Membrane-bound succinate dehydrogenase of Bacillus pumilus strain 5: effects of modulators of monoelectron transfer

The membrane-bound succinate dehydrogenase (SDH; EC 1.3.99.1) of Bacillus pumilus strain 5 was investigated as succinate:ferricyanide oxidoreductase activity at 27 degrees C. A Km of 8.3 x 10(-3) M was obtained, and the Vmax was 1.8 x 10(-6) mole succinate dehydrogenated min-1 mg-1 membrane protein, at a substrate (succinate) concentration below 40 x 10(-3) M. Above this succinate concentration the Km was 102 x 10(-3) M and the Vmax was 3.7 x 10(-6) mole succinate min-1 mg-1 membrane protein. Para-benzoquinone or 2,4-dinitrophenylhydrazine, in micromolar amounts inhibited the enzyme by serving as an electron sink. Hydroxyl radical (OH.) scavengers, mannitol and benzoate, activated the enzyme, while superoxide dismutase (SOD) had no effect on the enzyme. Thus, the mechanism of electron transfer from succinate to Fe(CN)3-(6) through SDH does not involve superoxide (O2-) as a rate-limiting intermediate.     

Purification and some properties of the extracellular protease ofBacillus pumilus

Supernatant of a culture ofBacillus pumilus D 78 was precipitated with ethanol and chromatographed on DEAE- and CM-cellulose to isolate and purify a neutral protease with fibrinolytic and caseinolytic activity. Analysis by ultracentrifugation and immunoelectrophoresis indicate the homogeneity of the purified enzyme with the sedimentation constant s_20,w equal to 2.3. The fibrinolytic activity had a lower heat stability and was also more sensitive to pH higher than 8.0. The caseinolytic activity was stable over a wide range of pH (4.5 to 11.0). The enzyme binds acid dyes and is inhibited by Cu²⁺, Zn²⁺, Ca²⁺ and Fe³⁺, as well as byL-cysteine and KCN at a concentration of 10mM. Likewise, EDTA andp-chloromercuribenzoate show an inhibitory effect.     

Morpholine-induced formation of l-alanine dehydrogenase activity in Mycobacterium strain HE5

An NAD-dependent, morpholine-stimulated l-alanine dehydrogenase activity was detected in crude extracts from morpholine-, pyrrolidine-, and piperidine-grown cells of Mycobacterium strain HE5. Addition of morpholine to the assay mixture resulted in an up to 4.6-fold increase of l-alanine dehydrogenase activity when l-alanine was supplied at suboptimal concentration. l-Alanine dehydrogenase was purified to near homogeneity using a four-step purification procedure. The native enzyme had a molecular mass of 160 kDa and contained one type of subunit with a molecular mass of 41 kDa, indicating a tetrameric structure. The sequence of 30 N-terminal amino acids was determined and showed a similarity of up to 81% to that of various alanine dehydrogenases. The pH optimum for the oxidative deamination of l-alanine, the only amino acid converted by the enzyme, was determined to be pH 10.1, and apparent K _m values for l-alanine and NAD were 1.0 and 0.2 mM, respectively. K _m values of 0.6, 0.02, and 72 mM for pyruvate, NADH, and NH₄ ⁺, respectively, were estimated at pH 8.7 for the reductive amination reaction. Received: 25 September 1998 / Accepted: 11 March 1999     

Copper retention by a strain ofBacillus

Summary A survey has been made of the copper accumulation by resting cells of bacteria selected as copper-resistant, isolated from activated sludges. The best selected strain, classified asBacillus, retained copper at up to 3.8% of its cell dry weight. These values were lower in the presence of glucose, unlike a type culture ofBacillus cereus, in which the retention of copper was higher when glucose was present. Possible reasons for these changes in uptake of both strains are suggested.     

Distribution of myo-inositol dehydrogenase in algae

The enzyme myo-inositol dehydrogenase (InDH; EC 1.1.1.18) catalyses the NAD-dependent oxidation of myo-inositol to scyllo-inosose (2-keto-inositol). A survey within different algal groups showed that this enzyme is present in rhodophytes, glaucocystophytes, phaeophytes, xanthophytes and haptophytes but not in green algae, euglenophytes and chrysophytes. Anion-exchange chromatography of crude homogenates resulted in two distinct peaks of activity. Both isoenzymes were specific for myo-inositol and scyllo-inosose. epi- and scyllo-inositol as well as epi-inosose were only poor substrates, while all other polyols and sugars tested did not serve as substrates. A possible role of the InDH isoenzymes is the shuttling of reducing power between the mitochondrion and the cytosol.     

