Two distinct site-dependent biosynthetic pathways for the incorporation of sarcosine into actinomycins.

Actinomycins II and III, containing sarcosine residues in two adjacent sites of their peptide moieties were produced by Streptomyces antibioticus in the presence of exogenous sarcosine labeled with deuterium in the N-methyl group. Combined gas chromatography-mass spectrometry of the cyclodipeptides derived by thermal degradation of these actinomycins demonstrated specific incorporation of the labeled sarcosine into the 3-site, implicating some other biosynthetic precursor, presumably glycine, for the sarcosine in the 4-site. The same conclusion emerged from proton nuclear magnetic resonance spectroscopy of these deuterium-labeled actinomycins.     

Characterization of glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase

Glycine betaine is accumulated in cells living in high salt concentrations to balance the osmotic pressure. Glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase (SDMT) of Ectothiorhodospira halochloris catalyze the threefold methylation of glycine to betaine, with S-adenosylmethionine acting as the methyl group donor. These methyltransferases were expressed in Escherichia coli and purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificities and pH optima near the physiological pH. No evidence of cofactors was found. The enzymes showed Michaelis-Menten kinetics for their substrates. The apparent K(m) and V(max) values were determined for all substrates when the other substrate was present in saturating concentrations. Both enzymes were strongly inhibited by the reaction product S-adenosylhomocysteine. Betaine inhibited the methylation reactions only at high concentrations.     

Studies on the biological activities of actinomycins Z1 and Z5

The biological activities of actinomycins Z₁, Z₅, and IV (D) were compared. No consistent pattern was observed between the in vitro and in vivo results. Inhibition of E. coli DNA-dependent RNA polymerase in vitro followed the sequence IV > Z₁ > Z₅; however, for inhibition of RNA synthesis in B. subtilis and in HeLa cells the sequence was IV > Z₅ > Z₁ and the latter relationship was seen in the antimicrobial activity also. Physicochemical data did not agree with any of these findings. Difference spectra obtained with B. subtilis and M. lysodeikticus DNA followed the sequence Z₁ > IV > Z₅, while the relative thermal denaturation profiles were the exact opposite, Z₅ > IV > Z₁. These physicochemical criteria appear to be less reliable quantitative guides to biological activity. The relative ineffectiveness of actinomycin Z₁in vivo is probably the result of permeability differences associated with the presence of an hydroxylated proline residue (3-hydroxy-4-oxo-5-methylproline).     

Kinetic studies on the reaction mechanism of sarcosine oxidase

A sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating), EC 1.5.3.1) isolated from Corynebacterium sp. U-96 contains both covalently bound FAD and noncovalently bound FAD. The noncovalent FAD reacts with sarcosine, the covalent FAD with molecular oxygen (Jorns, M.S. (1985) Biochemistry 24, 3189-3194). To clarify the reaction mechanism of the enzyme, kinetic investigations were performed by the stopped-flow method as well as by analysis of the overall reaction. The absorption spectrum of the enzyme in the steady state was very similar to that of the oxidized enzyme, and no intermediate enzyme species, such as a semiquinoid flavin, was detected. The rate for anaerobic reduction of the noncovalently bound FAD and the covalently bound FAD by sarcosine were 31 and 6.7 s-1, respectively. The latter value was smaller than the value of respective Vmax/e0 obtained by the overall reaction kinetics (Vmax/e0: the maximum velocity per enzyme concentration). Both rate constants for oxidation of the two FADs by molecular oxygen were 100 s-1. A reaction scheme of sarcosine oxidase is proposed to account for the data obtained; 70% of the enzyme functions via a fully reduced enzyme, and 30% of the enzyme goes along a side-path, without forming the fully reduced enzyme. In addition, it is suggested that the reactivity of noncovalently bound FAD with sarcosine is affected by the oxidation-reduction state of the covalently bound FAD, in contrast to the reactivity of the covalently bound FAD with molecular oxygen, which is independent of the oxidation-reduction state of the noncovalently bound FAD.     

Origin of amphiphilic molecules and their role in primary structure formation

Attempts to solve two fundamental questions are described: the first concerns which mechanisms were responsible for the self-assembly of membrane structures on the prebiotic Earth, and the second concerns the routes by which considerable amounts of membrane amphiphiles formed from simpler hydrocarbons. The physicochemical properties of several amphiphilic compounds extracted from the Murchison carbonaceous chondrite were studied, using infra-red and fluorescent spectroscopy, measurements of surface activity, chromato-mass spectrometry, and polarization and electron microscopy. The results supported previous observations that amphiphilic and aromatic hydrocarbons were present in significant quantities, and the first demonstration of surface activity among a number of acidic derivatives of hydrocarbons is reported. In addition, one fraction of the surface-active compounds can form bilayer structures, showing that membranes could have self-assembled on the prebiotic Earth. Photochemical oxidation of hydrocarbons is shown to be a likely source of the amphiphilic molecules required for the self-assembly of primary membrane structures.     

Origin and direction of replication in mitochondrial DNA molecules from the genus Drosophila.

Mitochondrial DNA (mtDNA) obtained from ovaries of Drosophila simulans, D. mauritiana, D. takahashii, D. yakuba and D. virilis was examined by electron microscopy. From a consideration of the structural properties of replicative intermediates, it was concluded that in mtDNA molecules of each species, synthesis on one strand can be up to 97% complete before synthesis on the complementary strand is initiated. MtDNA molecules of each species contain a single A+T-rich region which shows species-specific size variation from 1.0 kb (D. virilis) to 4.8 kb (D. simulans), and maps at the same position in all molecules relative to three common EcoRI sites. The structural properties of complex forms, interpreted as having originated from replicative intermediates, and produced by either partial denaturation or EcoRI digestion, are consistent with the hypothesis that replication is initiated within the A+T-rich region and proceeds unidirectionally around the molecule towards the nearest common EcoRI site. The replication origin is located near the center of the A+T-rich region in D. simulans and D. mauritiana, but lies closer to that end of the A+T-rich region which is distal to the nearest common EcoRI site in D. takahashii, D. yakuba and D. virilis.