The secretory activity of algal cells in buffered media

Summary The effect of the activity of algal cells on the buffering characteristics of suspending fluid was studied onChlorella 7-11-05 cells suspended in Tris buffer or in sodium citrate-citric acid buffer. It was observed that changes in the buffering capacity of the suspending fluid depended on the concentration of the original buffer, pH, and the nature of the buffer.In Tris buffer, which is not consumed byChlorella cells, the buffering capacity, measured in terms of the van Slyke's buffer index (), increased with time. In sodium citrate-citric acid buffer, which is used byChlorella cells in the course of their metabolism, not only the buffer index () changed, but also the pH at which the suspending fluid had maximum buffering capacity shifted to a new value. In both Tris and citrate buffers the newly formed buffer systems had a maximum buffering capacity at pH 6.4 corresponding to the pK₁ of carbonic acid.This work was supported by funds from the National Aeronautics and Space Administration.Scientific Article A 1237, Contribution No. 3742 of the University of Maryland Agricultural Experiment Station.     

β-1,3-Glucanase and dimorphism in Paracoccidioides brasiliensis

Mycelial and yeast forms of P. brasiliensis were tested for several glucohydrolases. In addition to high levels of -blucanases, low amounts of -glucanase, chitinase and maltase were found. Tests for invertase, amylase and lactase were negative. The levels of -1,3-glucanase were higher in the mycelial form. The shift to the mycelial phase correlated with an increase in the levels of -1,3-glucanase. The enzyme was present in the cytoplasm, cell wall and culture medium. The extracellular enzyme was purified 42 fold by ammonium sulphate precipitation and gel filtration. Maximal activity was obtained at 60°C and pH of 5.0 acetate buffer or pH 6.0 (phosphate buffer). Its K _m was 0.205 mg/ml. The cell wall-bound enzyme showed a higher temperature optimum. Optimum pH and K _m were also slightly different. Following treatment of the cell walls with chitinase, -1,3-glucanase was released into the medium.     

Some effects of polyvinyl alcohol and polyvinyl pyrrolidone on the activity of lactate dehydrogenase and its isoenzymes

Summary The activity of Sigma type II LDH (containing all LDH isoenzymes), type VII (LDH 1) and type V (LDH 5) has been evaluated in 0.1 M phosphate buffer and 0.4 M tris-HCl buffer, both at pH 7.4, before and after addition of PVP and PVA. A slight reduction of reaction velocity occurred when PVP and PVA were added, probably because of the increased viscosity of the medium. More specifically, PVA seemed to reduce the LDH 1 activity in phosphate buffer, but it seemed to counteract, together with PVP, conformational changes due to tris-HCl buffer of high molarity. None of these effects were found to influence the results of section histochemistry, but it could be shown that PVA was able to reduce urea inhibition of LDH 5 in tris-HCl buffer.The following Abbreviations are used in the Article PVA Polyvinyl alcohol - PVP Polyvinyl pyrrolidone - tris tris (hydroxymethyl)-aminomethan - LDH Lactate dehydrogenase - -NADH -Nicotinamide Adenine Dinucleotide, reduced form - PMS Phenazine methosulfate - BSA Bovine serum albumin     

Structure and non-enzymatic light emission of two luciferin precursors isolated from the luminous mushroom Panellus stipticus

The chemical structure of two luciferin precursors PS-A and PS-B, isolated from the luminous mushroom Panellus stipticus, were determined as 1-O-decanoylpanal (2) and 1-O-dodecanoylpanal (3), respectively. Both PS-A and PS-B were converted into chemiluminescent luciferins by treatment with 50 mmol/l methylamine in a pH 3.5 buffer solution containing an anionic surfactant Tergitol 4 at 25–35ºC. The luciferins emitted chemiluminescence in a pH 7–8 buffer solution containing a cationic surfactant in the presence of O₂ and O.     

