Crude Cell-free Extracts Research Articles

Isolation of proteins comprising native gene-specific messenger ribonucleoprotein particles using paramagnetic beads

A method for the rapid isolation of proteins comprising native messenger ribonucleoprotein particles (mRNPs), that utilises streptavidin-coated paramagnetic beads, coupled with biotinylated oligodeoxyribonucleotides, to pull down general and transcript-specific mRNP complexes from crude cell-free extracts, is described. Two biotinylated oligo probes were tested for their efficacy in isolating mRNPs from tobacco pollen extracts; a 25-mer oligo(dT) probe for the isolation of total mRNPs, and a transcript-specific oligo complementary to the pollen-specific ntp303 mRNA sequence. Following annealing to the target mRNAs, the oligo probes, with associated ribonucleoprotein particles, were captured by streptavidin-coated paramagnetic particles and pelleted in a magnetic field. After several washing steps, the bound RNPs were released and analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot immunodetection. This one-step mRNP enrichment method could be used either to enhance the detection of low-abundance mRNA-binding proteins in subsequent northwestern and RNA UV cross-linking experiments or the direct identification and biochemical characterisation of RNA-binding proteins.

The intracellular pH of the thermophilic bacterium Thermoanaerobacter wiegelii during growth and production of fermentation acids.

The thermophilic glycolytic anaerobe Thermoanaerobacter wiegelii grows over the pH range 5.1-7.7, and no growth is observed below pH 5.0 or above 7.7. When T. wiegelii was grown in pH-uncontrolled batch culture, glucose was fermented to ethanol, acetate, and lactate. Small amounts of lactic acid were produced once the external pH reached 6.0, and a fructose-1.6-diphosphate (FDP) activated lactate dehydrogenase (LDH) was detected in cell-free crude extracts. Maximal activation of LDH by FDP was observed at pH 6.2. As the pH of the medium declined from 6.7 to 5.1 due to the production of acetate and lactate, the total protonmotive force (deltap) remained between - 110 and - 130mV, and the membrane potential (dekltapsi) decreased from -104 to -65mV. This decrease in deltapsi was paralleled by an increase in the chemical gradient of protons (ZdeltapH) from -31 to -62mV at pH 5.1. Based on these results, T. wiegelii maintained a small deltapH (0.3-0.9 units, inside alkaline) as the medium pH declined and interconverted deltapsi to ZdeltapH to maintain the total deltap relatively constant. Intracellular potassium decreased from 150 mM at pH 6.70 to 50 mM at pH 5.1, and this represented a 33-mV decline in the transmembrane chemical potential of potassium. The ability to synthesize ATP remained constant as the external pH declined, and therefore metabolic energy per se was not a critical aspect of pH sensitivity.

Enzymes Leading to the Nucleotide Sugar Precursors for Exopolysaccharide Synthesis in Burkholderia cepacia

Based on the chemical composition of the exopolysaccharide produced by the cystic fibrosis bacterial isolate Burkholderia cepacia IST408, we postulated and confirmed, based on the specificity of enzymes detected in crude cell-free extracts, the pathway leading to the presumptive activated sugar precursors: UDP-D-glucose, UDP-D-galactose, UDP-D-glucuronic acid, GDP-D-mannose, and GDP-D-rhamnose. Results also suggest that regulation of the expression of the mucoid phenotype in B. cepacia does not occur at the level of synthesis of any of these enzymes.

Partial purification of proteins involved in the bioconversion of arteannuin B to artemisinin

Artemisinin is an important antimalarial drug that is presently being used for controlling the resurgence of malaria. Attempts have been made to partially purify the proteins involved in the bioconversion of arteannuin B to artemisinin by affinity chromatography and precipitation techniques. Our studies indicate that a Tris–HCl buffer system is better than a phosphate buffered saline buffer system. Highest specific activity (0.1 units/mg) with crude cell-free extract has been obtained with this system. A threefold and fivefold increase in specific activity was observed when the proteins were partially purified by acetone precipitation and affinity chromatography, respectively. This is the first report on the study of proteins involved in the bioconversion of arteannuin B to artemisinin.

