Preparation Of Cell-free Extracts Research Articles

DNA Damage Response in Xenopus laevis Cell-Free Extracts.

The DNA damage response (DDR) is a coordinated cellular response to a variety of insults to the genome. DDR initiates the activation of cell cycle checkpoints preventing the propagation of damaged DNA followed by DNA repair, which are both critical in maintaining genome integrity. Several model systems have been developed to study the mechanisms and complexity of checkpoint function. Here we describe the application of cell-free extracts derived from Xenopus eggs as a model system to investigate signaling from DNA damage, modulation of DNA replication, checkpoint activation, and ultimately DNA repair. We outline the preparation of cell-free extracts, DNA substrates, and their subsequent use in assays aimed at understanding the cellular response to DNA damage. Cell-free extracts derived from the eggs of Xenopus laevis remain a robust and versatile system to decipher the biochemical steps underlying this essential characteristic of all cells, critical for genome stability.

The cell free protein synthesis system from the model filamentous fungus Neurospora crassa.

Cell-free protein synthesis (CFPS) can be used in many applications to produce polypeptides and to analyze mechanisms of mRNA translation. Here we describe how to make and use a CPFS system from the model filamentous fungus Neurospora crassa. The extensive genetic resources available in this system provide capacities to exploit robust CFPS for understanding translational control. Included are procedures for the growth and harvesting of cells, the preparation of cell-free extracts that serve as the source of the translational machinery in the CFPS and the preparation of synthetic mRNA to program the CFPS. Methods to accomplish cell-free translation and analyze protein synthesis, and to map positions of ribosomes on mRNAs by toeprinting, are described.

A simple method for isolation of cell-associated viral particles from cell culture

A common method for cell-associated virus isolation involves disruption of infected cells by a combination of hypotonic burst, freeze-thaw cycles (F-T) and sonication. This protocol was also originally used for the preparation of cell-free extract containing the MX strain of lymphocytic choriomeningitis virus (LCMV), which is preferentially propagated by cell-to-cell contact and does not release distinct virions into the medium. In the present study, we compared different approaches to virus isolation. Based on virus yield, we show that deionized water lysis is the fastest and most effective method for releasing LCMV MX infectious viral particles from persistently infected cells. Moreover, we demonstrate that freeze-thaw cycles and sonication do not improve virus isolation. This simple protocol could be used for isolation of other viruses, the life cycle of which is strictly cell-associated and therefore are difficult to release in large amounts from host cells.

Orderly Inactivation of the Key Checkpoint Protein Mitotic Arrest Deficient 2 (MAD2) during Mitotic Progression

Anaphase is promoted by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) only when all the chromosomes have achieved bipolar attachment to the mitotic spindles. Unattached kinetochores or the absence of tension between the paired kinetochores activates a surveillance mechanism termed the spindle-assembly checkpoint. A fundamental principle of the checkpoint is the activation of mitotic arrest deficient 2 (MAD2). MAD2 then forms a diffusible complex called mitotic checkpoint complex (designated as MAD2(MCC)) before it is recruited to APC/C (designated as MAD2(APC/C)). Large gaps in our knowledge remain on how MAD2 is inactivated after the checkpoint is satisfied. In this study, we have investigated the regulation of MAD2-containing complexes during mitotic progression. Using selective immunoprecipitation of checkpoint components and gel filtration chromatography, we found that MAD2(MCC) and MAD2(APC/C) were regulated very differently during mitotic exit. Temporally, MAD2(MCC) was broken down ahead of MAD2(APC/C). The inactivation of the two complexes also displayed different requirements of proteolysis; although APC/C and proteasome activities were dispensable for MAD2(MCC) inactivation, they are required for MAD2(APC/C) inactivation. In fact, the degradation of CDC20 is inextricably linked to the breakdown of MAD2(APC/C). These data extended our understanding of the checkpoint complexes during checkpoint silencing.

