Thermal stabilities of membrane-bound,solubilized, and artificially immobilized hydrogenase from Chromatium vinosum

Thermal inactivation (at 65 °C) of both membrane-bound and solubilized hydrogenase from Chromatium vinosum has been studied. Membrane binding greatly increases thermostability relative to the solubilized enzyme. Covalent attachment (but not simple adsorption) of the solubilized enzyme to ω-NH₂-alkyl-agaroses sharply increases thermal stability, which almost attains that of membrane-bound hydrogenase. It appears that there are at least two requirements for such stabilization—the enzyme should be in a hydrophobic medium and should be firmly bound to a hydrophobic support.     

Phosphorylation of brain specific membrane proteins of developing chick embryo

Phosphorylation of plasma membrane proteins in various tissues of chick embryos was investigated during the development. A polypeptide of Mr 22,000 was found to be the major phosphorylated plasma membrane protein in embryonic brain; this protein was absent in embryonic muscle, liver, and gizzard tissues. Extraction of plasma membranes with Triton X-100 (1%) or Nonidet p40 (1%) or sodium deoxycholate (1%) resulted in the solubilization of most membrane proteins including the 22 KDa phosphoprotein suggesting that the 22 KDa protein is a membrane-bound protein. Maximum phosphorylation of the 22 KDa protein by [gamma-32P]ATP was observed at 0.01 mM Ca2+. Higher concentrations of Ca2+ (2.5 mM) inhibited the phosphorylation of the 22 KDa protein whereas 3.5 mM Mg2+ stimulated the phosphorylation.     

Solubilization of benzodiazepine binding site from rat cortex.

The high-affinity binding site for [³H] diazepam has been solubilized from rat brain using 0.5% Lubrol-PX. Using a polyethylene glycol (PEG)-γ-globulin assay, it has been possible to demonstrate solubilization of about 60% of the binding sites in a single step. The solubilized binding site possesses a K_D of 11 nM for [³H] diazepam compared to approximately 4 nM for the membrane-bound form, and binding is to a single class of sites. The order of potency of benzodiazepines is identical for the solubilized receptor and the membrane-bound form. Binding of [³H] diazepam is temperature dependent and higher at 4° than 37°C. Both urea and guanidine-HC1 were capable of totally inhibiting binding, and this inhibition was partly reversible; neither sulfhydryl groups nor carbohydrate moieties seem to be important for binding. γ-Aminobutyric acid which enhanced [³H] diazepam binding to membrane fractions was without effect on the solubilized binding site.     

Membrane-bound tubulin in brain and thyroid tissue.

Brain and thyroid tissue contain membrane-bound colchicine-binding activity that is not due to contamination by loosely bound cytoplasmic tubulin. This activity can be solubilized to the extent of 80 to 90% by treatment with 0.2% Nonidet P-40 with retention of colchicine binding. Extracts so obtained contain a prominent protein band in disc gel electrophoresis that co-migrates with tubulin. Membranes, and the solubilized protein therefrom, exhibit ligand binding properties like tubulin; for colchicine the KA is approximately 1 X 10(6) M-1 in brain and approximately 0.6 X 10(6) M-1 in thyroid; for vinblastine the KA is approximately 8 X 10(6) M-1 for both tissues; and for podophyllotoxin the Ki is approximately 2 X 10(-6) M for both tissues. Displacement by analogues of colchicine is of the same order as for soluble tubulin. Although membrane-bound colchicine-binding activity shows greater thermal stability and a higher optimum binding temperature (54 degrees versus 37 degrees) than soluble tubulin, this appears to be the result of the membrane environment since the solubilized binding activity behaves like the soluble tubulin. Antibody against soluble brain tubulin reacts with membranes and solubulized colchicine-binding activity from both brain and thyroid gland. We conclude that brain and thyroid membrane preparations contain firmly bound tubulin or a very similar protein.     

