Premeiotic DNA synthesis in fission yeast

Sporulating and various non-sporulating strains of S. pombe, especially several mutants deficient in conjugation or meiosis, were compared with respect to DNA synthesis under sporulation conditions. Meiosis and sporulation were induced by a transfer to nitrogen-free medium. As synchronized mitotic division was observed in all the strains as a first response to the shift, reducing the DNA amount per cell from the replicated state in G2 to the unreplicated state in the G1 phase of the cell cycle. Cells of the heterothallic wild-type strains ($\text{h}{}^{\mathrm{+}}\text{h}{}^{\mathrm{+}}$ or $\text{h}{}^{\mathrm{?}}\text{h}{}^{\mathrm{?}}$) accumulated in G1 with respect to DNA synthesis when they were incubated separately. In a mixed culture of these strains a period of enhanced DNA synthesis was observed after the start of zygote formation. This period of synthesis was absent in mutant fus1, where only prezygotes accumulated. Hence we conclude that in zygotic meiosis the premeiotic DNA synthesis is confined to zygotes after conjugation has been completed. In the diploid sporulating wild-type strain ($\text{h}{}^{\mathrm{+}}\text{h}{}^{\mathrm{?}}$), capable of azygotic meiosis without prior conjugation, premeiotic DNA synthesis occurred between $\text{2}\text{1}\text{2}$ and 5 h after the shift to the sporulation medium. There was no significant premeiotic DNA synthesis observed in diploid cells of the meiosis-deficient mutants mei1 or mei3, whereas premeiotic DNA synthesis proceeded normally in mutant mei4, which is blocked at a stage after commitment to meiosis in opposition to both the other mutants.     

Replication of repeated DNA in human cells

The replication pattern of the repeated sequence families of human DNA has been studied by means of DNA reassociation curves. Early- and late-replicating DNA fractions were obtained from synchronized cultures of KB cells by labeling cells with bromodeoxyuridine (BUdR) early or late in the DNA synthesis period and isolating the BUdR-containing DNA by CsCl density-gradient centrifugation. Highly repeated and moderately repeated sequence classes labeled with ${}^{14}\text{C-deoxycytidine}$ either early or late in the DNA synthesis period were also prepared. The effect of the isolated early- or late-replicating BUdR-DNA on the rate of reassociation of the ${}^{14}\text{C-labeled}$ repeated sequences was then tested. Increasing concentrations of early- or late-replicating BUdR-DNA were added to a constant amount of either ${}^{14}\text{C-labeled}$ early- or late-replicating repeated sequences, and the fraction of label in double-stranded DNA was determined. Analysis of the DNA reassociation curves so obtained indicates that some repeated sequence families are replicated throughout the DNA synthesis period whereas others are replicated primarily in the second half. This is true for both the highly-repeated and moderately-repeated sequence classes.     

The kinetics of myofibrillar protein breakdown in perfused rat skeletal muscle

N^t-Methylhistidine, a non-reutilised amino acid present in some myofibrillar proteins, was radioactively labelled in vito with $\text{[}{}^{\mathrm{Me}}\text{-}{}^3\text{H}\text{]}\text{methionine}$. The specific radioactivities of protein-bound methylhistidine and free methylhistidine in perfusate after perfusion of rat hind limbs taken from prelabelled rats was determined. The decrease in urinary methylhistidine activity with time was determined for rats similarly labelled. Comparison of the specific activities of free and bound methylhistidine and the non-linear semilogarithmic plot of urinary methylhistidine activity suggest that the myofibrillar protein catabolism, as indicated by methylhistidine release, may not be a simple exponential process. The possibility of non-random decay is discussed and an alternative model proposed.     

Effects of 5-hydroxylysine on acetylene reduction and NH4+-assimilation in the cyanobacterium Anabaena cylindrica.