Involvement of a Putative [Fe-S]-cluster-binding Protein in the Biogenesis of Quinohemoprotein Amine Dehydrogenase

Quinohemoprotein amine dehydrogenase (QHNDH) of Paracoccus denitrificans contains a peptidyl quinone cofactor, cysteine tryptophylquinone, as well as intrapeptidyl thioether cross-links between Cys and Asp/Glu residues within the smallestgamma-subunit of the alphabetagamma heterotrimeric protein. A putative [Fe-S]-cluster-binding protein (ORF2 protein) encoded between the structural genes for the alpha- and gamma-subunits of QHNDH in the n-butylamine-utilizing operon likely belongs to a Radical SAM (S-Ado-Met) superfamily that includes many proteins involved in vitamin biosynthesis and enzyme activation. In this study the role of ORF2 protein in the biogenesis of QHNDH has been explored. Although the wild-type strain of Paracoccus denitrificans produced an active, mature enzyme upon induction with n-butylamine, a mutant strain in which the ORF2 gene had been mostly deleted, neither grew in the n-butylamine medium nor showed QHNDH activity. When the mutant strain was transformed with an expression plasmid for the ORF2 protein, n-butylamine-dependent bacterial growth and QHNDH activity were restored. Site-specific mutations in the putative [Fe-S]-cluster or SAM binding motifs in the ORF2 protein failed to support bacterial growth. The alpha- and beta-subunits were both detected in the periplasm of the mutant strain, whereas the gamma-subunit polypeptide was accumulated in the cytoplasm and stained negatively for redox-cycling quinone staining. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that the gamma-subunit isolated from the mutant strain had not undergone posttranslational modification. These results unequivocally show that the putative [Fe-S]-cluster- and SAM-binding ORF2 protein is necessary for the posttranslational processing of gamma-subunit, most likely participating in the formation of the intrapeptidyl thioether cross-links.

The Cytotoxic Lymphocyte Protease, Granzyme B, Targets the Cytoskeleton and Perturbs Microtubule Polymerization Dynamics

Granzyme B, a serine protease derived from cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell granules, plays an important role in coordinating apoptosis of CTL and NK target cells. Here, we report that granzyme B targets the cytoskeleton by cleaving and removing the acidic C-terminal tail of alpha-tubulin. Consistent with this, Granzyme B markedly enhanced rates of microtubule polymerization in vitro, most likely by removal of an autoinhibitory domain within the tubulin C terminus. Moreover, delivery of Granzyme B into HeLa target cells promoted dramatic reorganization of the microtubule network in a caspase-independent manner. These data reveal that granzyme B directly attacks a major component of the cell cytoskeleton, which may contribute to the incapacitation of target cells during CTL/NK-mediated killing.

A Novel Monothiol Glutaredoxin (Grx4) from Escherichia coli Can Serve as a Substrate for Thioredoxin Reductase

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750-2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3',5'-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.

Molecular Ordering of the Caspase Activation Cascade Initiated by the Cytotoxic T Lymphocyte/Natural Killer (CTL/NK) Protease Granzyme B

Granzyme B is a major cytotoxic T lymphocyte/natural killer (CTL/NK) granule protease that can activate members of the caspase family of cysteine proteases through processing of caspase zymogens. However, the molecular order and relative importance of caspase activation events that occur in target cells during granzyme B-initiated apoptosis has not been established. Here, we have examined the hierarchy of granzyme B-initiated caspase activation events using a cell-free system where all caspases are present at physiological levels. We show that granzyme B initiates a two-tiered caspase activation cascade involving seven caspases, where caspase-3 is required for the second tier of caspase activation events. Using a two-dimensional gel-based proteomics approach we have also examined the scale of granzyme B-initiated alterations to the proteome in the presence or absence of effector caspase-3 or -7. These studies indicate that granzyme B targets a highly restricted range of substrates and orchestrates cellular demolition largely through activation of caspase-3.

Detection of farnesyl diphosphate accumulation in yeast ERG9 mutants

A sensitive method was developed for measuring farnesyl diphosphate (FPP) accumulation in a mutant strain of Saccharomyces cerevisiae. The strain was blocked at squalene synthase ( ERG9 gene) in the isoprenoid pathway and had the catalytic domain of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene integrated into the chromosome. It required ergosterol for growth and produced E, E-farnesol. The method was based on the isolation of FPP using the anion exchanger Macro Prep High Q and conversion of FPP to E, E-farnesol with alkaline phosphatase. Farnesol was measured using gas chromatography–mass spectrometry. Background farnesol in the cell-free extract was also retained by the anion exchanger, but was removed with repeated washing with methanol. Both 1 M NaCl and 40% (v/v) methanol were required in the elution buffer to effectively elute FPP. The preparation of cell-free extract in Bis-Tris propane/HCl, pH 7, buffer containing 0.025% (w/v) Triton X-100 and 15 mM MgCl 2 provided optimum conditions for the stabilization of FPP.

Phenylmethanesulfonyl fluoride inactivates an archaeal superoxide dismutase by chemical modification of a specific tyrosine residue. Cloning, sequencing and expression of the gene coding for Sulfolobus solfataricus superoxide dismutase.