Thermal adaptation in biological membranes: interacting effects of temperature and pH

Summary The interacting effects of pH and temperature on membrane fluidity were studied in plasma membranes isolated from liver of rainbow trout (Oncorhynchus mykiss) acclimated to 5 and 20°C. Fluidity was determined as a function of temperature under conditions of both constant (in potassium phosphate buffer) and variable pH (in imidazole buffer, consistent with imidazole alphastat regulation) from the fluorescence anisotropy of two probes: 1,6-diphenyl-1,3,5-hexatriene, which intercalates into the bilayer interior, and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene which is anchored at the membrane/water interface. The temperature dependence of the anisotropy parameter for 1,6-diphenyl-1,3,5-hexatriene in plasma membranes of 20°C-acclimated trout was greater when determined in phosphate (AP per °C=-0.047) than in imidazole buffer (AP per °C=-0.022); similar, but less significant, trends were noted with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene. In contrast, the temperature dependence of fluidity (AP/°C in the range-0.0222 to-0.027) did not vary with buffer composition in membranes of 5°C-acclimated trout. In phosphate buffer, anisotropy parameter values for 1,6-diphenyl-1,3,5-hexatriene were significantly lower in 5°C-than 20°C-acclimated trout, indicating a less restricted probe environment following cold acclimation and nearly perfect compensation (91%) of fluidity. Temperature-dependent patterns of acid-base regulation were estimated to account for 11–40% of the fluidization evident in membranes of 5°C-trout, but a period of cold acclimation was required for complete fluidity compensation. In contrast, no homeoviscous adaptation was evident in imidazole buffer, indicating that membrane fluidity is sensitive to buffer composition. Accordingly, vesicles of bovine brain phosphatidylcholine, suspensions of triolein, and plasma membranes of 5°C-acclimated trout were consistently more fluid in imidazole than phosphate buffer. Membranes of 5°C-acclimated trout were enriched in molecular species of phosphatidylcholine containing 22:6n3 (at the expense of species containing 18:1n9 and 18:2n6) compared to membranes of 20°C-trout; consequently, the unsaturation index was significantly higher (3.29 versus 2.73) in trout maintained at 5 as opposed to 20°C. It is concluded that: 1) the chemical composition of the internal milieu can significantly influence the physical properties of membrane lipids; 2) temperature-dependent patterns of intracellular pH regulation may partially offset the ordering effect of low temperature on membrane fluidity in 20°C-acclimated trout transferred to 5°C, but not in 5°C-acclimated trout transferred to warmer temperatures; 3) the majority of the thermal compensation of plasma membrane fluidity resulting from a period of temperature acclimation most likely reflects differences in membrane composition between acclimation groups; 4) imidazole apparently interacts with trout hepatocyte plasma membranes in a unique way.Abbreviations _im netcharge stateofproteins - AP anisotropyparameter - bw body weight - DPH 1,6-diphenyl-1,3,5-hexatriene - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonicacid - PC phosphatidylcholine - pH_e pHofarterial blood - pH_i intracellular pH - TMA-DPH 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene - TRIS tris(hydroxymethyl)aminomethane     

Light-induced phase shifting of the circadian clock in Neurospora crassa requires ammonium salts at high pH

Effects of external ionic conditions on light induced phase shifting of the circadian rhythm of conidiation in Neurospora crassa were examined in simple buffer solutions for discerning effects of individual ions. Mycelia were cultured to liquid media of different pHs and then transferted to 10 mM piperazine-N,N-bis(2-ethanesulmonic acid) (Pipes) buffer of various pHs and irradiated with while light. The phase of the rhythm of dark controls was not changed by transfer from medium to buffer. When mycelia were cultured in media of pH above 6.7, light did not advance the phase of the clock in Pipes buffer alone. However, light-induced phase advance was restored when an ammonium salt was added to buffer of pH higher than 7.6. An amination-defective mutant, bd am, showed the same response to ammonium nitrate as the wild-type strain, bd. Ammonium must be present before light irradiation for restoration of phase shifting. Free-amino-acid pools in the cells were changed by treatment with Pipes buffer: aspartle acid, glutamic acid, ammonia, glutamine and ornithine levels decreased, while lysine and histidine increased. Addition of ammonium nitrate to Pipes buffer resulted in further changes in amino-acid pools; lysine, histidine, arginine, alanine and ornithine decreased, and glutamine levels increased. Irradiation did not result in significant changes in amino acid pools.Abbreviation Pipes piperazine-N,N-bis(2-ethanesulfoniccid)     

