Marcia Brody

Fatty acids as model systems for the action of Ricinus leaf extract on higher plant chloroplasts and algae

Abstract The ability of long-chain unsaturated fatty acids to serve as models for the action of the protein fraction of Ricinus leaf extract on chloroplasts (and algae) has been determined experimentally for several parameters. These include steady-state fluorescence emission and excitation (measured at −196 °), fluorescence induction, light-induced absorption changes of chlorophylls a II and a I , Hill activity, and chloroplast ultrastructure. In addition, the sequential inhibition of system II- and system I-associated electron flow has been demonstrated as a function of increasing concentration of exogenous fatty acid.

THE MECHANISM OF THE FLAVIN SENSITIZED PHOTODESTRUCTION OF INDOLEACETIC ACID

Abstract— A detailed in vitro study was made of the flavin sensitized photoinactivation of indoleacetic acid, using primarily riboflavin as sensitizer. The dependence of the quantum yield on reactant concentrations, pH, presence of oxygen, viscosity, temperature, KI concentration, and solvent was determined. The involvement of a limiting dark reaction was demonstrated, using an intermittent light technique. The results are consistent with a mechanism involving a metastable state of riboflavin as the photochemically reactive species. The calculated rate constant for intersystem crossing to this state was found to be 2.5 times 108/sec. Riboflavin, in the metastable state, is believed to oxidize indoleacetic acid to indolealdehyde, with subsequent recovery of riboflavin by autoxidation. The maximum quantum yield of the photoinactivation of IAA is 0.71, indicating a highly efficient process, approaching 100% when energy loss due to riboflavin fluorescence is taken into account. Both carotenoids and pure chlorophyll‐a were found to be inactive as sensitizers.

Environmental regulation of enzymes in the microbodies and mitochondria of dark-grown, greening, and light-grown Euglena gracilis

Abstract Catalase activity is demonstrated histochemically in the microbodies of aerated cultures of Euglena gracilis strain Z grown on inorganic media supplemented with acetate or glucose. Although this enzyme can also be assayed photometrically in cell-free extracts of acetate-supplemented cells, it is below the level of detectability in extracts of glucose-supplemented cells, there being an order of magnitude fewer microbodies in the latter than the former. Even acetate-supplemented cultures (dark-grown, greening, or continuously light-grown) fail to exhibit detectable catalase activity when CO 2 is removed from the air by Ascarite. Negative results were obtained with histochemical techniques considered optimal for the demonstration of cytochrome oxidase; under other conditions, however, a KCN-sensitive enzyme was revealed in the mitochondrial matrix. This (unidentified) enzyme is first observed in mitochondria after 20–24 hr of greening, reaches a maximum intensity at about 48 hr, and becomes undetectable by 72 hr of greening. Poisoning of photosynthesis by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) results in loss of activity of this mitochondrial enzyme.

Environmental factors controlling enzymatic activity in microbodies and mitochondria of Euglena gracilis

Glyoxylate by-pass enzymes have been demonstrated in Euglena gracilis strain Z grown heterotrophically on acetate [ 1,2]. In the present work, catalase activity in microbodies is observed cytochemically by the enzymatic oxidative polymerization of 3,3’-diaminobenzidine (DAB) in the presnece of HzOz [3] in aerated cultures of E. gracilis strain Z grown on inorganic medium supplemented with acetate or glucose. Catalase activity is also detected photometrically in cell-free extracts of acetate-supplemented cells, but cannot be detected by this method in glucose-supplemented cells in which the number of microbodies is an order of magnitude fewer. In contrast with aerated cultures of acetate-supplemented cells, those grown in COs-free air fail to exhibit detectable levels of catalase activity. Although cytochrome oxidase could not be detected using conditions of incubation considered optimal [4] , a KCN-sensitive mitochondrial enzyme is revealed under different conditions of DAB incubation. This (unidentified) enzyme is first observed in the mitochondrial matrix after 20-24 hr of greening, attaining a maximum intensity at 40-48 hr, and falling below the limits of detectability sometime before 72 hr of greening. The mitochondrial enzyme seems to be photosynthesis-dependent; in the presence of 3 (3,4dichlorophenyl)-l,l-dimethylurea (DCMU) or in COzfree air, the DAB stain is no longer observed in the matrix.

Enzymatic characterization of sucrose-gradient microbodies of dark-grown, greening and continuously light-grown Euglena gracilis.

In earlier works [ 1,2] we reported that catalase activity may be detected cytochemically, by the oxidative polymerization of 3,3’diaminobenzidine (DAB), in the microbodies of Euglena grucilis strain Z cultured in aerated, acetate-supplemented media, but not in the microbodies of cells grown in CO,-free air; catalase activity was additionally demonstrated by application of Luck’s photometric assay [3] to cell-free fractions. In these studies [ 1,2] we noted that both the number of microbodies in situ and specific catalase activity in cell-free fractions of dark-grown cells begin to increase after about lo-12 hr in the light, and that with further greening (-24 hr), both microbody population (per cell) and catalase activity double. Microbodies may be classified as glyoxysomes or peroxisomes on the basis of enzymatic composition. The former contain the enzymes of the glyoxylate cycle [4] and the latter contain the enzymes of the glycolate pathway of metabolism [S] ; catalase is found in both types of particles. In the present work, cells from aerated, acetate-supplemented cultures of Euglena gracilis strain Z were grown under three light regimes (dark, greening and continuous light), and microbodycontaining fractions, isolated by means of discontinu-

ASYMMETRIC AND SYMMETRIC BIMOLECULAR MEMBRANES CONTAINING PHOTOSYNTHETIC PIGMENTS

Although chlorophyll a has been incorporated into black lipid films (Tien, 1968; Van and Tien, 1970; Berns, 1974), chlorophyll containing bimolecular membranes have not yet been made from “solventfree” (or low solvent containing) monolayers (Montal, 1974b). The advantages of bimolecular membranes over black lipid films have been described elsewhere (Montal, 1974b), in particular, the former have a more clearly defined chemical composition and readily allow the formation of asymmetric membranes. The ubiquitous co-occurrence of chlorophyll a, carotenoids, non-pigmented lipids and proteins in the photosynthetic apparatus of higher plants makes it especially desirable to work with such asymmetric membranes. In the present note we report both the formation and the relative [see below] electrical properties of symmetric, asymmetric, and mixed bimolecular membranes of a-lecithin, chlorophyll a, p-carotene and HP700 (a chlorophyll-protein-detergent complex).