The Isolation of Nucleoli from Ungerminated Pea Embryos

A method is described for the isolation of nuclei and nucleoli from ungerminated pea embryos. Electron micrographs of the nucleolar preparation indicate a high degree of purity but only a partial preservation of composition, because of a loss of material from the nucleoli which occurs during the isolation procedure.     

Some Properties of an Invertase-liberating Enzyme Isolated from Zymolyase

An enzyme which released invertase from cell ghosts of Candida utilis was isolated in an electrophoretically pure state from “Zymolyase.” The molecular weight of the purified enzyme was estimated to be 5.8 × 10⁴, and its isoelectric point was pH 6.9. The enzyme was stable in a pH range from 6.0 to 9.0, and the optimal pH for liberation of invertase from cell ghosts was around 6.0. The activity of the enzyme was competitively inhibited by glucose, mannose, and sucrose. Unlike the starting enzyme preparation, “Zymolyase,” the purified enzyme released invertase without making holes on the surface of the cell ghosts. Various tests were applied, but the specificity of the enzyme was not defined.     

Some Biochemical Properties of Mitochondria Isolated from Euglena gracilis*

SYNOPSIS. Mitochondria were isolated from Euglena gracilis strain Z by pressure-breakage of the cells and sucrose-cushion centrifugation. Multiple peaks (2-4) were observed in the rate of phosphorylation with Mg-ADP-phosphate concentration curves. The phosphorylative and oxidative activities were highest with NADH as the substrate, moderate with succinate, and lowest with glutamate. Inhibition of phosphorylation with 2,4-dinitrophenol and carbonyl cyanide, m-chlorophenylhydrazone gave sigmoidal concentration curves, with the extent of inhibition by DNP depending on the substrate used. Inhibition of phosphorylation by valinomycin, atractyloside, or carboxyatractyloside was only ～ 60%. Oligomycin inhibited phosphorylation in 2 phases at low and high concentrations; it inhibited Mg-ATPase in a sigmoidal fashion. Both phosphorylation and oxidation had discontinuities in Arrhenius plots at 34 C and 18 C. The relative Mg²⁺-dependent nucleoside triphosphatase activity was: 1 for ATP and GTP, 0.6 for ITP, 0.15 for CTP and and UTP; with Ca²⁺ in place of Mg²⁺ this activity was 0.35. Both DNP and CCCP stimulated the Mg-ATPase 50-200%. The optimal pH for the stimulation was ～ 7 regardless of the uncoupler used, and ～ 8 without the uncouplers. The few differences observed between mitochondria from Euglena and those from other sources are probably due to the fragmentation of the reticular mitochondrial structure during isolation and not to unique characteristics of these mitochondria.     

Light-dependent Assimilation of Nitrite by Isolated Pea Chloroplasts

Chloroplasts were prepared from peas (Pisum sativum) in glucose-phosphate medium. In the presence of dl-glyceraldehyde, they catalyzed nitrite-dependent O₂ evolution (mean of 13 preparations, 17.5 μmole per mg chlorophyll per hour, sd 3.64). The optimum concentration of nitrite was 0.5 mm; 0.12 mm nitrite supported V_max/2. The reaction was accompanied by the consumption of nitrite; 55 to 80% of the nitrite-N consumed was recovered as ammonia. In short experiments (less than 10 minutes) the O₂ to nitrite ratio approached 1.5, but thereafter decreased. There was no nitrite-dependent O₂ evolution with chloroplasts from plants grown without added nitrate but such chloroplasts could assimilate ammonia at about the usual rate. The results are consistent with the reduction of nitrite to ammonia involving nitrate-induced nitrite reductase and a reductant generated by the chloroplast electron transport chain.     

Physical and Chemical Properties of Cilia Isolated from Tetrahymena pyriformis*

SYNOPSIS. Cilia devoid of their basal granules and isolated from Tetrahymena pyriformis strain W, by the method of Watson et al. (66) have been characterized in terms of physical, chemical and immunologic properties. The general chemical composition of cilia, based on mean values from these determinations is 66.3% protein, 24.3%“lipid,” 3.7 or 4.8% carbohydrate, and 0.4% nucleic acid. The amino acid composition of cilia includes hydroxyproline and is not significantly different from the amino acid composition of whole cells. Chloroformmethanol extracts of ciliary material contain free amino acids as well as neutral lipids and phospholipids. The lipid composition of cilia also is not significantly different from that of whole cells. Pentose, hexose and small but significant amounts of adenine nucleotide and ribonucleic acid are present. Cilia are a greater stimulus for the production of antibody than an equivalent amount of cellular antigen, and the ciliary antigen is strain specific.     

