Crude Cell-free Extracts Research Articles

Reversible inhibition of bacterial bioluminescence by long-chain fatty acids

Long-chain unsaturated fatty acids, as well as certain saturated fatty acids such as lauric acid, are inhibitors of the in vivo luminescence of wild-type strains of four species of luminous bacteria (Beneckea harveyi, Photobacterium phosphoerum, P. fischeri, andP. leiognathi) as well as the myristic acid-stimulated luminescence in the aldehyde dim mutant M17 ofB. harveyi. Based on studies with the system in vivo, the principal site of action of all the fatty acids appears to be the reductase activity that converts myristic acid to myristyl aldehyde. This was confirmed by in vitro studies: Reductase activity in crude cell-free extracts is strongly inhibited by oleic acid.

Kinetic parameters of lactate dehydrogenases of some rumen bacterial species, the anaerobic ciliate Isotricha prostoma and mixed rumen microorganisms.

A number of kinetic parameters of the lactate dehydrogenases of three rumen bacterial species (Peptostreptococcus productus, Propionibacterium acnes and Actinomyces viscosus), the rumen ciliate Isotricha prostoma and mixed rumen microorganisms (MRM) with respect to NADH, pyruvate, fructose-1,6-diphosphate (FDP) as well as the effects of several nucleotide phosphates were studied. Partially purified LDH of Peptostr. productus had the same kinetic parameters as in crude cell free extracts. Values for Km, determined by Michaelis-Menten kinetics with pyruvate as the substrate, were in the same range for all lactate dehydrogenases. After feeding a cow, changes in the apparent Km and Vmax values for NADH of the total LDH activity in MRM were followed. It is suggested that of the factors studied the ratio NADH/NAD(H) and ATP are the most important regulatory factors for the lactate dehydrogenases of mixed rumen microorganisms.

The purification of inosine 5′-monophosphate dehydrogenase from Escherichia coli by affinity chromatography on immobilized Procion dyes

The application of a number of immobilized Procion dyes to the purification of inosine 5′-monophosphate dehydrogenase (EC 1.2.1.14) from Escherichia coli has been investigated. The enzyme is adsorbed to a number of these immobilized dyes and can be eluted by salt gradients with very substantial increases in specific activity. For example, adsorption of the enzyme from a crude cell-free extract of E. coli to immobilized Procion yellow MX-8G in the presence of 15% ( v v ) ethylene glycol and subsequent elution with a salt gradient yields an enzyme preparation approximately 90% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is quantitatively recovered with an overall increase in specific activity of 14-fold compared to the enzyme in the cell-free extract.

Ribulose-1,5-bisphosphate Carboxylase from Rhodomicrobium vannielii

The effect of carbon growth substrate on the level of ribulose-1,5-bisphosphate (RuBP) carboxylase in crude cell-free extracts of Rhodomicrobium vannielii (RM5) was investigated. The highest specific activity followed growth with sodium hydrogen malate as carbon source. Cells grown autotrophically or on the reduced carbon substrates, butyrate and ethanol, gave extracts of lower activity. RuBP carboxylase was purified from R. vannielii (RM5) grown on a mixture of pyruvate and malate as carbon source. The enzyme was homogeneous as judged by electrophoresis on polyacrylamide gels of several acrylamide concentrations and had an apparent molecular weight of 430000 as measured by gel filtration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis suggested the presence of three subunit types of molecular weights 56200, 53300 and 15700. Mg2+ was required for maximum enzyme activity although this could be completely replaced by Ni2+ and, to a lesser extent, by Mn2+ or Co2+. As with other high molecular weight RuBP carboxylases, the enzyme was sensitive to inhibition by 6-phosphogluconate.

Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum.

In crude cell-free extracts of Azotobacter chroococcum, nitrogenase was much less sensitive to irreversible inactivation by O2 than was the purified enzyme. When nitrogenase was partially purified by anaerobic discontinuous sucrose-density-gradient centrifugation, O2-tolerance was retained. This preparation was considerably enriched in four polypeptides, three of which were derived from the Mo-Fe(molybdenum-iron) protein and Fe (iron) protein of nitrogenase. The fourth was purified to homogeneity and shown to be an iron-sulphur protein (mol.wt. 14000) probably containing a 2Fe--2S centre. When this protein was added to purified nitrogenase, the enzyme was rendered O2-tolerant, through stabilization was Mg2+-dependent. The isolated O2-tolerant nitrogenase was an equimolar stoicheiometric complex between the MO--Fe, Fe and protective proteins. It is likely that the formation of this complex in vivo is the mechanism of 'conformational protection' in this organism.