Photosynthetic activities of a membrane preparation of the thermophilic cyanobacterium Mastigocladus laminosus

Photosynthetically active membranes have been prepared from the thermophilic cyanobacterium Mastigogladus laminosus by treatment with lysozyme. The membranes were active in electron transport through photosystem I and II as well as in photophosphorylation and proton uptake. Cells were grown at 40°, 45° and 55°C respectively. The temperature optimum of oxygen evolution of whole cells was about 10°C higher than the growth temperature. In isolated membranes the temperature optimum for cyclic photophosphorylation was identical to the growth temperature of the cells whereas the optimum for photosystem II electron transport never exceeded 40°C. Photophosphorylation was inhibited by N, N-dicyclohexyl carbodiimide (DCCD), carbonyl-cyanide-m-chlorophenylhydrazone and NH₄Cl, whereas proton uptake was enhanced by DCCD. Electron transport was slightly inhibited by these treatments. The membranes could be stored for several weeks at-20°C in 50% glycerol without any loss in the activities.Abbreviations DPIP 2,6-dichlorophenolindophenol - CCCP Carbonyl-cyanide-m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - PMS N-methylphenazonium methosulfate - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - TMP 20 mM Tris-HCl buffer pH 7.8, 10 mM MgCl₂, 5 mM phosphate buffer pH 7.8     

Inhibition of Peroxidase-Catalyzed Oxidation of Aromatic Amines by Substituted Phenols

Peroxidase-catalyzed oxidation of o-phenylene diamine (OPD) was competitively inhibited by trimethylhydroquinone (TMHQ), 4-tert-butylpyrocatechol (InH5), and 4,6-di-tert-butyl-3-sulfanyl-1,2-dihydroxybenzene (InH6). InH6 was the most efficient inhibitor (K _i = 11 M at 20°C in 0.015 M phosphate–citrate buffer, pH 6.0). The effects of InH5 and InH6 were not preceded by periods of induction of OPD oxidation products (contrary to TMHQ). Peroxidase-catalyzed oxidation of tetramethylbenzidine (TMB) was noncompetitively inhibited by InH6 and 3-(2-hydroxyethylthio)-4,6-di-tert-butylpyrocatechol (InH4), whereas o-aminophenol acted as a mixed-type inhibitor. The effects of all three inhibitors were preceded by an induction period, during which TMB oxidation products were formed. Again, InH6 was the most efficient inhibitor (K _i = 16 M at 20°C in 0.015 M phosphate–citrate buffer supplemented with 5% ethanol, pH 6.0). Judging by the characteristics of the inhibitors taken in aggregate, it is advisable to use the pairs OPD–InH5 and OPD–InH6 in systems for testing the total antioxidant activity of human biological fluids.     

A histochemical study of the oxidation of 17β-estradiol in human term placenta at different incubation conditions

