Isolation of 3-(2-carboxyethyl)thymine following in vitro reaction of β-propiolactone with calf thymus DNA

3-(2-Carboxyethyl)thymine (3-CET) was synthesized from β-propiolactone (BPL) and dThd5′P at pH 9.0–9.5 via the intermediate 3-(2-carboxyethyl)thymidine-5′-monophosphoric acid (3-CEdThd5′P). 3-CEdThd5′P was converted to 3-CET by hydrolysis in 1.5 N HCl at 100°C for 2 h. The structure of 3-CET was assigned on the basis of UV spectra, electron impact (EI) and isobutane chemical ionization mass spectra and the EI mass spectrum of a trimethylsilyl derivative of 3-CET. BPL was reacted in vitro with calf thymus DNA at pH 7.5. 100 A units of BPL-reacted DNA yielded, following perchloric acid hydrolysis and preparative paper chromatography, 3 A units of 3-CET. Reaction of BPL with the phosphodiester thymidylyl-(3′-5′)thymidine gave 3-(2-carboxyethyl)thymidylyl-(3′-5′)-3-(2-carboxyethyl)thymidine (～3%). Phosphotriester formation was not detected.     

Fasciola hepatica: A proteolytic digestive enzyme

A proteolytic enzyme in the gut exudate of the common liver fluke has been purified. The enzyme is specific for globin with a pH optimum of 3.9–4.0 and breaks the protein down to peptides and a small percentage of free amino acids. Collagenase activity at pH 7.5 copurifies with the main globinolytic enzyme. The enzyme has a molecular weight of 12,000 and does not appear to be antigenic in fluke-infected animals.     

Monolayer properties of chloroplast lipids

The properties of seven monogalactosyldiacylglycerols and six digalactosyldiacylglycerols, isolated from photosynthetic membranes and possessing different levels of fatty acid unsaturation, have been studied by the monolayer technique and compared with those of the fully saturated compounds. In addition, the monolayer properties of sulphoquinovosyldiacylglycerols and phosphatidylglycerols from higher plant chloroplasts, and several hexadecenoic acids have been measured.Monogalactosyldiacylglycerols containing saturated fatty acids form a condensed monolayer similar to that of saturated phosphatidylcholines. The naturally occurring monogalactosyldiacylglycerols, of which the double bond index ranged from 0.6 to 3.9, possessed comparable force-area curves suggesting that headgroup interactions play a more important role in packing behaviour than in phosphatidylcholines. Although digalactosyldiacylglycerols containing fully saturated fatty acids form a more expanded monolayer than the corresponding monogalactosyldiacylglycerols, the degree of expansion of the monolayer due to the presence of unsaturated fatty acids in the naturally occurring digalactosyldiacylglycerols is much less than in monogalactosyldiacylglycerols. Monogalactosyldiacylglycerols and digalactosyldiacylglycerols from a single species have very similar monolayer properties, and the presence of sulphoquinovosyldiacylglycerols and phosphatidylglycerols in the proportions in which they occur in higher plant chloroplasts does not have any condensing effect on a monolayer of galactolipids     

Pressure-induced changes in the molecular organization of a lipid-peptide complex. Polymyxin binding to phosphatidic acid membranes

The effect of 100 atm pressure on the organization of the lipid-peptide complex formed between polymyxin and dipalmitoyl phosphatidic acid has been investigated. Phase transition curves were obtained by electron paramagnetic resonance by measuring the partition coefficient of the spin label, 2, 2, 5, $\text{5-}\text{tetramethylpiperidine}\text{-N-}\text{oxyl}$. The three-step phase transition curve previously obtained with fluorescence polarization measurements was confirmed, demonstrating three distinct phosphatidic acid domains in the bilayer. Pressure increases binding of polymyxin to phosphatidic acid bilayers and alters the proportions of the two domains that differ in the mode of binding between phosphatidic acid and polymyxin. The binding curves of polymyxin to phosphatidic acid bilayers were determined and it was shown that application of pressure reduces the cooperativity of the binding curve.     