5-hydroxylysine, an analogue of glutamate and lysine, causes $\text{NH}{}_4{}^{\mathrm{+}}$ production by N₂-fixing A. cylindrica; it also reversibly inhibits GS activity in vitro but has no effect on alanine dehydrogenase or GOGAT. On adding 5-hydroxylysine intracellular pools of glutamine, glutamate and aspartate decrease; those of alanine and serine increase. 5-hydroxylysine alleviates the inhibitory effect of $\text{NH}{}_4{}^{\mathrm{+}}$ on heterocyst production and C₂H₂ reduction and in $\text{NH}{}_4{}^{\mathrm{+}}\text{-grown}$ cultures results in heterocyst synthesis and in C₂H₂ reduction. The data suggest that the GS-GOGAT pathway is the sole route of importance in primary $\text{NH}{}_4{}^{\mathrm{+}}$ assimilation in A. cylindrica, that $\text{NH}{}_4{}^{\mathrm{+}}$ alone does not inhibit nitrogenase and heterocyst production, and that GS and/or a product is involved in regulating the production of both.     

Preferential solvation of methylmercurated calf thymus deoxyribonucleic acid.

The changes in polymer-solvent interactions that occur when native calf thymus DNA is dialyzed against Na₂SO₄ solutions of a given ionic strength and buffer concentration but of varying concentrations in methylmercuric hydroxide have been investigated with the help of solution density measurements at 25 °C and pH 6.8–7.0. From measurements executed under equilibrium dialysis conditions at the three salt levels 5 mm, 0.05 m, and 0.5 m Na₂SO₄ (m refers to molality) and in the presence of 5 mm cacodylic acid buffer, the density increments $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ for native calf thymus DNA were determined as a function of CH₃HgOH concentration. $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ was found not to vary with organomercurial concentration, irrespective of the concentration of supporting electrolyte, until a certain CH₃HgOH concentration level has been reached, viz., , beyond which $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ increases strongly with increasing concentration of CH₃HgOH. As is shown by optical melting, $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ becomes a function of organomercurial concentration the moment DNA undergoes denaturation brought about by the complexing of CH₃HgOH with the various N-binding sites of the base residues in the DNA double helix.Polymer-solvent interactions, expressed in terms of preferential water interactions (“net hydration”) and preferential salt interactions (“salt solvation”), were derived from the $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ data in combination with data obtained on the preferential interaction of CH₃HgOH with denatured DNA and data on the partial specific volumes of all major solution components, gathered from density measurements on solutions with fixed concentrations of diffusible components. Evidence is presented which shows that denaturation in general decreases the net hydration while salt becomes preferentially associated with the polyelectrolyte. This process is further amplified by the interaction of CH₃HgOH with denatured DNA: Methylmercurated DNA alters the redistribution of diffusible components at dialysis equilibrium to such an extent that in a formal sense large amounts of water are rejected from the immediate vicinity of the polymer. The molecular implications of these findings are explored. The results are further discussed in the light of previous findings where the methylmercury-induced denaturation of DNA had been studied with the help of buoyant density measurements in a Cs₂SO₄ density gradient and by velocity-sedimentation in a variety of sulfate media.     

Estimation of DNA, RNA and 125 I- and 3 H-labelled DNA in the same sample

Estimation of DNA, RNA, and the specific activity of DNA after labelling with [³H]thymidine and/or [¹²⁵I]iodeoxyuridine has been accomplished using a recently developed procedure for the estimation of DNA with p-nitrophenylhydrazine (pNPH). Samples of the pNPH reaction mixture are used for RNA estimation by th orcinol procedure and for ¹²⁵I and tritium measurement. Correction for ¹²⁵I contribution to the tritium measurement in double labelling experiments is accomplished either by use of a simple calibration curve (for high ${}^3\text{H}{}^{125}\text{I}$ ratios) or by removal of ¹²⁵I by hydrolysis and precipitation as AgI; in the latter procedure the efficiency of removal of ¹²⁵I was greater than 99%.     

The repetitive sequence structure of component alpha DNA and its relationship to the nucleosomes of the African green monkey.