Summary The present study deals with the histochemical demonstration of 17-estradiol dehydrogenase in human term placenta using the polyvinyl alcohol method to reduce diffusion artefacts. Incubations took place with both NAD⁺ and NADP⁺ as coenzymes and at different pH values of the incubation medium. The NAD⁺ linked enzyme reaction showed a greater activity than the NADP⁺ linked, both in the trophoblast as well as in connective tissue. There were differences in staining intensity at the different pH values, and strongest reaction was observed using glycine-NaOH buffer pH 10 in the incubation medium. Owing to a non-enzymatically reduction of nitro blue tetrazolium by reduced NAD⁺, the demonstration of 17-estradiol dehydrogenase is independent of diaphorase at this high pH. The findings are discussed in relation to data about nothing dehydrogenase and biochemically determined pH optima for the enzymatic reactions dealt with in this work.The following Abbreviations are used in this Article NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NBT nitro blue tetrazolium - PVA polyvinyl alcohol - tris tris (hydroxymethyl)-aminomethane - 17-OH-SDH 17-OH-steroid-dehydrogenase Supported by The Norwegian Research Council for Science and the Humanities. Skilful assistance of Mrs. E. Alvestad, Mrs. Aa. Flatnes and Mrs. F. Sørensen is greatfully acknowledged.     

Cherry fruit development: The use of a microassay for studying indoleacetic acid oxidase

An improved procedure is described for extraction and assay of indoleacetic acid oxidase from seeds of sour cherry (Prunus cerasus L.). The extraction procedure was optimized for pH, buffer, polyvinylpolypyrrolidone (PVP) and tissue: buffer ratio. Greatest extraction efficiency was obtained at pH 4.0, 0.2 M acetate buffer, tissue: PVP ratio of 1:2.5 and tissue: buffer ratio of 50 ml per g of seed. The enzyme was assayed at 30°C using indoleacetic acid-1-¹⁴C as substrate and radioassaying the ¹⁴CO₂ evolved. Mn²⁺ and 2,4-dichlorophenol enhanced enzyme activity but were not obligatory. A minimum substrate concentration of 60 M was needed for quantitative evaluation. This assay was sensitive and reproducible, enzyme activity being demonstrated in as little as 0.8 mg of seed tissue with a coefficient of variation of 1 to 9%.     

Surcose transport in isolated plasma-membrane vesicles from sugar beet (Beta vulgaris L.) Evidence for an electrogenic sucrose-proton symport

An analysis of the molecular mechanism of sucrose transport across the plasmalemma was conducted with isolated plasma-membrane (PM) vesicles. Plasma membrane was isolated by aqueous two-phase partitioning from fully expanded sugar beet (Beta vulgaris L.) leaves. The isolated fraction was predominantly PM vesicles as determined by marker-enzyme analysis, and the vesicles were oriented right-side-out as determined by structurally linked latency of the PM enzyme, vanadate-sensitive Mg²⁺-ATPase. Sucrose uptake was investigated by equilibrating PM vesicles in pH 7.6 buffer and diluting them 20-fold into pH 6.0 buffer. Using this pH-jump technique, vesicles accumulated acetate in a pH-dependent, protonophore-sensitive manner, which demonstrated the presence of a pH gradient (pH) across the vesicle membrane. Addition of sucrose to pH-jumped PM vesicles resulted in a pH-dependent, protonophoresensitive uptake of sucrose into the vesicles. Uptake was sucrose-specific in that a 10-fold excess of mannose, glucose, fructose, mannitol, melibiose, lactose or maltose did not inhibit sucrose accumulation. The rate of pH-dependent uptake was saturable with respect of sucrose concentration and had an apparent K _m, of 0.45 mM. Sucrose uptake was stimulated approximately twofold by the addition of valinomycin and K⁺, which indicated an electrogenic sucrose-H⁺ symport. Membrane potentials () were imposed across the vesicle membrane using valinomycin and K⁺. A membrane potential, negative inside, stimulated pH-dependent sucrose uptake while a , positive inside, inhibited uptake. Conditions that produce a negative in the absence of a pH gradient supported, although weakly, sucrose uptake. These data support an electrogenic sucrose-H⁺ symport as the mechanism of sucrose transport across the PM in Beta leaves.Abbreviations and symbols CCCP carbonyl cyanide m-chlorophenylhydrazone - cyt cytochrome - PM plasma-membrane(s) - electrical potential difference     

