On the action of hydroxylamine,hydrazine and their derivatives on the water-oxidizing complex

Photosynthetic water oxidation proceeds by a four-step sequence of one-electron oxidations which is formally described by the transitions S₀ S₁, S₁ S₂, S₂ S₃, S₃ (S₄) S₀. State S₁ is most stable in the dark. Oxygen is released during S₃ (S₄) S₀. Hydroxylamine and hydrazine interact with S₁. They cause a two-digit shift in the oxidation sequence as observed from the dark equilibrium, i.e. from S₁ S₂ : S₂ S₃ : S₃ (S₄) S₀ : S₀ S₁ :... in the absence of the agents, to S₁ ^* S₀ : S₀ S₁ : S₁ S₂ : S₂ S₃ :... in the presence of hydroxylamine or hydrazine.We measured the concentration dependence of this two-digit shift via the pattern of proton release which is associated with water oxidation. At saturating concentrations hydroxylamine and hydrazine shift the proton-release pattern from OH⁺(S₁ S₂) : 1H⁺(S₂ S₃) : 2H(S₃ S₀) : 1H⁺(S₀ S₁) :... to 2H⁺(S₁ ^* S₀) : 1H⁺(S₀ S₁) : OH⁺(S₁ S₂) : 1H⁺(S₂ S₃) : 2H⁺(S₃ S₀) :... The 2H⁺ were released upon the first excitation with a half-rise time of 3.1 ms, both with hydroxylamine and withydrazine. The concentration dependence of the shift was rather steep with an apparent Hill coefficient at half saturation of 2.43 with hydroxylamien (Förster and Junge (1985) FEBS Lett. 186, 53–57) and 1.48 with hydrazine. The concentration dependence could be explained by cooperative binding of n3 molecules of hydroxylamine and of n2 molecules of hydrazine, respectively. Tentatively, we explain the interaction of hydroxylamine and hydrazine with the water-oxidizing complex (WOC) as follows: Two bridging ligands, possible Cl^- or OH^-, which normally connect two Mn nuclei, can be substituted by either 4 molecules of hydroxylamine or 2 molecules of hydrazine when the WOC resides in state S₁.Abbreviations DNP-INT dinitrophenylether of iodonitrothymol - FWHM full width at half maximum - NR neutral red (3-amino-7-dimethylamino-2-methylphenazine-HCI) - PS II photosystem II - WOC or (in formulas:) W water-oxidizing complex Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Monoclonal antibody specific for α2→8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing 28-linked polysialic acid (8Neu5Gc2) _n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (8KDN2) _n 6(KDN23Gal13GlNAc13) GalNAc1 residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.kdn8kdn was shown specifically to recognize the 28-linked oligo/polyKDN sequences, (8KDN2) _n , and to be able to distinguish specifically (8KDN2) _n chains from (8Neu5Ac2) _n and (8Neu5Gc2) _n chains. The antibody was used successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several controls verified the specificity of the immunohistochemical staining, thus providing the first demonstration of (8KDN2) _n sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (8KDN2) _n chains and will facilitate studies on their biosynthesis, intracellular localization and function.     

Electron transport-linked proton translocation at nitrite reduction inCampylobacter sputorum subspeciesbubulus

Campylobacter sputorum subspeciesbubulus contains a membrane-bound nitrite reductase which catalyses the six-electron reduction of nitrite to ammonia. Formate andL-lactate are used as hydrogen donors. Cells ofC. sputorum grown with nitrate or nitrite contain cytochromes of theb-andc-type and a carbon monoxide-binding cytochromec. In addition, a special membrane-bound carbon monoxide-binding pigment is found. Nitrite reduction with formate orL-lactate as a hydrogen donor is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Nitrite reduction by bacterial suspensions with lactate as a hydrogen donor is strongly inhibited by carbonylcyanide-m-chlorophenyl-hydrazone (CCCP) whereas nitrite reduction with formate as a hydrogen donor is not inhibited at all. H⁺/O values and H⁺/NO ₂ ^- values were measured with ascorbate + N,N,N,N-tetramethyl-p-phenylenediamine (TMPD), formate (in the absence and presence of carbonic anhydrase) andL-lactate as a hydrogen donor. The results are summarized in a scheme for electron transport from formate or lactate to oxygen or nitrite which shows a periplasmic orientation of formate dehydrogenase and nitrite reductase and a cytoplasmic orientation of lactate dehydrogenase and oxygen reduction, and which shows proton translocation with a H⁺/2e value of 2.0. The H⁺/O and H⁺/NO ₂ ^- values predicted by this scheme are in good agreement with the experimental values.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - MTPP⁺ methyltriphenylphosphonium cation - TMPD N,N,N,N-tetramethyl-p-phenylenediamine; H⁺/O (H⁺/NO ₂ ^- ), number of protons liberated in the outer bulk phase at the reduction of one atom O (one ion NO ₂ ^- ); H⁺/2e (q⁺/2e), number of protons (charges) translocated across the cytoplasmic membrane during flow of two electrons to an acceptor     