A simple approach for the simultaneous isolation and immobilization of invertase using crude extracts of yeast and Jack bean meal

Crude cell-free extract of yeast cells was mixed with sufficient amount of Jack bean meal extract so as to precipitate all the invertase. The precipitate was then cross-linked using 2% glutaraldehyde retaining over 60% of the activity. The immobilized invertase could be reused for over ten batches without loss in activity.

Peptide hydrolase system of lactic acid bacteria isolated from Serra da Estrela cheese

Abstract Lactobacillus paracasei subsp. paracasei ESB 230, Leuconostoc mesenteroides subsp. mesenteroides ESB 136, Lactococcus lactis subsp. lactis ESB 117 and Enterococcus faecium ESB 50, previously isolated from certified Serra da Estrela cheeses, were tested for their aminopeptidase, dipeptidyl aminopeptidase, endopeptidase, dipeptidase and carboxypeptidase activities. The crude cell-free extracts (CFE) of Lb. paracasei ESB 230 exhibited the highest aminopeptidase activity, followed by CFE of Leuc. mesenteroides ESB 136 and, at last, by CFE of L. lactis ESB 117; the aminopeptidase activity in CFE of Ent. faecium was practically non-existent. The four CFE studied also showed appreciable carboxypeptidase activities, although these were lower than their dipeptidase counterparts; in addition, their dipeptidyl aminopeptidase and endopeptidase activities were lower than their aminopeptidase activities. Dipeptides consisting of hydrophobic amino acid residues (i.e. leucine, methionine and phenylalanine) were more rapidly attacked by all CFE than those with hydrophilic amino acid residues. The peptide hydrolase system of CFE of Lb. paracasei ESB 230 was qualitatively quite similar to, but quantitatively more active than that of CFE of Leuc. mesenteroides ESB 136 (except for the endopeptidase); additionally, the CFE of L. lactis ESB 117 and of Ent. faecium ESB 50 were quite distinct from each other, and from the other two CFE tested.

WLIP, a lipodepsipeptide of Pseudomonas ‘reactans’ , as inhibitor of the symptoms of the brown blotch disease of Agaricus bisporus

A cell-free crude extract containing the white line inducing principle (WLIP), a lipodepsipeptide produced by Pseudomonas ‘reactans’, could inhibit browning of mushrooms caused by Pseudomonas tolaasii. Mushrooms inoculated with Ps. tolaasii at concentrations of 2·7 × 106 cfu ml−1 or higher showed the symptoms of the disease after 2 d of incubation. Mushroom caps treated with various concentrations of a crude WLIP preparation, and later inoculated with bacterial concentrations higher than the threshold value, did not develop the symptoms of the disease. One milligram of a crude WLIP preparation could block 50% of the symptoms caused by 1·2 × 107 cfu. The inhibition of browning was effective when incubating at low temperatures for 4 d. A suspension containing 1·6 mg ml−1 of pure WLIP was also able to inhibit the symptoms of brown blotch disease induced by 7·6 × 106 cfu ml−1 of Ps. tolaasii.

17Beta-hydroxysteroid dehydrogenases in human bone cells.

