Cell-free Particulate Fraction Research Articles

Macrophage colony-stimulating factor-induced bone marrow macrophages do not synthesize or release prostaglandin E2

Previously, we found that murine bone marrow-derived macrophages (MO) induced in vitro by MO-specific colony-stimulating factor (M-CSF) have little capacity to release prostaglandin E2 (PGE2) and other eicosanoids. This work focused on the functional and transcriptional expression of the key enzymes for the PGE2 synthesis in the MO. Nonadherent bone marrow cells were cultured with RPMI1640 plus 10% fetal bovine serum (FBS) further supplemented with either M-CSF or granulocyte-macrophage (GM)-CSF and interleukin-3 (IL-3). Cellular PGG/H synthase (cyclooxygenase) levels were quantified by cytometric analysis with antibodies specific for the two isozymes of PGG/H synthase (PGG/H synthases 1 and 2). The enzyme activity was monitored by adding exogenous arachidonic acid (AA) substrate to the bone marrow MO cultures and to the cell-free particulate fractions. The levels of PGE2 converted were quantitated by radioimmunoassay (RIA). mRNA levels of the enzymes were detected by Northern blot analysis hybridized with mouse PGG/H synthase cDNA probes, 2.7 kb (PGG/H synthase 1) and 4.2 kb (PGG/H synthase 2). In addition, cellular phospholipase A2 (PLA2) activities were detected with sn-2-14C-arachidonyl phosphatidylcholine as a substrate. Cells proliferating in the presence of GM-CSF and IL-3 for more than 4 days showed significant release of PGE2 (> 7 ng/10(6) cells) when stimulated by AA. These cells also expressed significant amounts of PGG/H synthase 1 protein, its mRNA (2.7 kb) and cellular PLA2. M-CSF-induced MO, in sharp contrast, expressed little PGG/H synthase protein, mRNA, cellular enzyme activity, or PGE2 release, despite comparable levels of cellular PLA2 activity. These data suggest that the capacity of differentiating marrow-derived MO to form PGE2 is growth factor-dependent.

Macrophage Colony-Stimulating Factor-Induced Bone Marrow Macrophages Do Not Synthesize or Release Prostaglandin E2

Abstract Previously, we found that murine bone marrow-derived macrophages (MO) induced in vitro by MO-specific colony-stimulating factor (M-CSF) have little capacity to release prostaglandin E2 (PGE2) and other eicosanoids. This work focused on the functional and transcriptional expression of the key enzymes for the PGE2 synthesis in the MO. Nonadherent bone marrow cells were cultured with RPMI1640 plus 10% fetal bovine serum (FBS) further supplemented with either M-CSF or granulocyte-macrophage (GM)-CSF and interleukin-3 (IL-3). Cellular PGG/H synthase (cyclooxygenase) levels were quantified by cytometric analysis with antibodies specific for the two isozymes of PGG/H synthase (PGG/H synthases 1 and 2). The enzyme activity was monitored by adding exogenous arachidonic acid (AA) substrate to the bone marrow MO cultures and to the cell-free particulate fractions. The levels of PGE2 converted were quantitated by radioimmunoassay (RIA). mRNA levels of the enzymes were detected by Northern blot analysis hybridized with mouse PGG/H synthase cDNA probes, 2.7 kb (PGG/H synthase 1) and 4.2 kb (PGG/H synthase 2). In addition, cellular phospholipase A2 (PLA2) activities were detected with sn-2–14C-arachidonyl phosphatidylcholine as a substrate. Cells proliferating in the presence of GM-CSF and IL-3 for more than 4 days showed significant release of PGE2 (> 7 ng/10(6) cells) when stimulated by AA. These cells also expressed significant amounts of PGG/H synthase 1 protein, its mRNA (2.7 kb) and cellular PLA2. M-CSF-induced MO, in sharp contrast, expressed little PGG/H synthase protein, mRNA, cellular enzyme activity, or PGE2 release, despite comparable levels of cellular PLA2 activity. These data suggest that the capacity of differentiating marrow-derived MO to form PGE2 is growth factor-dependent.

Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli. In vitro and in vivo incorporation into phospholipids.

sn-Glycerol 3-phosphorothioate, a bacteriocidal analog of sn-glycerol 3-phosphate in strains of Escherichia coli with a functioning glycerol phosphate transport system, was investigated for its ability to be incorporated into phospholipid under in vitro and in vivo conditions. A cell-free particulate fraction from E. coli strain 8 catalyzes the transfer of sn-[3H]glycerol 3-phosphoro[35S]thioate to chloroform-soluble material in the presence of either CDP-diglyceride or palmitoyl coenzyme A. With CDP-diglyceride as the co-substrate, the product of the reaction was tentatively identified as phosphatidylglycerol phosphorothioate. No formation of phosphatidylglycerol was observed, suggesting that the specific phosphatase required for the synthesis of phosphatidylglycerol does not catalyze, or else at a greatly reduced rate, the hydrolysis of the phosphorothioate monoester linkage. The kinetics of incorporation of sn-[3H]glycerol 3-phosphate and phosphorothioate into chloroform-soluble material in the presence of CDP-diglyceride are almost identical. In the presence of palmitoyl coenzyme A, sn-[3H]glycerol 3-phosphoro[35S]thioate was converted to the phosphorothioate analog of phosphatidic acid. Kinetic analysis showed that the apparent Km values for the incorporation of the phosphate and the phosphorothioate derivatives into phospholipid were 0.4 and 0.8 mM, respectively. The Vmax for the phosphorothioate analog was approximately half that for the phosphate derivative. Chemically synthesized thiophosphatidic acid was not a substrate for CTP:phosphatidic acid cytidylyltransferase. sn-[3H]Glycerol 3-phosphoro[35S]thioate was incorporated into phospholipid by cultures of E. coli strain 8. The major phosphorothioate-containing phospholipid synthesized in vivo was identified as 1,2-diacyl-sn-[3H]glycerol 3-phosphoro[35S]thioate. The phosphorothioate analog of phosphatidylglycerol phosphate was not observed despite our observations that this analog can be synthesized in vitro. Our results indicate that the phosphorothioate analog is an effective sn-glycerol 3-phosphate surrogate and suggest that a major reason for its toxicity toward E. coli strain 8 may be due to a total blockade of endogenous phospholipid biosynthesis.

Elicitation of Casbene Synthetase Activity in Castor Bean

Endopolygalacturonase isolated from culture filtrates of the fungus Rhizopus stolonifer was shown previously to act as an elicitor of biosynthetic capacity for the antifungal agent, casbene, in castor bean (Ricinus communis L.) seedlings (S.-C. Lee, C.A. West 1981 Plant Physiology 67:633-639). Selective amidation of exposed carboxyl groups of the pure fungal endopolygalacturonase using intermediate activation with a water-soluble carbodiimide under mild conditions leads to inactivation of its enzymic activity. Tests of active and partially inactivated preparations of the enzyme reveal a close correlation between the levels of catalytic and elicitor activities. This suggests that the catalytic activity of the enzyme is necessary for its function as an elicitor. Treatment of the cell-free particulate fraction of homogenates of castor bean seedlings with the active fungal endopolygalacturonase results in the production of a heat-stable, water-soluble component which is highly active as an elicitor of casbene synthetase activity. Several additional lines of evidence, including the susceptibility of the heat-stable elicitor fraction to partial inactivation following prolonged treatment with endopolygalacturonase, indicate that the heat-stable elicitor is most likely a pectic fragment of the plant cell wall and that it is a required intermediate in the process of elicitation of casbene synthetase activity by the fungal endopolygalacturonase.

Microbial oxidation of gaseous hydrocarbons: production of alcohols and methyl ketones from their corresponding n-alkanes by methylotrophic bacteria.