A missense mutation (G197→A) in theα-l-fucosidase gene of fucosidosis patients leads to loss ofα-l-fucosidase

Fucosidosis is an autosomal recessive lysosomal storage disease resulting from the absence of -l-fucosidase activity. Two natural missense mutations (G¹⁹⁷A) and (A⁸⁶⁰G) within the -l-fucosidase gene have been reported to be homozygous in four patients with fucosidosis. Expression of wild-type and mutated -l-fucosidase cDNAs in COS-1 cells revealed complete deficiency of -l-fucosidase for the G¹⁹⁷A transition and a normal level of enzyme for A⁸⁶⁰G. We therefore conclude that the change of G¹⁹⁷A is responsible for fucosidosis in the patients while A⁸⁶⁰G is a normal polymorphic variant of -l-fucosidase.     

A new base substitution in the 5′ regulatory region of the humanAγ globin gene is linked with the βs gene

A new TA base substitution, identified inside the 5 regulatory region of the human^A globin gene (^A –499 T A), is reported. This nucleotide change was found to be linked incis with the mutation producing sickle cell anemia (CD6 GAGGTG: ^s gene).     

Molecular characterization of β-thalassemia in Czechoslovakia

Summary We have identified different -thalassemia mutations in 93 members of 34 families of Czech or Slovakian descent using gene amplification, hybridization with specific ³²P-labeled oligonucleotide probes, sequencing of amplified DNA, and gene mapping. The GA mutation at IVS-I-1 was found in 18 families; other Mediterranean mutations were IVS-II-1 (GA), IVS-II-745 (CG), IVS-I-110 (GA), and codon 39 (CT); these were present in 9 additional families. The GT mutation at codon 121, known to cause Heinzbody -thalassemia, was present in 3 families, and the frameshift at codons 82/83 (-G), first described in the Azerbaijanian population, in 2 families. A newly discovered allele was a frameshift at codons 38/39 (-C). One -thalassemia allele was incompletely characterized. We observed in 2 families a TC mutation at position +96 UTR (untranslated region) relative to the termination codon; this mutation likely is a rare polymorphism, -Thalassemia was rare; only one person carried the -^3.7 heterozygosity, and one other had a yet to be identified -thalassemia-1, while seven had the ^{anti 3.7} triplication.     

Differential expression of enzyme activities initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA

The regulation of the expression of enzyme activities catalyzing initial reactions in the anoxic metabolism of various aromatic compounds was studied at the whole cell level in the denitrifying Pseudomonas strain K 172. The specific enzyme activities were determined after growth on six different aromatic substrates (phenol, 4-hydroxybenzoate, benzoate, p-cresol, phenylacetate, 4-hydroxyphenylacetate) all being proposed to be metabolized anaerobically via benzoyl-CoA. As a control cells were grown on acetate, or aerobically on benzoate. The expression of the following enzyme activities was determined.Phenol carboxylase, as studied by the isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate; 4-hydroxybenzoyl-CoA reductase (dehydroxylating); p-cresol methylhydroxylase; 4-hydroxybenzyl alcohol dehydrogenase; 4-hydroxybenzaldehyde dehydrogenase; coenzymeA ligases for the aromatic acids benzoate, 4-hydroxybenzoate, phenylacetate, and 4-hydroxyphenylacetate; phenylglyoxylate: acceptor oxidoreductase and 4-hydroxyphenylglyoxylate: acceptor oxidoreductase; aromatic alcohol and aldehyde dehydrogenases.The formation of most active enzymes is strictly regulated; they were only induced when required, the basic activities being almost zero. The observed whole cell regulation pattern supports the postulate that the enzyme activities play a role in anoxic aromatic metabolism and that the compounds are degraded via the following intermediates: Phenol 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; benzoate benzoyl-CoA; p-cresol 4-hydroxybenzaldehyde 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; phenylacetate phenylacetyl-CoA phenylglyoxylate benzoyl-CoA plus CO₂; 4-hydroxyphenylacetate 4-hydroxyphenylacetyl-CoA 4-hydroxyphenylglyoxylate 4-hydroxybenzoyl-CoA plus CO₂ benzoyl-CoA.     