Interconversion of estrogens by osteoblasts may play a role in regulating bone mass. As a first step toward exploring this possibility, we investigated the expression and activity of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) in cultured human osteoblasts (HOB) and osteoblast-like osteosarcoma cells (MG63, TE85, and SaOS-2). Significant 17beta-HSD activity was detected in cell-free extracts of all bone cells with oxidation of estradiol to estrone predominating over reduction. Reverse transcription-polymerase chain reaction (RT-PCR) experiments showed that the mRNA for 17beta-HSD I was detectable only in MG63 cells, albeit at low levels, while 17beta-HSD II was present in MG63, TE85, and HOB, but not SaOS-2, and 17beta-HSD III was absent from each bone cell type. 17Beta-HSD IV was the only isoform present in all bone cells analyzed. Further analysis of the expression of 17beta-HSD IV in these bone cells by immunoblotting revealed both the full-length 83 kDa protein and the proteolytic 38 kDa form. The kinetic parameters for estradiol oxidation by purified recombinant 17beta-HSD IV (Km = 49.7 microM, Vmax = 79.4 nmol/minute/mg of protein) and its HSD-domain (Km = 79.4 microM, Vmax = 476 nmol/minute/mg of protein) were significantly higher than previously reported, but consistent with the values obtained with crude cell-free extracts of SaOS-2 cells (Km = 98.8 microM, Vmax = 0.07 nmol/minute/mg of protein) which contain only 17beta-HSD IV based on RT-PCR. These studies show that bone cells have the capacity to interconvert circulating estrogens and suggest that bone cell 17beta-HSDs serve primarily to attenuate the continuing actions of estradiol through conversion to its less potent form, estrone, under certain conditions.

Hypoxanthine, Guanine, Xanthine Phosphoribosyltransferase Activity inCryptosporidium parvum

Doyle, P. S., Kanaani, J., and Wang, C. C. 1998. Hypoxanthine, guanine, xanthine phosphoribosyltransferase activity inCryptosporidium parvum. Experimental Parasitology89,9–15. All parasitic protozoa examined to date are incapable ofde novosynthesis of purine nucleotides and rely on salvage mechanisms for survival. We have identified hypoxanthine, guanine, xanthine phosphoribosyl-transferase activities in crude cell-free extracts ofCryptosporidiumsporulated oocysts utilizing radiolabeled substrates. Guanine, hypoxanthine, and xanthine were converted to their corresponding mononucleotides with specific activities of 346, 280, and 108 nmol/min/ mg protein, respectively. The conversion of the radiolabeled purines was examined in the presence of another, unlabeled, purine base. These competition assays showed that both hypoxanthine and guanine were capable of inhibiting conversion of hypoxanthine, guanine, and xanthine to the corresponding nucleotides. Xanthine had a much lower inhibitory effect on the conversion of guanine and hypoxanthine to the nucleotides, whereas adenine had no effect at all. Autoradio-graphic studies ofCryptosporidium-infected Madin—Darby canine kidney (MDCK) cells showed that radiolabeled hypoxanthine, guanine, and adenine were primarily incorporated by intracellularCryptosporidiumas well as by MDCK nuclei. No apparent incorporation of xanthine by either host cells or intracellular parasites occurred. Radiolabeled glycine and formate were incorporated only into the nuclei of MDCK cells, suggesting a lack ofde novosynthesis of purine nucleotides inCryptosporidium. Radiolabeled hypoxanthine and guanine were also incorporated by excystingCryptosporidiumsporozoites. Altogether, our results indicate the presence of HPRTase, GPRTase, and XPRTase activities. These activities may play an important role in purine salvage, and may localize to a single HGXPRTase enzyme, as in the case ofEimeria, Toxoplasma,and Plasmodium.

ApbA, the Ketopantoate Reductase Enzyme of Salmonella typhimurium Is Required for the Synthesis of Thiamine via the Alternative Pyrimidine Biosynthetic Pathway

The apbA gene of Salmonella typhimurium was shown to encode ketopantoic acid reductase. ApbA was purified from crude cell-free extracts to greater than 95% homogeneity after two chromatographic steps. N-terminal amino acid sequencing (first 15 amino acids) and Western blot analysis confirmed the isolated protein was ApbA. The functional protein was a monomer with a molecular mass of 31.1 kDa. Optimal reaction conditions for the reduction of ketopantoic acid were established at a pH of 6.25, and a temperature of 42 degreesC. The preferred electron source was NADPH, and the apparent Km constants of the enzyme for NADPH and ketopantoic acid were determined to be 0.776 +/- 0.09 mM and 0.742 +/- 0.01 mM, respectively. The homogeneous enzyme had a specific activity of 64.3.