A Ceratocystis Associated with an Ambrosia Beetle in Dendroctonus-Killed Pines

Hirsch, H. M. 1954. Environmental factors influencing the differentiation of protoperithecia and their relation to tyrosinase and melanin formation in Neurospora crassa. Physiol. PI. (Copenhagen) 7: 72-97. Leonard, T. J. 1971. Phenoloxidase activity and fruiting body formation in Schisophyllum coZmmune. J. Bactcriol. 106: 162-167. . 1972. Phenoloxidase activity in mycelia carrying modifier mutations that affect sporocarp development in Schizophylluml coltmmune. J. Bacteriol. 111: 292-293. --, and L. E. Phillips. 1973. Study of phenoloxidase activity during the reproductive cycle in Schizophyllum commune. J. Bacteriol. 114: 7-10. Leslie, J. F. 1979. Genetic and physiologic aspects of monokaryotic fruiting in the basidiomycetous fungus Schisophylllm commune. Ph.D. thesis, University of Wisconsin-Madison, Madison, Wisconsin. --, and T. J. Leonard. 1979. Three independent genetic systems that control initiation of a fungal fruiting body. Molec. Gen. Genet. 171: 257-260. Perkins, J. H., and J. R. Raper. 1970. Morphogenesis in Schizophyllum colmmune: III. A mutation that blocks initiation of fruiting. Molec. Ge. Genet. 106: 151-154. Phillips, L. E. 1974. Studies of phenoloxidase activity during development in Schizophyllum co01mmun7e. Ph.D. thesis, University of Kentucky, Lexington, Kentucky. --, and T. J. Leonard. 1976a. Extracellular and intracellular phenoloxidase activity during growth and development in Schisophyllum. Mycologia 68: 268-276. --, and . 1976b. Benzidene as a substrate for measuring phenoloxidase activity in crude cell-free extracts of Schizophyllum commune. Mycologia 68: 277-285. Raper, J. R., and P. G. Miles. 1958. The genetics of Schizophyllum, . Genetics 43: 530-546.

Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli.

NAD(+)-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose-hexane-NAD(+) and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD(+) and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.

Further studies on a human lung tumor-associated antigen. Comparison of antigens from different tumors.

A human lung tumor-associated antigen was purified to homogeneity from a crude cell-free extract of a human lung adenocarcinoma using standard biochemical procedures. In order to facilitate monitoring the recovery of antigen, trace amounts of previously purified and radioiodinated antigen from another lung tumor were added to the crude extract. The purified antigen was a glycoprotein and contained sialic acid. The antigen had a molecular weight of 76,000 and appeared to contain three subunits, each with a molecular weight of 25,000. The antigen had the following physical properties: Stokes radius, 39.4 A; S20,w, 4.24 S; D20,w, 5.15 x 10(-7) cm2 S-1; and a frictional ratio of 1.40. In addition, the purified, radioiodinated antigen retained complete immune reactivity since it could be quantitatively precipitated with specific immune serum. All of these properties were in close agreement with the properties of another antigen which was purified from a separate human lung tumor. Thus, it appeared from the biochemical and immunochemical criteria presented in this report that a common and identical antigen was isolated from two distinct human lung tumor extracts.

Nitroalkane oxidation by streptomycetes.

