Donald S. Berns

ULTRASTRUCTURE OF THE THERMOPHILIC BLUE-GREEN ALGA, SYNECHOCOCCUS LIVIDUS COPELAND(1).

The ultrastructure of Synechococcus lividus Copeland, a thermophilic blue‐green alga, was studied in thin sections. The cell envelope reveals striking similarities with that of some gram‐negative bacteria. In contrast to bacteria and to many other species of blue‐green algae, ribosomes are predominantly found in the central nuclear region and appear to be associated with the DNA fibrils. Thylakoids (photo‐synthetic lamellae) are arranged as concentric shells, around the nuclear equivalent, lying nearly parallel to one another and to the plasma membrane. Both plasma and thylakoidal membranes, as described by other authors for different Cyanophyceae, are of the unit membrane dimension and morphology. Various types of intracellular inclusions are found: (1) Lipid inclusions, located in the cytoplasm are similar to the osmiophilic globules of higher plant chloroplasts. (2) Polyphosphate inclusions (or volutin) resembling those of other species are generally found at the cell poles but within the nuclear region. (3) Polyhedral inclusions also located in the nuclear region are clearly recognized to be different from the polyphosphate bodies, but their function remains unknown.

Electron micrographic investigations of C-phycocyanin☆

Abstract C-Phycocyanin extracted from the blue-green alga Plectonema calothricoides was investigated with the electron microscope and the contrasting technique. Potassium phosphotungstate and uranyl acetate were used as contrast agents. Round structures with a central hole were evident and were interpreted as being the hexamer structure postulated from independent physical studies. Higher magnification demonstrated the hexamer to indeed consist of six globular monomer units. The morphology of the monomer unit and a possible trimer unit are also discussed.

The relationship of the quaternary structure of allophycocyanin to its spectrum

Abstract Allophycocyanin II in its trimer form (α3β3) at pH 7.0 has an absorption maximum at 652 nm. This band is selectively reduced in intensity at pH 7.0 when various salts are added. The loss of 652 nm absorption follows the order: NaClO4 ⪢ NaNO3 > NaBr > NaCl. When the NaClO4 concentration is in the range 0.6-1.0 m the 652-nm band is entirely lost, and sedimentation equilibrium and velocity studies suggest that the trimer is completely dissociated to monomers (αβ). Hydrophobic interactions appear to be important in maintaining the trimer. The monomer absorption maximum is at 616 nm. A series of experiments using these salts demonstrated at intermediate 652-nm intensities and the two extrema that an isobestic point at 626 nm is present which indicates an equilibrium between two species. Corresponding to the loss of 652 nm absorption is the disappearance of 661 nm fluorescence emission and the appearance of a new band at 642 nm. Removal of the NaClO4 by dialysis essentially restores the 652-nm absorption and 661-nm emission and the trimeric protein structure. The near ultraviolet region is only slightly perturbed during the loss of 652 nm absorption. In the absence of any additional salts these spectral changes also occur in pH 7.0 buffer at very low protein concentrations.

The use of Coomassie brilliant blue for critical micelle concentration determination of detergents

Abstract The spectral changes of Coomassie brilliant blue G-250 dye have been studied in the presence of detergent micelles. Association of detergents with the dye produced an absorbance change at 618 nm. At cetyltrimethylammonium bromide concentrations well below the critical micelle concentration a large decrease in absorbance is observed. This is due to formation of a 1:1 water insoluble complex between the anionic dye and the cationic detergent. At sufficiently low dye concentration, the 618-nm absorbance is significantly increased with micelle concentration of the detergent. The onset of enhanced absorbance may be used to determine the critical micelle concentration of cetyltrimethylammonium bromide. The critical micelle concentration of anionic, neutral, and zwitterionic detergents has been determined also by plotting the absorbance at 618 nm as a function of detergent concentration.

