A soluble chloroplast protein catalyzes ribulosebisphosphate carboxylase/oxygenase activation in vivo

Ribulosebisphosphate carboxylase/oxygenase (EC 4.1.1.39) (rubisco) must be fully activated in order to catalyze the maximum rates of photosynthesis observed in plants. Activation of the isolated enzyme occurs spontaneously, but conditions required to observe full activation are inconsistent with those known to occur in illuminated chloroplasts. Genetic studies with a nutant of Arabidopsis thaliana incapable of activating rubisco linked two chloroplast polypeptides to the activation process in vivo. Using a reconstituted light activation system, it was possible to demonstrate the participation of a chloroplast protein in rubisco activation. These results indicate that a specific chloroplast enzyme, rubisco activase, catalyzes the activation of rubisco in vivo.     

The relationship of hormone-sensitive and hormone-insensitive phosphatidylinositol to phosphatidylinositol 4,5-bisphosphate in the WRK-1 cell

We have previously characterized two distinct pools of phosphatidylinositol (PI) in the WRK-1 rat mammary tumor cell, one whose metabolism is enhanced in response to vasopressin and another which is insensitive to hormonal manipulation. The purpose of the present study was to examine the relationship between cellular phosphatidylinositol 4,5-bisphosphate (PIP2) and each of the two PI pools. We have found that in WRK-1 cells, vasopressin induces the rapid loss of PIP2 and the accumulation of inositol phosphates. By making use of kinetic differences in 32Pi uptake into the two pools of PI and assessing radioactivity levels in the 1-phosphate of PIP2, we have determined that hormone-sensitive PI is the precursor of approximately 60% of the cellular PIP2; the remainder is synthesized from the hormone-insensitive pool. Additional data indicate that PIP2 derived from hormone-sensitive PI is likewise hormone-sensitive, while that synthesized from hormone-insensitive PI remains stable over a long period of time and is not affected by the presence of vasopressin.     

Demonstration of choleragen-dependent ADP-ribosylation in whole cells and correlation with the activation of adenylate cyclase

3T3C₂ mouse fibroblasts rendered permeable to (α^?32P)NAD⁺ show cholera toxin-dependent labeling of a 45,000 m.w. protein and of a doublet of polypeptides around 52,000 m.w. These same bands are ADP-ribosylated in broken cells. Membranes prepared from pigeon erythrocytes pretreated with choleragen show a decrease in subsequent cholera toxin-specific ADP-ribosylation of a 43,000 m.w. polypeptide. Both whole cell and broken cell adenylate cyclase activation and toxin-specific ADP-ribosylation are reversed specifically by low pH and high concentrations of toxin and nicotinamide in all systems. Thus ADP-ribosylation appears to be relevant to the molecular action of choleragen in whole cells as well as in broken cells.     

Different secretory pathways of renin from mouse cells transfected with the human renin gene

Mammalian cells in culture, transfected with human renin gene, can provide a useful tool for studying renin biosynthesis and secretion. We transfected fibroblast cells (mouse L929 and Chinese hamster ovary cells) and pituitary tumor cells (mouse AtT-20) with the human renin gene and a selectable plasmid (pSV2Neo). Transfected fibroblasts synthesize prorenin only. Prorenin is secreted by fibroblasts constitutively and the secretion is not influenced by 8-bromo-cAMP. On the other hand, transfected AtT-20 cells synthesized both prorenin and mature active renin. Transfected AtT-20 cells release prorenin by constitutive secretion but mature renin is secreted by a regulated mechanism since the secretion of the former is not influenced by 8-bromo-cAMP but the release of the latter is significantly stimulated. Our studies demonstrate that human renin may be secreted by at least two cellular pathways: prorenin by a constitutive pathway and mature renin by a regulated pathway. These transfected cells may provide useful models for studies of human renin synthesis, processing, and secretion.     

The effectiveness of constructed wetland for treatment of woodwaste leachate

Percolation of rainfall through woodwaste piles leaches natural chemicals from the wood residuals that can have adverse impacts on the environment. A study was conducted on a woodwaste storage site, adjacent to the Lower Fraser River, near Mission, BC, Canada. The objective of this research was to evaluate the effectiveness of constructed wetland for treatment of this woodwaste leachate. The leachate was characterized by high oxygen demand, tannin and lignin, and volatile fatty acids (VFAs), but low pH and nutrients. Diluted leachate passed through six pilot-scale wetland cells, four planted with cattail (Typha latifolia) and two unplanted controls, with a hydraulic retention time of 7 days and an average depth of 40 cm. Nutrient addition and pH adjustments were made to improve contaminant removal. Reductions in contaminants were consistently achieved, with average removals for BOD, COD, VFAs and tannin and lignin of 60, 50, 69 and 42%, respectively. Climatic conditions had an impact on the performance of the constructed wetland. Further operation of the system will help to elucidate the seasonal fluctuations. Aging of the constructed wetland system increased the treatment performance.     

Cytochrome c peroxidase compound I: formation of covalent protein crosslinks during the endogenous reduction of the active site

Cytochrome c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) was oxidized by hydrogen peroxide in the absence of exogenous electron donor. Higher molecular weight species were observed in the decay products at pH 4.5. Monomer and dimer were separated by gel filtration and purified by anion-exchange chromatography. Peptide mapping of tryptic digests of the dimer indicated a tyrosine crosslink localized between residues 32 and 48 of the native enzyme.