Decarboxylative metabolism of [1′-14C]indole-3-acetic acid by tomato pericarp discs during ripening

The rate of decarboxylation of [1′-¹⁴C]indole-3-acetic acid (IAA) infiltrated into tomato (Lycopersicon esculentum Mill.) pericarp discs was much more rapid in green than in breaker and pink tissues. Studies were carried out in order to determine whether the decarboxylative catabolism occurring in the green pericarp discs was associated with ripening or was a consequence of wound-induced peroxidase activity and/or ethylene production. After a 2-h lag, the decarboxylative capacity of the green pericarp discs increased exponentially during a 24-h incubation period. This increase was accompanied by increases in IAA-oxidase activity in cell-free preparations from the intercellular space and cut surface of the discs. Although higher IAA-oxidase activity was detected in extracts from the tissue residue, which comprises mainly intracellular peroxidases, this activity did not increase during the 24-h incubation period. Analysis of the cell-free preparations by isoelectric focusing revealed the major component in all samples was a highly anionic peroxidase (pI=3.5) the levels of which did not increase during incubation. However, the intercellular and cut-surface preparations contained additional anionic and cationic peroxidases which increased in parallel with the increases in both the IAA-oxidase activity of the preparations and the decarboxylative capacity of the green pericarp discs from which they were derived. Treatment of green discs with the ethylene-biosynthesis inhibitors aminooxyacetic acid and CoCl₂, inhibited the development of an enhanced capacity to decarboxylate [1′-¹⁴C]IAA but the inhibition was not counteracted by exogenous ethylene. Another ethylene-biosynthesis inhibitor, aminoethoxyvinyl glycine, also reduced ethylene levels but did not affect IAA decarboxylation, indicating that the decarboxylation was not a consequence of wound-induced ethylene production. The data obtained thus demonstrate that the enhanced capacity to decarboxylate [1′-¹⁴C]IAA that develops in green tomato pericarp discs following excision is not associated with ripening but instead is attributable to a wound-induced increase in anionic and cationic peroxidase activity in the intercellular fluid and at the cut surface of the excised tissues.     

Aldrin epoxidation by plant root extracts

Aldrin epoxidase activity of the cell-free pea and bean root preparations was located in the particulate fraction. High speed centrifugation at 250 000 g for 2 hr resulted in a pellet with almost all the activity of the crude cell-free preparations. While the epoxidase was stimulated by NADPH generating system in these cell-free root preparations, that in high speed centrifugation pellets was not. Aldrin epoxidase activity of the dwarf bean root homogenates was increased by addition of p-aminobenzoic acid (10^?4 M). This increase in activity is above that already manifested by Polyclar AT. No activity was detected in the dormant or germinating Alaska peas or dwarf beans until the 5th and 6th day, respectively.     

Covalent binding of peroxidase to CH- and AH-Sepharose 4 B]

The properties of peroxidase insolubilized by covalent binding to CH- and AH-Sepharose 4 B in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) are described. CH-Sepharose 4 B bound peroxidase yields an enzyme preparation with a residual specific activity of 60.6%. When bound to AH-Sepharose 4 B, the residual specific activity is to 78%. The reasons of these differences in the catalytic activity of the two insolubilized enzyme preparations are discussed. By covalent binding on CH- and AH-Sepharose 4 B, peroxidase exibits no changes in its pH optimum; it virtually keeps the same activity after being used ten times. Insolubilized peroxidase preparations, dried and reimbibed after being stored for 6 weeks at room temperature still display 50% of the initial specific activity of the insolubilized enzyme. Stored in acetate buffer, the enzyme preparations maintain their activity during all this interval.     

An extraordinary accumulation of (-)-pinoresinol in cell-free extracts of Forsythia intermedia: evidence for enantiospecific reduction of (+)-pinoresinol

Stereoselective and enantiospecific transformation mechanisms in lignan biogenesis are only now yielding to scientific inquiry: it has been shown that soluble cell-free preparations from Forsythia intermedia catalyse the formation of the enantiomerically pure lignan, (-)-secoisolariciresinol, when incubated with coniferyl alcohol in the presence of NAD(P)H and H2O2. Surprisingly, (-)-pinoresinol also accumulates in this soluble cell-free assay mixture in > 96% enantiomeric excess, even though it is not the naturally occurring antipode present in Forsythia sp. But these soluble cell-free preparations do not engender stereoselective coupling; instead, racemic pinoresinols are first formed, catalysed by an H2O2-dependent peroxidase reaction. An enantiospecific NAD(P)H reductase then converts (+)-pinoresinol, and not the (-)-antipode, into (-)-secoisolariciresinol. Stereoselective synthesis [correction of syntheis] of (+)-pinoresinol from E-coniferyl alcohol is, however, catalysed by an insoluble enzyme preparation in F. suspensa, obtained following removal of readily soluble and ionically bound enzymes; no exogenously supplied cofactors were required other than oxygen, although the reaction was stimulated by NAD-malate addition. Thus, the overall biochemical pathway to enantiomerically pure (-)-secoisolariciresinol has been delineated.     