Crude cell-free extracts of nine strains of Streptomyces tested for nitroalkane-oxidizing activity showed production of nitrous acid from 2-nitropropane, 1-nitropropane, nitroethane, nitromethane, and 3-nitropropionic acid. These substrates were utilized in most strains but to a decreasing extent in the order given, and different strains varied in their relative efficiency of oxidation. p-Nitrobenzoic acid, p-aminobenzoic acid, enteromycin, and omega-nitro-l-arginine were not attacked. d-Amino acid oxidase, glucose oxidase, glutathione S-transferase, and xanthine oxidase, enzymes potentially responsible for the observed oxidations in crude cellfree extracts, were present at concentrations too low to play any significant role. A nitroalkane-oxidizing enzyme from streptozotocin-producing Streptomyces achromogenes subsp. streptozoticus was partially purified and characterized. It catalyzes the oxidative denitrification of 2-nitropropane as follows: 2CH(3)CH(NO(2))CH(3) + O(2) --> 2CH(3)COCH(3) + 2HNO(2). At the optimum pH of 7.5 of the enzyme, 2-nitropropane was as good a substrate as its sodium salt; t-nitrobutane was not a substrate. Whereas Tiron, oxine, and nitroxyl radical acted as potent inhibitors of this enzyme, superoxide dismutase was essentially without effect. Sodium peroxide abolished a lag phase in the progress curve of the enzyme and afforded stimulation, whereas sodium superoxide did not affect the reaction. Reducing agents, such as glutathione, reduced nicotinamide adenine dinucleotide, and nicotinamide adenine dinucleotide phosphate, reduced form, as well as thiol compounds, were strongly inhibitory, but cyanide had no effect. The S. achromogenes enzyme at the present stage of purification is similar in many respects to the enzyme 2-nitropropane dioxygenase from Hansenula mrakii. The possible involvement of the nitroalkane-oxidizing enzyme in the biosynthesis of antibiotics that contain a nitrogen-nitrogen bond is discussed.

Superoxide dismutase and hydrogen peroxide formation in Campylobacter sputorum subspecies bubulus

Cell-free extracts of Campylobacter sputorum subspecies bubulus contained superoxide dismutase. The enzyme was located in the cytoplasmic fraction and insensitive to cyanide. After centrifuging a cell-free extract at 144000 x g for 1.5 h the total activity in the supernatant fraction was threefold higher than in the crude cell-free extract. The pellet fraction thus obtained was shown to have a lowering effect on superoxide dismutase activities from different sources in the assay method used here. C. sputorum responded to a raised oxygen tension in the culture by an increase in the superoxide dismutase activity. The ability to produce superoxide anion radicals (O2-.) during oxidation of formate and lactate was demonstrated. Furthermore C. sputorum was found to produce H2O2 while oxidizing formate. In experiments in which the reduction of cytochrome c by formate was followed, step-wise kinetics were observed. One of the steady states then obtained was attributed to the oxidizing action of H2O2, because it was abolished by the addition of catalase and lengthened by H2O2 added in addition to H2O2 formed as a product of formate oxidation. An overall reaction for formate oxidation by C. sputorum is discussed.

Oxygen and the Metabolism of Peptostreptococcus anaerobius VPI4330-1

Peptostreptococcus anaerobius VPI4330-1 is an anaerobic organism which has no superoxide dismutase, catalase or peroxidase. It can be protected from the toxic effects of oxygen by catalase in the culture medium. In order to elucidate its mechanisms of oxygen tolerance, the effect of oxygen on the metabolic activity of the organism was studied. In salt solution supplemented with glucose or pyruvate the organism had a more rapid metabolic rate under aerobic conditions than under anaerobic conditions. There were also significant differences in metabolic end-products obtained under aerobic and anaerobic conditions. The crude cell-free extract had NADH oxidase activity, which reduced oxygen to water, and NADPH oxidase activity, which reduced oxygen to superoxide radicals and hydrogen peroxide. The former specific activity was much higher than the latter. The results indicate that the main product of intracellular oxygen reactions was water. Deleterious products such as superoxide radicals and hydrogen peroxide were only formed to a limited extent. NADH oxidase may fulfil an important protective role in this organism.

Characterization and in vitro processing of transfer RNA precursors accumulated in a temperature-sensitive mutant of Escherichia coli

A temperature-sensitive mutant (TS241) of Excherichia coli was shown to be defective in RNAase P activity. In this mutant, many precursor molecules of E. coli transfer RNAs accumulated at the restrictive temperature. These precursors were purified by two-dimensional polyacrylamide pel electrophoresis, and characterized by fingerprint analysis and by in vitro cleavage reactions using E. coli cell-free extracts. There were two types of monomeric tRNA precursors which had extra nucleotides at the 5′ and 3′ termini of single tRNA molecules; the smaller monomeric precursors (4 to 4.5 S) carried short segments of extra nucleotides at the 5′ termini of tRNA molecules and represented the predominant species of tRNA precursors accumulated in the mutant, while the larger monomeric precursors (4.5 to 5 S) carried longer 5′ leader segments. The majority of the larger monomeric precursors had ribonucleoside triphosphates at their 5′ termini, whereas most of the smaller precursors had ribonucleoside monophosphates at the 5′ ends. In addition to the monomeric precursors, various multimeric tRNA precursors containing two or more tRNA sequences within a single molecule were detected. Many of the multimeric precursors, except some dimeric precursors, did not accumulate as stably as the monomeric precursors in the mutant. The modes of the in vitro processing of these precursors were studied using crude cell-free extracts or purified enzyme preparations. The results showed that the precursor molecules were converted to mature size molecules in specific manners and that at least three different enzymatic activities were involved in the process.