Tyrosinemia type 1 — complex splicing defects and a missense mutation in the fumarylacetoacetase gene

Two mutations are reported in six tyrosinemia type 1 patients from northern Europe. In four patients, a G to A transition at nucleotide position 1009 (G¹⁰⁰⁹A) of the fumarylacetoacetase (FAH) coding sequence caused aberrant splicing by introducing an acceptor splice site within exon 12, thereby deleting the first 50 nucleotides of this exon. The following exon-intron boundary was frequently missed, and a cryptic donor splice site within intron 12 caused a partial intron 12 retention of 105 bp. This point mutation alternatively gave a glycine 337 to serine substitution in instances of correct splicing. The mutation is rapidly detected by PvuII digestion of polymerase chain reaction (PCR)-amplified genomic DNA. Another mutation, g⁺⁵a in the intron 12 donor splice site consensus sequence (IVS12 g⁺⁵a), was found in five of the patients. This caused alternative splicing with retention of the first 105 nucleotides of intron 12, exon 12 skipping, and a combined deletion of exons 12 and 13. Rapid detection of this mutation is achieved by restriction digestion of PCR-amplified genomic DNA; a mismatch primer combined with the point mutation creates a Tru9I restriction site. One patient who was homozygous for the G¹⁰⁰⁹A mutation had a chronic form of tyrosinemia. Three patients were combined heterozygotes for G¹⁰⁰⁹A and TVS12 g⁺⁵a. Their clinical phenotypes varied from acute to chronic, indicating the impact of background genes and/or external factors on the presentation of typrosinemia type 1.     

Respiration-driven proton translocation in Thiobacillus versutus and the role of the periplasmic thiosulphate-oxidizing enzyme system

The periplasmic location of enzymes A and B of the thiosulphate-oxidizing multienzyme system of Thiobacillus versutus has been further confirmed by differential radiolabelling of periplasmic and cytoplasmic proteins. The stoichiometries of respiration-driven proton translocation in T. versutus were determined using the oxygen pulse and the initial rate methods. A value for the H⁺/O quotient (number of protons translocated per oxygen atom reduced) of about 2.8 was found for the oxidation of thiosulphate, and of about 2.5 for sulphite. The H⁺/O quotient for endogenous respiration was about 5.7. The data are shown to be in good agreement with the scheme proposed previously for thiosulphate oxidation by this organism. Proton generation during the oxidation of thiosulphate or sulphite is indicated to occur in the periplasm rather than by pumping across the cytoplasmic membrane. The results also suggest that a H⁺/O quotient of six occurs during NADH oxidation (from endogenous metabolism measurements) and that the terminal cytochrome oxidase, aa₃, does not function as a proton pump.Abbreviations DCCD dicyclohexyl carbodiimide - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - HQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - IEF isoelectric focusing - HIC hydrophobic interaction chromatography - EAI ethyl acetimidate hydrochloride - IAI isethionyl acetimidate     

High misses after odd flashes in oxygen evolution in thoroughly dark-adapted thylakoids from pea and Chenopodium album