An efficient cyanide-degrading Bacillus pumilus strain

A Gram-positive, aerobic, endospore-forming bacterium was isolated by an enrichment technique for the ability to degrade cyanide and was identified as a Bacillus pumilus strain. The bacterium rapidly degraded 100 mg l-1 of free cyanide in the absence of added inorganic and organic substances. The ability to degrade cyanide was linked to the growth phase and was not exhibited before late exponential/early stationary phase. Cyanide-degrading activity could not be induced before this time by the addition of 20 mg cyanide l-1. Production of the cyanide-degrading activity required 0.01 mg Mn2+ l-1 and did not occur at Mn2+ concentrations below 0.002 mg l-1. Cyanide-degrading activity was intracellular and cell-free extracts rapidly degraded cyanide.     

Surfactant activity of a naphthalene degrading Bacillus pumilus strain isolated from oil sludge

We studied the growth, biosurfactant activities and petroleum hydrocarbon compounds utilisation of strain 28-11 isolated from a solid waste oil. The isolate was identified as Bacillus pumilus. It grew well in the presence of 0.1% (w/v) of crude oil and naphthalene under aerobic conditions and utilised these substances as carbon and energy source. The capacity of strain 28-11 to emulsify crude oil and its ability to remove hydrocarbons looks promising for its application in environmental technologies.     

Colony switching in an alpha-amylase-producing strain ofBacillus subtilis

Four colony variants (two stable and two unstable phenotypes) were observed inBacillus subtilis -10, an -amylase-hyperproducing strain. The stable variants lost the ability to produce -amylase, while the unstable ones reverted to the typical morphology after restreaking. Unstable expanding sectors appeared in typical colonies, and their appearance was influenced by the culture origin and age.     

Membrane-associated hyaluronate-binding activity of chondrosarcoma chondrocytes

The association of hyaluronate with the surface of chondrocytes was examined by several approaches using primary cultures of chondrocytes derived from the Swarm rat chondrosarcoma. In culture, chondrosarcoma chondrocytes produced large pericellular coats, which can be visualized by particle exclusion, and which can be removed by Streptomyces hyaluronidase. Exposure of chondrocytes, which had been metabolically labelled with 3H-acetate, to exogenous hyaluronate or to Streptomyces hyaluronidase resulted in the release of 36-38% of the endogenous, labelled chondroitin sulfate from the cell layer into the incubation solution. These results imply that at least 37% of the cell layer chondroitin sulfate proteoglycan is retained there by an interaction with hyaluronate. Thus membranes were prepared from cultured chondrocytes and examined for sites which bind 3H-hyaluronate. Binding was observed and found to be saturable, specific for hyaluronate, of high affinity (Kd = approximately 10(-10) M), and destroyed by treating the membranes with trypsin. The 3H-hyaluronate-binding activity was inhibited competitively by hyaluronate decasaccharides but not by hexasaccharides or octasaccharides, indicating that the binding sites recognize a sequence of hyaluronate composed of five disaccharide repeats. The binding activity was partially purified from a detergent extract of chondrocyte membranes by ion exchange chromatography on DEAE-cellulose, followed by affinity chromatography on wheat germ agglutinin-agarose. Analysis of the partially purified binding activity by SDS-PAGE revealed five protein bands of 48,000-66,000 daltons in silver-stained gels. SDS-PAGE followed by Western blotting and exposure to monoclonal antibodies which recognize epitopes present in link protein and in the hyaluronate-binding region of cartilage proteoglycan revealed no immunoreactive protein bands in the partially purified material. We conclude that one mechanism by which hyaluronate associates with the chondrocyte surface may be via interaction with a membrane-bound hyaluronate-binding protein which is distinct from link protein and proteoglycan.     

Production of extracellular lysine by a strain ofBacillus coagulans

A naturally deficient thiamine and methionine requiring strain ofBacillus coagulans (Ms15) accumulates lysine in medium only when exogenous pyridoxine (optimal concentration, 0.1 μg/ml) and threonine (optimal concentration, 100 μg/ml) are supplied. Threonine exerts an inhibitory effect at higher concentrations but pyridoxine does not.     

Effect of dimethyl sulfoxide on lysine production by a mutant ofBacillus subtilis with low homoserine dehydrogenase activity

SomeBacillus subtilis mutants with different levels of homoserine dehydrogenase were described. Strains that do not accumulate methionine have a high homoserine dehydrogenase activity. Low activity was detected in mutants where cell growth was completely inhibited by 0.7 mmol/L methionine. A low concentration of dimethyl sulfoxide had a stimulatory effect on lysine production by the methionine-sensitive mutant ofBacillus subtilis.     