A comparison of the sites of phytoalexin accumulation and of biosynthetic activity in potato tuber tissue inoculated with biotic elicitors

Aged discs cut from Kennebec potato tubers were inoculated with one of the following: an elicitor preparation from mycelia of Phytophthora infestans race 4, zoospores from either race 4 or race TY complex of this fungus, or sodium arachidonate. At 24 hr intervals after inoculation, four successive 0.5 mm thick layers of tissue were cut from the discs. This tissue was analysed for accumulated phytoalexins and also used to prepare cell-free enzyme systems for lubimin biosynthesis. In tissue treated with either the elicitor preparation or race 4 zoospores, levels of phytoalexin accumulation were highest in the first layer of tissue. Surprisingly, however, cell-free lubimin biosynthesis from [1-¹⁴C]isopentenyl pyrophosphate was also generally greater in preparations derived from the first 0.5 mm of tissue. Accumulation of phytoalexins in tissue inoculated with zoospores from race TY complex was very low, whereas cell-free biosynthetic activity was initially comparable to that seen in preparations from tissue treated with the elicitor preparation. By the end of the experimental period lower layers of tissue from discs treated with sodium arachidonate contained the highest levels of phytoalexins and yielded cell-free enzyme preparations with the greatest lubimin biosynthetic activity.     

Nuclease activity in preparations of creatine phosphokinase: effect on mRNA stability

Creatine phosphokinase is used to generate ATP with creatine phosphate for $\text{in vitro}$ protein synthesis. Some preparations of this enzyme contain nuclease activity, which can be demonstrated by a sensitive assay of the cleavage of poly(A)-containing RNA. These preparations of creatine phosphokinase support protein synthesis poorly in a cell-free system prepared from HeLa cells. Poly(A)-containing RNA is quite stable in this cell-free system when the phosphorylated sugar fructose 1,6-bisphosphate with no addition of enzyme is used to generate ATP.     

A Noninvasive Technique for Monitoring Peroxidative and H2O2-Scavenging Activities during Interactions between Bacterial Plant Pathogens and Suspension Cells

Stimulation of active oxygen metabolism occurs during the early stages of interactions involving bacteria and plant cell suspensions. Although many cellular processes are known to affect active oxygen metabolism in plants, it is not known which of these factors affect active oxygen levels during plant-bacteria interactions. Extracellular peroxidases have been shown to participate in both the production and utilization of active oxygen species such as H2O2 and superoxide. Catalase and other scavenging mechanisms also affect the overall level of active oxygen. In this study the luminol-dependent chemiluminescent reaction previously used to measure H2O2 levels in suspension cells was modified to allow the assay of both peroxidase and H2O2-scavenging activity. The early stages of the interactions between tobacco (Nicotiana tabacum) and Pseudomonas syringae pv syringae, as well as between soybean (Glycine max) and P. syringae pv glycinea, were investigated. This method of monitoring peroxidase and H2O2-scavenging activity proved to be rapid, sensitive, and nonintrusive, allowing the processing of multiple samples using intact cells or cell-free preparations. The results from the study demonstrate that the scavenging activities can be significant and must be considered when studying active oxygen production in biological interactions.     

Protein synthesis in 3T3 and SV40-transformed 3T3 cells. Activity of ribosomes and cytosol proteins in cell-free protein synthesis.

