Effective cotranslational folding of firefly luciferase without chaperones of the Hsp70 family

Molecular chaperones of the Hsp70 family (bacterial DnaK, DnaJ, and GrpE) were shown to be strictly required for refolding of firefly luciferase from a denatured state and thus for effective restoration of its activity. At the same time the luciferase was found to be synthesized in an Escherichia coli cell-free translation system in a highly active state in the extract with no chaperone activity. The addition of the chaperones to the extract during translation did not raise the activity of the enzyme. The abrupt arrest of translation by the addition of a translational inhibitor led to immediate cessation of the enzyme activity accumulation, indicating the cotranslational character of luciferase folding. The results presented suggest that the chaperones of the Hsp70 family are not required for effective cotranslational folding of firefly luciferase.     

The recBC enzyme of Escherichia coli K12: premature cessation of catalytic activities in vitro and reactivation by potassium ions.

It is shown that in vitro the degradation of native and single-stranded DNA as well as the hydrolysis of ATP by purified recBC enzyme ceases 2-3 min after the start of the reaction. The presence of potassium ions (60-100 mM), bovine serum albumin (1 mg/ml) or protein from cell-free Escherichia coli extract (10 microgram/ml) prevents the cessation of the activity. Once the cessation has occurred, the activity of the enzyme can be completely restored by the addition of potassium ions, but not by bovine serum albumin. Sedimentation studies revealed that, in contrast to the active recBC enzyme, the 'silent' enzyme is no longer associated with substrate DNA of high molecular weight. On the basis of these results and other observations it is hypothesized that during the degradation of DNA in the absence of potassium ions or bovine serum albumin the recBC enzyme is subject to an alteration of its molecular conformation which results in an inactive form.     

Biological properties of sherry yeasts in the presence of antimicrobial preparations

The wine yeasts Cheres were not sensitive to high concentration (500 gamma/ml) of benzylpenicillin, streptomycin, tetracycline, oxytetracycline, chlortetracycline, morphocycline, erythromycin, oleandomycin, monomycin, neomycin, kanamycin, polymyxin, ampicillin, oxacillin, methicillin, ceporin, ristomycin, levomycetin, furadonin and furazolidone. In concentrations of 50 to 500 gamma-ml oleadomycin, chlortetracycline, oxytetracycline, kanamycin and ristomycin inhibited synthesis of the Cheres yeast biomass. Benzylpenicillin, polymyxin, neomycin and ampicillin in concentrations of 50 to 100 gamma/ml had a stimulating effect on the yeast biomass synthesis.     

Purification of the NADP+:F420 oxidoreductase of Methanosphaera stadtmanae

Methanosphaera stadtmanae (DSM 3091) is a methanogen that requires H2 and CH3OH for methanogenesis. The organism does not possess an F420-dependent hydrogenase and only low levels of F420. It does however possess NADP+:F420 oxidoreductase activity. The NADP+:F420 oxidoreductase, the enzyme which catalyses the electron transfer between NADP+ and F420 in this organism, was purified and characterized. NAD+, NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F420 reduction was 6.0, and 8.5 for NADP+ reduction. During the purification process, it was noted that precipitation with (NH4)2SO4 increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)2SO4 pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of alpha, beta, and gamma subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The Km values were 370 microM for NADP+, 142 microM for NADPH, 62.5 microM for F420, and 7.7 microM for F420H2. These values were different from the Km values observed in the cell-free extract.     

Reduction of methionine sulfoxide to methionine by Escherichia coli.

L-Methionine-dl-sulfoxide can support the growth of an Escherichia coli methionine auxotroph, suggesting the presence of an enzyme(s) capable of reducing the sulfoxide to methionine. This was verified by showing that a cell-free extract of E. coli catalyzes the conversion of methionine sulfoxide to methionine. This reaction required reduced nicotinamide adenine dinucleotide phosphate and a generating system for this compound. The specific activity of the enzyme increased during logarithmic growth and was maximal when the culture attained a density of about 10(9) cells per ml.     

Two pathways in the biosynthesis of cadystins (gamma EC)nG in the cell-free system of the fission yeast

Small metal-binding peptides, cadystins, with the general structure of (gamma-Glu-Cys)n-Gly ((gamma EC)nG), were synthesized in a cell-free system of fission yeast to examine the in vivo synthetic pathway. The crude enzyme for cadystin synthesis was prepared by ammonium sulfate precipitation (75% saturation) from the 120,000 x g supernatant of the cell extract, and the excess salt in the enzyme fraction was removed by Sephadex gel filtration. Using this crude enzyme fraction, it was shown that there were two pathways for cadystin biosynthesis. One pathway is gamma-Glu-Cys (gamma EC) dipeptidyl transfer from both glutathione (gamma ECG) and cadystins to glutathione and cadystins. The other one is gamma EC polymerization from (gamma EC)n and glutathione to (gamma EC)n + i, followed by glycine addition with glutathione synthetase.     

