Isolated and purified plasma and thylakoid membranes of the cyanobacteriumAnacystis nidulans contain immunologically cross-reactive aa3-type cytochrome oxidase

Cell-free extracts ofAnacystis nidulans were fractionated by discontinuous sucrose density gradient centrifugation resulting in the separation of two distinct types of membranes, the heavier one containing the chlorophyll and the lighter one devoid of chlorophyll. Identity of the latter with plasma membrane was confirmed by labeling of intact cells with impermeant marker,³⁵S-diazobenzenesulfonate, prior to cell disruption. Both membrane fractions were purified individually by repeated recentrifugation on identical gradients. Purified membranes were subjected to dissociating polyacrylamide gel electrophoresis, either type of membranes yielding a distinct polypeptide pattern. After transfer of the polypeptides to nitrocellulose by Western blotting, two of the proteins, with molecular weights of approximately 55,000 and 32,000, respectively, gave strong and specifically complementary cross-reactions with antibodies raised against subunits I and II of the aa₃-type cytochrome oxidase fromParacoccus denitrificans. The findings will be discussed in terms of the presence of aa₃-type cytochrome oxidase in both plasma and thylakoid membranes ofAnacystis nidulans.     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase of Actinomadura R39.

By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown to have promoter activity in Streptomyces lividans. The same 100-200-copy-number plasmid was used to clone in S. lividans TK24, the gene that encodes the Actinomadura R39 beta-lactamase. Gene cloning resulted in an amplified expression of the beta-lactamase when compared with the amounts of enzyme produced by the original strain (1 mg versus 0.008 mg.litre of culture-1).     

Regio- and stereoselective regulation of monooxygenase activities by isoenzyme-selective phosphorylation of cytochrome P450

The phosphorylation of the two major phenobarbital-inducible cytochrome P450 isoenzymes IIB1 and IIB2 was increased in hepatocytes by the action of the membrane permeating cAMP derivatives N6-dibutyryl-cAMP and 8-thiomethyl-cAMP. Under these conditions the dealkylation of 7-pentoxyresorufin, a selective substrate of cytochrome P450IIB1 and P450IIB2 was markedly reduced. 16 beta-Hydroxylation of testosterone which is catalyzed specifically only by cytochrome P450IIB1 and IIB2 was strongly reduced; for 16 alpha-hydroxylation which is also catalyzed by cytochrome P450IIB1 and IIB2 but additionally by 3 further cytochrome P450 isoenzymes, this reduction was less pronounced; for the oxidation of the 17 beta-hydroxyl group which besides cytochromes P450IIB1 and IIB2 is additionally catalyzed not only by other cytochromes P450 but also by 17 beta-hydroxysteroid dehydrogenase there was a clear tendency of reduction which, however, no longer reached statistical significance. Hydroxylation at other positions of testosterone which are catalyzed by other cytochrome P450 isoenzymes were not significantly changed. Hence isoenzyme-selective phosphorylation of cytochrome P450 leads to a corresponding isoenzyme-selective modulation of monooxygenase activity which holds promise to be especially important as a fast regulation of the control of genotoxic metabolites.     

Characterization of the cytochrome c oxidase in isolated and purified plasma membranes from the cyanobacterium Anacystis nidulans

Functionally intact plasma membranes were isolated from the cyanobacterium (blue-green alga) Anacystis nidulans through French pressure cell extrusion of lysozyme/EDTA-treated cells, separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation, and purified by repeated recentrifugation. Origin and identity of the chlorophyll-free plasma membrane fraction were confirmed by labeling of intact cells with impermeant protein markers, [35S]diazobenzenesulfonate and fluorescamine, prior to membrane isolation. Rates of oxidation of reduced horse heart cytochrome c by purified plasma and thylakoid membranes were 90 and 2 nmol min-1 (mg of protein)-1, respectively. The cytochrome oxidase in isolated plasma membranes was identified as a copper-containing aa3-type enzyme from the properties of its redox-active and EDTA-resistant Cu2+ ESR signal, the characteristic inhibition profile, reduced minus oxidized difference spectra, carbon monoxide difference spectra, photoaction and photodissociation spectra of the CO-inhibited enzyme, and immunological cross-reaction of two subunits of the enzyme with antibodies against subunits I and II, and the holoenzyme, of Paracoccus denitrificans aa3-type cytochrome oxidase. The data presented are the first comprehensive evidence for the occurrence of aa3-type cytochrome oxidase in the plasma membrane of a cyanobacterium similar to the corresponding mitochondrial enzyme (EC 1.9.3.1).     