The gene encoding the superoxide dismutase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsSOD) was cloned and sequenced and its expression in Escherichia coli obtained. The chemicophysical properties of the recombinant SsSOD were identical with those of the native enzyme. The recombinant SsSOD possessed a covalent modification of Tyr41, already observed in native SsSOD [Ursby, T., Adinolfi, B.S., Al-Karadaghi, S., De Vendittis, E. & Bocchini, V. (1999) J. Mol. Biol. 286, 189--205]. HPLC analysis of SsSOD samples prepared from cells treated or not with phenylmethanesulfonyl fluoride (PhCH(2)SO(2)F), a protease inhibitor routinely added during the preparation of cell-free extracts, showed that the modification was caused by PhCH(2)SO(2)F. Refinement of the crystal model of SsSOD confirmed that a phenylmethanesulfonyl moiety was attached to the hydroxy group of Tyr41. PhCH(2)SO(2)F behaved as an irreversible inactivator of SsSOD; in fact, the specific activity of both native and recombinant enzyme decreased as the percentage of modification increased. The covalent modification caused by PhCH2SO2F reinforced the heat stability of SsSOD. These results show that Tyr41 plays an important role in the enzyme activity and the maintenance of the structural architecture of SsSOD.

Preparation of cell-free extracts from mycobacteria.

AbstractMuch work in the mycobacterial field has focused on the identification and characterization of antigenic proteins (1,2); many have now been identified and assigned a function; for example, the immunodommant 65kDa antigen of Mycobacterzum tuberculosis has been identified as a chaperonin (3) and the 28-kDa antigen of Mycobacterzum leprae was shown to be superoxide dismutase (4). In the beginnings of mycobactertal molecular biology, antigenic proteins were identified by screening M. leprae and M tuberculosis expression libraries in Escherichia coli (5–9) with mouse monoclonal antibodies (MAbs) (7,8) and polyclonal sera from rabbits (10,11) and patients (12,13). Although E. coli is still being used for the overexpression and purification of mycobacterial proteins (14), the use of nonpathogenic mycobacterta, such as Mycobacterium smegmatis as surrogate hosts may be preferable (15). For example, several mycobacterial proteins have been shown to undergo post-translational modification, such as glycosylation (15,16). Because glycosylation does not occur in E. coli, the study of such proteins in their native state requires the use of mycobacterial hosts. The preparation of cell-free extracts and protein purification from mycobacteria is a prerequisite for this kind of work.KeywordsCell DisruptionAntigenic ProteinMycobacterium SmegmatisPolyclonal SeronInsoluble PelletThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Fad-dependent epoxidase as a key enzyme in fungal metabolism of prenylated flavonoids

Crude protein extracts from Botrytis cinerea preincubated with 6-prenylnaringenin (6-PN) for 20 hr catalysed the prenyl epoxidation of 7- O-methyl-luteone. The resulting epoxide was non-enzymatically and slowly converted into the corresponding dihydrofurano derivative in a buffer solution at pH 7.5. Preparation of cell-free extracts in the presence of 6-PN from the mycelia without preincubation with 6-PN hardly showed the epoxidizing activity. These facts revealed that the substrate analogue 6-PN has a role as an enzyme inducer rather than stabilizer. The enzyme reaction depends on molecular oxygen and NADPH. Low amounts of FAD were necessary for maximal enzyme activity. The enzymatic activity was not inhibited by various inhibitors of cytochrome P-450 tested, in addition to carbon monoxide and cytochrome c. The results indicated that this enzyme does not belong to the monooxygenases dependent on cytochrome P-450, but to those dependent on FAD. About half of the total enzyme activity was found in the 125 000 g supernatant, but the specific activity for the epoxidation reaction in the 125 000 g pellet was 3.7-fold higher than in the soluble fraction. The enzyme showed high specificity to monoprenyl isoflavones. Finally, a preliminary experiment using a cell-free system from white lupin hypocotyls resulted in formation of small amounts of an epoxide corresponding to 7- O-methyl-luteone used as the substrate.

Proteolysis of hexokinase PII is not the triggering signal of carbon catabolite derepression in Saccharomyces cerevisiae.