The activity of specific components involved in protein synthesis in 3T3 cells and its SV40-transformed derivative, SV3T3, were examined in a cell-free protein synthetic system, and the results correlated with previous studies, indicating that a decreasing rate of protein synthesis does not accompany the stationary phase of growth. We found that 3T3 and SV3T3 polysome preparations containing endogenous mRNA were equally efficient in supporting cell-free protein synthesis in this system. Further, the net protein synthesis observed was not altered by an increase in the population density of the cellular polysome source. The activity of the aminoacyl-tRNA synthetase enzymes from 3T3 and SV3T3 cells was examined in vitro after isolation by pH 5 precipitation and by ammonium sulfate fractionation. The activity of these preparations from stationary phase 3T3 and nonexponential phase SV3T3 cells was found to be approximately 3 times higher than the activity of fractions from the homologous exponential phase cell. However, at both growth stages, the SV3T3 preparations were 30 to 40 times more active than the 3T3 preparations. These findings may have implications for the different growth properties observed in the two cell types.     

Characterization of adenylate cyclase fromEscherichia coli

Adenylate cyclase activity was detected and characterized in cell-free preparations of different strains ofEscherichia coli; it was localized not only in the membrane fraction but also in the cytoplasm, the localization differing from strain to strain. The adenylate cyclase activity is highly dependent on the method used for disintegration of cells. The best results were obtained when using vortexing of the cell suspension with ballotini beads. The pH optimum of adenylate cyclase in cell-free preparations was found to be 9.0 –9.5. The enzyme has an absolute requirement for Mg²⁺ and is inhibited by sodium fluoride and inorganic diphosphate. Release of adenylate cyclase from the membrane leads to an immediate loss of the activity; it was found that adenylate cyclase is quite labile and hence it could not yet been purified. The method used to determine adenylate cyclase activity and cyclic AMP is described.     

Release of cell-free ice nuclei by Erwinia herbicola.

Several ice-nucleating bacterial strains, including Erwinia herbicola, Pseudomonas fluorescens, and Pseudomonas syringae isolates, were examined for their ability to shed ice nuclei into the growth medium. Only E. herbicola isolates shed cell-free ice nuclei active at -2 to -10 degrees C. These cell-free nuclei exhibited a freezing spectrum similar to that of ice nuclei found on whole cells, both above and below -5 degrees C. Partially purified cell-free nuclei were examined by density gradient centrifugation, chemical and enzymatic probes, and electron microscopy. Ice-nucleating activity in these cell-free preparations was associated with outer membrane vesicles shed by cells and was sensitive to protein-modifying reagents.     

Effects of Ca2+ ions on the formation of metaphase chromosomes and sperm pronuclei in cell-free preparations from unactivated Rana pipiens eggs

Nuclei transplanted into unactivated amphibian eggs are known to condense into metaphase chromosomes whereas those transplanted into activated eggs decondense and enlarge. We have made cell-free cytoplasmic preparations from Rana pipiens eggs which can induce demembranated Xenopus laevis sperm to undergo changes similar to those seen in intact eggs. Sperm chromatin which is incubated for 3 hr in unactivated egg preparations made using a buffer containing 3 mM EGTA is induced to form metaphase chromosomes. However, decondensed interphase nuclei are formed when chromatin is incubated in unactivated egg preparations made without EGTA as well as in activated egg preparations. When Ca2+ ions are added to unactivated egg preparations made with EGTA, the preparations lose the ability to induce metaphase chromosome formation and become capable of decondensing sperm chromatin. Once the ability to decondense chromatin has developed, either in unactivated or activated egg preparations, it cannot be suppressed by the addition of EGTA. However, decondensation of sperm chromatin in activated egg preparations can be suppressed by the addition of unactivated egg preparations made with EGTA. In this case, the incubated sperm chromatin is induced to form metaphase chromosomes. These results may indicate that the chromosome condensation activity of unactivated egg cytoplasm can be sustained in cell-free preparations when Ca2+ ion levels are kept low, but when Ca2+ ion levels increase this activity is lost and replaced by a new activity which can decondense chromatin. Since this change in cytoplasmic activities is comparable to that occurring in the intact egg following fertilization, these results suggest that Ca2+ ions play a crucial role during activation in altering the cytoplasmic activities which control nuclear behavior.     

Oxidation of ethane by an Acremonium species.

Ethane oxidation was studied in ethane-grown resting cells (mycelia) of an Acremonium sp. and in cell-free preparations of such mycelia. From resting cell experiments evidence was found for a pathway of ethane oxidation via ethanol, acetaldehyde, and acetic acid. In vitro studies indicated that ethane-oxidizing activity in such mycelia occurred predominantly in the microsomal fraction of crude homogenates. Microsomal preparations were inactive in the absence of added coenzyme. Marked stimulation of activity was obtained in such preparations with reduced nicotinamide adenine dinucleotide phosphate and to a much lesser degree with nicotinamide adenine dinucleotide phosphate. Ethane oxidation was inhibited by sodium azide and carbon monoxide.     

