Transferase-deficiency galactosemia: evidence for the lack of a transferase protein in galactosemic red cells

Red blood cell lysates from normal individuals, a homozygous Duarte variant, and a patient with transferase-deficiency galactosemia were challenged with rabbit antibody to pure human placental galactose-1-phosphate uridylyltransferase. Although the antibody quantitatively precipitated the enzymatically active proteins in the normal and Duarte hemolysates, the Duarte sample absorbed only about one-half as much antibody as did the normal. In contrast, the antibody did not react with the galactosemic hemolysate.     

Tetrachloroplatinate(II) and hexachloroplatinate(IV) salts of N,N,N′,N′-tetramethylethylenediammonium: significant differences in the conformation of the cation, as shown by infrared and x-ray diffraction data

The structures of (H₂tmen)[PtCl₄], (1), (H₂tmen = N,N,N′,N′-tetramethylethylenediammonium), [triclinic, P ; A = 7.344(3), B = 8.345(3), C = 6.216(2) Å, α = 84.53(3), β = 109.22(3), γ = 69.43(3)°, Z = 1] and (H₂tmen)[PtCl₆], (2), [monoclinic, P2₁/a; A = 14.409(4), B = 12.736(7), C = 8.601(3) Å, β = 99.58(3)°, Z = 4] were determined from diffractometric data by Patterson and Fourier methods and refined by full-matrix least-squares to R = 0.027 and 0.039 for (1) and (2) respectively. In both cases the anions and cations are joined in polymeric chains through hydrogen bonds involving the protonated nitrogens and the co-ordinated chlorine atoms. The square-planar [PtCl₄]²⁻ and octahedral [PtCl₆]²⁻ anions are centrosymmetric; the H₂tmen²⁺ cations are centrosymmetric in (1) with a N-C-C-N dihedral angle of exactly 180°, while in (2) the dihedral angle is 166°. The different symmetry of the organic moieties/C_i and C₁ in (1) and (2) respectively] results in a different i.r. spectrum which is more complex the lower the symmetry; moreover the spectrum of a KBr pellet of (2) changes with time, finally resembling that of (1) and of the (H₂tmen)Cl₂ salt.     

Regulation of methionine biosythesis in Neurospora crassa.

The regulation of serine hydroxymethyltransferase, methylenetetrahydrofolate reductase, and methyltetrahydropteroylpolyglutamate:homocysteine methyltransferase was investigated in Neurospora crassa. Adding choline to the medium decreased the specific activity of these enzymes. Methionine potentiated the choline effect, but, when added alone, was without effect. Neither choline, methionine, nor S-adenosylmethionine appears to be the immediate corepressor of synthesis of these enzymes.Several nonallelic mutants were examined for the enzymes of methionine methyl group synthesis. The formate-requiring mutant for lacks serine hydroxymethyltransferase. The methionine-requiring mutants me-1 and me-8 lack, respectively, the reductase and the methyltransferase. The methionine-requiring mutants me-1, me-6 (folate polyglutamate synthetase deficient) and me-8 were found to have significantly higher serine hydroxymethyltransferase specific activities than did the wild-type strain.     

Hydrolysis of neutral phosphate and phosphonate esters catalysed by Co2+-chelates of tris-imidazolyl phosphines

The hydrolysis of two neutral phosphate esters, (tris-2-pyridylphosphate and tris-p-nitrophenylphosphate, 5 and 4 respectively) and a phosphonate ester (ethyl-p-nitrophenylmethylphosphonate (6)) were studied in the presence of Co²⁺-complexes of two tris- imidazolylphosphines. In the hydrolyses of both 5 and 6, the Co²⁺-complex of bis-[4,5-diisopropyl- imidazol-2-yl]imidazole-2-yl phosphine (2) appears to act as the anionic form, generated by ionization of a Co²⁺-bound H₂O having a pK_a of ～8 at 37 °C in 80% ethanol-H₂O. On the other hand, the Co²⁺-complex of bis-[4,5-diisopropylimidazol-2-yl]-4(5)- hydroxyethylimidazol-2-yl phosphine (3) catalyses the hydrolyses of 5 and 6 with an activity which increases linearly with pH. In this case, the active form of Co²⁺:3 has a pK_a in excess of 8.3 which apparently obtains from ionization of the Co²⁺- associated hydroxyethyl group. Evidence is presented that Co²⁺:3 functions as a general base catalyst in the hydrolysis of 5.     