The role of hexokinase PII in mediating carbon catabolite derepression in yeast has been examined. Hexokinase isoenzyme PII (EC 2.7.1.1) was partially degraded when protease inhibitors were omitted from the buffer used for preparation of cell-free extracts. The hexokinase PII inactivation induced by D-xylose was correlated with derepression of maltase (EC 3.2.1.20) in the wild-type strain Saccharomyces cerevisiae G-517 and in D.308.3, a strain that contains the cloned hexokinase PII gene on a multicopy plasmid. This inactivation was not correlated with the loss of hexokinase PII protein as assayed by immunoblotting. We conclude that during the derepression process there is no release of proteolytic peptides from hexokinase PII.

Preparation of cell-free extracts and the enzymes involved in fatty acid metabolism in Syntrophomonas wolfei.

Syntrophomonas wolfei is an anaerobic fatty acid degrader that can only be grown in coculture with H2-using bacteria such as Methanospirillum hungatei. Cells of S. wolfei were selectively lysed by lysozyme treatment, and unlysed cells of M. hungatei were removed by centrifugation. The cell extract of S. wolfei obtained with this method had low levels of contamination by methanogenic cofactors. However, lysozyme treatment was not efficient in releasing S. wolfei protein; only about 15% of the L-3-hydroxyacyl-coenzyme A (CoA) dehydrogenase activity was found in the lysozyme supernatant. Cell extracts of S. wolfei obtained with this method had high specific activities of acyl-CoA dehydrogenase, enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase. These activities were not detected in cell extracts of M. hungatei grown alone, confirming that these activities were present in S. wolfei. The acyl-CoA dehydrogenase activity was high when a C4 but not a C8 or C16 acyl-CoA derivative served as the substrate. S. Wolfei cell extracts had high CoA transferase specific activities and no detectable acyl-CoA synthetase activity, indicating that fatty acid activation occurred by transfer of CoA from acetyl-CoA. Phosphotransacetylase and acetate kinase activities were detected in cell extracts of S. wolfei, indicating that S. wolfei is able to perform substrate-level phosphorylation.

17] Glutamate-aspartate transaminase from microorganisms

Publisher Summary Various methods have been developed for the assay of aspartate aminotransferase. Most of these depend on the determination of oxalacetate or L-glutamate. Out of these, the Karmen method is used most widely, particularly in the field of clinical analysis, in which oxalacetate is determined with malate dehydrogenase and NADH by following a decrease in absorbance at 340 nm. A new method introduced recently, determines 2-oxoglutarate produced in the reverse reaction is with 2-hydroxyglutarate dehydrogenase. A method for the determination of aspartate aminotransferase activity after electrophoretic separation of the isoenzymes has also been developed. The purification procedure consists of several steps: preparation of cell-free extract, ammonium sulfate fractionation, first Diethylaminoethyl (DEAE)-cellulose column chromatography, heat treatment, Sephacryl S-200 chromatography, second DEAE-cellulose chromatography, and hydroxyapatite chromatography.

Studies on the microsomal 11α-hydroxylation of progesterone inAspergillus ochraceus: characterization of the hydroxylase system

From the induced vegetative cell cultures ofA. ochraceus, a procedure for the preparation of cell-free extract with high 11α-hydroxylase activity was developed. To obtain optimal hydroxylase activity,edta (10mM), glycerol (10%) anddtt (5mM) were required in the grinding medium. Although the optimum pH for the grinding medium was 8·3, the hydroxylase has a pH optimum of 7·7. Microsomes (2mg) isolated from the crude cell-free extract, hydroxylated progesterone in high yields (85–90% in 30 min) in the presence ofnadph and O2. The apparentK m for NADPH and progesterone were 0·052 mM and 0·625 mM respectively. The involvement of cytochrome P-450 system in the hydroxylation reaction was established by various inhibition studies. The hydroxylase activity was inhibited by metyrapone, carbon monoxide,skf-525A andp-cmb. The presence of high levels ofnadph-cytochromec reductase in the microsomal fraction and the strong inhibition of the hydroxylase system by cytochromec indicated that the reductase could be one of the components of the hydroxylase system. Progesterone has the ability to induce the 11α-hydroxylase system significantly, whereas deoxycorticosterone and phenobarbital failed to bring about the induction. However, deoxycorticosterone acted as a good substrate for the 11α-hydroxylase system. The membrane-bound hydroxylase was solubilized using various ionic and non-ionic detergents. Solubilized membrane fraction contained considerable levels of cytochrome P-450 andnadph-cytochromec reductase, besides hydroxylase activity.