Purification and characterization of tert-butyl ester-hydrolyzing lipase from Burkholderia sp. YY62.

An intracellular novel lipase which can hydrolyze t-butyl octanoate (TBO) was purified to homogeneity from crude cell-free extracts of Burkholderia (formerly Pseudomonas) sp. YY62 with 9% overall yield. Seventy-four-fold purification was achieved by ammonium-sulfate precipitation, three consecutive open-column chromatographies (DEAE anion-exchange, Sepharose CL-6B gel-filtration, and the second DEAE anion-exchange columns), and two HPLCs (TSK G2000SWXL gel-filtration and phenyl 5PW hydrophobic interaction columns). Enzymes hydrolyzing p-nitrophenyl acetate were separated into two peaks (peak I and II) on the hydrophobic HPLC, and only peak II was found to have TBO-hydrolyzing activity. The peak preparation showed a single band of 40 kDa on SDS-PAGE and a molecular mass of 39 kDa on gel-filtration under non-denatured conditions, indicating the monomeric nature of the TBO-hydrolyzing lipase. The lipase showed maximum activity at pH 7.0 and 28 degrees C. The N-terminal 15 amino acid residues were determined as Met-Asp-Phe-Tyr-Asp-Ala-Asn-Glu-Thr-Arg-His-Pro-Glu-Gln-Arg, which showed no homology to known proteins, suggesting that the purified enzyme may belong to a novel class of hydrolase.

Cloning and expression of β-galactosidase gene from Bifidobacterium infantis into Escherichia coli

Bifidobacterium infantis HL96 produces three β-galactosidases (β-gal I, II and III). A genomic bank of B. infantis was constructed in E. coli by using pBR322 as a cloning vector. Two E. coli transformants, BIG1 and BIG4, possessing β-galactosidase activity, were selected from X-gal plates. They contained two different recombinant plasmids with insert DNA fragments of approx. 4.6 and 4.4 kb, respectively. The restriction maps of pBIG1 and pBIG4 were constructed. β-Galactosidases from crude cell-free extracts of B. infantis and of two E. coli recombinants were analyzed by native PAGE and characterized by activity staining. pBIG1 and pBIG4 were shown to carry the genes for β-gal I and β-gal III, respectively. Optimal pH and temperature for hydrolytic activity of the native enzyme were 7.5 and 40°C, while those for recombinant BIG1 and BIG4 were 7.5, 50°C and 8.0, 40°C, respectively. © Rapid Science Ltd. 1998

Inhibitory effects of human α2-macroglobulin on Trypanosoma cruzi epimastigote proteinases

The inactivation of Trypanosoma cruzi proteinases by human α2-macroglobulin ( α2-M), a major plasma proteinase inhibitor was studied. Evidences regarding the interaction between α2-M and proteolytic enzymes contained in crude cell-free extracts of T. cruzi were derived from electrophoretic and enzymatic assays. The former showed conformational and structural changes ocurring in α2-M, as judged by the appearance of transformed `fast' form on native PAGE; generation of bands of ≈90 kDa on reduced SDS-PAGE and formation of covalent complexes enzyme-inhibitor on SDS-PAGE. On the other hand, the total proteolytic activity on azocasein dropped significantly in the presence of α2-M, although partial activity was still maintained. The proteinases detected as a double band of 44 and 53 kDa on gelatin SDS-PAGE were also inhibited by α2-M. Results suggest that the study of specific interactions between α2-M and T. cruzi-proteinases, probably with cruzipain, could be biologically important in the fate of T. cruzi-infection and Chagas' disease.

Entrapment of atrazine-degrading enzymes in sol-gel glass

Cell-free crude extracts of Pseudomonas sp. strain ADP (ADP) that could rapidly degrade atrazine were successfully entrapped in a tetramethyl orthosilicate (TMOS) sol-gel. The immobilized crude extract maintained atrazine-degrading activity for at least a few days. However, compared with the non-entrapped crude extract, the sol-gel immobilization resulted in significant loss of activity. Part of the loss in activity is due to methanol produced during gel formation.