Purification and properties of trehalase from the thermophilic fungus Humicola lanuginosa

Trehalase (α,α-Trehalosee gludohydrolase, EC 3.2.1.28) was partially solubilized from the thermophilic fungus Humicola lanuginosa RM-B, and purified 184-fold. The purified enzyme was optimally active at 50°C in acetate buffer at pH 5.5. It was highly specific for α,α-trehalose and had an apparent K m = 0.4 mM at 50°C. None of the other disaccharides tested either inhibited or activated the enzyme. The molecular weight of the enzyme was around 170000. Trehalase from mycelium grown at 40 and 50°C had similar properties. The purified enzyme, in contrast to that in the crude-cell free extract, was less stable. At low concentration, purified trehalase was afforded protection against heat-inactivation by “protective factor(s)” present in mycelial extracts. The “protective factor(s)” was sensitive to proteolytic digestion. It was not diffusable and was stable to boiling for at least 30 min. Bovine serum albumin and casein also protected the enzyme from heat-inactivation.

The enzymes of proline biosynthesis in Escherichia coli. Their molecular weights and the problem of enzyme aggregation.

1. By using Bio-Gel A1.5M and Sephadex G-150 columns, crude cell-free extracts of Escherichia coli were fractionated to demonstrate the existence of a proline-biosynthetic aggregate. 2. Sephadex G-150 resolves two glutamyl kinases that are inhibited by proline, with mol.wts. of 125000 and 38000, the reactions of which are Mg2+-dependent. The heavier species is more sensitive to inhibition by proline. 3. Gamma-Glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase (EC 1.5.1.2) have mol.wts. of approx. 125000 and 190000 respectively, the specific activity of the latter being 5 X 10(3)-fold greater than either of the other two biosynthetic enzymes or of the total pathway in vivo. 4. Bio-Gel A1.5M chromatography gave a single glutamyl kinase of mol.wt. 250000, and the possibility of this being a constituent of an enzyme complex is discussed.

Glucose-6-phosphate dehydrogenase of Anabaena sp. Kinetic and molecular properties.

The kinetic and molecular properties of cyanobacterial glucose-6-phosphate dehydrogenase, partly purified from Anabaena sp. ATCC 27893, show that it undergoes relatively slow, reversible transitions between different aggregation states which differ in catalytic activity. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis reveal three pincipal forms, with approximate molecular weights of 120 000 (M1), 240 000 (M2) and 345 000 (M3). The relative catalytic activities are: M1 less than M2 less than M3. In concentrated solutions of the enzyme, the equilibrium favors the more active, oligomeric forms. Dilution in the absence of effectors shifts the equilibrium in favor of the M1 form, with a marked diminution of catalytic activity. This transition is prevented by a substrate, glucose-6-phosphate, and also by glutamine. The other substrate, nicotinamide adenine dinucleotide phosphate (NADP+), and (in crude cell-free extracts) ribulose-1,5-diphosphate are negative effectors, which tend to maintain the enzyme in the M1 form. The equilibrium state between different forms of the enzyme is also strongly dependent on hydrogen ion concentration. Although the optimal pH for catalytic activity is 7.4, dissociation to the hypoactive M1 form is favored at pH values above 7; a pH of 6.5 is optimal for maintenance of the enzyme in the active state. Reduced nicotamide adenine dinucleotide phosphate (NADPH) and adenosine 5'-triphosphate (ATP), inhibit catalytic activity, but do not significantly affect the equilibrium state. The relevance of these findings to the regulation of enzyme activity in vivo is discussed.

Migration Inhibition by an Agarose Microdroplet Assay: Monitoring of Tumor-Associated Antigens on a Simian Virus 40-Induced Sarcoma

Macrophage migration inhibition assays, with a direct agarose microdroplet method, were used to monitor TAA activity of preparations of SV-40-induced mKSA cells. These preparations included cell-free crude membranes, papain-solubilized and NP40 detergent-solubilized membrane extracts from mKSA tumor cells. The assay was extremely sensitive and could detect migration inhibition reactivity with all three types of antigenic preparations with concentrations as low at 250 ng protein/ml. The reactivities were quite reproducible from experiment to experiment using the same or different lots of these antigen preparations, and the reactivities were specific in that peritoneal exudate cells from BALB/c mice, immunized with antigenically unrelated but syngeneic plasmacytomas, were not inhibited by these antigens. The results demonstrated the usefulness of this assay in rapidly detecting small concentrations of partially purified TAA preparations by using small number of immune cells.