An analysis of the repeat structure of the highly repetitive sequence, component α DNA of the African green monkey, shows that the DNA contains restriction sites for EcoRI, $\text{Eco}\text{RI}{}^1$, HindIII and HaeIII. All four restriction enzyme activities indicate a basic repeat length of 176 ± 4 base-pairs. In addition to primary $\text{Eco}\text{RI}{}^1$ and HindIII sites, about 59% of the repeat sequences contain secondary $\text{Eco}\text{RI}{}^1$ sites and about 36% of the repeat sequences contain secondary HindIII sites. The secondary sites are located less than 176 base-pairs from the primary sites and their cleavage yields several complex series of minor, intermediate segments in gels of the partial $\text{Eco}\text{RI}{}^1$ or HindIII digests. Cleavage at the secondary sites yields segments shorter than the unit monomer in the limit digests. The sites for EcoRI, $\text{Eco}\text{RI}{}^1$, HindIII and HaeIII have been mapped within the repeat unit.Treatment of the monkey nuclei with micrococcal nuclease at 2 °C and in the presence of 80 mm-NaCl reveals two distinct populations of nucleosomes. One population contains bulk DNA sequences, and after cleavage with micrococcal nuclease this population yields heterogeneous segments of DNA spanning 180 to 200 base-pairs in length. The other population contains component α sequences and after cleavage with micrococcal nuclease yields homogeneous segments of component α DNA that are exact multiples of the basic sequence repeat unit of 176 base-pairs. Thus, the cleavage by micrococcal nuclease of nucleosomal arrays containing component α sequences is as regular and precise as the cleavage of the purified DNA by the restriction enzymes. The resolution of the two distinct subsets of nucleosomes in the monkey nuclei is dependent upon the conditions of ionic strength and temperature employed during the nuclear isolation and the micrococcal nuclease digestion.These observations are consistent with a phase relation between the component α repeat sequences and the associated nucleosomal proteins (Musich et al., 1977b). They are also in accord with the hypothesis that the subunit structure of constitutive heterochromatin modulates or determines the repeat sequence structure and hence, the evolution of many highly repetitive mammalian DNAs (Maio et al., 1977).     

The aminoacyl-tRNA synthetase-tRNA complex: detection by differential labelling of lysine residues involved in complex formation

The interaction of tRNA^Tyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by differential acetylation of lysine residues. The synthetase was trace-labelled in the free form and as the synthetase-tRNA^Tyr complex with [³H]acetic anhydride. In a second step the two ³H-labelled enzyme preparations were fully acetylated with cold reagent under denaturing conditions and were mixed with synthetase that had been homogeneously labelled with excess [¹⁴C]acetic anhydride. Peptides containing labelled lysine residues were isolated after chymotryptic digestion and their ${}^{14}\text{C}{}^3\text{H}$ ratios were determined. These ratios reflect the reactivity of primary amino groups towards acetic anhydride.Involvement of lysine side-chains in complex formation with tRNA^Tyr was suggested from altered ${}^{14}\text{C}{}^3\text{H}$ ratios. Out of the 22 primary amino groups of tyrosyl-tRNA synthetase at least three showed reduced reactivities towards acetic anhydride in the synthetase-tRNA^Tyr complex by factors of 1.6, 1.9 and 6.8, respectively. The sequences around these lysine residues have been determined enabling their placement when the primary and tertiary structure of the enzyme are available (G. L. E. Koch, to be published). No lysine residue of increased reactivity in the synthetase-tRNA^Tyr complex has been detected.Only one molecule of tRNA^Tyr binds to the dimeric synthetase molecule under the conditions of the differential labelling. If the binding site for the tRNA is on one of the two identical subunits, any observed decrease in chemical reactivity of a particular lysine residue should not exceed a factor of two. The detection of a lysine residue which reacts about seven times more slowly in the synthetase-tRNA complex could therefore indicate that the single binding site is formed by both enzyme subunits.     