The regulation of one-carbon oxidation in the rat by nitrous oxide and methionine

Formate is oxidized to CO₂ in the rat by folate-dependent reactions. Nitrous oxide treatment inhibited hepatic methionine synthetase activity, reduced hepatic S-adenosyl-l-methionine (Ado-Met) and tetrahydrofolate (H₄ folate) concentrations and decreased the rate of formate oxidation in the rat. The administration of methionine to nitrous oxide-treated rats increased hepatic Ado-Met concentrations and restored hepatic H₄folate levels and formate oxidation to control values but did not reverse the inhibition of methionine synthetase. Positive correlations were observed between hepatic Ado-Met levels and H₄folate concentrations and between hepatic H₄folate concentrations and formate oxidation. These results suggest that alterations in hepatic H₄folate concentrations may profoundly influence the oxidation of one-carbon compounds. They confirm the importance of the methionine synthetase reaction as a major source of regeneration of H₄folate. These findings also indicate that methionine acts at a site other than the methionine synthetase reaction to restore hepatic H₄folate concentrations and formate oxidation to control values in nitrous oxide-treated rats.     

Subsite mapping of enzymes. Use of subsite map to simulate complete time course of hydrolysis of a polymeric substrate.

In this paper we extend our earlier work on subsite mapping and show that our model for depolymerase action can be used to accurately predict product ratios vs the extent of reaction when a polymer is hydrolyzed. The experimental product ratios for Bacillus amyloliquefaciens α-amylase acting on reducing end-labeled ¹⁴C-maltodextrins ranging in chain length 3 to 10 are reported. These data and Michaelis parameters are used with a depolymerase computer model (J. D. Allen, 1977, Ph.D. thesis, University of Arkansas; J. D. Allen and J. A. Thoma, 1976, Biochem. J.159, 105) to compute an optimized subsite map. The depolymerase computer model generates a 10-subsite map for B. amyloliquefaciens α-amylase with the catalytic site located to the left of subsite 7. The binding affinities of the subsites are then used as the sole input in another computer program to quantitatively predict the mole fraction of products vs the extent of hydrolysis for substrates of varying chain length. Excellent agreement is obtained between the computed and experimental data for seven maltodextrins examined.     

Role of ATP in the cleavage mechanism of the EcoP15 restriction endonuclease

The EcoP15 restriction endonuclease forms complexes at specific sites on unmodified DNA both in the presence and in the absence of S-adenosyl-l-methionine. ATP acts as an allosteric effector of EcoP15 and induces DNA cleavage followed by release of the enzyme from the DNA. The efficiency of endonucleolytic scission varies from site to site. The nucleotide sequences at sites that are cleaved at a high frequency were compared.     

Effect of adenosylhomocysteine and other analog thioethers on a prokaryotic tRNA (guanine-7)-methyltransferase

S-Adenosylhomocysteine (SAH), a potent inhibitor of methyltransferases, and several thioethers structurally related to SAH, have been tested as potential inhibitors of tRNA (guanine-7)-methyltransferase from Salmonella typhimurium. The tested compounds are l-, d-, dl-S-adenosylhomocysteine, S-adenosylcysteine, methylthioadenosine, butylthioadenosine, thioethanoladenosine, isobutylthioadenosine, S-inosylhomocysteine, and methylthioinosine. Among them the highest inhibitory activity has been shown by SAH (K_i = 8 μM), whereas butylthioadenosine, isobutylthioadenosine, and thioethanoladenosine are almost inactive as inhibitors. The other compounds inhibit the enzyme with K_i values ranging between 400 and 800 μm. From these data it is possible to evaluate the importance of the -NH₂ and -COOH groups of the substrate in the binding to the enzyme molecule, as well as other features such as the chirality at the α-carbon atom and the length of the hydrocarbon chain connecting the -NH₂ and -COOH groups to the aromatic ring of adenosine. The aminic group of the adenosine is also critical, because S-inosylhornocysteine and methylthioinosine are poorer inhibitors in comparison with SAH and methylthioadenosine.     