Absorption of nitrate and ammonium ions by Lolium perenne from flowing solution cultures at low root temperatures

Lolium perenne L. cv. 23 (perennial ryegrass) plants were grown in flowing solution culture and acclimatized over 49 d to low root temperature (5°C) prior to treatment at root temperatures of 3, 5, 7 and 9°C for 41 d with common air temperature of 20/15°C day/night and solution pH 5·0. The effects of root temperature on growth, uptake and assimilation of N were compared with N supplied as either NH₄ or NO3 at 10 mmol m^?3. At any given temperature, the relative growth rate (RGR) of roots exceeded that of shoots, thus the root fraction (Rf) increased with time. These effects were found in plants grown with the two N sources. Plants grown at 3 and 5°C had very high dry matter contents as reflected by the fresh weight: freeze-dried weight ratio. This ratio increased sharply, especially in roots at 7 and 9°C. Expressed on a fresh weight basis, there was no major effect of root temperature on the [N] of plants receiving NHJ but at any given temperature, the [N] in plants grown with NHJ was significantly greater than in those grown with NO₃. The specific absorption rate (SAR) of NH⁺₄ was greater at all temperatures than SAR-NO₃. In plants grown with NH⁺, 3–5% of the total N was recovered as NH⁺₄, whereas in those grown with NO^?₃ the unassimilated NO^?₃ rose sharply between 7 and 9°C to become 14 and 28% of the total N in shoots and roots, respectively. The greater assimilation of NH⁺₄ lead to concentrations of insoluble reduced N (= protein) which were 125 and 20% greater, in roots and shoots, respectively, than in NO^?₃-grown plants. Plants grown with NH⁺₄ had very much greater glutamine and asparagine concentrations in both roots and shoots, although other amino acids were more similar in Concentration to those in NO^?₃ grown plants. It is concluded that slow growth at low root temperature is not caused by restriction of the absorption or assimilation of either NH⁺₄ or NO^?₃. The additional residual N (protein) in NH⁺₄ grown plants may serve as a labile store of N which could support growth when external N supply becomes deficient.     

Effect of concanavalin A on the activity of membrane-bound and detergent-solubilized Mg2+-ATPase

Plasma membranes were isolated after binding liver and hepatoma cells to polylysine-coated polyacrylamide beads, and the effect of concanavalin A on the membrane-bound Mg²⁺-ATPase and the Mg²⁺-ATPase solubilized by octaethylene glycol monododecyl ether (C₁₂E₈) was studied. In the experiment of membranebound Mg²⁺-ATPase, plasma membranes were pretreated with Concanavalin A and the activity was assayed. Concanavalin A stimulated the activity of both liver and hepatoma enzymes assayed above 20°C. Concanavalin A abolished the negative temperature dependency characteristic of liver plasma membrane Mg²⁺-ATPase. On the other hand, Concanavalin A prevented the rapid inactivation due to storage at ?20°C, which was characteristic of hepatoma plasma membrane Mg²⁺-ATPase. With solubilized Mg²⁺-ATPase from liver plasma membranes, the negative temperature dependency was not observed. Concanavalin A, which was added to the assay medium, stimulated the activity of the enzyme solubilized in C₁₂E₈ at a high ionic strength. However, Concanavalin A failed to show any effect on the enzyme solubilized in C₁₂E₈ at a low ionic strength. With solubilized Mg²⁺-ATPase from hepatoma plasma membranes, Concanavalin A could not prevent the inactivation of the enzyme during incubation at ?20°C.     

Protein isoaspartate methyltransferase prevents apoptosis induced by oxidative stress in endothelial cells: role of Bcl-Xl deamidation and methylation

Background

Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free α-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal α-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s).

Methodology/Principal Findings

Endothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H₂O₂; 0.1–0.5 mM range). Results show that A) Cells overexpressing “wild type” human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H₂O₂ concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H₂O₂ treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative stress conditions leading to programmed cell death. These proteins, including Hsp70, Hsp90, actin, and Bcl-xL, are recognized and methylated by PCMT, according to the general repair mechanism of this methyltransferase.

Conclusion/Significance

Apoptosis can be modulated by “on/off” switch partitioning the amount of specific protein effectors, which are either in their active (native) or inactive (deamidated) molecular forms. Deamidated proteins can also be functionally restored through methylation. Bcl-xL provides a case for the role of PCMT in the maintenance of functional stability of this antiapoptotic protein.     

Characterization of a soluble follitropin receptor from porcine testis.