α-Galactosidase Activity of Lactobacilli

alpha-Galactosidase (EC 3.2.1.22) activity was observed in cell-free extracts of Lactobacillus fermenti, L. brevis, L. buchneri, L. cellobiosis, and L. salivarius subsp. salivarius. The cultural conditions under which the enzyme activity was detected suggest that the enzyme is constitutive and present in the soluble fraction in the cell. The enzyme preparations readily hydrolyzed melibiose and other oligosaccharides containing alpha(1 --> 6) linked galactose. Although the cell-free extracts of L. fermenti and L. brevis are negative for beta-fructofuranosidase (EC 3.2.1.26), they hydrolyzed melibiose, stachyose, and raffinose in decreasing order of activity. The beta-fructofuranosidase-positive L. buchneri, L. cellobiosis, and L. salivarius preparations hydrolyzed melibiose, raffinose, and stachyose in decreasing rates of activity. The alpha-galactosidases from different lactobacilli showed optimum activity in pH range 5.2 to 5.9. L. fermenti and L. salivarius preparations exhibited maximum activity between 40 to 44 C and 48 to 51 C, respectively, whereas a 38 to 42 C range was observed for other lactobacilli. Cell-free extract of L. cellobiosis was studied for transgalactosylase activity. When incubated with melibiose, a new compound was detected and tentatively identified as manninotriose.     

Oxidation of ethane by an Acremonium species.

Ethane oxidation was studied in ethane-grown resting cells (mycelia) of an Acremonium sp. and in cell-free preparations of such mycelia. From resting cell experiments evidence was found for a pathway of ethane oxidation via ethanol, acetaldehyde, and acetic acid. In vitro studies indicated that ethane-oxidizing activity in such mycelia occurred predominantly in the microsomal fraction of crude homogenates. Microsomal preparations were inactive in the absence of added coenzyme. Marked stimulation of activity was obtained in such preparations with reduced nicotinamide adenine dinucleotide phosphate and to a much lesser degree with nicotinamide adenine dinucleotide phosphate. Ethane oxidation was inhibited by sodium azide and carbon monoxide.     

Effect of Sublethal Heat on the Metabolic Activity of Staphylococcus aureus

Cells of Staphylococcus aureus MF-31 which have been heat-injured at 52 C have an altered metabolic activity. Analyses of whole-cell preparations by means of the Thunberg technique and Warburg manometry showed decreased dehydrogenase activity and oxygen uptake on a variety of substrates. In cell-free extracts prepared from injured cells, it was demonstrated that the specific activity of fructose diphosphate aldolase, lactate dehydrogenase, and butanediol dehydrogenase was less than that of extracts prepared from normal unheated cells. Recovery of the heat-injured cells in a suitable medium supported a return of the dehydrogenase activity and oxygen uptake, but the activity of the enzymes in cell-free extracts prepared from such partially recovered cells did not fully return to the level of normal (unheated) preparations. Addition of chloramphenicol or actinomycin D to the recovery medium, singly or in combination, retarded the return of the normal metabolic activity. Radiorespirometric experiments indicated that the percentage participation of the Embden-Meyerhoff Parnas and hexose monophosphate pathways remained the same for normal and heat-injured cells. The sublethal heat treatment decreased the catabolic capabilities of S. aureus and the production of selected end products associated with the metabolism of glucose.     

Activation of 5-lipoxygenase by guanosine 5'-O-(3-thiotriphosphate) and other nucleoside phosphorothioates: redox properties of thionucleotide analogs