Intracellular proteolytic activity during sporulation ofBacillus megaterium

Intracellular proteolytic activity increased during incubation of the sporogenic strain ofBacillus megaterium KM in a sporulation medium together with excretion of an extracellular metalloprotease. The exocellular protease activity in a constant volume of the medium reached a 100-fold value with respeot to the intracellular activity. Maximal values of the activity of both the extracellular and intracellular enzyme were reached after 3 – 5 h of incubation. After 7 h 20 – 50% cells formed refractile spores. The intracellular proteolytic system hydrolyzed denatured proteinsin vitro at a rate up to 150 μg mg_-1 h_-1 and native proteins at a rate up to 70 μg mg^-1 h^-1. Degradation of proteinsin vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 μg mg^-1 h^-1 and the inactivation of beta-galactosidase at a rate of 70 μg mg^-1 h^-1. The intracellular proteolytic activity was inhibited to 65 – 88% by EDTA, to 23 – 76% by PMSF. Proteolysis of denatured proteins was inhibited both by EDTA and PMSF more pronouncedly than proteolysis of native proteins; 50 – 65% of the activity were localized in protoplasts. Another strain ofBacillus megaterium (J) characterized by a high (up to 90%) and synchronous sporulation activity was found to behave in a similar way, but the rate of protein turnover in this strain was almost twice as high. The asporogenic strain ofBacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3 – 4 h. The intraeellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.     

Membrane-associated chromate reductase activity from Enterobacter cloacae.

Washed cells of Enterobacter cloacae HO1 reduced hexavalent chromium (chromate: CrO4(2-) anaerobically. Chromate reductase activity was preferentially associated with the membrane fraction of the cells. Right-side-out membrane vesicles prepared from E. cloacae cells showed high chromate reductase activities when ascorbate-reduced phenazine methosulfate was added as an electron donor.     

The pathway of myo-inositol 1,3,4-trisphosphate phosphorylation in liver. Identification of myo-inositol 1,3,4-trisphosphate 6-kinase, myo-inositol 1,3,4-trisphosphate 5-kinase, and myo-inositol 1,3,4,6-tetrakisphosphate 5-kinase

Inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) metabolism has been studied in liver homogenates and in 100,000 x g supernatant and particulate fractions. When liver homogenates were incubated in an "intracellular" medium containing 5 mM MgATP, equal proportions of Ins(1,3,4)P3 were dephosphorylated and phosphorylated. Two inositol tetrakisphosphate (InsP4) products and an inositol pentakisphosphate (InsP5) were detected. The InsP4 isomers were unequivocally identified as inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) and inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) by high performance liquid chromatography separation of inositol phosphates, periodate oxidation, alkaline hydrolysis, and stereo-specific polyol dehydrogenase. Ins(1,3,4)P3 5-kinase is a novel enzyme activity and accounted for 16% of the total Ins(1,3,4)P3 phosphorylation. Ins(1,3,4,6)P4 was also shown to be further phosphorylated to inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) by a kinase not previously known to occur in liver. About 75% of Ins(1,3,4)P3 kinase activities were soluble and were partly purified by anion-exchange fast protein liquid chromatography. The two Ins(1,3,4)P3 kinase activities eluted as a single peak that was well resolved from Ins(1,3,4)P3 phosphatase, Ins(1,3,4,6)P4 5-kinase, and Ins(1,3,4,5)P4 5-phosphatase activities. A further novel observation was that 10 microM Ins(1,3,4,5)P4 inhibited Ins(1,3,4)P3 kinase activities by 60%.     

Protease activity in cells ofBacillus megaterium during derepression

A proteolytic activity hydrolyzing denatured proteins of Bacillus megaterium labelled with 35S or 14C amino acids was detected in cells of the asporogenic strain of Bacillus megaterium. The substrate is hydrolyzed by the enzyme or enzymes at optimum pH around 7, their activity being almost completely inhibited by EDTA and o-phenanthroline. PMSF, the inhibitor of serine proteases, is slightly inhibitory. Gel filtration on a Sephadex column separated the protease activity to two or three fractions. The protease activity in cells with the repressed synthesis of protease corresponds to 5-20 mug of substrate degraded per hour by 1 mg of protein at 37 degrees C. It increases five to ten-fold during the derepression. When the intracellular protease activity increases the extracellular enzyme begins to be excreted into the medium. The intracellular protease activity rapidly decreases after the addition of chloramphenicol or of a mixture of amino acids to the derepressed culture. Half or even more of the protease activity is released from the cells during their conversion to protoplasts by means of lysozyme. This "periplasmic" activity remains mostly in the supernatant also after mesosomes have been centrifuged down from the periplasm. A portion of the activity bound in protoplasts sediments together with membrane fraction after their lysis.     