Studies on the microsomal 11α-hydroxylation of progesterone in Aspergillus ochraceus: isolation, characterization, and solubilization of the hydroxylase system

A suitable procedure for the preparation of cell-free extract with high 11α-hydroxylase activity from the induced vegetative cell cultures of Aspergillus ochraceus was developed. The presence of EDTA (10 mM), glycerol (10%), and dithiothreitol (5 mM), in the grinding medium was found to be necessary for optimal hydroxylase activity. Although the pH optimum of the hydroxylase was found to be 7.7, the ideal pH for preparing the cell-free extract from mycelium was 8.3. Microsomes (2.0 mg) isolated from the cell-free extract hydroxylated progresterone in very high yields (85–90% in 30 min) in the presence of NADPH and O2. Cytosolic involvement in the hydroxylation reaction was not noticed. The apparent Km for NADPH and progesterone were 0.052 and 0.625 mM, respectively. The hydroxylase activity was inhibited by metyrapone, carbon monoxide, SKF-525A, and p-chloromercuribenzoate indicating the involvement of cytochrome P-450 system in the reaction. Inhibition of the hydroxylase activity by cytochrome c and the presence of significant levels of NADPH–cytochrome c reductase in the microsomal fraction suggested that the reductase could be one of the components of the hydroxylase system. The membrane-bound hydroxylase was solubilized using sodium cholate. Solubilized membrane fraction contained considerable levels of cytochrome P-450 and NADPH–cytochrome c reductase, besides hydroxylase activity.

A simple method for the preparation of extracts from animal cells which catalyze efficient in vitro protein synthesis.

A rapid, simple procedure is described for the preparation of cell-free extracts of both uninfected and virus-infected pig kidney and baby hamster kidney cells which are very active in the in vitro translation of not only endogenous mRNA, but also of exogenous mRNA added to extracts that had been depleted of endogenous mRNA by treatment with micrococcal nuclease. This procedure appears to be adaptable with only minor variations to many eukaryotic cell lines and should greatly facilitate in vitro molecular studies of protein synthesis and gene regulation at the level of translation.

Studies of nitrate reductase in the fresh water dinoflagellatePeridinium cinctum

Nitrate reduction was studied in the dinoflagellatePeridinium cinctum collected from extensive algal blooms in Lake Kinneret (Israel). Among several methods tested for the preparation of cell free extracts, only the use of a ground-glass tissue culture homogenizer was found to be efficient. The assimilatory nitrate reductase ofP. cinctum was located in a particulate fraction. In this respect,P. cinctum did not behave like other eukaryotes, such as green algae, but as a prokaryote. Nitrite reductase activity was found in the soluble fraction. Nitrate reductase used NADH as a preferable electron donor; it reacted also with NADPH but only to give 16.5% of the NADH dependent rate. Methyl viologen and benzyl viologen could also serve as electron donors, with rates higher than the NADH dependent activity (3–6 times and 1.5–3 times, respectively). The Km of nitrate reductase for NADH was 2.8×10−4 M and for NO3-1.9×10−4 M. Flavins did not stimulate the activity, nor was ferricyanide able to activate it. Carboxylic anions stimulated nitrate reductase activity 3–4 fold, an effect which was not mimicked by other anions. Chlorate, azide and cyanide were competitive inhibitors ofP. cinctum, nitrate reductase withK i values of 1.79×10−3 M, 2.1×10−5 M and 8.9×10−6 M respectively.

29] d-Galactose dehydrogenase from Pseudomonas fluorescens

Publisher Summary D-Galactose dehydrogenase catalyzes the pyridine nucleotide-dependent dehydrogenation of β -D-galactopyranose at C-1 with the formation of nicotinamide adenine dinucleotide dehydrogenase (NADH) and D-galactono-l,5-1actone. Depending on the pH value, the immediately formed D-galactono-l,5-1actone rearranges intramolecularly to the corresponding 1,4-1actone or hydrolyzes to D-galactonate, or both. The assay is based on the spectrophotometric determination of NADH formed with D-galactose as substrate at high pH. This chapter discusses the assay method and properties of D-galactose dehydrogenase. The steps involved in the purification procedure are (1) the preparation of cell-free extract (2) ammonium-sulfate fractionation, (3) bulk separation on CM-Sephadex, (4) diethylaminoethyl (DEAE)-sephadex chromatography, and (5) hydroxyapatite chromatography. All operations are carried out at 0-4°C, unless stated otherwise. The purified enzyme with a specific activity of 849units/mg is homogeneous by ultracentrifugation, by disc gel electrophoresis, by sodium dodecyl sulfate gel electrophoresis, and by gel isoelectric focusing. D-Galactose dehydrogenase from P. fluorescens has a molecular weight of 64,000. The enzyme is composed of two identical subunits and has two binding sites.