Biotransformation of warfarin by the fungus Beauveria bassiana

Summary Biotransformation of racemic warfarin by the fungus Beauveria bassiana IMI 12939 was studied as part of an investigation into alternative methods for the production of drug metabolites. Resting cell suspensions in buffer were able to biotransform warfarin. High activity occurred within a broad pH range (6.0–9.0) with an optimum at 7.0. The major metabolites were 4-hydroxywarfarin (metabolite I), warfarin alpha-diketone (metabolite B) and diastereomers of novel tautomeric ring-cleaved warfarin (metabolites E and F). The exact quantitative metabolite profile obtained following warfarin metabolism was shown to be highly dependent upon the cell concentration. Kinetic evidence suggested the operation of multiple enzyme activities.     

Interaction of batrachotoxinin-A benzoate with voltage-sensitive sodium channels: The effects of pH

The binding of labeled batrachotoxinin-A benzoate (BTX-B) to voltage-sensitive sodium channels in broken membrane preparations of mouse cerebral cortex has been measured as a function of the pH. Specific binding is negligible at pH <6.0, maximum at pH 8.5, and decreases again at pH 9.0. A major component of nonspecific binding, however, increases linearly in the pH range 7.0-9.0. The pK _a of batrachotoxinin-A, an analogue of BTX-B, was found by titrimetric methods to be 8.2. Analysis of the data shows that at least part of the pH dependence of BTX-B binding is due to the titration of a sodium channel residue(s) associated in some way with the BTX-B recognition site. The possible involvement of a histidine residue is suggested. Abbreviations used BTX batrachotoxin - BTX-A batrachotoxinin-A - BTX-B batrachotoxinin-A-20--benzoate - DEP diethylpyrocarbonate     

Antimicrobial proteins from cowpea root exudates: inhibitory activity against Fusarium oxysporum and purification of a chitinase-like protein

Plants exude a variety of substances through their external surfaces and from germinating seeds, some of which have an inhibitory action against plant pathogens. The aim of this study was the investigation and characterization of defense proteins present in exudates from roots of cowpea seedlings (Vigna unguiculata (L.) Walp.). Root exudates were collected from seedlings that were grown hydroponically in three different media, including, 100 mM sodium acetate buffer pH 4.5, water pH 6.0 and 100 mM sodium phosphate buffer pH 7.5. The proteins from these exudates were analyzed by SDS–PAGE and SDS–Tricine–PAGE and the presence of antimicrobial proteins in the exudates was investigated by immunological and enzymatic assays. Results showed that roots from cowpea seedlings contained -1,3-Glucanases, chitinases and lipid transfer proteins (LTPs), all of which may potentially function as plant defense proteins. Immunolocalization of one of these proteins, chitinase, revealed its presence in the xylem cell wall vessel elements. These exudates also demonstrated an inhibitory effect on the growth of the fungus, Fusarium oxysporum, in vitro. The results suggest that plant roots may exude a variety of proteins that may function to repress the growth of root pathogenic fungi.     

Purification and characterization of a sialidase fromClostridium chauvoei NC08596