Protein aggregation in C-phycocyanin. Studies at very low concentrations with the photoelectric scanner of the ultracentrifuge

Solutions of C-phycocyanin of very low concentrations were examined by sedimentation-velocity studies in the Spinco model E ultracentrifuge equipped with a photoelectric scanning system and a monochromator. At sufficiently low concentrations complete disaggregation from the hexamer to the monomer was observed. The equilibrium constant of monomer to hexamer was estimated to be approx. 10(30). For studies of aggregation over the complete range of concentration, C-phycocyanins from Phormidium luridum and Lyngbya sp. were used. Sedimentation-velocity studies at high concentration with schlieren optics are reported for C-phycocyanins from Anabaena variabilis and Lyngbya sp. The pH-dependence of aggregation and the temperature-dependence of trimer-hexamer equilibrium for phycocyanins from these algae were found to be similar to those of other C-phycocyanins. The principal feature of the pH-dependence is the dominance of hexamers at the isoelectric point. Increasing temperature increased the amount of hexamer and decreased the amount of trimer.

C-Phycocyanin: Minimum Molecular Weight

Sedimentation and immunodiffusion experiments indicate that the molecular weight for the minimum molecular unit of C-phycocyanin is 30,000. This result agrees with an analysis of available data on amino acid content for C-phycocyanins from several different algae.

Effect of salts on C-phycocyanin

Abstract The effect of a number of inorganic anions on the quaternary structure of C-phycocyanin has been investigated by fluorescence polarization. Dissociation to monomer occurred in the order: SCN− > ClO4− > NO3− > Br− > Cl−. These results suggest that hydrophobic interactions are important in the hexamer-monomer equilibrium of C-phycocyanin.

Photoresponse of chlorophyll-containing bileaflet membranes and the effect of phycocyanin as extrinsic membrane protein

SummaryArtificial bileaflet membranes were formed from extracts of chloroplasts. Gradients of a redox potential were created across the membranes by adding various concentrations of ceric-cerous ions, ferric-ferrous ions, and ascorbic acid to the aqueous solutions on either side of the membrane. When a membrane interposed between solutions of different redox potential was irradiated with light, a potential difference of up to 50 mV was recorded. Analysis of the photoresponse allowed its separation into two components: a photoelectromotive driving force dependent upon the redox potential gradient, and a photoconductive pathway dependent upon the amount of light absorbed by the membranes. There appeared to be a limit to the photocurrent that could be drawn from a membrane at a particular intensity of irradiation; i.e., it did not increase indefinitely with increase of the redox potential gradient. Conductance of the photoconductive pathway was independent of temperature. Phycocyanin added to the aqueous solution participated in the photoresponse in a unidirectional manner that suggested facilitation of electron transport from membrane to acceptors in the aqueous solution.

Urease Activity in Blue-Green Algae

The apparent enzymatic hydrolysis of urea has been detected in whole blue-green algae and in cell extracts. Urease is present as an intracellullar component in cultures in which no bacterial contaminants are found. The activity in the cells was recovered from the extracts.

Comparison of the stability of phycocyanins from thermophilic, mesophilic, psychrophilic and halophilic algae

Protein unfolding of eight different phycocyanins was investigated utilizing circular dichroism and visible spectra. The phycocyanin samples were extracted from algae that are normally found in vastly different environments, and are classified as mesophilic, thermophilic, halophilic and psychrophilic. The ability of these proteins to resist the denaturant urea is in the order of thermophile greater than mesophile, halophile greater than psychrophile. Based on a two-state approximation the apparent free energies of protein unfolding at zero urea denaturant concentration, deltaGH2Oapp, were found to range from 2.4 to 8.8 kcal/mole for the eight phycocyanins at pH 6 and 25 degrees C. The proteins from the thermophile are generally more stable than those from the mesophile. An extra stability of the halophile is believed due to the specific interaction of the proteins and the ions in solution. A correction for deltaGH2Oapp due to minor amino acid differences reveals that the stability and the structural properties of these proteins are primarily affected by this minor difference in amino acid compositions.