Glycerol is not an inhibitor of mitochondrial citrate oxidation by Aspergillus niger.

We have re-assessed the hypothesis that an accumulation of intracellular glycerol triggers the accumulation of citric acid by Aspergillus niger by inhibiting the activity of the mitochondrial NADP-dependent isocitrate dehydrogenase isoenzyme. To this end, we have incubated mycelia of A. niger with 0.5 M glycerol, which resulted in a maximal intracellular glycerol pool level of 0.92 M, comparable to that determined during the early phase of citrate accumulation. This addition affected neither the uptake of [1,5-14C] citrate from the medium nor the rate of the subsequent release of 14CO2 by the mycelia, indicating no effect on citrate oxidation. Mitochondria isolated from mycelia previously loaded with 0.5 M glycerol contained 8% of the total mycelial glycerol. They released 14CO2 from exogenously added [1,5-14C]citrate at the same rate as mitochondria isolated from mycelia not loaded with 0.5 M glycerol. The addition of glycerol had no effect on the activity of the purified NADP-specific isocitrate dehydrogenase, but appeared to inhibit the activity in crude cell-free extracts of A. niger. We conclude that the intracellular accumulation of glycerol does not affect the rate of mitochondrial citrate oxidation and is therefore, in contrast to previous claims, not a trigger of citrate accumulation.

A Novel Invertase from a Thermophilic FungusThermomyces lanuginosus:Its Requirement of Thiol and Protein for Activation

Unlike the invertases from the mesophilic fungi and yeasts, invertase from a thermophilic fungus,Thermomyces lanuginosus,was unusually unstable bothin vivoandin vitro.The following observations suggested that the unstable nature of the enzyme activity in the cell-free extracts was due to the oxidation of the cysteine residue(s) in the enzyme molecule: (a) the addition of dithiothreitol or reduced glutathione stabilized invertase activity during storage of the extracts and also revived enzyme activity in the extracts which had become inactive with time; (b)N-ethylmaleimide, iodoacetamide, oxidized glutathione, cystine, or oxidized coenzyme A-inactivated invertase; (c) invertase activity was low when the ratio reduced/oxidized glutathione was lower and high when this ratio was higher, suggesting regulation of the enzyme by thiol/disulfide exchange reaction. In contrast to the activation of invertase by the thiol compounds and its inactivation by the disulfides in the cell-free extracts, the purified enzyme did not respond to these compounds. Following its inactivation, the purified enzyme required a helper protein in addition to dithiothreitol for maximal activation. A cellular protein was identified that promoted activation of invertase by dithiothreitol and it was called “PRIA” for theprotein which helps inrestoringinvertaseactivity. The revival of enzyme activity was due to the conversion of the inactive invertase molecules into an active form. A model is presented to explain the modulation of invertase activity by the thiol compounds and the disulfides, both in the crude cell-free extracts and in the purified preparations. The requirement of free sulfhydryl group(s) for the enzyme activity and, furthermore, the reciprocal effects of the thiols and the disulfides on invertase activity have not been reported for invertase from any other source. The finding of a novel invertase which shows a distinct mode of regulation demonstrates the diversity in an enzyme that has figured prominently in the development of biochemistry.

Metabolism of methanesulfonic acid involves a multicomponent monooxygenase enzyme.

A novel methylotroph, strain M2, capable of utilizing methanesulfonic acid (MSA) as a sole source of carbon and energy was the subject of these investigations. The initial step in the biodegradative pathway of MSA in strain M2 involved an inducible NADH-specific monooxygenase enzyme (MSAMO). Partial purification of MSAMO from cell-free extracts by ion-exchange chromatography led to the loss of MSAMO activity. Activity was restored by the mixing of three distinct protein fractions designated A, B and C. The reconstituted enzyme had a narrow substrate specificity relative to crude cell-free extracts. Addition of FAD and ferrous ions to the reconstituted enzyme complex resulted in a fivefold increase in enzyme activity, suggesting the loss of FAD and ferrous ion from the multicomponent enzyme on purification. Analysis of mutants of strain M2 defective in the metabolism of C1 compounds indicated that methanol was not an intermediate in the degradative pathway of MSA and also confirmed the involvement of a multicomponent enzyme in the degradation of MSA by methylotroph strain M2.