The crystal and molecular structure of [Co(NH3)5PO4]·3H2O,a Na+-K+ ATPase probe

The title compound, Co(NH₃)₅PO₄, prepared by a modified literature procedure, was used to study the inhibition of Na⁺-K⁺ ATPase and to serve as a structural model for ML₄(nucleotide) complexes. The structure was determined by single crystal X-ray diffraction techniques. The crystals are monoclinic, space group P2₁/n, a = 8.638(3), b = 14.517(2), c = 9.145(2) Å, and β = 112.71(2)°. The structure, solved by the heavy atom method to an R value of 3.3% for 1924 reflections, consists of a slightly distorted octahedron with the cobalt bound to the five amines and a monodentate phosphate. Solution structural data is taken from ³¹P NMR measurements. From comparison with other metal phosphate complexes it is concluded that multiple monodentate coordination of a di- or triphosphate closely resembles the coordination of a monophosphate This is based on the similarity of the MO bond angle which is 129.6° in the present example.     

The effect of temperature on the self-association of S5 and on the association of S5 with S8 as determined by sedimentation equilibrium

Solutions of proteins S5 and S8 from the Escherichia coli 30 S ribosomal subunit have been examined by sedimentation equilibrium methods as a function of temperature for their behavior in solution as isolated components and in mixtures. The standard enthalpy and entropy at 4 °C for the isodesmic self-association of S5 were determined from a study over the temperature range of 3 to 33 °C to be 0.1 ± 0.9 kcal/mol and 18 ± 3 cal/(mol × deg), respectively. The protein S8 remained monomeric over the same range of temperature. The standard enthalpy and entropy at 4 °C for the association of S5 and S8 were determined on mixtures from a study over the temperature range of 3 to 27 °C to be ?0.4 ± 1.6 kcal/mol and 20 ± 6 cal/(mol × deg), respectively. Based on these values and the previously determined standard Gibbs free energies (S. H. Tindall and K. C. Aune, 1981, Biochemistry20, 4861–4866), the driving force for the self-association of S5 and the association of S5 with S8 could be interpreted as being derived from the expulsion of water upon ion pair formation at the interaction sites.     

5-Nitro-2'-deoxyuridylate: a mechanism-based inhibitor of thymidylate synthetase.

5-Nitro-2′-deoxyuridylate is a potent reversible inhibitor of thymidylate synthetase. Once the reversible binary complex is formed, a nucleophile of the enzyme covalently binds to the 6-position of this inhibitor. The interaction does not require the cofactor of the normal enzymic reaction, and spectrophotometric properties of the complex are ideal for detailed studies of the interaction.     

Studies on carnitine palmitoyl transferase: The similar nature of CPTI (inner form) and CPTO (outer form)

Rat liver mitochondrial carnitine palmitoyl transferase (CPT) was found to reside in two mitochondrial locations. Twenty to twenty-five percent of the total CPT activity was easily released and solubilized by digitonin. This activity appeared to be the outer form of CPT (CPT_O). The remainder of the activity or the inner CPT (CPT_I) was tightly membrane bound. Trypsin digestion of the digitonin prepared mitoplast did not affect residual CPT activity indicating that this activity probably resided on the inner side of the membrane. Following their separation by digitonin treatment, CPT_O and CPT_I were partially purified 14.7-and 16.7-fold, respectively. The purification of each enzyme involved extraction from the membrane with Tween 80, ammonium sulfate fractionation, gel filtration, and another ammonium sulfate fractionation. The partially purified CPT_O and CPT_I were found to have identical elution volumes from a G-200 column corresponding to a molecular weight of 430,000. Also they both were found to have nearly identical K_m values for palmitoyl-CoA, palmitoyl-carnitine, CoA, and carnitine suggesting they were identical enzymes. The V values could not be compared due to differences in purity, but the ratio of V in the forward direction to V in the reverse direction was identical for CPT_I and CPT_O again suggesting enzyme identity. Assay of the CPT system “in situ” by following the reduction of the acyl-CoA dehydrogenase, a flavoprotein, suggested that the activity of CPT_I was 450-fold greater than the activity of CPT_O when both were present in the intact membrane. These data suggest that “in situ” factors exist which greatly change the catalytic properties of CPT_I compared to CPT_O.     

