Atypical pulmonary thrombosis caused by a toxic cyanobacterial peptide.

Parenteral injection into mice of a toxic pentapeptide isolated from the cyanobacterium Microcystis aeruginosa induced thrombocytopenia, pulmonary thrombi, and hepatic congestion. The lethality of the toxin was unaffected by several anticoagulants. The acute liver damage that follows injection of the toxin has been attributed to direct action on liver cells but may be due to hypoxemia, heart failure, and shock.

Membrane-bound ATPase of intact vacuoles and tonoplasts isolated from mature plant tissue.

Intact vacuoles were isolated from petals of Hippeastrum and Tulipa (Wagner G.J. and Siegelman, H.W. (1975) Science 190, 1298--1299). The ATPase activity of fresh vacuole suspensions was found to be 2--3 times that of protoplasts from the same tissue. 70--80% of the ATPase activity of intact vacuoles was recovered in tonoplast preparations. The antibiotic Dio-9 at 6mug/10(6) vacuoles or protoplasts causes 40% inhibition. However, only the protoplast ATPase is sensitive to oligomycin. N,N'-dicyclohexylcarbodiimide (DCCD) slightly stimulates ATPase activity in both vacuole and protoplast suspensions, whereas ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDAC) strongly inhibits. Spectrophotometric studies show that in the petal the vacuolar contents have a pH of 4.0 for Tuplipa and 4.3 for Hippeastrum, whereas the intact isolated vacuole has an internal pH of 7.0 (in pH 8.0 buffer) for (Tulipa and about 7.3 for Hippeastrum. Internal ion concentrations of 150, 46, 30, 30 and 6 mM were found for K+, Na+, Mg2+, Cl-, and Ca2+ respectively, which are about the same as those in protoplasts.

The effects of single L-amino acid substitutions on the lethal potencies of the microcystins.

Microcystin-LR and -LA are more toxic than microcystin-LY and -RR in adult mice. They induce different degrees of thrombocytopenia and leukopenia, and the lethalities of their binary and ternary mixtures are addictive. Postnatal mice are resistant to doses of microcystin-LR that are lethal to adults but they are susceptible to higher doses. Substitution of a single L-amino acid for another in a microcystin markedly affects the dosimetric potency, but not the pathophysiology of its toxicity.

Pathophysiologic effects of a toxic peptide from Microcystis aeruginosa.

Toxin-LR, a hexapeptide produced by Microcystis aeruginosa, causes marked hepatic vascular congestion, thrombocytopenia, microscopic pulmonary thrombi and death in 50-70 min when injected into mice. Although it is considered an hepatotoxin, we report that sublethal hepatocellular damage produced by CCl4 given 24 hr prior to toxin-LR administration prevents the acute deaths. However, CCl4-treated mice surviving toxin-LR acute effects often died during the subsequent three days. Pretreatment of mice with the microsomal enzyme inhibitors SKF 525A or cobaltous chloride did not alter the acute lethality of toxin-LR, but pharmacologic doses of hydrocortisone prevented both the acute and delayed deaths. X irradiation-induced thrombocytopenia or thrombocytopenia and leukopenia did not significantly affect the toxin's lethality. In vitro platelet aggregation or lysis did not occur during incubation with toxin-LR, nor was a humoral aggregating factor detected in plasma from toxin-injected mice.

Cyclosporine A inhibition of microcystin toxins.

Cyclosporine A (CyA) given i.v. at a dose of 1.25 mg/mouse blocks a subsequent i.v. lethal dose (1.7-1.8 x LD50) of microcystin-LR for 24 hr, and is about 50% protective at 48 hr. Conversely, the fraction of mice that can be rescued by CyA (0.2 mg/mouse) after a lethal dose of microcystin-LR decreases rapidly with a pharmacodynamic half-time of only about 100 sec. The prophylactic action of CyA was tested against lethal doses of four microcystins. The acute lethality of 1.7-1.8 x LD50 dose of microcystin-LR, -RR, -LY, or -LA given 1 hr after administration of 0.2 mg of CyA is 0%, 0%, 58%, or 100%, respectively. Even a 0.6 mg/mouse dose of CyA is ineffective prophylaxis against a lethal dose of microcystin-LA. The inhibitory potency of CyA on microcystin toxicity can be completely reversed by the single L-amino acid substitution of alanine for arginine in the microcystin.

Molecular morphology of cyanobacterial phycobilisomes.

Phycobilisomes were isolated from several cyanobacteria following cell lysis with Triton X-100. They were purified by phosphate precipitation and hydrophobic-interaction chromatography. Their phycobiliprotein compositions were quantitatively determined by application of sets of simultaneous absorbance equations to gel chromatographic separations of the chromoproteins. Phycobilisomes purified from several cyanobacteria had characteristic elution times on agarose gel chromatography. Combining electron microscope observations of phycobilisome structure, phycobiliprotein composition, and agarose gel chromatography estimates of molecular weight permitted the calculation of many details of phycobilisome molecular structure. Complementary chromatic adaptation resulted in a change of phycobilisome composition and structure. The polypeptide compositions of phycobilisomes were examined by sodium dodecyl sulfate-agarose gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The phycobilisomes were composed of phycobilipeptides derived from the constituent phycobiliproteins. Higher molecular-weight phycobilipeptide aggregates were also observed. The dominant forces responsible for the maintenance of phycobilisome structure are concluded to be hydrophobic interactions.