Temperature-sensitive mutations in Drosophila melanogaster. 8. Temperature-sensitive periods of the lethal and morphological phenotypes of selected combinations of notch-locus mutations

Temperature-shift experiments were performed on five Notch-locus genotypes with temperature-sensitive phenotypes. The results show that temperature-sensitive periods (TSPs) for lethality may occur at any developmental stage: (1) $\text{N}{}^{\mathrm{g11}}\text{N}{}^{\mathrm{g11}}$;Dp^51b7 having a short embryonic TSP for lethality, (2) $\text{Ax}{}^{16172}\text{N}{}^{\mathrm{?40}}$ having a second-instar TSP for lethality, and (3) $\text{N}{}^{\mathrm{?103}}\text{fa}{}^{\mathrm{no}}$ with a long, possibly polyphasic, TSP, beginning in the embryonic stage and ending in the pupal stage. On the other hand, TSPs for adult morphological phenotypes appear to be restricted to the third larval instar: (1) $\text{Ax}{}^{16172}\text{N}{}^{\mathrm{?40}}$ having third-instar TSPs for wing vein gapping and ocellar bristle loss, and (2) $\text{N}{}^{\mathrm{?103}}\text{spl}$ having third-instar TSPs for eye facet disarray, wing notching, bristle number variation, and fusion of tarsal segments. The significance of these results is discussed in terms of the role of the Notch locus in development.     

A simple logarithmic amplifier for the photographic quantitation of DNA in gel electrophoresis

For the quantitative determination of nonradioactive DNA fragments by gel electrophoresis, it is usually necessary to photograph the gel after staining with ethidium bromide and evaluate the negative by densitometry. It has previously been shown that, because of the logarithmic nature of the photographic process, it is not the optical density (E) of the film which is proportional to the amount of DNA in the gel but instead the value $\text{10}{}^{\mathrm{<mtext>E</mtext><mtext>\gamma </mtext>}}$, γ being a film constant. We describe the design of a simple instrument that converts E into $\text{10}{}^{\mathrm{<mtext>E</mtext><mtext>\gamma </mtext>}}$. The instrument can be built in any electronic workshop at low cost. When it is used together with a standard recording densitometer, densitometric tracings of $\text{10}{}^{\mathrm{<mtext>E</mtext><mtext>\gamma </mtext>}}$ are obtained directly. These tracings can be quantitated by simple peak area measurements, thereby circumventing complicated mathematical transformations. Quantitative analyses of a linear and an exponential densitogram of restriction nuclease digested plasmid DNA are presented to demonstrate the usefulness of the instrument.     

The effect of experimentally induced triploidy on the lethal expression of the t12 mutation in mouse embryos

Diploid embryos which are homozygous for the t¹² mutation die at the morula stage. In the current studies, ova from heterozygous ($\text{+}\text{t}{}^{12}$) females were fertilized in vitro with spermatozoa from $\text{+}\text{t}{}^{12}$ males. The fertilized ova were immediately placed into media containing cytochalasin B to prevent second polar body formation, producing +/+/+, +/+/t¹², +/t¹²/t¹², and t¹²/t¹²/t¹² embryos. The subsequent development of these triploid embryos was compared with that of diploid +/+, $\text{+}\text{t}{}^{12}$, and $\text{t}{}^{12}\text{t}{}^{12}$ embryos developing from ova which were also fertilized in vitro with spermatozoa from $\text{+}\text{t}{}^{12}$ males but which were not treated with cytochalasin B immediately following gamete coincubation. The data show that those triploid embryos which possess a wild-type allele and two mutant alleles are phenotypically wild type while those possessing three mutant alleles are not phenotypically distinguishable from their diploid ($\text{t}{}^{12}\text{t}{}^{12}$) counterparts. Like $\text{t}{}^{12}\text{t}{}^{12}$ embryos, t¹²/t¹²/t¹² embryos die at the morula stage, prior to blastocoelic cavity formation.     

Studies on (K+ + H+)-ATPase IV. Effects of phospholipase C treatment

(1) The total phospholipid content of a gradient purified ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase preparation from pig gastric mucosa is 105 μmol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 μmol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase and K⁺ stimulated $\text{p-}\text{nitrophenylphosphatase}$ activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP and temperature dependence of the gastric ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase, but slightly decreases the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ value for K⁺. (5) Phospholipase C treatment lowers the $\text{Ado}\text{PP[}\text{NH}\text{]P}$ binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules.     

On the effect of rfe mutation on the biosynthesis of the 08 and 09 antigens of E. coli.