Cell suspensions of methane-utilizing bacteria oxidized n-alkanes (propane, butane, pentane, and hexane) to their corresponding alcohols and methyl ketones. The product alcohols and methyl ketones accumulated extracellularly. Methanol-grown cells of methane-utilizing bacteria did not oxidize n-alkanes. The product primary alcohol was detected in a cell-free system but only in a trace amount in the whole cell system due to further oxidation. The optimum conditions for in vivo formation of secondary alcohol and methyl ketone from n-alkanes were compared between two distinct types of C1-utilizing microbes: Methylococcus capsulatus M1 (type I membrane) and Methylosinus trichosporium OB3b (type II membrane). The production of acetone or 2-butanone from n-alkanes ceased after 3 h of incubation for strain OB3b and 5 h for strain M1. The amount of these methyl ketones did not decline during 30 h of incubation. The optimum pH for the in vivo production of methyl ketones from n-alkanes by both strains was around 7.0. However, secondary alcohols were accumulated at higher amounts around pH 6.0. The optimum temperature for the in vivo production of methyl ketones from n-alkanes was around 40 degrees C for strain M1 and around 30-35 degrees C for strain OB3b. Higher accumulation of secondary alcohol was detected at 30-40 degrees C for strain M1 and 25 degrees C for strain OB3b. The alkane hydroxylation enzyme was located in the cell-free particulate fraction precipitated between 10 000 and 40 000 X g centrifugation. The yield of primary and secondary alcohols from n-alkane in the cell-free system was about equal. Evidence obtained indicates that the hydroxylation of n-alkanes (both terminal and subterminal oxidations) is also catalyzed by the methane hydroxylation - alkene epoxidation enzyme system.

Microbial Oxidation of Gaseous Hydrocarbons: Production of Secondary Alcohols from Corresponding n -Alkanes by Methane-Utilizing Bacteria

Over 20 new strains of methane-utilizing bacteria were isolated from lake water and soil samples. Cell suspensions of these and of other known strains of methane-utilizing bacteria oxidized n-alkanes (propane, butane, pentane, hexane) to their corresponding secondary alcohols (2-propanol, 2-butanol, 2-pentanol, 2-hexanol). The product secondary alcohols accumulated extracellularly. The rate of production of secondary alcohols varied with the organism used for oxidation. The average rate of 2-propanol, 2-butanol, 2-pentanol, and 2-hexanol production was 1.5, 1.0, 0.15, and 0.08 mumol/h per 5.0 mg of protein in cell suspensions, respectively. Secondary alcohols were slowly oxidized further to the corresponding methylketones. Primary alcohols and aldehydes were also detected in low amounts (rate of production were 0.05 to 0.08 mumol/h per 5.0 mg of protein in cell suspensions) as products of n-alkane (propane and butane) oxidation. However, primary alcohols and aldehydes were rapidly metabolized further by cell suspensions. Methanol-grown cells of methane-utilizing bacteria did not oxidize n-alkanes to their corresponding secondary alcohols, indicating that the enzymatic system required for oxidation of n-alkanes was induced only during growth on methane. The optimal conditions for in vivo secondary alcohol formation from n-alkanes were investigated in Methylosinus sp. (CRL-15). The rate of 2-propanol and 2-butanol production was linear for the 40-min incubation period and increased directly with cell protein concentration up to 12 mg/ml. The optimal temperature and pH for the production of 2-propanol and 2-butanol were 40 degrees C and pH 7.0. Metalchelating agents inhibited the production of secondary alcohols. The activities for the hydroxylation of n-alkanes in various methylotrophic bacteria were localized in the cell-free particulate fractions precipitated by centrifugation between 10,000 and 40,000 x g. Both oxygen and reduced nicotinamide adenine dinucleotide were required for hydroxylation activity. The metal-chelating agents inhibited hydroxylation of n-alkanes by the particulate fraction, indicating the involvement of a metal-containing enzyme system in the oxidation of n-alkanes. The production of 2-propanol from the corresponding n-alkane by the particulate fraction was inhibited in the presence of methane, suggesting that the subterminal hydroxylation of n-alkanes may be catalyzed by methane monooxygenase.

Microbial oxidation of gaseous hydrocarbons. II. Hydroxylation of alkanes and epoxidation of alkenes by cell-free particulate fractions of methane-utilizing bacteria

Cell-free particulate fractions derived from methylotrophic bacteria catalyze the oxygen- and reduced nicotinamide adenine dinucleotide-dependent epoxidation of alkenes and hydroxylation of alkanes. Evidence presented indicates that the hydroxylation and epoxidation reactions are catalyzed by the same or a similar metal-containing monooxygenase.