Protochlorophyllide resynthesis in dark-grown bean leaves.

The protochlorophyllide content of dark-grown bean leaves was determined at various ages. It was detectable the second day after germination and reached a maximum on about the tenth day.The resynthesis of protochlorophyllide following a single irradiation and the rate of the 685 to 673 mmu shift of the newly formed chlorophyll was examined. Leaves up to 5-days old show no lag phase in protochlorophyllide resynthesis. By the sixth day, a lag phase is evident which increases with age. The rate of the 685 to 673 mmu shift is similarly age dependent.

Purification of protochlorophyllide holochrome.

Phototransformable protochlorophyllide holochrome was prepared from etiolated bean leaves. The detergent Triton X-100 in the presence of glycerol and tricine-KOH buffer (pH 8) enhanced the extractability, specific activity, and phototransformability of the holochrome. Purification was achieved by polyethylene glycol-6000 precipitation and hydroxyl-apatite, DEAE-cellulose, and agarose chromatography. The presence of Triton X-100 permitted removal of the carotenoid contamination from the holochrome. The 678-nm absorption maximum of newly formed chlorophyllide a holochrome shifts to 672 nm in a temperature-dependent manner. The purified holochrome contains 0.24 g of protein per mumole of protochlorophyllide. Estimation of the molecular weight of the holochrome by gel filtration on agarose revealed the presence of aggregates of approximately 550,000 and 300,000. There are at least 2 chromophores per 550,000 molecular weight.

Localization of Acid hydrolases in protoplasts: examination of the proposed lysosomal function of the mature vacuole.

The development of techniques to isolate and purify relatively large quantities of intact vacuoles from mature tissues permits direct biochemical analysis of this ubiquitous mature plant cell organelle. Vacuoles and a fraction enriched in soluble cytoplasmic constituents were quantitatively prepared from Hippeastrum flower petal protoplasts. Vacuolar lysate and soluble cytoplasmic fractions were examined for acid hydrolase activities commonly associated with animal lysosomes, and pH optima were determined. Esterase, protease, carboxypeptidase, beta-galactosidase, alpha-glycosidase and beta-glycosidase, not found in the vacuole lysate fraction, were components of the soluble cytoplasmic fraction. Acid phosphatase, RNase and DNase were present in both fractions. Vacuolar enzyme activities were also examined as a function of flower development from bud through senescent stages. The data obtained are not consistent with the concept that the mature plant cell vacuole functions as a generalized lysosome.

Mass-spectral identification and purification of phycoerythrobilin and phycocyanobilin.

The bile-pigment chromophores of C-phycoerythrin (phycoerythrobilin) and C-phycocyanin (phycocyanobilin) were cleaved from their respective proteins with boiling methanol or butan-1-ol. They were purified as dicarboxylic acids by preparative reverse-phase liquid chromatography. Each pigment existed in two principal forms, which were characterized by using 20 pmol samples by proton-transfer chemical-ionization mass spectroscopy. These two principal forms were isomeric species, and all had protonated parent molecular ions with m/e 587, corresponding to a molecular weight of 586.

Lipopolysaccharides of the cyanobacterium Microcystis aeruginosa.

Lipopolysaccharides (LPS) of two isolates of Microcystis aeruginosa were extracted with phenol/water and purified. Cesium chloride gradient ultracentrifugation of these preparations yielded only one fraction. The LPS contained significant amounts of 3-deoxy-D-manno-octulosonic acid, glucose, 3-deoxy sugars, glucosamine, fatty acids, fatty acid esters, hexoses, and phosphate. Heptose, a characteristic sugar component of the polysaccharide moiety of LPS of most gram-negative bacteria was absent. Lipopolysaccharides and lipid A hydrolysate of LPS preparations were active in mouse lethality and Limulus lysate gelation. The lipid A moiety was slightly less active in toxicity and Limulus lysate gelation assays than the intact LPS. The LPS and lipid A moiety of the two isolates of M. aeruginosa were less active in toxicity in mice and Limulus test than LPS of Salmonella abortus equi.

Chromophores of allophycocyanin and R-phycocyanin.

The biliprotein allophycocyanin was purified from Phormidium luridum, Anabaena variabilis and Plectonema boryanum. R-phycocyanin was purified from Rhodymenia palmata. The chromophores were cleaved from the denatured protein by methanol hydrolysis. They were purified and crystallized as the dimethyl esters. Chromatographic and absorption-spectral (visible-ultraviolet and infrared) comparisons with reference material have established phycocyanobilin as the chromophore of allophycocyanin. Phycocyanobilin and phycoerythrobilin were shown to be the chromophores of R-phycocyanin.

The chromophore of phytochrome.

The chromophore of phytochrome, the plant photomorphogenic pigment, was cleaved from the associated protein. Chromatographic and spectral properties indicated that it was a bilitriene closely similar to but distinct from the chromophore of C-phycocyanin and allophycocyanin.

