On the Reversal of the Inhibition of Photosynthesis Induced by Sodium Dimethyldithiocarbamate and Tetramethylthiuram disulphide

The reversal of 80–100 per cent inhibition of photosynthesis in the green alga Enteromorpha linza (L.) J. Ag., obtained with tetramethylthiuram disulphide (TMTD) and the corresponding sodium dithiocarhamate (Na-DMDT), was investigated. The inhibition obtained by the two compounds proves to be almost completely reversible by simply washing away the inhibitors. Copper(II) sulphate partially reversed the inhibition obtained with Na-DMDT while reduced glutathione (GSH) partially reversed inhibition obtained with both Na-DMDT and TMTD. The time to complete the reversal varied between 12 and 33 min. Almost no reversal of the TMTD induced inhibition was achieved with CuSO₄. Na-DMDT and CuSO₄ reacted instantaneously with each other. GSH most probably reduced TMTD stoichiometrically to DMDT. This reaction was complete- within 6 seconds. No reaction took place between GSH and Na-DMDT. The interpretation of The results eliminates the most conventional explanation of the mechanism of the inhibition of photosynthesis in E. linza with Na-DMDT, viz. complex formation with functional metals. The reaction between TMTD and functional thiol groups in the alga is the most plausible inhibition mechanism. It is inferred from the results that Na-DMDT is most probably oxidized by E. linza to TMTD and effects inhibition in this form. (PDF DAMAGE)     

Protein Arp and protein H from group A streptococci. Ig binding and dimerization are regulated by temperature.

Cell surface proteins that bind to the Fc part of Ig are expressed by many strains of group A streptococci, an important human pathogen. Two such bacterial strains, AP4 and AP1, were shown to bind IgA and IgG, respectively, in a temperature-dependent manner. The binding of radiolabeled Ig to the bacterial cells was lower at 37 degrees C than at 22 and 4 degrees C. Similarly, protein Arp, the IgA-binding protein isolated from strain AP4, and protein H, the IgG-binding protein isolated from strain AP1, displayed a strong Ig-binding at 22 degrees C and lower temperatures, and virtually no binding at all at 37 degrees C. The effect was reversible: lowering of the temperature restored the binding and vice versa. A gradual shift between binding and nonbinding took place between 27 and 37 degrees C. Gel chromatography and velocity sedimentation centrifugation showed that protein Arp and protein H appeared as noncovalently associated dimers at 10 and 22 degrees C, and as monomers at 37 degrees C. These results strongly suggest that the dimerization of protein Arp and protein H, rather than the low temperature itself, yielded the strong Ig-binding of the proteins at 10 and 22 degrees C. Indeed, after covalent cross-linking of the dimers at 10 degrees C by incubation with low concentrations of glutaraldehyde, full Ig-binding was achieved even at 37 degrees C. A carboxyl-terminal proteolytic fragment of protein Arp, which completely lacked the IgA-binding capacity at any temperature, showed the same temperature-dependent dimerization as intact protein Arp, suggesting that the Ig-binding part of the protein is not required for dimerization. The implications of these results for the function of Ig-binding group A streptococcal proteins, and their role in the host-parasite relationship are discussed.     

Mammalian DNA ligases. Serological evidence for two separate enzymes.

Mammalian cells contain two DNA ligase activities with different chromatographic properties, referred to as DNA ligase I and II. The major ligase activity present in calf thymus cell extracts, DNA ligase I, has been purified 1000-fold. After repeated injections of this enzyme with complete Freund's adjuvant into a rabbit, antibodies were induced that inhibit DNA ligase I from calf, human, mouse, and rabbit tissues. This antiserum did not affect DNA ligase II from the same sources to a detectable extent, even at a concentration 10-fold higher than that required for 98% inhibition of DNA ligase I. These data strongly indicate that the two mammalian DNA ligase activities are due to two separate enzymes, and not to two forms of the same enzyme. Both enzymes are present in the nuclear fraction, but are also found in the cytoplasmic fraction. Rapidly dividing cells (mouse ascites tumor cells and calf thymus) contain higher amounts of DNA ligase I than other cells (calf liver and spleen, human placenta, and rabbit spleen), while no such correlation was observed for DNA ligase II.     