Characterization of Tannin Acylhydrolase Activity in the Ruminal Bacterium Selenomonas Ruminantium

A strain of the anaerobe Selenomonas ruminantium subsp. ruminantium that is capable of growing on tannic acid or condensed tannin as a sole energy source has been isolated from ruminal contents of feral goats browsing tannin-rich Acacia sp. Growth on tannic acid was accompanied by release of gallic acid into the culture medium but the bacterium was incapable of using gallic acid as a sole energy source. Tannin acylhydrolase (EC 3.1.1.20) activity was measured in crude cell-free extracts of the bacterium. The enzyme has a pH optimum of 7, a temperature optimum of 30-40°C and a molecular size of 59 kDa. In crude extracts, the maximal rate of gallic acid methyl ester hydrolysis was 6.3 μmol min-1 mg-1 of protein and the Km for gallic acid methyl ester was 1.6 mM. Enzyme activity was displayed in situ in polyacrylamide and isoelectric focusing gels and was demonstrated to increase 17-fold and 36-fold respectively when cells were grown in the presence of gallic acid methyl ester or tannic acid.

Assay of Serum Glutamic-Oxaloacetic Transaminase Using Malate Dehydrogenase Preparation Obtained fromStreptomyces aureofaciens

A modified spectrophotometric method for serum glutamic-oxaloacetic transaminase (SGOT) assay was developed. A crude cell-free extract from Streptomyces aureofaciens which showed a high level of malate dehydrogenase (MDH) activity (E.C. 1.1.1.37) was used as the enzymatic indicator. The lyophilized microbial preparation was used without previous purification and was quite stable under refrigeration for one year. Serum sample assays using both the method utilizing the crude cell extract and an enzymatic commercial kit showed good correlation.

Nitrite reductase from the magnetotactic bacterium Magnetospirillum magnetotacticum. A novel cytochrome cd1 with Fe(II):nitrite oxidoreductase activity.

Cytochrome cd1 nitrite reductase was isolated from magnetite-containing cells of the magnetotactic bacterium Magnetospirillum (formerly Aquaspirillum) magnetotacticum, which was microaerobically cultivated under denitrifying conditions. The enzyme showed absorption maxima at 643 nm and 409 nm in the oxidized form, and at 663, 551, 522, and 418 nm in the reduced form. A distinctive split absorption band did not occur at about 550 nm. The pyridine ferrohemochrome spectra suggested the presence of heme c and heme d1 in the molecule. The enzyme was composed of two identical subunits each with a molecular mass of 54 kDa; each subunit contained one c-type and one d-type heme. The isoelectric point was 9.2. The redox potentials of heme c and heme d1 were estimated to be +191 mV and +180 mV, respectively. Although the enzyme showed cyanide-sensitive N,N,N',N'-tetramethyl-p-phenylenediamine-O2 oxidoreductase activity and N,N,N',N'-tetramethyl-p-phenylenediamine-nitrite oxidoreductase activity, the enzyme did not oxidize M. magnetotacticum ferrocytochrome c-550 and Pseudomonas aeruginosa ferrocytochrome c-551 in the presence of nitrite. Furthermore, sodium succinate did not cause the reduction of cytochrome cd1 in the crude cell-free extract prepared from the magnetite-containing bacterial cells. However, M. magnetotacticum cytochrome cd1 showed a novel Fe(II):nitrite oxidoreductase activity whereas P. aeruginosa cytochromes cd1 had no Fe(II):nitrite oxidoreductase activity. These results suggest that M. magnetotacticum cytochrome cd1 may function as a Fe(II)-oxidizing enzyme under microaerobic conditions using nitrite as electron acceptor.