DT-diaphorase as a quinone reductase: a cellular control device against semiquinone and superoxide radical formation

Rat liver microsomes incubated in the presence of NADPH catalyze the oxidation of menadione (2-methyl-1,4-naphthoquinone) by two pathways: NADPH-cytochrome P-450 reductase and DT-diaphorase. The former pathway gives rise to labile semiquinones which are readily autooxidized as revealed by a nonstoichiometric NADPH oxidation and a concomitant O₂ consumption. The reduction of menadione catalyzed by DT-diaphorase on the other hand results in a relatively stable hydroquinone accompanied by a stoichiometric oxidation of NADPH and no O₂ consumption. The total amount of NADPH oxidized by a given amount of menadione reflects the relative contributions of the two pathways which can be demonstrated by the addition of selective inhibitors of the two enzymes or by treatment of the rats with phenobarbital or 3-methylcholanthrene which preferentially induces NADPH-cytochrome P-450 reductase and DT-diaphorase, respectively. Addition of cytosol, which contains the bulk of cellular DT-diaphorase, minimizes the formation of semiquinones and the concomitant O₂ consumption. Data relating to other quinones are also presented. The results support the earlier proposal that DT-diaphorase serves as a cellular control device against quinone toxicity.     

Crystal structure of the low-dimensional uranium pentatelluride: UTe5

The crystal structure of UTe₅ (a = 17.915(5), b = 10.407(3), c = 4.220(2) Å, Pnma, Pn2₁a, Z =4) was refined from 822 intensities with I>3σ(I) to a conventional R factor of 0.060. The uranium coordination polyhedron is a three capped tellurium trigonal prism, and all the Te atoms are involved in TeTe bonds. The structure is built up with infinite chains of prisms stacked in the $\text{c}$ direction. The chains are linked into (b, c) layers by a single Te atom which exhibits some positional disorder.     

Detecting the bonding type of dithiocarbamate ligands in their complexes as inferred from the asymmetric CS mode

The IR spectra of a number of dithiocarbamate (dtc) complexes (M(R₂dtc)₂, n = 2, M = Ni, Cu, Zn, Cd, Pb, Hg, Se, Te; n = 3, M = Cr, Fe, Co, As, Sb, Bi, R = Et, Prⁿ, Prⁱ, Buⁿ, Brⁱ, as well as the laser Raman spectra of a few colourless compounds (M(Et₂dtc)₂ M = Zn, Cd, Pb, Hg), have been recorded and discussed as to the validity of the Bonati-Ugo (BU) criterion for discerning the dtc bonding type from its ν_as(CS) band (ca. 1000 cm^?1), By comparing these bands for dtc complexes containing different N-substituted ligands, their splittings can be proved to be due to interligand coupling of the CS ligand modes. Further comparison with X-ray diffraction data shows that the dtc ligands, irrespective of the host complex or the ligand bonding type, are at sites of C₁ symmetry, thus ruling out the possibility to detect the ligand bonding type from the solid state vibrational spectra. New evidence is presented that the RN modes are present in the 1000 cm^?1 region, thus making it unsuitable for the determination of the ligand bonding type.     

Binding study of the drug cis-dichlorodiammineplatinum(II)to G p5′ and dGp5′ by high resolution proton and carbon-13 NMR spectroscopy

The molecular mode of action leading to the anticancer activity of the drug cis-diamminedichloroplatinum(II), cis-DDP or cis-platinum is still the subject of speculation. In the present high field (400 MHz) ¹H NMR study the results on coupling constants for cis- and trans-diammine bis(guanosine- 5′-monophosphate) and (d-guanosine-5′-monophosphate)platinum(II) complexes are presented and discussed. The ¹H and ¹³C NMR chemical shifts obtained are consistent with the drug binding to N7 of each guanine. It has been found that the drug induces different conformational changes in the nucleotide from the trans-DDP isomer.     

An aromatic schiff base stabilised as a coordinated enol iminium zwitterion by complex formation with a series of divalent metal ions

The Schiff base N-(2-hydroxy-3-carboxy-1-naphthylidine)-4-methyl-2-sulphonic acid aniline (bonsaH₃) has been found to react with a range of divalent metal ions (Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Zn²⁺ and UO₂²⁺ to give red-yellow insoluble complexes (bonsaH)_m(H₂O)_n. The solid state diffuse reflectance spectra of all the complexes have an intense visible band at ca. 470 nm. This fact, together with evidence from infrared spectra and room-temperature magnetic-moment measurements, suggests that in all cases the ligand is coordinated to the metal ion in the solid state in the enol-iminium zwitterionic form. The ¹H NMR spectra of the Mg²⁺ and Zn²⁺ complexes in DMSO-d₆ indicate that a different structure is adopted in this solvent. Comparisons with the spectra of bonsa-H₃ and (bonsa-H₂)K·H₂O suggest that the solution structure is that of an enol-imine.