Microbial oxidation of gaseous hydrocarbons: epoxidation of C2 to C4 n-alkenes by methylotrophic bacteria.

Over 20 new cultures of methane-utilizing microbes, including obligate (types I and III) and facultative methylotrophic bacteria were isolated. In addition to their ability to oxidize methane to methanol, resting cell-suspensions of three distinct types of methane-grown bacteria (Methylosinus trichosporium OB3b [type II, obligate]; Methylococcus capsulatus CRL M1 NRRL B-11219 [type I, obligate]; and Methylobacterium organophilum CRL-26 NRRL B-11222 [facultative]) oxidize C2 to C4 n-alkenes to their corresponding 1,2-epoxides. The product 1,2-epoxides are not further metabolized and accumulate extracellularly. Methanol-grown cells do not have either the epoxidation or the hydroxylation activities. Among the substrate gaseous alkenes, propylene is oxidized at the highest rate. Methane inhibits the epoxidation of propylene. The stoichiometry of the consumption of propylene and oxygen and the production of propylene oxide is 1:1:1. The optimal conditions for in vivo epoxidation are described. Results from inhibition studies indicate that the same monooxygenase system catalyzes both the hydroxylation and the epoxidation reactions. Both the hydroxylation and epoxidation activities are located in the cell-free particulate fraction precipitated between 10,000 and 40,000 x g centrifugation.

On the nature of the presumed receptor for IgE on mast cells. V. Enhanced binding of 125I-labeled IgE to cell-free particulate fractions in the presence of protease inhibitors.

Cell-free particulate preparations derived from sonicated, purified rat peritoneal mast cells can be radiolabeled with IgE. The IgE concentration-dependence and the kinetics of the binding reaction at pH 6.6 were very similar to those observed with intact mast cells. The number of IgE molecules bound per mast cell equivalent at saturation varied somewhat from one particulate preparation to the next and, on average, was about 50% of the binding capacity of the intact cells. Incubation of intact mast cells or of particulate fractions with low concentrations of IgE in serum-free media resulted in a decrease in the amount of bindable IgE which remained in the supernatants in excess over the amount of IgE which was bound to the cells. Specific binding of IgE to both particles and to intact cells at limiting IgE concentrations was stimulated up to approximately twofold by a variety of antibiotic, synthetic or high molecular weight (protein) protease inhibitors of which soybean trypsin inhibitor and p-nitrophenyl-p'-guanidinobenzoate were the most active. These substances also markedly protected the IgE, leaving more of it bindable to rat basophil leukemic cells in a second incubation. Binding to particulate fractions also occurred at pH 4.8 when bovine serum albumin was added to the incubations. The results are consistent with the view that normal peritoneal mast cells have a membrane-bound protease which has high selectivity for IgE.

Methane production by cell-free particulate fraction of rumen bacteria

Rumen bacteria retained methanogenic activity when stored at −60° under H 2. This activity, which resides in Methanobacterium ruminantium and Methanobacterium mobilis , is not lost when the cells are broken, as has been suggested. Unlike in Methanosarcina barkerii and Methanobacterium M.o.H. , in rumen bacteria methanogenic enzymes are not soluble but readily precipitated at 15,000 g. Methane was synthesized from tetrahydrofolate derivatives but at slower rates than from CO 2. From the data, it was not possible to determine if methyl- and methylene tetrahydrofolate were oxidized to CO 2 prior to reduction to CH 4. In room light, CH 3-B 12 was reduced to CH 4 non-enzymatically in the presence of protein. When the reactions were carried out in the dark, very little CH 4 was formed from CH 3-B 12 by rumen bacterial enzymes. The cell-free particulate fraction did not require added ATP for methanogenesis but showed an absolute requirement for H 2.

