), b = 17444(2) and c = 27.642(2) A. The structure was solved by high resolution Patterson and Fourier methods and refined to R = 0.136. There are two d(CpG) molecules in the asymmetric unit forming a mini left handed Z-DNA helix. This is in contrast to the earlier reported forms of d(CpG) where the molecules form self base paired duplexes. There are two ammonium ions in the asymmetric unit. The major groove NH+4 ion interacts with N7 of guanines through water bridges besides making H-bonded interactions directly with the phosphate oxygen atoms. A second NH+4 ion is found in the minor groove interacting directly with the phosphate oxygen atoms. Symmetry related molecules pack in such a way that the cytosine base stacks on cytosine and guanine base on guanine. Our structure demonstrates that alternating d(CpG) sequences have the ability to adopt the left handed Z-DNA structure even at the dimer level i.e., in a sequence which is only two base pairs long.

Interaction of metal ions with 6-oxopurine nucleotides. Part 2. X-Ray structures of ternary nickel(II) complexes with 2'-deoxyguanosine 5'-monophosphate or guanosine 5'-monophosphate

The ternary metal nucleotide complexes [Ni(en)1.3(H2O)1.4(H2O)2][Ni(5?-dGMP)2(en)0.7-(H2O)0.6(H2O)2]·7H2O (1) and [Ni(en)2(H2O)2][Ni(5?-GMP)2(H2O)4]·6H2O (2)(en = ethylenediamine, 5?-dGMP = 2?-deoxyguanosine 5?-monophosphate, 5?-GMP = guanosine 5?-monophosphate) have been prepared and their structures analyzed by X-ray diffraction methods. Both compounds crystallise in the space group C2221 with a= 8.810(1), b= 25.090(4), c= 21.084(1)Å, and Z= 4 for (1) and a= 8.730(1), b= 25.691(4), c= 21.313(5)Å, and Z= 4 for (2). The structures were deduced from the analogous CoIII complexes and refined by full-matrix least-squares methods to final R values of 0.087 and 0.131 for 1 211 and 954 reflections for (1) and (2) respectively. An interesting feature of the deoxyribonucleotide complex (1) is that en is not totally labilized from the metal centre on nucleotide co-ordination, as observed in corresponding ribonucleotide complexes. Apart from extensive intra- and inter-molecular hydrogen bonding, the structures are stabilized by significant intracomplex base�base and base�sugar interactions. The nucleotides in both complexes have an anti base, C(2?)-endo sugar pucker, and gauche�gauche conformation about the C(4?)�C(5?) bond.