Structural properties of the zinc site in Cu,Zn-superoxide dismutase; Perturbed angular correlation of gamma ray spectroscopy on the Cu, 111Cd-superoxide dismutase derivative

The Zn(II) site of the dimeric Cu(II),Zn(II)-superoxide dismutase from Saccharomyces cerevisiae has been examined by means of perturbed angular correlation of gamma rays (PAC) on the Cu(II),Cd(II)- and Cu(I),Cd(II)-superoxide dismutase. The PAC spectrum for the Cu(II),Cd(II) enzyme reveals two different, pH independent, coordination geometries for the Cd site. Removal of Cu(II) does not affect the PAC spectrum, which suggests that Cu(II) and Cd(II) do not share a common histidine side chain as ligand. The results are consistent with either an equilibrium between two coordination geometries for Cd(II) in each subunit or a difference in the structure of the Cd(II) site in the two subunits. In contrast, in the reduced enzyme only one structure is present, identical for the two subunits.     

Identification of rat brain polysomes synthesizing the brain specific enolase (14.3.2 protein), S100 protein and alpha and beta tubulin subunits

Polysomes prepared from frozen rat brain powder were fractionated by centrifugation in a sucrose gradient. Individual fractions were used to program a reticulocyte lysate in a run-off reaction. The products of cell-free synthesis were assayed for the brain-specific enolase (14.3.2 protein) and S100 protein by immunoprecipitation with specific antisera and for tubulin by two-dimensional electrophoresis in polyacrylamide slab gels. The relative synthesis of these proteins by unfractionated free brain polysomes were 0.1 per cent, 0.05 per cent and 0.7 per cent respectively. After centrifugation in a sucrose gradient polysomes synthesizing S100 protein were separated from those synthesizing the other two markers. There was a threefold enrichment in the specific messenger RNA activity for each of the three proteins studied in their respective peak fractions of polysomes.     

Structure of bovine beta-lactoglobulin at 6A resolution.

Bovine β-lactoglobulin is a dimer with a molecular weight of 2 × 18,400. In solution it undergoes a pH-dependent transition at pH 7.0 between two alternative structures, named N and R. The structures of four different crystal forms have been determined by multiple isomorphous replacement with heavy-atoms. Two of them, lattices K and X, were crystallised at pH 6.5, corresponding to the N state in solution; and the other two, lattices Y and Z, were crystallised at pH 7.5, corresponding to the R state in solution. The figures of merit of the phase angles determined for these lattices were 0.76, 0.77, 0.80 and 0.80, respectively. The four structures that emerged are similar and show certain features suggestive of α-helices and pleated sheets, but the resolution is insufficient to trace the entire course of the polypeptide chain. No clear distinction can yet be made between the structures above or below pH 7.0, nor between the native molecule and the molecule from which the C-terminal leucine and histidine residues have been cleaved. Analyses at higher resolution are in progress.     

The formation of dipeptides from amino acids and the 2′(3′)-glycyl ester of an adenylate

Summary The yields of dipeptide obtained from the reaction of 0.2M 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) and 0.2M amino acid at pH 8.2 ranged from 0.1% to 35.5% for a group of 15 amino acids. The yields of glyser (35.3%), gly-cys (11.8%) and gly-thr (5.4%) were considerably greater than dipeptide yields obtained from any of the other 12 amino acids ( 1.7%). Aminolysis of 0.05M 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by 0.4M serine ethyl ester yielded 53% glycylserine diketopiperazine, via N-(glycyl)-serine ethyl ester as a transient intermediate. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - serOEt serine ethyl ester - gly-serOEt N-(glycyl)-serine ethyl ester - Boc-gly N-tertbutyloxycarbonylglycine - cyclo-(gly-ser-) glycylserine diketo-piperazine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - gly-ser N-glycylserine     

Nitrous acid mutagenesis of duplex DNA as a three-component system.

Purified native Hemophilus influenzae DNA is relatively insusceptible to nitrous acid (NA) mutagenesis in vitro, but is readily mutated following denaturation. NA mutagenicity for duplex DNA is significantly increased in the presence of various alcohols, glycols, phenols or primary amines. Phenol-extracted DNA contains dissociable contaminants of low molecular weight that enhance NA mutagenesis. Enhancement of NA mutagenesis by phenol and by spermine is due to the formation of unstable molecular species. We propose that reactive organic nitroso compounds are formed which then serve as delivery vehicles to promote mutagenicity of native DNA, perhaps via transnitrosation reactions. Similar reactions probably occur in vivo to promote NA-induced base substitution (but not frameshift) mutations in Salmonella typhimurium and in Escherichia coli. The possible significance of these observations to carcinogenesis is discussed.