The sialidase secreted byClostridium chauvoei NC08596 was purified to apparent homogeneity by ion-exchange chromatography, gel filtration, hydrophobic interaction-chromatography, FPLC ion-exchange chromatography, and FPLC gel filtration. The enzyme was enriched about 10 200-fold, reaching a final specific activity of 24.4 U mg^–1. It has a relatively high molecular mass of 300 kDa and consists of two subunits each of 150 kDa. The cations Mn²⁺, Mg²⁺, and Ca²⁺ and bovine serum albumin have a positive effect on the sialidase activity, while Hg²⁺, Cu²⁺, and Zn²⁺, chelating agents and salt decrease enzyme activity. The substrate specificity, kinetic data, and pH optimum of the enzyme are similar to those of other bacterial sialidases.Abbreviations FPLC fast protein liquid chromatography - NCTC National Collection of Type Cultures - ATCC American Type Culture Collection - MU-Neu5Ac 4-methylumbelliferyl--d-N-acetylneuraminic acid - buffer A 0.02m piperazine, 0.01m CaCl₂, pH 5.5 - buffer B 0.02m piperazine, 0.01m CaCl₂, 1.0m NaCl, pH 5.5 - buffer C 0.1m sodium acetate, 0.01m CaCl₂, pH 5.5 - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - Neu5Ac N-acetylneuraminic acid - BSM bovine submandibular gland mucin - GD1a IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer - GM1 II³Neu5Ac-GgOse₄Cer - MU-Neu4,5Ac₂ 4-methylumbelliferyl--d-N-acetyl-4-O-acetylneuraminic acid - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - EDTA ethylenediamine tetraacetic acid - EGTA ethylene glycol bis(2-aminoethyl-ethen)-N,N,N,N-tetraacetic acid - BSA bovine serum albumin - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - IEF isoelectric focusing - IEP isoelectric point     

Selection methods in bacilli for recombinants and transformants of intra- and interspecific fused protoplasts

The intraspecific fusion frequencies obtained with the direct selection method on a semi-synthetic regeneration medium between strains of B. subtilis and B. licheniformis were distribution from 9.9×10^-2 to 4.5×10^-3, which was one or two orders higher than those of interspecific recombinations between B. subtilis and B. licheniformis.The regeneration media were also useful for selecting interspecific transformants from plasmid carrier to non-carrier. This selection could be used as a primary selection method for inter-and intraspecific recombinants obtained by protoplast fusion.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - Cm chloramphenicol - SMM 0.5 M sucrose-0.02 M MgCl₂-0.02 M maleate buffer, pH 6.5     

Intracellular pH during daily torpor inPeromyscus maniculatus

Summary Intracellular and extracellular acid-base parameters during normothermy and daily torpor were examined in deer mice (Peromyscus maniculatus). [¹⁴C]Dimethyloxazolidinedione and [³]inulin were used to assess intracellular pH in liver, heart, skeletal muscle, and brain. Buffering capacities were determined using tissue homogenates. A significant increase in plasma and during daily torpor indicates a respiratory acidosis. All tissues experienced a reduction in the calculated dissociation ratio of histidine imidazole groups (_imid) during daily torpor (16.5% for brain, approximately 10% for other tissues). Based on comparisons with physicochemical tissue buffering capacities, metabolic compensation of the respiratory acidosis occurred in liver, heart, and plasma, while brain was more acidotic than predicted. The more extensive change in brain _imid might influence a regulated decrease in body temperature. Comparison of acid-base parameters during daily torpor and hibernation suggests that the magnitude of acid-base modifications in mammals may be associated with the level of dormancy.Abbreviations _imid dissociation ratio of histidine imidazole groups - physicochemical non-bicarbonate buffer value - ' apparent (in vivo) buffer value - bicarbonate bicarbonate values corrected to a temperature of 25 °C - pH pH values corrected to a temperature of 25 °C - pH _i intracellular pH - pK _imid pK of histidine imidazole groups - T _b body temperature     

Mechanism of the self-assembly of apoferritin from horse spleen

Apoferritin from horse spleen can be reversibly dissociated at pH 2 or in 7.2 M G-HCl (pH 3.5). Reconstitution of the native icositetramer in 0.1 M TEA buffer (pH 7.9) in the presence of 1 mM EDTA and 3 mM dithioerythritol leads to yields higher than 80%. To monitor the kinetic mechanism, intrinsic fluorescence, far-UV circular dichroism, and covalent cross-linking with glutaraldehyde were applied.The overall mechanism of assembly is characterized by a sequence of concentration-dependent association reactions involving structured monomers and a dimeric intermediate as the most prominent species, apart from trimers and dodecamers. The parallel decrease in monomers, dimers and trimers indicates that association equilibria precede the formation of the final assembly product.The assembly reaction is accompanied by characteristic changes in fluorescence emission and dichroic absorption. To a first approximation, renaturation and reassociation may be quantitatively described by one single rate-determining second-order process, subsequent to fast folding steps at the monomer level.Abbreviations CD circular dichroism - DTE dithioerythritol - G-HCl guanidinium chloride - SDS sodium dodecylsulfate - TEA triethanolamine - Tris tris hydroxymethylamino methane Dedicated to Professor Harold A. Scheraga on the occasion of his 65^th birthday     