Impact of salt adaptation on esterified fatty acids and cytochrome oxidase in plasma and thylakoid membranes from the cyanobacterium Anacystis nidulans

During adaptation of photoautotrophically growing fresh water cyanobacterium Anacystis nidulans to high salinity the cells showed a pronounced increase of proton-sodium antiporter activity, and of cytochrome c oxidase in isolated and purified plasma membrane. At the same time the concentrations of plasma membrane-bound EDTA-resistant copper and iron (determined by inductively coupled plasma atomic emission spectrometry) rose proportionately, accompanied by an increase in whole cell respiration. In plasma membranes from salt adapted cells lipid/protein ratios were markedly higher than in control cells, levels of esterified saturated and long-chain fatty acids being significantly higher than the respective levels of unsaturated and short-chain fatty acids which explains the higher lipid-phase transition temperatures derived from Arrhenius plots. Immunoblotting of the membrane proteins with antisera raised against the cytochrome c oxidases from Paracoccus denitrificans and A. nidulans gave two cross-reacting bands with apparent molecular weights around 50000 and 30000 (subunits I and II, respectively) which were more pronounced in plasma membranes from salt adapted cells when compared to control cells. The protein pattern of plasma membranes from salt adapted cells also showed the appearance of bands at apparent molecular weights of 44000–48000 and 54000–56000 which might stem from the proton/sodium-antiporter in this membrane.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - cyt cytochrome - DCCD N,N-dicyclohexylcarbodiimide - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - ICP-AES inductively coupled plasma atomic emission spectrometry - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - EPR electron paramagnetic resonance spectrometry     

Identification and characterization of a porcine parvovirus nonstructural polypeptide.

Sera from porcine parvovirus (PPV)-infected swine fetuses immunoprecipitated and 84- to 86-kilodalton polypeptide in addition to the A and B virion structural proteins. This polypeptide, designated NS-1, was present in PPV-infected cell lysates but not in purified virions. Partial proteolysis mapping revealed that NS-1 was not related to the A and B viral structural proteins. All three proteins in infected cells were phosphorylated at serine residues, and NS-1 also contained phosphothreonine. From pulse-labeling experiments with either 32Pi or [35S]methionine, NS-1 was found to first appear 5 to 7 h postinfection, whereas the viral structural polypeptides were first synthesized 9 to 11 h postinfection. Pulse-chase experiments revealed that NS-1 initially appeared as an 84-kilodalton protein and was subsequently structurally modified to forms of slower electrophoretic mobilities. The time of appearance of NS-1 after virus infection coincided with the initiation of viral DNA synthesis, suggesting that this polypeptide (and the modified forms thereof) may be involved in PPV replication.     

KBSH parvovirus: comparison with porcine parvovirus.

We compared the molecular, antigenic, and pathogenic properties of KBSH parvovirus to those of porcine parvovirus (PPV) isolate NADL-8. KBSH, propagated in swine testes cells in culture, possessed two major capsid polypeptides of 83 and 64 kilodaltons that were similar in size to those of PPV. KBSH-infected cells also contained an 86-kilodalton nonstructural polypeptide that was identical in size to the PPV nonstructural polypeptide (NS-1). The KBSH polypeptides were structurally similar but not identical to the corresponding PPV polypeptides, as revealed by partial proteolysis mapping. Viral replicative-form DNA from KBSH-infected cells was similar in size to PPV replicative-form DNA and exhibited similar but not identical restriction endonuclease cleavage patterns to that of PPV replicative-form DNA. Antigenically, the two viruses were also very closely related. By using heterologous and homologous antisera, the two viruses were indistinguishable in hemagglutination inhibition and immunoprecipitation assays. However, pathogenically these viruses were dramatically different. NADL-8 caused fetal death when injected into swine fetuses in utero and viremia and high persisting antibody titers when administered orally to weaning-age swine. KBSH-inoculated fetuses were normal in appearance, and pigs orally exposed to KBSH failed to establish viremia and demonstrated only transient antibody titers. Thus, KBSH appears to be a PPV that is very closely related to a highly pathogenic PPV isolate, yet is itself nonpathogenic in swine. This reduced pathogenic potential of KBSH may be attributable to its poor ability to replicate in swine.     