Membrane-associated IL 1-like activity on rat dendritic cells

The secretion of interleukin 1 (IL 1) by rat dendritic cells (DC) was studied in relation to their ability to induce the production of interleukin 2 (IL 2) and to induce IL 2 responsiveness. IL 1 (or IL 1-like activity) was measured by its capacity to enhance IL 2 production by EL4 cells. In contrast to peritoneal exudate cells (PEC) or splenic adherent cells, DC from thoracic duct lymph (TD-DC) or from spleen did not secrete detectable amounts of IL 1 on stimulation with LPS/Silica. However, TD-DC and splenic DC were able to enhance IL 2 production by EL4 cells directly, and were only two times less effective than PEC. By preventing cell-to-cell contact between stimulator cells and EL4 cells, it was demonstrated that most of the IL 2-inducing activity of TD-DC and PEC was associated with the cell membrane. Treatment with 1% paraformaldehyde (PFA) to abolish metabolic activity resulted in a 50% decrease (or inactivation) of IL 2-inducing activity of TD-DC in the EL4 assay. Moreover, UVB-irradiation (300 mJ/cm2) of TD-DC, which has been described to inhibit the release of IL 1 by macrophages, caused a 70% decrease in IL 2-inducing activity. In a primary allogeneic mixed leukocyte reaction, neither PFA-treated TD-DC nor UV-irradiated TD-DC were able to induce T cell proliferation or IL 2 production. The cells were, however, able to induce IL 2 responsiveness, i.e., T cell proliferation in the presence of excess human recombinant IL 2. The finding that IL 1 enhanced T cell responses to PFA-treated or UV-irradiated TD-DC in the absence and in the presence of excess IL 2 indicates that loss of stimulatory activity of TD-DC may be due in part to loss or inactivation of IL 1. These results suggest that membrane-associated structures, that are identical to or mimic IL 1, are involved in the activation of T cells by DC.     

Regulation of 5-hydroxyeicosanoid dehydrogenase activity in monocytic cells

The requirement of DAG (diacylglycerol) to recruit PKD (protein kinase D) to the TGN (trans-Golgi network) for the targeting of transport carriers to the cell surface, has led us to a search for new components involved in this regulatory pathway. Previous findings reveal that the heterotrimeric Gbetagamma (GTP-binding protein betagamma subunits) act as PKD activators, leading to fission of transport vesicles at the TGN. We have recently shown that PKCeta (protein kinase Ceta) functions as an intermediate member in the vesicle generating pathway. DAG is capable of activating this kinase at the TGN, and at the same time is able to recruit PKD to this organelle in order to interact with PKCeta, allowing phosphorylation of PKD's activation loop. The most qualified candidates for the production of DAG at the TGN are PI-PLCs (phosphatidylinositol-specific phospholipases C), since some members of this family can be directly activated by Gbetagamma, utilizing PtdIns(4,5)P2 as a substrate, to produce the second messengers DAG and InsP3. In the present study we show that betagamma-dependent Golgi fragmentation, PKD1 activation and TGN to plasma membrane transport were affected by a specific PI-PLC inhibitor, U73122 [1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. In addition, a recently described PI-PLC activator, m-3M3FBS [2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide], induced vesiculation of the Golgi apparatus as well as PKD1 phosphorylation at its activation loop. Finally, using siRNA (small interfering RNA) to block several PI-PLCs, we were able to identify PLCbeta3 as the sole member of this family involved in the regulation of the formation of transport carriers at the TGN. In conclusion, we demonstrate that fission of transport carriers at the TGN is dependent on PI-PLCs, specifically PLCbeta3, which is necessary to activate PKCeta and PKD in that Golgi compartment, via DAG production.     

Direct assay method for inosine 5'-monophosphate dehydrogenase activity

A rapid microassay method for the accurate measurement of the activity of inosine 5'-monophosphate dehydrogenase in crude tissue extracts was described. [8-14C]IMP and the radioactive products were separated by high-voltage electrophoresis in 0.1 M potassium phosphate buffer, pH 7.0, for 45 min. This separation method provides an analysis of the possible interfering reactions such as the metabolic conversion of the substrate IMP to inosine and adenylosuccinate, and the loss of the product XMP to xanthosine or GMP and to other metabolites. Low blank values were consistently obtained with this method because the XMP spot moves faster than the IMP spot. The major advantages of this assay method are direct measurement of IMP dehydrogenase activity in crude extracts, high sensitivity (with a limit of detection of 5 pmol of XMP production), high reproducibility (less than +/- 3.6%), low blank values (60-80 cpm), speed (2 h per 30 assays), and capability to measure activity in small amounts of tissue (10-50 mg wet wt).