Porcine testis receptors for follitropin (FSH) were solubilized by treatment with the non-ionic detergent Nonidet P-40 and receptor-bound and free ¹²⁵I-porcine FSH were separated by ammonium sulfate precipitation. The soluble receptor retained both its high affinity and specificity for FSH. The soluble hormone-receptor complex exhibited an equilibrium association constant of 4.7 × 10¹⁰ M^?1 at 4°C. Its hydrodynamic properties were consistent with those obtained for other solubilized peptide hormone receptors, and its molecular weight estimated to 244,000.     

Effects of Photosystem II extrinsic proteins on microstructure of the oxygen-evolving complex and its reactivity to water analogs

We used Triton-prepared PS II membranes in studies of the inactivation of O₂ evolution and solubilization of Mn and specific PS II polypeptides by NH₂OH, N- and O-substituted NH₂OH derivatives, NH₂NH₂ and NH₄Cl. The inactivation of O₂-evolution, solubilization of Mn and the solubilization of the extrinsic PS II polypeptides (17, 23 and 33 kDa) proved closely correlated, half-maximal effects occurring with only 100 μM NH₂OH. NH₂OH (2 mM) and NaCl (1 M) extractions solubilized about one-half the amount of protein solubilized by 0.8 M Tris-HCl (pH 8.0). The inactivation of the Mn-S-state complex proceeded by apparent first-order kinetics, the rate constant dependent on NH₂OH (CH₃NHON) concentration and pH. In the range of micromolar concentrations of NH₂OH, this inactivation did not occur via a cooperative type mechanism. Depletion of the 17 and 23 kDa proteins modified the pH dependency of inactivation (from pH 7.8 to 6.5) and also resulted in an approx. 2-fold maximum increase in the inactivation rate constant. Significantly, reconstitution of such NaCl-TMF-2 membranes with the 17 and 23 kDa proteins reverted both the pH dependency and the inactivation rate constant to that of TMF-2. A hierarchy of effectivity for solubilization of Mn and protein, which was highly correlated with inactivation of the Mn-S-state enzyme, was observed among NH₂OH and its derivatives. This same hierarchy was observed irrespective of prior depletion of the 17 and 23 or the 17, 23 and 33 kDa proteins from TMF-2. The hierarchy of effectivity among derivatives was: NH₂OH > CH₃NHOH > NH₂NH₂, NH₂OSO₃ > NH₂OCH₃ ⪢ CH₃NHOCH₃, NH₄Cl. The function(s) of the extrinsic PS II proteins as determinants of the reactivity of the Mn-S-state complex with polar amine vs other type compounds is discussed.     

Biosynthesis of glycoproteins in Candida albicans: activity of mannosyl and glucosyl transferases

The enzymes dolichol phosphate glucose synthase and dolichol phosphate mannose synthase (DPMS), which catalyze essential steps in glycoprotein biosynthesis, were solubilized and partially characterized in Candida albicans. Sequential incubation of a mixed membrane fraction with increasing concentrations of Nonidet P-40 released a soluble fraction that transferred glucose from UDP-Glc to dolichol phosphate glucose and minor amounts of glucoproteins in the absence of exogenous dolichol phosphate. Studies with the soluble fraction revealed that some properties were different from those previously determined for the membrane-bound enzyme. Accordingly, the soluble enzyme exhibited a twofold higher affinity for UDP-Glc and a sixfold higher affinity over the competitive inhibitor UMP, and the transfer reaction was fourfold more sensitive to inhibition by amphomycin. On the other hand, a previously described protocol for the solubilization of mannosyl transferases that rendered a fraction exhibiting both DPMS and protein mannosyl transferase (PMT) activities operating in a functionally coupled reaction was modified by increasing the concentration of Nonidet P-40. This resulted in a solubilized preparation enriched with DPMS and nearly free of PMT activity which remained membrane bound. DPMS solubilized in this manner exhibited an absolute dependence on exogenous Dol-P. Uncoupling of these enzyme activities was a fundamental prerequisite for future individual analysis of these transferases.     