Changes of some physical properties of isolated and purified plasma and thylakoid membrane vesicles from the freshwater cyanobacterium Synechococcus 6301 (Anacystis nidulans) during adaptation to salinity

Photoautotrophically growing cultures of the freshwater cyanobacterium Anacystis nidulans (Synechococcus sp.) became adapted to the presence of 0.4-0.5 M NaCl in the growth medium (about seawater level) with a lag phase of 2 days after which time the growth rate resumed at 80-90% of the control. Major changes in structure and function of the plasma membranes (and, to a much lesser extent, of the thylakoid membranes) were found to accompany the adaptation process. Plasma and thylakoid membranes were separated from crude cell-free extracts of French pressure cell-treated Anacystis by discontinuous sucrose density gradient centrifugation and purified by repeated recentrifugation on fresh gradients. Concentrations of copper, iron, calcium, and magnesium ions were determined by inductively coupled plasma atomic emission spectrometry with EDTA-washed and dialyzed membrane preparations; salt adaptation was found to increase (decrease) the concentration of membrane-bound calcium in plasma (thylakoid) membranes, qualitatively reciprocal results being obtained for magnesium. Levels of plasma membrane-bound copper and iron roughly tripled during the adaptation process; by contrast, corresponding effects on thylakoid membranes were negligible. The size of the membrane vesicles was measured by quasi-elastic laser light-scattering and the electric surface charge of the membranes was measured by laser Doppler velocimetry. Salt adaptation decreased the mean diameter of plasma membrane vesicles to a much higher extent than that of thylakoid membrane vesicles. Overall surface charge densities of resting vesicles were only slightly affected by the salt treatment as was also seen from titration of the electrophoretic mobility of the vesicles with electrolytes. Yet, induction of (photosynthetic or respiratory) electron transport provoked a charge separation across the membrane which was easily measurable in terms of electrophoretic mobility. The results will be discussed with particular emphasis on the stimulated cytochrome c oxidase activity of plasma (but not thylakoid) membranes from salt-adapted cells compared to control cells and also with respect to the decreased ion permeability of the plasma membrane of salt grown cells.     

Metabolic analysis of the synthesis of high levels of intracellular human SOD in Saccharomyces cerevisiae rhSOD 2060 411 SGA122

The synthesis of human superoxide dismutase (SOD) in batch cultures of a Saccharomyces cerevisiae strain using a glucose-limited minimal medium was studied through metabolic flux analysis. A stoichiometric model was built, which included 78 reactions, according to metabolic pathways operative in these strains during respirofermentative and oxidative metabolism. It allowed calculation of the distribution of metabolic fluxes during diauxic growth on glucose and ethanol. Fermentation profiles and metabolic fluxes were analyzed at different phases of diauxic growth for the recombinant strain (P+) and for its wild type (P-). The synthesis of SOD by the strain P+ resulted in a decrease in specific growth rate of 34 and 54% (growth on glucose and ethanol respectively) in comparison to the wild type. Both strains exhibited similar flux of glucose consumption and ethanol synthesis but important differences in carbon distribution with biomass/substrate yields and ATP production 50% higher in P-. A higher contribution of fermentative metabolism, with 64% of the energy produced at the phosphorylation level, was observed during SOD production. The flux of precursors to amino acids and nucleotides was higher in the recombinant strain, in agreement with the higher total RNA and protein levels. Lower specific growth rates in strain P+ appear to be related to the decrease in the rate of synthesis of nonrecombinant protein, as well as a decrease in the activities of the pentose phosphate (PP) pathway and TCA cycle. A very different way of entry into the stationary phase was observed for each strain: in the wild-type strain most metabolic fluxes decreased and fluxes related to energy reserve synthesis increased, while in the P+ strain the flux of 22 reactions (including PP pathway and amino acids biosynthesis) related to SOD production increased their fluxes. Changes in SOD production rates at different physiological states appear to be related to the differences in building blocks availability between respirofermentative and oxidative metabolism. Using the present expression system, ideal conditions for SOD synthesis are represented by either active growth during respirofermentative metabolism or transition from a growing to a nongrowing state. An increase in SOD flux could be achieved using an expression system nonassociated to growth and potentially eliminating part of the metabolic burden.     

A retro-evolution study of CDP-6-deoxy-D-glycero-L-threo-4-hexulose-3-dehydrase (E1) from Yersinia pseudotuberculosis: implications for C-3 deoxygenation in the biosynthesis of 3,6-dideoxyhexoses

CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase (E1), which catalyzes C-3 deoxygenation of CDP-4-keto-6-deoxyglucose in the biosynthesis of 3,6-dideoxyhexoses, shares a modest sequence identity with other B6-dependent enzymes, albeit with two important distinctions. It is a rare example of a B6-dependent enzyme that harbors a [2Fe-2S] cluster, and a highly conserved lysine that serves as an anchor for PLP in most B6-dependent enzymes is replaced by histidine at position 220 in E1. Since alteration of His220 to a lysine residue may produce a putative progenitor of E1, the H220K mutant was constructed and tested for the ability to process the predicted substrate, CDP-4-amino-4,6-dideoxyglucose, using PLP as the coenzyme. Our data showed that H220K-E1 has no dehydrase activity, but can act as a PLP-dependent transaminase. However, the reaction is not catalytic since PLP cannot be regenerated during turnover. Reported herein are the results of this investigation and the implications for the role of His220 in the catalytic mechanism of E1.