EVIDENCE FOR A CRYPTOMONAD SYMBIONT IN THE CILIATE, CYCLOTRICHIUM MEUNIERI(1).

Extracts of the marine ciliate Cyclotrichium meunieri contained chlorophylls a and c, carotenoids, and a phycoerythrin with a single absorbance maximum at 542 nm. This assemblage of pigments suggests that the numerous photosynthetic symbionts present in each ciliate cell belong to the Cryptophyceae.

Cleavage of phycocyanobilin from C-phycocyanin. Separation and mass spectral identification of the products.

The chromophore of C-phycocyanin, phycocyanobilin, was cleaved from the protein with methanol, concentrated hydrochloric acid, or subtilisin BPN'. The pigments obtained were converted to their dimethyl esters and purified by preparative high pressure liquid chromatography and examined for purity by analytical high pressure chromatography on silica gel. They were characterized by proton transfer and electron impact mass spectroscopy. The principal product obtained by the three cleavage procedures was phycocyanobilin. Methanol and hydrochloric acid adducts of phycocyanobilin were obtained with methanol and concentrated hydrochloric acid cleavages, respectively. Methanol adduct formation of phycocyanobilin can occur subsequent to cleavage and requires acid catalysis. No adduct formation was observed with mesobiliverdin under similar conditions. These results and mass spectral data support the conclusion that adduct formation takes place at the exocyclic olefin linkage of ring A in phycocyanobilin. The ease of co-valent adduct formation strongly suggests that the ethylidene side chain is an important binding site of phycocyanobilin to the polypeptide chain.

Peridinin-Chlorophyll a Proteins of the Dinoflagellate Amphidinium carterae (Plymouth 450).

The marine dinoflagellate Amphidinium carterae (Plymouth 450) releases several water-soluble peridinin-chlorophyll a proteins after freezethawing. These chromoproteins have a molecular weight of 39.2 x 10(3) and are comprised of noncovalently bound peridinin and chlorophyll a and a nonoligomeric protein. They have distinct isoelectric points and may be resolved into six components by either isoelectric focusing on polyacrylamide gel or ion exchange chromatography. The predominant chromoprotein, which has a pI of 7.5, constitutes about 90% of the extractable peridinin-chlorophyll a protein. It consists of an alanine-rich apoprotein of molecular weight 31.8 x 10(3) stoichiometrically associated with 9 peridinin and 2 chlorophyll a molecules. Additionally, the peridinin-chlorophyll a proteins with pI values of 7.6 and 6.4 were purified and found to have amino acid and chromophore composition essentially identical with the pI 7.5 protein. Peridinin-chlorophyll a protein, pI 7.5, after treatment at alkaline pH was transformed into several more acid pI forms of the protein, strongly suggesting that the naturally occurring proteins are deamidation products of a single protein. Fluorescence excitation and emission spectra demonstrate that light energy absorbed by peridinin induces chlorophyll a fluorescence presumably by intramolecular energy transfer. The peridinin-chlorophyll a proteins presumably function in vivo as photosynthetic light-harvesting pigments.

Cyanobacterial phycobilisomes: Selective dissociation monitored by fluorescence and circular dichroism.

Phycobilisomes are supramolecular assemblies of phycobiliproteins responsible for photosynthetic light collection in red algae and cyanobacteria. They can be selectively dissociated by reduction of temperature and buffer concentration. Phycobilisomes isolated from Fremyella diplosiphon transfer energy collected by C-phycoerythrin and C-phycocyanin to allophycocyanin. The energy transfer to allophycocyanin is nearly abolished at 2 degrees C, as indicated by a blue shift in fluorescence emission, and is accompanied by a decrease in the circular dichroism in the region of allophycocyanin absorbance. Further dissociation of the phycobilisomes can be attained by reduction of buffer concentration and holding at 2 degrees C. Energy transfer to C-phycocyanin is nearly abolished, and decreases occur in the circular dichroism in the region of C-phycocyanin and C-phycoerythrin absorbance. Complete dissociation of the phycobilisomes at low buffer concentration and 2 degrees C requires extended time. Energy transfer to C-phycocyanin is further reduced and the circular dichroism maximum of C-phycoerythrin at 575 nm is lost. Circular dichroism provides information on the hexamer-monomer transitions of the phycobiliproteins, whereas fluorescence is indicative of hexamer-hexamer interactions. We consider that hydrophobic interactions are fundamental to the maintenance of the structure and function of phycobilisomes.

Adaptation of the Cyanobacterium Microcystis aeruginosa to Light Intensity.

Light intensity adaptation (20 to 565 microeinsteins per square meter per second) of Microcystis aeruginosa (UV-027) was examined in turbidostat culture. Chlorophyll a and phycocyanin concentrations decreased with increasing light intensity while carotenoid, cellular carbon, and nitrogen contents did not vary. Variation in the number but not the size of photosynthetic units per cell, based on chlorophyll a/P(700) ratios, occurred on light intensity adaptation. Changes in the numbers of photosynthetic units partially dampened the effects of changes in light intensity on growth rates.