Characterization of a functional recombinant rat liver aldehyde dehydrogenase: expression as a non-fusion protein in E. coli

A cDNA encoding a rat liver inducible aldehyde dehydrogenase carried in a pUC8 plasmid is expressed in E. coli as a dimeric enzyme molecule functionally and physically identical to the authentic rat enzyme. The cDNA appears to be transcribed using the lac promoter, but is translated from an initiator codon 174 base pairs from the 5' end of the cDNA. The aldehyde dehydrogenase polypeptide is not produced as a fusion protein. This is the first example of the production by E. coli of a catalytically active, multimeric eukaryotic protein which is not a fusion protein.     

Different substrate specificities of the two DNA ligases of mammalian cells

Mammalian cells contain the DNA ligases I and II. These enzymes show different molecular weights and heat labilities, and antibodies against ligase I do not inhibit ligase II. Here, the nonidentical substrate specificities of the enzymes are described. Under standard reaction conditions DNA ligase I, but not ligase II, catalyzes blunt-end joining of DNA, while ligase II is the only activity that joins oligo(dT) molecules hydrogen-bonded to poly(rA). These differences facilitate the distinction between the two enzymes and should permit further analysis of their functions.     

Nonimmunodominant regions are effective as building blocks in a streptococcal fusion protein vaccine

Identification of antigens that elicit protective immunity is essential for effective vaccine development. We investigated the related surface proteins of group B Streptococcus, Rib and alpha, as potential vaccine candidates. Paradoxically, nonimmunodominant regions proved to be of particular interest as vaccine components. Mouse antibodies elicited by Rib and alpha were directed almost exclusively against the C-terminal repeats and not against the N-terminal regions. However, a fusion protein derived from the nonimmunodominant N-terminal regions of Rib and alpha was much more immunogenic than one derived from the repeats and was immunogenic even without adjuvant. Moreover, antibodies to the N-terminal fusion protein protected against infection and inhibited bacterial invasion of epithelial cells. Similarly, the N-terminal region of Streptococcus pyogenes M22 protein, which is targeted by opsonic antibodies, is nonimmunodominant. These data indicate that nonimmunodominant regions of bacterial antigens could be valuable for vaccine development.     

Amino-acid solvation structure in transmembrane helices from molecular dynamics simulations

Understanding the solvation of amino acids in biomembranes is an important step to better explain membrane protein folding. Several experimental studies have shown that polar residues are both common and important in transmembrane segments, which means they have to be solvated in the hydrophobic membrane, at least until helices have aggregated to form integral proteins. In this work, we have used computer simulations to unravel these interactions on the atomic level, and classify intramembrane solvation properties of amino acids. Simulations have been performed for systematic mutations in poly-Leu helices, including not only each amino acid type, but also every z-position in a model helix. Interestingly, many polar or charged residues do not desolvate completely, but rather retain hydration by snorkeling or pulling in water/headgroups--even to the extent where many of them exist in a microscopic polar environment, with hydration levels corresponding well to experimental hydrophobicity scales. This suggests that even for polar/charged residues a large part of solvation cost is due to entropy, not enthalpy loss. Both hydration level and hydrogen bonding exhibit clear position-dependence. Basic side chains cause much less membrane distortion than acidic, since they are able to form hydrogen bonds with carbonyl groups instead of water or headgroups. This preference is supported by sequence statistics, where basic residues have increased relative occurrence at carbonyl z-coordinates. Snorkeling effects and N-/C-terminal orientation bias are directly observed, which significantly reduces the effective thickness of the hydrophobic core. Aromatic side chains intercalate efficiently with lipid chains (improving Trp/Tyr anchoring to the interface) and Ser/Thr residues are stabilized by hydroxyl groups sharing hydrogen bonds to backbone oxygens.