Oxidation of C1 Compounds by Particulate fractions from Methylococcus capsulatus: distribution and properties of methane-dependent reduced nicotinamide adenine dinucleotide oxidase (methane hydroxylase)

Cell-free particulate fractions of extracts from the obligate methylotroph Methylococcus capsulatus catalyze the reduced nicotinamide adenine dinucleotide (NADH) and O2-dependent oxidation of methane (methane hydroxylase). The only oxidation product detected was formate. These preparations also catalyze the oxidation of methanol and formaldehyde to formate in the presence or absence of phenazine methosulphate with oxygen as the terminal electron acceptor. Methane hydroxylase activity cannot be reproducibly obtained from disintegrated cell suspensions even though the whole cells actively respired when methane was presented as a substrate. Varying the disintegration method or extraction medium had no significant effect on the activities obtained. When active particles were obtained, hydroxylase activity was stable at 0 C for days. Methane hydroxylase assays were made by measuring the methane-dependent oxidation of NADH by O2. In separate experiments, methane consumption and the accumulation of formate were also demonstrated. Formate is not oxidized by these particulate fractions. The effects of particle concentration, temperature, pH, and phosphate concentration on enzymic activity are described. Ethane is utilized in the presence of NADH and O2. The stoichiometric relationships of the reaction(s) with methane as substrate were not established since (i) the presumed initial product, methanol, is also oxidized to formate, and (ii) the contribution that NADH oxidase activity makes to the observed consumption of reactants could not be assessed in the presence of methane. Studies with known inhibitors of electron transport systems indicate that the path of electron flow from NADH to oxygen is different for the NADH oxidase, methane hydroxylase, and methanol oxidase activities.

Effects of serotonin and catecholamines on the adenylate cyclase of molluscan heart

1. 1. Sensitivity to serotonin, dopamine and noradrenaline of membrane-bound adenylate cyclase has been studied in cell-free particulate fractions obtained from mussel ( Anodonta cygnea) and snail ( Helix pomatia) hearts. 2. 2. In freshly made preparations the basal level of adenylate cyclase was relatively high and a significant neurohormonal activation occurred only after depletion of biogenic amines with reserpine or following storage for 24 hr at 4°C in molluscan Ringer. 3. 3. Serotonin and dopamine activated cardiac adenylate cyclase in both species, whereas noradrenaline increased adenylate cyclase activity in the snail and inhibited it in mussel heart.

Subcellular localization of the leucine biosynthetic enzymes in yeast.

When baker's yeast spheroplasts were lysed by mild osmotic shock, practically all of the isopropylmalate isomerase and the beta-isopropylmalate dehydrogenase was released into the 30,000 x g supernatant fraction, as was the cytosol marker enzyme, glucose-6-phosphate dehydrogenase. alpha-Isopropylmalate synthase, however, was not detected in the initial supernatant, but could be progressively solubilized by homogenization, appearing more slowly than citrate synthase but faster than cytochrome oxidase. Of the total glutamate-alpha-ketoisocaproate transaminase activity, approximately 20% was in the initial soluble fraction, whereas solubilization of the remainder again required homogenization of the spheroplast lysate. Results from sucrose density gradient centrifugation of a cell-free particulate fraction and comparison with marker enzymes suggested that alpha-isopropylmalate synthase was located in the mitochondria. It thus appears that, in yeast, the first specific enzyme in the leucine biosynthetic pathway (alpha-isopropylmalate synthase) is particulate, whereas the next two enzymes in the pathway (isopropylmalate isomerase and beta-isopropylmalate dehydrogenase) are "soluble," with glutamate-alpha-ketoisocaproate transaminase activity being located in both the cytosol and particulate cell fractions.

In vitro product of an RNA polymerase induced in broadbean by infection with broadbean mottle virus

The RNA polymerase activity of a cell-free particulate fraction extracted from broadbean leaves infected with broadbean mottle virus was investigated. The RNA product labeled in vitro during a brief pulse of [ 3H]UTP in the presence of actinomycin D and exogenous RNA was shown by reannealing interference to have sequences homologous to parental viral RNA. The product was fractionated in 2 M LiCl and analysed by centrifugation in sucrose gradients. The major part of the acid-insoluble radioactivity did not precipitate in 2 M LiCl and was largely resistant to pancreatic ribonuclease in high salt. Some of the radioactivity associated with the LiCl-insoluble fraction of a 1.75-min pulse-labeled product was resistant to ribonuclease in high salt and had sedimentation properties expected for a replicative intermediate. After a 1-min chase, more of the LiCl-insoluble product became sensitive to ribonuclease and a portion of it sedimented in the region of viral RNA.