Oxygen-dependent inactivation of gramicidin S synthetase in Bacillus brevis.

Incorporation of L-[14C]ornithine into gramicidin S by crude, unfractionated lysozyme extracts of Bacillus brevis ATCC 9999 was shown to represent the activity of the gramicidin synthetase complex. Frozen-thawed cells were the source of active extracts, but when cells were shaken in air at 37 degrees C, they rapidly lost activity in a first-order reaction with a half-life of 13 min. Protease inhibitors and inhibitors of energy metabolism had no effect on the inactivation process in frozen-thawed cells. Stabilization was achieved when the cells were shaken in nitrogen or helium instead of air. The addition of dithiothreitol produced a moderate degree of stabilization. The L-ornithine- and D-phenylalanine-activating activities of the gramicidin S synthetase complex were also lost during aeration of the cells. Crude cell-free extracts also lost activity when they were shaken in oxygen, but, in this case, inactivation was slower (half-life of 80 min). Nitrogen also stabilized these cell-free extracts.

A Carbon Monoxide-Binding Hemoprotein Formed by Heme Accumulation in Escherichia coli

A CO-binding hemochrome was accumulated in Escherichia coli cells, when intracellular heme concentration was increased by aerobic incubation of resting cell suspensions with ALA. Reduced minus oxidized difference spectrum of the hemochrome showed peaks at 560, 530, and 430 nm and a shoulder at 575 nm. The peaks of CO reduced minus reduced difference spectrum were located at 572, 540, and 422 nm. The CO spectrum was similar to but not identical with the spectrum of cytochrome o, a known terminal oxidase in E. coli. SDS-polyacrylamide gel electrophoresis of the CO-binding hemochrome showed its molecular weight to be about 33,000. The hemochrome in crude cell-free extracts was oxidized by aeration and reduced by the addition of succinate or NADH. The reduction by succinate was inhibited by inhibitors of succinate dehydrogenase [EC 1.3.99.1], and the reduction by NADH was inhibited by 2-heptyl-4-hydroxy-quinolin-N-oxide, which is an inhibitor of electron transport in E. coli cells.

Isolation and Characterization of a Membrane-associated Thiosulphate-oxidizing System of Thiobacillus novellus

Summary: A thiosulphate-oxidizing system of Thiobacillus novellus grown on thiosulphate was studied using intact cells, crude cell-free extracts and isolated membrane vesicles. All active preparations oxidized thiosulphate to sulphate, without intermediary accumulation of polythionates, consuming two moles of oxygen for every mole of thiosulphate oxidized. Less active cell-free preparations required reduced glutathione, NADH or sulphite for thiosulphate oxidation. An active membrane-associated thiosulphate-oxidizing system from crude cell-free extracts was isolated by either Sepharose 4B column chromatography or differential centrifugation. The isolated multi-enzyme complex system exhibited high specificity for thiosulphate with an apparent K m value of 0·12 mm. Enzyme activity was optimum at pH 7·5 and 25 °C. The system was sensitive to oxygen, storage at certain temperatures and freezing. Inhibition studies indicated that active sulphydryl groups and an electron transport chain were involved during thiosulphate oxidation. Electron micrographs of active crude extracts and the isolated membrane complex after negative staining showed the presence of spherical structures with a diameter of about 0·1 to 0·4 μm. An ultra-thin section of the membrane complex revealed that the large spherical particles observed in the negatively stained preparations were aggregated structures consisting of smaller vesicles 0·02 to 0·05 μm in diameter.

Differential binding of cyclic adenosine 3' ,5'-monophosphate to the cyclic adenosine 3' ,5'-monophosphate receptor protein in Escherichia coli

Binding of cyclic adenosine 3' ,5'-monophosphate (cAMP) by the cAMP receptor protein in crude cell-free extracts of Escherichia coli was characterized. When cell were grown in glucose, binding was inhibited 50% relative to extracts from cells grown with succinate as carbon source . This inhibition could be relieved by dialysis.