In Photosystem II (PS II), water is oxidized to molecular oxygen and plastoquinone is reduced to plastoquinol. The oxidation of water requires the accumulation of four oxidizing equivalents, through the so-called S-states of the oxygen evolving complex; the production of plastoquinol requires the accumulation of two reducing equivalents on a bound plastoquinone, Q_B. It has been generally believed that during the flash-induced transition of each of the S-states (S_n S_n+1, where n=0, 1, 2 and 3), a certain small but equal fraction of the PS II reaction centers are unable to function and, thus, miss being turned over. We used thoroughly dark-adapted thylakoids from peas (Pisum sativum) and Chenopodium album (susceptible and resistant to atrazine) starting with 100% of the oxygen evolving complex in the S₁ state. Thylakoids were illuminated with saturating flashes, providing a double hit parameter of about 0.07. Our experimental data on flashnumber dependent oscillations in the amount of oxygen per flash fit very well with a binary pattern of misses: 0, 0.2, 0, 0.4 during S₀ S₁, S₁ S₂, S₂ S₃ and S₃ S₀ transitions. Addition of 2 mM ferricyanide appears to shift this pattern by one flash. These results are consistent with the bicycle model recently proposed by V. P. Shinkarev and C. A. Wraight (Oxygen evolution in photosynthesis: From unicycle to bicycle, 1993, Proc Natl Acad Sci USA 90: 1834–1838), where misses are due to the presence of P⁺ or Q_A ^- among the various equilibrium states of PS II centers.Abbreviations miss parameter - double hit parameter - PS II Photosystem II - Q_A primary one-electron acceptor of PS II, a plastoquinone molecule - Q_B secondary plastoquinone two-electron acceptor of PS II - S-states (S_n, where n=0, 1, 2, 3 or 4) redox states of the oxygen evolving complex     

Point mutations in the upstream region of the α-galactosidase A gene exon 6 in an atypical variant of Fabry disease

Summary Single point mutations in the upstream region of exon 6 of the -galactosidase A gene were found in two Japanese cases of the cardiac form of Fabry disease; ³⁰¹ArgGln (⁹⁰²GA) in a case that has already been published and ²⁷⁹GlnGlu (⁸³⁵CG) in a new case. They both expressed markedly low, but significant, amounts of residual activity in COS-1 cells. In contrast, two unrelated cases with classic Fabry disease were found to have different point mutations, which showed a complete loss of enzyme activity in a transient expression assay; ³²⁸GlyArg (⁹⁸²GA) in the downstream region of exon 6 in one case and two combined mutations, ⁶⁶GluGln (¹⁹⁶GC)/¹¹²ArgCys (³³⁴CT), in exon 2 in the other. We conclude, on the basis of the results recorded in this study and those in previous reports, that the pathogenesis of atypical Fabry disease is closely associated with point mutations in the upstream region of exon 6 of the -galactosidase A gene.     

Energy conservation in whole cells of the methylotrophic bacterium Methylophilus methylotrophus

Respiratory chain phosphorylation has been investigated in the methylotrophic bacterium Methylophilus methylotrophus following the addition of oxidisable substrates to aerobic, whole cell suspensions. Initial-rate experiments showed that ATP synthesis occurred at the overall expense of AMP and inorganic phosphate via the sequential action of the ATP phosphohydrolase and adenylate kinase; some of the nascent ATP was rapidly used to synthesis nonadenine nucleoside triphosphates. After being corrected for ATP turnover, Pi/O quotients of 0.46 to 0.54, 0.77 and 1.37 nmol/ng-atom O were obtained for the oxidation of methanol dehydrogenase-linked substrates (methanol, ethanol and acetaldehyde), duroquinol and formate (NAD⁺-linked) respectively. These values were proportional to the H⁺/O and/or K⁺/O quotients exhibited by these substrates, and yielded an average H⁺/ATP (H⁺/Pi) quotient of 4.2 ng-ion H⁺/nmol. Steady-state experiments showed that the extent of cellular energisation varied with the respiration rate but was always in the order methanol > duroquinol > acetaldehyde, thus indicating that under these longer-term conditions methanol was completely oxidised to yield PQQH₂ and 2NAD(P)H. These results are discussed in terms of the various reactions which lead to the generation or utilisation of the protonmotive force in this organism.Abbreviations FCCP carbonylcyanide p-trifluoromethyxyphenyl-hydrazone - bulk phase, transmembrane electrochemical potential difference of protons ( ) - pH bulk phase, transmembrane pH difference (pH_in–pH_out) - bulk phase, transmembrane electrical potential difference (_in - _out) - [P] concentration of anhydride phosphate bonds in adenine nucleotides (2[ATP]+[ADP]) - FPLC fast protein liquid chromatography - PQQ pyrroloquinoline quinone - Gp phosphorylation potential     