Isolation and characterization of a <Emphasis Type="Italic">Mastigocladus</Emphasis> species capable of growth,N<Subscript>2</Subscript>-fixation and N-assimilation at elevated temperature

A Mastigocladus species was isolated from the hot spring of Jakrem (Meghalaya) India. Uptake and utilization of nitrate, nitrite, ammonium and amino acids (glutamine, asparagine, arginine, alanine) were studied in this cyanobacterium grown at different temperatures (25°C, 45°C). There was 2–3 fold increase in the heterocyst formation and nitrogenase activity in N-free medium at higher temperature (45°C). Growth and uptake and assimilation of various nitrogen sources were also 2–3 fold higher at 45°C indicating that it is a thermophile. The extent of induction and repression of nitrate uptake by NO₃ ⁻ and NH₄ ⁺, respectively, differed from that of nitrite. It appeared that Mastigocladus had two independent nitrate/nitrite transport systems. Nitrate reductase and nitrite reductase activitiy was not NO₃ ⁻-inducible and ammonium or amino acids caused only partial repression. Presence of various amino acids in the media partially repressed glutamine synthetase activity. Ammonium (methylammonium) and amino acid uptake showed a biphasic pattern, was energy-dependent and the induction of uptake required de novo protein synthesis. Ammonium transport was substrate (NH₄ ⁺)-repressible, while the amino acid uptake was substrate inducible. When grown at 25°C, the cyanobacterium formed maximum akinetes that remained viable upto 5 years under dry conditions.     

Interaction of isolated components from mammalian sperm and egg

In order to identify those proteins from the plasma membrane of hamster spermatozoa that exhibit an affinity for components of the zona pellucida we have used the Western blot technique. Zonae pellucidae from postovulatory hamster oocytes were solubilized by exposure to an acidic pH and then radiolabeled using the Bolton-Hunter reagent. These ¹²⁵I-zona pellucida proteins retain their immunoreactivity and migrate in three heterogeneous bands when submitted to SDS-PAGE electrophoresis. Membrane proteins from epididymal spermatozoa of mature hamsters were extracted by treatment with Nonidet P-40 and then submitted to electrophoresis (SDS-PAGE). The proteins in the gel were electrophoretically transferred to a nitrocellulose membrane and then probed with the radiolabelled zone pellucida proteins. ¹²⁵I-zona pellucida proteins bind preferentially to a sperm protein with a molecular weight of 26,400 ± 1,400 daltons (n = 9). Using a similar procedure it was shown that this protein also binds ¹²⁵I-Concanavalin A. The interaction between the sperm protein and the ¹²⁵I-zona pellucida proteins shows species specificity as demonstrated by the fact that the hamster ¹²⁵I-zona pellucida proteins do not bind to proteins extracted from ram, bull, and stallion spermatozoa. Whether this sperm protein could be implicated be implicated in the process of sperm-egg interaction is under investigation.     

Partial purification and characterization of a mannosyl transferase involved in O-linked mannosylation of glycoproteins in Candida albicans

Incubation of a mixed membrane fraction of C. albicans with the nonionic detergents Nonidet P-40 or Lubrol solubilized a fraction that catalyzed the transfer of mannose either from endogenously generated or exogenously added dolichol-P-[14C]Man onto endogenous protein acceptors. The protein mannosyl transferase solubilized with Nonidet P-40 was partially purified by a single step of preparative nondenaturing electrophoresis and some of its properties were investigated. Although transfer activity occurred in the absence of exogenous mannose acceptors and thus depended on acceptor proteins isolated along with the enzyme, addition of the protein fraction obtained after chemical de-mannosylation of glycoproteins synthesized in vitro stimulated mannoprotein labeling in a concentration-dependent manner. Other de-mannosylated glycoproteins, such as yeast invertase or glycoproteins extracted from C. albicans, failed to increase the amount of labeled mannoproteins. Mannosyl transfer activity was not influenced by common metal ions such as Mg(2+), Mn(2+) and Ca(2+), but it was stimulated up to 3-fold by EDTA. Common phosphoglycerides such as phosphatidylglycerol and, to a lower extent, phosphatidylinositol and phosphatidylcholine enhanced transfer activity. Interestingly, coupled transfer activity between dolichol phosphate mannose synthase, i.e., the enzyme responsible for Dol-P-Man synthesis, and protein mannosyl transferase could be reconstituted in vitro from the partially purified transferases, indicating that this process can occur in the absence of cell membranes.     