Properties of Single-stranded RNA Synthesized by a Crude RNA Polymerase Fraction from Barley Leaves Infected with Brome Mosaic Virus

SUMMARY The RNA polymerase activity of a cell-free particulate fraction of barley leaves infected with brome mosaic virus was investigated in the presence of actinomycin D and EDTA. The RNA products were fractionated according to their solubility in 2 m-LiCl. Both LiCl-soluble and LiCl-insoluble fractions of the RNA synthesized during a 2 min. pulse of [3H]-UTP were largely resistant to RNase in high salt concentration; the radioactive RNA of the insoluble fraction had sedimentation properties expected for replicative intermediates. After a 2 min. chase, LiCl-insoluble radioactivity was essentially RNase-sensitive in high salt, and sedimented in sucrose gradients in association with all three components of brome mosaic virus RNA, provided protective exogenous RNA was added throughout the experimental procedure. The overall results suggest that the in vitro synthesis of the RNAs found in virus particles occurs by a continuous process resulting possibly from recycling activity of the polymerase.

Biosynthesis of sphingolipid bases: IV. The biosynthetic origin of sphingosine in Hansenula ciferri

The synthesis and properties of 3-ketodihydrosphingosine-3- 14C(VIII) are described. Cell-free particulate fractions from the yeast Hansenula ciferri convert VIII exclusively to dihydrosphingosine; no 3-ketosphingosine or sphingosine could be detected. Similar fractions convert, 14C-ammonium palmitate to trams-2-hexadecenoic acid indicating that, in yeast, the biosynthetic pathways of saturated and unsaturated sphingolipids diverge at the fatty acyl CoA level. Substrate preferences for the enzyme(s) which mediates the condensation of serine with fatty acyl CoA to produce ketosphingolipid bases are: palmityl CoA > stearyl CoA > myristyl CoA > decanoyl CoA > lauryl CoA. Under appropriate conditions trans-2-hexadecenoyl CoA is an excellent substrate and, like palmityl CoA, leads to a mixture of sphingosine and dihydrosphingosine. α-Hydroxypalmityl CoA is not a substrate for the condensing enzyme and appears, therefore, not to be a precursor of phytosphingosine in yeast preparations. Most of the fatty acyl CoA derivatives tested are potent inhibitors of the condensing enzyme; bovine serum albumin is effective in reducing this inhibition.

Biosynthesis of Cardiolipin in Escherichia coli

Cell-free particulate fractions from Escherichia coli have been found to catalyze the synthesis of cardiolipin from cytidine diphosphate diglyceride and l -glycerol 3-phosphate. Evidence is presented that phosphatidylglycerol is the immediate precursor of cardiolipin in a reaction involving the transfer of a phosphatidyl moiety from CDP-diglyceride.

Studies on thermophilic sulfate-reducing bacteria. II. Hydrogenase activity of Clostridium nigrificans.

Akagi, J. M. (Western Reserve University, Cleveland, Ohio) and L. Leon Campbell. Studies on thermophilic sulfate-reducing bacteria. II. Hydrogenase activity of Clostridium nigrificans. J. Bacteriol. 82:927-932. 1961.-The hydrogenase of Clostridium nigrificans has been found to be associated with the cell-free particulate fraction which can be sedimented at 105,000 x g in 1 hr. The specific activity of this fraction was increased 2 to 3 fold over that of the crude extract. It was not found possible to liberate the enzyme from the particulate fraction by methods of enzymatic digestion, chemical extraction, or physical disruption. The optimum temperature for H(2) utilization using benzyl viologen as an electron acceptor was found to be 55 C, and the optimum pH range was 7 to 8. Employing metal complexing agents it was found that the enzyme required Fe(++) ions for H(2) utilization. In contrast, Fe(++) ions were not required to catalyze the evolution of H(2) from reduced methyl viologen. The role of Fe(++) ions in the hydrogenase activity of this organism is discussed.