Effects of pH on the degradation of phenanthrene and pyrene by Mycobacterium vanbaalenii PYR-1

The effects of pH on the growth of Mycobacterium vanbaalenii PYR-1 and its degradation of phenanthrene and pyrene were compared at pH 6.5 and pH 7.5. Various degradation pathways were proposed in this study, based on the identification of metabolites from mass and NMR spectral analyses. In tryptic soy broth, M. vanbaalenii PYR-1 grew more rapidly at pH 7.5 (=0.058 h^–1) than at pH 6.5 (=0.028 h^–1). However, resting cells suspended in phosphate buffers with the same pH values displayed a shorter lag time for the degradation of phenanthrene and pyrene at pH 6.5 (6 h) than at pH 7.5 (48 h). The one-unit pH drop increased the degradation rates four-fold. Higher levels of both compounds were detected in the cytosol fractions obtained at pH 6.5. An acidic pH seemed to render the mycobacterial cells more permeable to hydrophobic substrates. The major pathways for the metabolism of phenanthrene and pyrene were initiated by oxidation at the K-regions. Phenanthrene-9,10- and pyrene-4,5-dihydrodiols were metabolized via transient catechols to the ring fission products, 2,2-diphenic acid and 4,5-dicarboxyphenanthrene, respectively. The metabolic pathways converged to form phthalic acid. At pH 6.5, M. vanbaalenii PYR-1 produced higher levels of the O-methylated derivatives of non-K-region phenanthrene- and pyrene-diols. Other non-K-region products, such as cis-4-(1-hydroxynaphth-2-yl)-2-oxobut-3-enoic acid, 1,2-dicarboxynaphthalene and benzocoumarin-like compounds, were also detected in the culture fluids. The non-K-region polycyclic aromatic hydrocarbon oxidation might be a significant burden to the cell due to the accumulation of toxic metabolites.     

On the course of thermal denaturation of deoxyribonucleic acids after γ-irradiation

Summary The changes which are caused by action of-irradiation onDNAs of various origin were followed by spectrophotometric method at differing thermal denaturation curves. It was found that all measured bacterialDNAs as well asDNA isolated from calf thymus, irradiated by exposures higher that 5×10⁴ R, produced significantly decreasedT _m values with concomittantly decreased hyperchromic effect and changed transition intervals obtained in 10^–2 M sodium phosphate, 10^–3 M EDTA medium at pH 7. It was also observed that higher-irradiation exposures caused the loss of renaturation ability ofEscherichia coli DNA.Abbreviations used DNA deoxyribonucleic acid - G guanine - C cytosine - E ₂₆₀ extinction (absorbancy) measured at 260m - T _m the temperature corresponding to the midpoint of the absorbance rise - 2/3 the transition interval of the denaturation curve corresponding to the temperature interval between 17–83% of the total absorbancy increment of the denaturation curve - SSC standard saline citrate buffer (0.15 M NaCl, 0.015 M sodium citrate, pH 7) - PE 10^–2 M sodium phosphate buffer [molarity related to (PO₄)] - PE 10^–3 M EDTA, pH 7. 1.000 ml prepared as follows: 0.608 g NaH₂PO₄.2 H₂O, 0.218 g Na₂HPO₄.12 H₂O, 0.372 g disodium salt of EDTA, 1.0 ml 1 M NaOH. Concentration of Na ions — 0.02 M.