Stoichiometry of Photosystem I, Photosystem II, and Phycobilisomes in the Red Alga Porphyridium cruentum as a Function of Growth Irradiance

Cells of the red alga Porphyridium cruentum (ATCC 50161) exposed to increasing growth irradiance exhibited up to a three-fold reduction in photosystems I and II (PSI and PSII) and phycobilisomes but little change in the relative numbers of these components. Batch cultures of P. cruentum were grown under four photon flux densities of continuous white light; 6 (low light, LL), 35 (medium light, ML), 180 (high light, HL), and 280 (very high light, VHL) microeinsteins per square meter per second and sampled in the exponential phase of growth. Ratios of PSII to PSI ranged between 0.43 and 0.54. About three PSII centers per phycobilisome were found, regardless of growth irradiance. The phycoerythrin content of phycobilisomes decreased by about 25% for HL and VHL compared to LL and ML cultures. The unit sizes of PSI (chlorophyll/P₇₀₀) and PSII (chlorophyll/Q_A) decreased by about 20% with increase in photon flux density from 6 to 280 microeinsteins per square meter per second. A threefold reduction in cell content of chlorophyll at the higher photon flux densities was accompanied by a twofold reduction in β-carotene, and a drastic reduction in thylakoid membrane area. Cell content of zeaxanthin, the major carotenoid in P. cruentum, did not vary with growth irradiance, suggesting a role other than light-harvesting. HL cultures had a growth rate twice that of ML, eight times that of LL, and slightly greater than that of VHL cultures. Cell volume increased threefold from LL to VHL, but volume of the single chloroplast did not change. From this study it is evident that a relatively fixed stoichiometry of PSI, PSII, and phycobilisomes is maintained in the photosynthetic apparatus of this red alga over a wide range of growth irradiance.     

A cell cycle-associated pathway for repair of DNA-protein crosslinks in mammalian cells

Bulky adducts to DNA including DNA-protein crosslinks formed with trans-platinum(II)diammine-dichloride are repaired largely by the nucleotide excision pathway in mammalian cells. The discovery in this laboratory that cells deficient in nucleotide excision repair, i.e., SV40-virus transformed SV-XP20S cells, can efficiently repair DNA-protein crosslinks implicates a second pathway. In this report, details concerning this pathway are presented. DNA-protein crosslinks induced with 20 microM trans-platinum were assayed by the membrane alkaline elution procedure of Kohn. DNA replication was measured by CsCl gradient separation of newly synthesized DNA that had incorporated 5-bromodeoxyuridine. The following results indicate that this new repair pathway is associated with cell cycling: Whereas rapidly proliferating human cells deficient in excision repair (SV40 transformed XP20S, group A) are proficient in repair of DNA-protein crosslinks, the more slowly growing untransformed parent line is deficient but can complete repair after prolonged periods of 4-6 days, the approximate doubling time of the cell population. Either "used" culture medium or cycloheximide (1 microgram/ml) inhibits cell proliferation, protein synthesis, DNA replication and crosslink repair. In the presence of increasing concentrations of cycloheximide (0.01-5 micrograms/ml) the percent of DNA replication decreases and is essentially equivalent to the percent of crosslink repair. The following results indicate that this new repair pathway, though associated with cell cycling, is independent of DNA replication per se. The rates of DNA-protein crosslink repair and DNA replication are essentially the same in mouse L1210 cells rapidly proliferating in 20% serum supplement; however, to slower proliferation rates in 1% serum rate of crosslink repair is slower but differs from that of DNA replication. In the presence of aphidicolin (10 micrograms/ml) cells can repair DNA-protein crosslinks in virtually the complete absence of DNA replication, though the rate is slower in both nucleotide excision-proficient and -deficient cells. Thus, DNA replication is not essential for repair of DNA-protein crosslinks. Comparison of the kinetics of replication and DNA-protein crosslink repair of pulse-labeled indicates that, in the absence of metabolic inhibitors, repair of the crosslinks is independent of replication per se and, therefore, DNA recombination events are not involved in this repair process. We conclude, therefore, that the new repair pathway is not coupled with DNA replication but is with cell cycling.     

A high molecular weight terminal pigment (“anchor polypeptide”) and a minor blue polypeptide from phycobilisomes of the cyanobacterium Nostoc sp. (MAC): Isolation and characterization

A 94 kD pigment-polypeptide, which is presumed to be involved in anchoring the phycobilisomes to the thylakoids, was isolated from Nostoc phycobilisomes by gel filtration in 63 mM formic acid. The isolation condition did not require detergents or denaturating reagents, as in previous procedures, and enzymatic degradation was not observed at the low pH of 2.5. The anchor polypeptide thus obtained had absorption (Abs) and fluorescence maxima (Em) at 658 and 673 nm, respectively, in 63 mM formic acid at room temperature. The maxima shifted to longer wavelengths in 100 mM potassium phosphate (pH 6.8), Abs 665 and Em 683 nm at room temperature, and Abs 665 and Em 684 nm at liquid nitrogen temperature. The fluorescence maxima at both temperatures correspond to the longest wavelength component resolved in phycobilisomes from second derivative spectra. A minor blue polypeptide was also found by this isolation method. The molecular weight of this polypeptide was ca. 18,000 and is probably similar to a polypeptide which has been found in the phycobilisome core of other cyanobacteria.Abbreviations used -APB -subunit of allophycocyanin B - APC allophycocyanin - kD kilodalton - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - PBS phycobilisome - PE phycoerythrin - PC phycocyanin - PS pigment system     