The stable nucleotide analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was found to be a very potent activator of 5-lipoxygenase in cell-free preparations from rat polymorphonuclear (PMN) leukocytes, causing a 10-fold stimulation of arachidonic acid oxidation at concentrations as low as 0.5-1 microM. The enhancement of enzyme activity was not directly related to G protein activation since the effect of GTP gamma S could not be abolished by GDP nor replaced by GTP or guanylyl-imidodiphosphate (up to 100 microM). Furthermore, other phosphorothioate analogs, such as guanosine 5'-O-(2-thiodiphosphate), adenosine 5'-O-(3-thiotriphosphate), adenosine 5'-O-(2-thiodiphosphate), and adenosine 5'-O-thiomonophosphate all stimulated 5-lipoxygenase activity at concentrations of 10 microM or lower. This effect could not be detected with any of the corresponding nucleoside phosphate derivatives. The stimulation of 5-lipoxygenase activity by nucleoside phosphorothioates was observed under conditions where the reaction is highly dependent on exogenous hydroperoxides, such as in the presence of beta-mercaptoethanol or using enzyme preparations pretreated with sodium borohydride or glutathione peroxidase. GTP gamma S stimulated arachidonic acid oxidation by 5-lipoxygenase to the same extent as the activating hydroperoxides but had no effect on the reaction measured in the presence of optimal concentrations of 13-hydroperoxyoctadecadienoic acid (1-5 microM). Finally, sodium thiophosphate, but not sodium phosphate, markedly stimulated 5-lipoxygenase activity with properties similar to those of GTP gamma S. These results indicate that GTP gamma S and other phosphorothioate derivatives have redox properties that can contribute to increase 5-lipoxygenase activity by replacing the effect of hydroperoxides.     

Peroxidases from marine microalgae

Peroxidase activity was detected in cell-free extractsof strains of three species of the marine microalgae,Porphyridium purpureum, Phaeodactylumtricornutum and Dunaliella tertiolecta. However, no bromo- or chloroperoxidase activity wasdetected in any, using the standard 2-chlorodimedoneassay. Only the extract from P. purpureumoxidized iodide and this peroxidase was partiallypurified via anion-exchange chromatography. KI ando-dianisidine assay of the fractions indicatedthat only one peroxidase was present. Characterization of the thermally labile enzymesuggested that it is a heme-containing peroxidase,with a molecular weight of approximately 36,000.     

Soybean Seed Coat Peroxidase (A Comparison of High-Activity and Low-Activity Genotypes)

Peroxidase activity in the seed coats of soybean (Glycine max [L.] Merr.) is controlled by the Ep locus. We compared peroxidase activity in cell-free extracts from seed coat, root, and leaf tissues of three EpEp cultivars (Harosoy 63, Harovinton, and Coles) to three epep cultivars (Steele, Marathon, and Raiden). Extracts from the seed coats of EpEp cultivars were 100-fold higher in specific activity than those from epep cultivars, but there was no difference in specific activity in crude root or leaf extracts. Isoelectric focusing of root tissue extracts and staining for peroxidase activity showed that EpEp cultivars had a root peroxidase of identical isoelectric point to the seed coat peroxidase, whereas roots of the epep types were lacking that peroxidase, indicating that the Ep locus may also affect expression in the root. In seed coat extracts, peroxidase was the most abundant soluble protein in EpEp cultivars, whereas this enzyme was present only in trace amounts in epep genotypes, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Histochemical localization of peroxidase activity in seed coats of EpEp cultivars shows that the enzyme occurs predominately in the cytoplasm of hourglass cells of the subepidermis. No obvious difference in the gross or microscopic structure of the seed coat was observed to be associated with the Ep locus. These results suggest that soybean seed coat peroxidase may be involved in processes other than seed coat biosynthesis.     

Calmodulin-dependent stimulation of the NADPH oxidase of human neutrophils

The NADPH oxidase of human neutrophils is highly sensitive to calcium concentration and is inhibited in intact cells and cell-free preparations by various phenothiazine drugs. Addition of calmodulin to preparations of NADPH oxidase stimulates enzymatic rates from 1.4-2.5-fold. Addition of calmodulin and calcium, but not calcium alone, to NADPH oxidase preparations which have been inactivated by EDTA results in the restoration of activity. No activation is observed when membrane preparations containing latent NADPH oxidase are exposed to calcium and calmodulin. These studies suggest a role for calmodulin in the control of NADPH oxidase but that calmodulin alone is not sufficient for activation.     

Calmodulin-dependents stimulation of the NADPH oxidase of human neutrophils

The NADPH oxidase of human neutrophils is highly sensitive to calcium concentration and is inhibited in intact cells and cell-free preparations by various phenothiazine drugs. Addition of calmodulin to preparations of NADPH oxidase stimulates enzymatic rates from 1.4–2.5-fold. Addition of calmodulin and calcium, but not calcium alone, to NADPH oxidase preparations which have been inactivated by EDTA results in the restoration of activity. No activation is observed when membrane preparations containing latent NADPH oxidase are exposed to calcium and calmodulin. These studies suggest a role for calmodulin in the control of NADPH oxidase but that calmodium alone is not sufficient for activation.