From phosphomannose isomerase-less mutants of $\text{E. coli}$ strains 08 and 09, $\text{rfe}{}^{\mathrm{?}}$ derivatives were constructed by recombination with a $\text{Salmonella rfe}{}^{\mathrm{?}}$ donor. In contrast to membranes from the parent $\text{E. coli}$ strains, those from the $\text{rfe}{}^{\mathrm{?}}$ recombinants did not synthesize the 08 or 09 mannan from GDP mannose $\text{in vitro}$. They could, however, be restored to biosynthetic activity with butanol extracts from the $\text{E. coli rfe}{}^{\mathrm{+}}$ bacteria. This indicated that the $\text{rfe}$ mutation affects the synthesis of a hydrophobic acceptor.     

Transfer of N, N'-diacetylchitobiose from dolichyl diphosphate into a gray matter membrane glycoprotein

Gray matter and white matter membranes catalyze the transfer of label from UDP-N-acetyl-[${}^{14}\text{C}$] glucosamine into N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, N,N′-diacetyl [${}^{14}\text{C}$]chitobiosyl-pyrophosphoryl-dolichol, and N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein. Gel filtration of the Pronase digests of gray matter N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein reveals two N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide fractions. One fraction (A) contains approximately eight glycose units. All of the radioactivity is at nonreducing termini and can be released by treatment with an exo-β-N-acetylglucosaminidase. A smaller N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (B) is recovered in the elution volume expected for an asparaginyl disaccharide. Structural studies show that the labeled saccharide unit in glycopeptide B is N,N′-diacetyl[${}^{14}\text{C}$]chitobiose. The linkage between the ${}^{14}\text{C}$-labeled disaccharide and the polypeptide has the properties of an N-glycosidic attachment to asparagine. Only the larger N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (A) is found in Pronase digests of white matter membrane N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein after incubation with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine. When gray matter membranes are incubated with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine in the presence of tunicamycin or UMP, the labeling of glycolipid and the asparaginyl disaccharide is inhibited. UMP and tunicamycin have no effect on the transfer of N-acetyl[${}^{14}\text{C}$]glucosamine to external acceptor sites of the larger glycopeptide (A). The transfer of N,N′-diacetyl[${}^{14}\text{C}$]-chitobiose from carrier lipid to protein is observed when extensively washed membranes containing endogenous, prelabeled ${}^{14}\text{C}$-labeled glycolipids are incubated in the presence or absence of unlabeled GDP-mannose. UMP treatment of the prelabeled membranes selectively discharged over 80% of the label from N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, but had no effect on the transfer of the ${}^{14}\text{C}$-labeled disaccharide to protein. All of these results are concordant with transfer of N,N′-diacetylchitobiose from dolichyl diphosphate to gray matter glycoprotein. The major membrane glycoprotein labeled by the lipid-mediated [${}^{14}\text{C}$]disaccharide transfer reaction has an apparent molecular weight of 24,000. Tunicamycin prevents the enzymatic labeling of the gray matter glycoprotein having an apparent molecular weight of 24,000.     

Effects of L-methionine-DL-sulfoximine and beta-N-oxalyl-L-alpha, beta-diaminopropionic acid on nitrogenase biosynthesis and activity in Rhodopseudomonas capsulata.

Inhibitors of glutamine synthetase cause derepression of nitrogenase biosynthesis in the presence of $\text{NH}{}_4{}^{\mathrm{+}}$ in the photosynthetic bacterium Rhodopseudomonas capsulata. A new derepressor of nitrogenase biosynthesis, β-N-oxalyl-L-α,β-diaminopropionic acid (ODAP), is here compared with the widely used L-methionine-DL-sulfoximine (MSX). With both compounds, a quantitative correlation has been observed between inhibition of glutamine synthetase and derepression of nitrogenase biosynthesis. We also find that both MSX and ODAP inhibit nitrogenase activity in vivo in R. capsulata. The latter effect seems to be indirect and related to the previously reported reversible inhibition of nitrogenase activity in vivo by $\text{NH}{}_4{}^{\mathrm{+}}$. As a control it was observed that neither $\text{NH}{}_4{}^{\mathrm{+}}$ nor MSX nor ODAP inhibit nitrogenase activity in vivo in Clostridium pasteurianum.     