Localization, solubilization and characterization of plant membrane-associated calcium-dependent protein kinases

Membrane fractions from mature silver beet (Beta vulgaris) deveined leaf and leaf stem homogenates have associated Ca²⁺ -dependent protein kinase. The Ca²⁺ -dependent protein kinase activity is associated with plasma membranes (density 1.14-1.18 grams per cubic centimeter) as determined from copurification on isopycnic centrifugation with plasma membrane markers such as β-glucan synthetase, eosin-5-maleimidelabeling, and specific naphthylphthalamic acid-binding. The Ca²⁺ -dependent protein kinase is not specifically associated with chloroplasts or mitochondria. The membrane-bound Ca²⁺ -dependent protein kinases were solubilized with 0.8% (volume/volume) Nonidet P40. The solubilized enzymes were extensively purified by a protocol involving binding to diethylaminoethyl-cellulose (Whatman DE-52), Ca²⁺ -dependent binding to phenyl-Sepharose CL-4B, gradient elution from diethylaminoethyl-Sephacel (resolving two distinct Ca²⁺ -dependent protein kinases), and gel filtration on Ultrogel AcA 44. These two membrane-derived enzymes have similar molecular weights but differ in protein substrate specificity, in K_m values for ATP, and in Ca²⁺ -independent activation by unsaturated fatty acids. The membrane-bound enzymes correspond closely in these properties to two Ca²⁺ -dependent protein kinases present in the soluble phase.     

The selective inhibitory effect of NH4Cl on estrogen-stimulated rat uterine in vitro RNA synthesis

Estrogen-stimulated in vitro RNA synthesis in rat uterine nuclear-myofibrilar fractions, isolated uterine nuclei, and nucleoli was selectively inhibited by NH₄Cl (400 μmoles) when added prior to the start of a 37 °C incubation. This inhibitory effect was not observed if the salt was added after the start (as early as 1 min) of a 37 °C incubation. The removal of NH₄Cl from reaction mixtures not yet incubated at 37 °C further reduced in vitro RNA synthesis in both control and hormone-treated nuclei. The data suggest that NH₄Cl (400 μmoles) added prior to, but not after, the start of a 37 °C incubation inhibited estrogen-stimulated rat uterine nucleolar RNA polymerase activity perhaps by removing a protein component(s) which is necessary for hormonal stimulation.     

Effect on digestibility and nitrogen content of barley straw of different ammonia treatments

The article discusses the effect on solubility in cellulolytic enzyme suspensions, digestibility in vitro and crude protein content (F × 6.25) of treating barley straw with various dosages of NH₃ (2.6–5.9%), at various temperatures (15–75°C) for various treatment times (1–14 days).An increase in any of the above factors resulted in an increased intensity of treatment, with an increase in temperature of 15°C being equal to an increased NH₃-dosage level of 1.5% or prolongation of the treatment time by a factor of 4.5Digestibility in vitro increased with increased intensity of treatment, until a maximum level was obtained. Beyond this point, an increase in NH₃-dosage, or especially in temperature, tended to decrease digestibility in vitro. Maximum digestibility could be obtained, for example, with 2.6% NH₃, 62°C and 4 days incubation, or 5.9% NH₃, 30°C and 3–7 days incubation.Likewise, both solubility in cellulolytic enzyme suspensions and crude protein content increased with increased intensity of treatment, up to a certain level. Thereafter, increased dosing with NH₃, higher temperatures or longer incubation times had little or no effect. However, maximum values were obtained with a greater intensity of treatment than the maximum digestibility in vitro, and no tendency towards decreased values was observed.Increased enzyme solubility, beyond that corresponding to maximum digestibility in vitro, was accompanied by an increased rate of fermentation and a decreased content of neutral detergent fibre.Treatment with heat (100°C) and pressure after incubation, to simulate pelleting before evaporation of surplus NH₃, was also investigated. Only after the lowest incubation temperatures, however, was there an obvious tendency towards increased digestibility. The enzyme solubility was, on the other hand, very obviously increased. Crude protein content was also slightly increased by the heat- and pressure-treatment.     