Some Physical and Chemical Properties of Nuclease P1

The inhibitory action of compressed hydrocarbon gases on the growth of the yeast Saccharomyces cerevisiae was investigated quantitatively by microcalorimetry. Both the 50% inhibitory pressure (IP₅₀) and the minimum inhibitory pressure (MIP), which are regarded as indices of the toxicity of hydrocarbon gases, were determined from growth thermograms. Based on these values, the inhibitory potency of the hydrocarbon gases increased in the order methane << ethane < propane < i-butane < n-butane. The toxicity of these hydrocarbon gases correlated to their hydrophobicity, suggesting that hydrocarbon gases interact with some hydrophobic regions of the cell membrane. In support of this, we found that UV absorbing materials at 260 nm were released from yeast cells exposed to compressed hydrocarbon gases. Additionally, scanning electron microscopy indicated that morphological changes occurred in these cells.     

The Photochemical and Surface-Active Properties of Melanins Isolated from Some Black Fungi

The absorption spectra of melanins isolated from some black ascomycetes, as well as of synthetic melanin and natural melanin from Sepia officinalis, were recorded in the long-wavelength ultraviolet region A (320 nm < < 400 nm) and in the blue–violet region of the electromagnetic spectrum at illumination intensities varying from 0.02 to 1 mW/cm². The photochemical properties of fungal melanins were found to be dependent on both the producing strain and the conditions of its cultivation. The fungal melanins are more susceptible to photomodification and more biologically active than the synthetic melanin, indicating that these properties may be related. The data obtained suggest that the fungal melanins susceptible to photomodification possess higher biological activity than commercial melanins.     

Some Physiological Properties of Protoplasts Isolated from Maize and Tobacco Tissues2

The stability and respiratory and permeability properties ofprotoplasts isolated from maize and tobacco tissues have beeninvestigated. Oxygen uptake rates of maize leaf protoplastsshowed the expected response to a variety of metabolic inhibitors.Studies of leakage from preloaded tissues during protoplastisolation and of phosphate and potassium uptake byprotoplastsindicated that isolation produced a marked increase in membranepermeability. Ageing of protoplasts for 24 h produced a markedchange in the ability of maize leaf protoplasts to absorb potassium.     

Characterization of Green Tissue-Specific Phytochrome Isolated Immunochemically from Pea Seedlings

Phytochrome was isolated and purified from light-grown pea (Pisumsativum) seedlings and compared with that from dark-grown seedlingsin terms of spectral and immunochemical properties. Approximately40% of phytochrome in the brushite eluate prepared from light-grownpea tissue bound with a monoclonal anti-pea phytochrome antibody(mAP3), but the remaining 60% did not. Both phytochrome fractionsshowed a typical photoreversible absorbance change after alternatered and far-red actinic irradiations, which was similar to thatof phytochrome from etiolated pea tissue. The peptide mappingof the mAP3-bound phytochrome from light-grown tissue was essentiallythe same as that of the mAP3-bound phytochrome from etiolatedtissue. However, the digestion pattern of the phytochrome thatwas prepared from light-grown tissue but which did not bindto mAP3 was obviously different from that of mAP3-bound phytochrome.Polyclonal anti-pea phytochrome antibodies and mAP5 and 10,however, bound to both the phytochromes. These results suggestthat light-grown tissue contains two phytochrome pools whichare distinct from each other with respect to the primary structureof the phytochrome polypeptide but which share a few commondeterminant sites. ¹ Permanent address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Fukazawa, Tokyo 158, Japan (H.A.), and Department of Botany, Faculty of Science, Universityof Tokyo, Hongo, Tokyo 113, Japan (M. F.).     

Proposed Enzymes of Auxin Biosynthesis and Their Regulation III. Some Properties of Pea Indolylacetaldehyde Oxidase

Indole-3-acetaldehyde oxidase (IAAld-oxidase) occurs in pea in two forms, of which the first, more active enzyme, has its pH optimum at 4.5, while the second, barely half as active, has a pH optimum at 7.0. Only the pH 4.5 oxidase can be resolved from the acetone powder. Besides IAA1d the more stable IA1d was used as substrate in testing the enzymatic activity. The pea enzyme seems not to be a dismutase since indolylmethanol or indolylethanol were not formed as products. Pyridine nucleotide coenzymes did not activate the partially purified enzyme. The pH 4.5 oxidase was inhibited by more than 50 % by IAA > L-asp > tryptophol > indoleacetylaspartic acid > 2,4-D (at 1 mM concentration). The pH 7.0 oxidase was inhibited relatively more weakly, a stronger than 50 % inhibition was caused only by NAA > L-asp. The oxidases were clearly distinguished by the response to L-asparagine (1 mM): the activity of the pH 4.5 oxidase was increased (+ 12 %), while the activity of the pH 7.0 oxidase was decreased (-71 %). In preliminaryin vitro experiments the phytohormones (1 mM) kinetin and GA₃ increased the conversion of IAAld to IAA, while ABA decreased it.     