Genetic transformation in E.coli: The inhibitory role of the recBC DNase

Genetic transformation of $\text{E.}\text{coli}$ for various chromosomal markers was accomplished by (i) using recipient cells that lack the $\text{recBC}$ DNase but were recombination proficient due to $\text{sbcA}$ or $\text{sbcB}$ mutations and (ii) treating the recipient cells with CaCl₂ at a concentration that facilitates transfection by λ DNA. Cotransformation of three markers ($\text{thr}{}^{\mathrm{+}}\text{ara}{}^{\mathrm{+.}}\text{leu}{}^{\mathrm{+}}$) was found to depend on the molecular weight of the transforming DNA.     

The dimerization of ferrihaems: I. The effect of buffer ions and specific cations on deuteroferrihaem dimerization

The dimerization of deuteroferrihaem in aqueous solution has been investigated using a parameter, named the dimerization index (R_obs). This is defined as the ratio of extinction coefficients at wavelengths corresponding to Soret band maxima for the monomeric and dimeric species, respectively. For solutions containing mainly monomeric species, R_obs > 2, whereas for solutions containing mainly dimeric species R_obs < 1.A computer programme has been applied to determine values of the dimerization constant, K, defined as: $\text{K =}\text{[}\text{dimer}\text{][}\text{H}{}^{\mathrm{+}}\text{]}\text{[}\text{monomer}\text{]}{}^2$.Phosphate buffer anions and Tris · HCl buffer enhanced dimerization. Monovalent and divalent cations also increased dimerization, but in a specific manner. The magnitudes of their effects increased in the order $\text{K}{}^{\mathrm{+}}\text{< Na}{}^{\mathrm{+}}\text{< Li}{}^{\mathrm{+}}\text{<}\text{Sr}{}^{\mathrm{2+}}\text{< Mg}{}^{\mathrm{2+}}\text{? Ca}{}^{\mathrm{2+}}$. Values of K were determined for several concentrations of Na⁺ and Sr²⁺. These data are interpreted in terms of a stabilization of the ferrihaem dimer by the formation of ion triplets with the added cation ‘sandwiched’ between carboxyl residues of the adjacent ferrihaem monomeric units. General guidelines are recommended for the choice of conditions which minimize dimerization.     

Reconstitution of b-type cytochrome oxidase from Rhodopseudomonas capsulata in liposomes and turnover studies of proton translocation

Purified b-type cytochrome oxidase from Rhodopseudomonas capsulata was incorporated into phospholipid vesicles to measure proton extrusion with pulses of ferrocytochrome c for one oxidase turnover. In accordance with the pH shift of its midpoint potential, the purified oxidase showed a proton extrusion of 0.24 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ with uptake of 1 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ from the liposomes for the reduction of oxygen to water. This proton translocation could only be observed in the presence of valinomycin +K⁺ and was not inhibited by DCCD. Oxidase preparations from the first purification step, which contain other protein compounds especially a membrane-bound cytochrome c but not the ubiquinol-cytochrome c₂-oxidoreductase showed a pumping activity of 0.9 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$, which was inhibited by DCCD for nearly 75%. Inhibition of the electron transfer was not observed, which could be explained by a ‘molecular slipping’ of proton extrusion and electron transfer. Proton extrusion from two oxidase-turnovers was only 80% of that from one turnover. The proton pumping of the b-type oxidase strongly depended on the enzyme/phospholipid ratio.     

Occurrence of selenocysteine in the selenium-dependent formate dehydrogenase of Methanococcus vannielii.

The selenium-dependent formate dehydrogenase of Methanococcus vannielii was isolated from bacteria grown in the presence of [${}^{75}\text{Se}$]selenite. Purification under strictly anaerobic conditions resulted in the simultaneous enrichment of formate dehydrogenase activity, ${}^{75}\text{Se}$, and a brown chromophore that absorbs maximally at 380 nm. Acid hydrolysis of the enzyme after reduction with borohydride and alkylation with iodoacetamide, released a radioactive selenoamino acid derivative that was identified as [${}^{75}\text{Se}$]carboxymethyl-selenocysteine. This is the third selenoenzyme shown to contain selenocysteine.