Energy conservation in the extreme thermophile Thermus thermophilus HB8

Whole cells of the extreme thermophile Thermus thermophilus HB8 contained a membrane-bound respiratory chain (comprised of nicotinamide nucleotide transhydrogenase, NADH dehydrogenase, menaquinone, and cytochromes b, c, aa₃, o), which exhibited a maximumH⁺/O quotient of approximately 8 g-ion H⁺·g-atom O^-1 for the oxidation of endogenous substrates. Whole cell respiration at 70° at the expense of endogenous substrates or ascorbate-TMPD generated a transmembrane protonmotive force (p) of up to 197 mV and an intracellular phosphorylation poteintial (Gp), measured under similar conditions, of approximately 43.9 kJ·mol^-1.The measured Gp/p ratio thus indicated anH⁺/ATP quotient of approximately 2.3 g-ion H⁺·mole ATP^-1. Glucose-limited continuous cultures of T. thermophilus at 60°, 70° and 78.5° exhibited extremely low moler growth yields (Y _O2 ^max 27.6 g cells·mol O ₂ ^-1 ; Y _glucose ^max 64.4 g cells ·mol glucose^-1) compared with mesophilic bacteria of similar respiratory chain composition and proton translocation efficiency. These low yields are probably at least partly explained by the extremely high permeability of the cytoplasmic membrane to H⁺, which thus causes the cells to respire rapidly in order to maintain the protonmotive force at a level commensurate with cell growth.Abbreviations TPMP⁺ triphenylmethylphosphonium cation - FCCP carbonylcyanide p-trifluoromethoxy phenythydrazone - TMPD N,N,N,N-tetramethyl-p-phenylene diamine     

Direct evidence of structural changes in reaction centers of Rb. sphaeroides containing suppressor mutations for Asp L213 → Asn: A FTIR study of QB photoreduction

In bacterial reaction centers (RCs), changes of protonation state of carboxylic groups, of quinone-protein interactions as well as backbone rearrangements occuring upon Q_B photoreduction can be revealed by FTIR difference spectroscopy. The influence of compensatory mutations to the detrimental Asp L213 Asn replacement on Q_B ^–/Q_B FTIR spectra of Rb. sphaeroides RCs was studied in three double mutants carrying a Asn M44 Asp, Arg M233 Cys, or Arg H177 His suppressor mutation. The proton uptake by Glu L212 upon Q_B ^– formation, as reflected by the positive band at 1728 cm^–1, is increased in the Asn M44 Asp and Arg H177 His suppressor RCs with respect to native RCs, and remains comparable to that observed in Asp L213 Asn mutant RCs. Only the Arg M233 Cys suppressor mutation affected the 1728 cm^–1 band, reducing its amplitude to near native level. Thus, there is no clear correlation between the apparent extent of proton uptake by Glu L212 and the recovery of the proton transfer RC function. In all of the mutant spectra, several protein (amide I and amide II) and quinone anion (C...O/C...C) modes are perturbed compared to the spectrum of native RCs. These IR data show that all of the compensatory mutations alter the semiquinone-protein interactions and the backbone providing direct evidence of structural changes accompanying the restoration of efficient proton transfer in RCs containing the Asp L213 Asn lesion.     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

Conformational analysis of the Sda determinant-containing tetrasaccharide and two mimics in aqueous solution by using 1H NMR ROESY spectroscopy in combination with MD simulations

The conformational behaviour of the spacer-linked synthetic Sd^a tetrasaccharide -d-GalpNAc-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (1) and the two mimics -d-Galp-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (2) and -d-GlcpNAc-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (3) were investigated by ¹H NMR spectroscopy in combination with molecular dynamics (MD) simulations in water. Experimental 2D ¹H ROESY cross-peak intensities (ROEs) of the tetrasaccharides were compared with calculated ROEs derived from MD trajectories using the CROSREL program. Analysis of these data indicated that the oligosaccharidic skeletons of the compounds 1–3 are rather rigid, especially the -d-Hex(NAc)-(14)-[-Neu5Ac-(23)]--d-Galp fragments. The - Neu5-Ac-(23)--d-Galp linkage occurred in two different energy minima in the three-dimensional structure of the compounds 1–3 in aqueous solution. Experimental data and dynamics simulations supported the finding that the higher energy rotamer (CHEAT forcefield) was abundant in compounds 1 and 3 due to the existence of a hydrogen bond between the carboxyl group of the sialic acid and the acetamido group of the terminal monosaccharide (GalNAc or GlcNAc) unit. The conformational similarity between 1 and 3 leads to the suggestion that also their activities will be alike.     