Enrichment of a 50-kilodalton polypeptide in a photosystem II-phycobilisome particle from Porphyridium cruentum

A 50-kDa polypeptide was obtained from photosynthetically active phycobilisome-photosystem II preparations from the red alga Porphyridium cruentum after removal of phycobiliproteins. Removal of phycobiliproteins caused destabilization of the structure of the phycobilisome-photosystem II preparations and was accompanied by a decline in photosystem II activity (oxygen-evolution and dichlorophenol-indophenol (DPIP) reduction). The treatments in increasing relative effectiveness were: addition of EDTA (10 mM), lowering the pH (6.8----4.4), and lowering the ionic strength (to ca. 1 mM phosphate). The lowering of the ionic strength by dialysis resulted in a preparation highly enriched in a 50-kDa polypeptide (apparent molecular mass on SDS-PAGE). This preparation retained photosystem II activity as evidenced by the photoreduction of DPIP in the presence of diphenylcarbazide (222 mumol DPIP/mg chlorophyll/h). Also it had a 698-nm (77K) fluorescence emission maximum, as compared to a 668-nm emission in the unfractionated preparation, which indicates enrichment of the photosystem II reaction center. Comparing our results with those obtained from green plants and a cyanobacterium leads us to suggest that the reaction center II polypeptides are highly similar in all chlorophyll alpha-containing plants.     

Phycobilisome-thylakoid Topography on Photosynthetically Active Vesicles of Porphyridium cruentum

Conditions are described for isolating functional phycobilisome-thylakoid vesicles from the red alga Porphyridium cruentum. Phycobilisome-thylakoid vesicles were prepared by brief sonication and centrifugation in a medium containing 0.5 molar sucrose, 0.5 molar potassium phosphate, and 0.3 molar sodium citrate (pH 7.0). They required ferricyanide as an oxidant and had O₂ evolution rates (about 450 micromoles O₂ per hour per milligram chlorophyll) higher than whole cells (about 250 micromoles O₂ per hour per milligram chlorophyll). Energy transfer to photosystem II chlorophyll was evident from a high F695 nanometer (−196 C) emission peak. Preparations could be stored for over 24 hours and were considerably more stable than those from the cyanobacterium Anabaena variabilis (Katoh T, E Gantt 1979 Biochim Biophys Acta 546: 383-393). In electron micrographs of negatively stained material, the active thylakoid vesicles were found covered by closely spaced phycobilisomes on their external surface. The phycobilisome number in negatively stained vesicles was 450 per square micrometer, which was in the same range as the 400 per square micrometer observed in surface sections. A cell containing 1.5 × 10⁻⁶ micrograms phycoerythrin and 1.3 × 10⁻⁶ micrograms chlorophyll was found to contain 5 to 7 × 10⁵ phycobilisomes on a thylakoid area of 1.1 to 1.6 × 10³ square micrometers.     

In vitro Protein Synthesis by Plastids of Phaseolus vulgaris. II. The Probable Relation Between Ribonuclease Insensitive Amino Acid Incorporation and the Presence of Intact Chloroplasts

Amino acid incorporation into protein by chloroplasts from primary leaves of Phaseolus vulgaris L., var. Black Valentine is only partially inhibited by 400 μg/ml ribonuclease. The rate of incorporation, in the presence of ribonuclease, is progressively inhibited with time, and ceases after about half an hour. Preincubation of chloroplasts at 25°, in the absence of ribonuclease, increases the inhibitory effect of ribonuclease on the initial rate of incorporation of amino acid into protein. Examination of electron micrographs of freshly prepared chloroplast suspensions shows that chloroplasts are largely intact. However, after incubation at 25° for 1 hour the chloroplasts are disrupted, as indicated by loss of their stroma contents. It is concluded that the intact chloroplast membrane is relatively impermeable to ribonuclease. Amino acid incorporating activity probably becomes inhibited as the inside of the chloroplast is made accessible to ribonuclease by breakage of membranes during incubation at 25°.     

GRANULES ASSOCIATED WITH THE CHLOROPLAST LAMELLAE OF PORPHYRIDIUM CRUENTUM

Small granules with a diameter of approximately 350 A are attached to the chloroplast lamellae of the red alga Porphyridium cruentum. To some extent, their size depends on the culture conditions and the age of the cell. It was possible to preserve the granules only with aldehyde prefixation. It can be seen that fixed or negatively stained granules are comprised of smaller subunits. The granules are arranged regularly on the lamellae in repeating rows with a center-to-center granule distance of 400 to 500 A. Attempts at characterization of these chloroplast granules revealed that they are resistant to hydrolysis by ribonuclease and appear to be structurally unaffected by methanol-acetone extraction. Because of their close association with the chloroplast lamellae, they are considered as possible sites of phycobilin concentration. This possibility is supported by two observations: when the phycobilins are removed, the granules disappear; and, when the chlorophyll and stainable membrane portions are selectively removed, the phycobilins and granules are still present. It was found that all other marine red algae examined had granules which were associated with the chloroplast lamellae.