The pH Dependence of Violaxanthin Deepoxidation in Isolated Pea Chloroplasts

The absorbance change at 505 nm was used to monitor the kinetics of violaxanthin deepoxidation in isolated pea (Pisum sativum) chloroplasts under dark conditions at various pH values. In long-term measurements (65 min) a fast and a slow exponential component of the 505-nm absorbance change could be resolved. The fast rate constant was up to 10 times higher than the slow rate constant. The asymptote value of the fast kinetic component was twice that of the slow component. The pH dependency of the parameters of the fast kinetic component was analyzed from pH 5.2 to pH 7.0. It was found that the asymptote value dropped slightly with increasing pH. The rate constant was zero at pH values greater than 6.3 and showed maximum values at pH values less than 5.8. Hill plot analysis revealed a strong positive cooperativity for the pH dependency of the fast rate constant (Hill coefficient nH = 5.3). The results are discussed with respect to published activity curves of violaxanthin deepoxidation.     

Effect of Tribenzylphosphate on the Phosphate Translocator of Isolated Pea Chloroplasts

The effect of tribenzylphosphate on the activity of the phosphatetranslocator of intact pea chloroplasts was tested. The translocatoractivity was followed by O₂ evolution, ¹⁴CO₂ fixation and ³²Pback-exchange. The reagent inhibited 3-phosphoglycerate dependent-photosyntheticactivities probably through an interaction with the PGA translocator. (Received September 11, 1985; Accepted November 21, 1985)     

Some Characteristics of Formyltetrahydrofolate Synthetase from Pea Seedlings

It has been found, that ammonium sulfate is effective not only in stabilizing, but also in stimulating the activity of formyltetrahydrofolate synthetase (E. C. 6, 3. 4. 3) purified approximately 500-fold from pea seedlings. Kinetic studies have indicated that the stimulation by ammonium sulfate is due to the enhancement of the binding of the substrate, formate, with the enzyme. The binding of the another substrate, FAH₄, with the enzyme was not affected by the addition of ammonium sulfate. The enzyme activity was inhibited by various sulfhydryl reagents, and the inhibition by PCMB was overcome by the addition of l-cysteine. The inhibition by PCMB was competitive with FAH₄, and the K_i value for PCMB was 0.8 × 10?⁶m.     

Wounding of Aged Pea Epicotyls Enhances the Reinitiating Ability of Isolated Ribosomes

Total ribosomes (monosomes plus polysomes) isolated from woundedpea epicotyls are more efficient at supporting protein synthesisin a wheat germ S₃₀ system (containing wheat ribosomes) thanare total ribosomes from aged (control) pea tissue. This increasedefficiency is seen when enriched large polysomes, almost devoidof monosomes, are used to program a wheat germ S₃₀₀ system,from which the wheat germ ribosomes have been removed. Reactionsprimed by enriched polysomes from wounded tissue, but not agedtissue, continue for at least 30 min, suggesting that reinitiationis occurring during the reaction, albeit in the initial absenceof monosomes from wheat or pea. Wheat germ ribosomes, but notmonosomes from either aged or wounded pea tissue, are able totranslate pea poly(A) RNA and globin mRNA. Aurintricarboxylicacid reduces protein synthesis in a rather indiscriminate manner,whereas, pactamycin seems to have an inhibitory effect specificfor initiation, and it is much more effective on wounded thanon control tissue polysomes. We interpret these results to implythat polysomal ribosomes from wounded tissue are more efficientat initiation than are polysomal ribosomes from control tissueor than non-polysomal ribosomes (monosomes) from either tissue. (Received May 7, 1985; Accepted July 4, 1985)     

Structure and Some Properties of Soluble 1,3-beta-Glucan Isolated from the Green Alga Caulerpa simpliciuscula

The properties of the soluble β-glucans formed during photosynthesis of the green siphonous alga Caulerpa simpliciuscula are described. There are two components in the soluble β-glucan fraction. One has an apparent degree of polymerization of 37 glucose units and the other of 270 glucose units. The β-glucan with the lower apparent molecular weight accounts for most of the mass in the β-glucan fraction and is similar in properties to soluble laminarins reported in other algal and fungal species. The β-glucan with the high apparent molecular weight contains most of the radioactivity accumulated in the β-glucan fraction during short periods of photosynthesis.