Formation of the imidazolides of dinucleotides under potentially prebiotic conditions

Summary Imidazolides of dinucleotides such as ImpApA can be formed from the corresponding dinucleotides in a two-stage process, which gives up to 15% yields under potentially prebiotic conditions. First a solution of the dinucleotide and sodium trimetaphosphate is dried out at constant temperature and humidity. This produces polyphosphates such as pnApA in excellent yield (80%). The products are dissolved in water, imidazole is added, and the solution is dried out again. This yields the 5-phosphorimidazolides.Abbreviations P₃! trimetaphosphate - A adenosine - U uridine - EDTA ethylenediaminetetraacetic acid - Ap adenosine 2(3)-phosphate - Ap! adenosine cyclic 2:3-phosphate - pA adenosine 5-phosphate - pA²p adenosine 2, 5-diphosphate - pA³p adenosine 3, 5-diphosphate - pAp! 5-phospho-adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - ImpA adenosine 5-phosphorimidazolide - A²pA adenylyl-[25]-adenosine - A³pA adenylyl-[35]-adenosine - A²pU adenylyl-[25]-uridine - A³pU adenylyl-[35]-uridine - pA²pA 5-phosphoadenylyl-[25]-adenosine - pA³pA 5-phospho-adenylyl-[35]-adenosine - pA²pU 5-phospho-adenylyl-[25]-uridine - pA³pU 5-phospho-adenylyl-[35]-uridine - pApN (N= A, U) 5-phosphate of a dinucleoside phosphate - p_nApN (N = A, U; n = 2, 3, 4.) 5-polyphosphate of a dinucleoside phosphate - ImpA²pA imidazolide of pA²pA - ImpA³pA imidazolide of pA³pA - ImpA²pU imidazolide of pA²pU - ImpA³pU imidazolide of pA³pU - ImpApN imidazolide of pApN     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

Production of N-glycolylneuraminic acid derived from chemically modifiedE. coli Kl bacterial polysaccharide by membrane enclosed enzymatic catalysis

Summary N-Glycolylneuraminic acid (Neu5Gc) has been prepared by enzymatic hydrolysis of its -(28) linked homopolymer. The rate of hydrolysis of the natural poly -(28)-(Neu5Ac) and the semi-synthetic poly -(28)-(Neu5Gc) were compared with the neuraminidases fromClostridium perfringens andVibrio cholerae. The natural Neu5Ac polysaccharide was a better substrate for both enzymes. For comparison, acid hydrolysis of the two polysaccharides showed extensive degradation.     

Mutational specificity of ethyl methanesulfonate in excision-repair-proficient and -deficient strains of Drosophila melanogaster

Summary The vermilion gene was used as a target to determine the mutational specificity of ethyl methanesulfonate (EMS) in germ cells of Drosophila melanogaster. To study the impact of DNA repair on the type of mutations induced, both excision-repair-proficient (exr ⁺) and excision-repair-deficient (exr ^–) strains were used for the isolation of mutant flies. In all, 28 mutants from the exr ⁺ strain and 24 from the exr ^– strain, were characterized by sequence analysis. In two mutants obtained from the exr ⁺ strain, small deletions were observed. All other mutations were caused by single base-pair changes. In two mutants double base-pair substitutions had occurred. Of the mutations induced in the exr ⁺ strain, 22 (76%) were GCAT transitions, 3 (10%) ATTA transversions, 2 (6%) GCTA transversions and 2 (6%) were deletions. As in other systems, the mutation spectrum of EMS in Drosophila is dominated by GCAT transitions. Of the mutations in an exr ^– background, 12 (48%) were GCAT transitions, 7 (28%) ATTA transversions, 5 (20%) GCTA transversions and 1 (4%) was a ATGC transition. The significant increase in the contribution of transversion mutations obtained in the absence of an active maternal excision-repair mechanism, clearly indicates efficient repair of N-alkyl adducts (7-ethyl guanine and 3-ethyl adenine) by the excision-repair system in Drosophila germ cells.