Enzymatic attributes of an l-isoaspartyl methyltransferase from Candida utilis and its role in cell survival

BackgroundsSpontaneous deamidation and isoaspartate (IsoAsp) formation contributes to aging and reduced longevity in cells. A protein-l-isoaspartate (d-aspartate) O-methyltransferase (PCMT) is responsible for minimizing IsoAsp moieties in most organisms.MethodsPCMT was purified in its native form from yeast Candida utilis. The role of the native PCMT in cell survival and protein repair was investigated by manipulating intracellular PCMT levels with Oxidized Adenosine (AdOx) and Lithium Chloride (LiCl). Proteomic Identification of possible cellular targets was carried out using 2-dimensional gel electrophoresis, followed by on-Blot methylation and mass spectrometric analysis.ResultsThe 25.4 kDa native PCMT from C. utilis was found to have a K_m of 3.5 µM for AdoMet and 33.36 µM for IsoAsp containing Delta Sleep Inducing Peptide (DSIP) at pH 7.0. Native PCMT comprises of 232 amino acids which is coded by a 698 bp long nucleotide sequence. Phylogenetic comparison revealed the PCMT to be related more closely with the prokaryotic homologs. Increase in PCMT levels in vivo correlated with increased cell survival under physiological stresses. PCMT expression was seen to be linked with increased intracellular reactive oxygen species (ROS) concentration. Proteomic identification of possible cellular substrates revealed that PCMT interacts with proteins mainly involved with cellular housekeeping. PCMT effected both functional and structural repair in aged proteins in vitro.General significanceIdentification of PCMT in unicellular eukaryotes like C. utilis promises to make investigations into its control machinery easier owing to the familiarity and flexibility of the system.     

Regulation of ammonium and potassium uptake by glutamine and asparagine in Pisum arvense plants

Pisum arvense plants were subjected to 5 days of nitrogen deprivation. Then, in the conditions that increased or decreased the root glutamine and asparagine pools, the uptake rates of 0.5 mM NH₄ ⁺ and 0.5 mM K⁺ were examined. The plants supplied with 1 mM glutamine or asparagine took up ammonium and potassium at rates lower than those for the control plants. The uptake rates of NH₄ ⁺ and K⁺ were not affected by 1 mM glutamate. When the plants were pre-treated with 100 μM methionine sulphoximine, an inhibitor of glutamine synthesis, the efflux of NH₄ ⁺ from roots to ambient solution was enhanced. On the other hand, exposure of plants to methionine sulphoximine led to an increase in potassium uptake rate. The addition of asparagine, glutamine or glutamate into the incubation medium caused a decline in the rate of NH₄ ⁺ uptake by plasma membrane vesicles isolated from roots of Pisum arvense, whereas on addition of methionine sulphoximine increased ammonium uptake. The results indicate that both NH₄ ⁺ and K⁺ uptake appear to be similarly affected by glutamine and asparagine status in root cells. The research was supported by grant of KBN No. 6PO4C 068 08     

Growth of Salvinia molesta as affected by water temperature and nutrition I. Effects of nitrogen level and nitrogen compounds

The growth of Salvinia molesta D.S. Mitchell was studied in a greenhouse using controlled-temperature water-baths at 16, 19 and 22°C and 4 different nitrogen compounds (NO₃^?, NH₄⁺, NH₄NO₃ and urea) at levels up to 60 mg N l^?1. Little growth occurred at 16°C even if 20 mg N l^?1 was supplied together with other nutrients including phosphorus (2 mg H₂PO₄-P l^?1). The highest relative growth rate and total dry matter production occurred at 22°C when plants were supplied with 20 mg NH₄-N l^?1. At this temperature, the NH₄⁺ ion was superior to the NO₃^? ion or urea as a nitrogen source (almost doubling the biomass), but was not significantly better than NH₄NO₃. Over a period of 19 days for plants receiving 0.02 mg NH₄-N l^?, biomass increased 4-fold at 16°C, 9-fold at 19°C and 10-fold at 22°C. In contrast, for plants receiving 20 mg NH₄-N l^?1, biomass increased 4-fold at 16°C, 18-fold at 19°C and 38-fold at 22°C.     

ESR-spectroscopy of cytochrome P-450 LM2 in the soluble and membrane-bound state

Influence of microenvironment on the structure of spin-labeled cytochrome P-450 LM2 was studied. The distance between the spin-label which is covalently bound to cysteine-152 and heme ferrum in soluble protein is 17 A and 23 A in the membrane-bound one. The spin-label was exposed in water solution in both cases. It was shown that solubilized cytochrome P-450 LM2 in water solution forms the aggregates consisting of 4-6 monomers.