Release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126: a comparative study

Nitrilases constitute an important class of hydrolases, however, cheap and ready availability of enzyme sources limit their practical synthetic applications. The present investigation was directed to compare the applicability of various physical cell disintegration methods namely, solid shear, liquid shear and sonication, for the release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126. Different parameters associated with each method were optimized in order to ensure maximal release of active nitrilase. The methods were also compared under optimal conditions for their efficiency of nitrilase release and extent of cell disruption, and enzyme release were visualized under a differential interference contrast microscope (DIC) and SDS-PAGE, respectively. Maximum release of the enzyme protein from the cells was observed in case of liquid shear method employing high-pressure homogenization, however, the specific activity of nitrilase was highest in cell-free extract (CFE) generated by sonication. Both the solid shear and liquid shear proved to be equally effective for maximum release of intracellular enzymes, however, from the specific activity point of view, sonication was found to be a better one compared to other two methodologies. The generated cell-free extract can be further employed for the production of enantiopure chiral carboxylic acids, which are important chiral building blocks.     

Factors Affecting the Activity of Cellulases Isolated from the Rumen Digesta of Sheep

Sodium phosphate buffer was used to extract cellulases from the plant solids fraction of rumen contents. The mixed cellulase preparation had maximal activity at pH 6.9 and 49°C. The V_max and the apparent K_m for wheaten hay cellulose were 19.8 glucose units/min and 6.35 mg/ml, respectively, and for microcrystalline cellulose (Sigmacell) at the same enzyme concentration, they were 33 glucose units/min and 27.5 mg/ml, respectively. For these assays a glucose unit was defined as nanomoles of glucose plus twice the nanomoles of cellobiose. Consideration of thermodynamic and kinetic data suggested that the hydrolysis of a relatively labile arabino-xylan comprising 3% of the wheaten hay cellulose was dependent on prior removal of the protecting β-1,4-glucose chains at the outer surface of the cellulose preparation. Sequential removal of structural polysaccharides from the plant cell wall rendered the latter more susceptible to cellulase activity. Cellulase activity was stimulated by increasing the concentration of phosphate from 5 to 50 mM. The stimulation was magnified in the presence of cell-free rumen fluid. Cellulase activity was not stimulated by calcium, magnesium, iron, zinc, manganese, copper, or cobalt ions and was unaffected by the chelators ethylenediaminetetraacetic acid and ethyleneglycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid. O-phenanthroline inhibited activity by 30 to 50%, but this may have been due to nonchelate properties. Anaerobic conditions or thiol protective agents were not essential for either the activity or stability of the cellulases during assay. An ultrafiltrable inhibitor of cellulase activity was detected in cell-free rumen fluid.     

Chromate reduction by cell-free extract of Bacillus firmus KUCr1

Microbial enzymatic reduction of a toxic form of chromium [Cr(VI)] has been considered as an effective method for bioremediation of this metal. This study reports on the in vitro reduction of Cr(VI) using cell-free extracts from a Cr(VI) reducing Bacillus firmus KUCr1 strain. Chromium reductase was found to be constitutive and its activity was observed both in soluble cell fractions (S12 and S150 and membrane cell fraction (P150). The reductase activity of S12 fraction was found to be optimal at 40 microM Cr(VI) with enzyme concentration equivalent to 0.493 mg protein/ml. Enzyme activity was dependent on NADH or NADPH as electron donor; optimal temperature and pH for better enzyme activity were 70 degrees C and 5.6, respectively. The Km value of the reductase was 58.33 microM chromate having a V(max) of 11.42 microM/min/mg protein. The metabolic inhibitor like sodium azide inhibited reductase activity of membrane fraction of the cell-free extract. Metal ions like Cu2+, Co2+, Ni2+ and As3+ stimulated the enzyme but others, such as Ag+, Hg2+, Zn2+, Mn2+, Cd2+ and Pb2+, inhibited Cr(VI) reductase activity.     

Properties of an inducible extracellular neuraminidase from an Arthrobacter isolate.

The elective isolation of a soil microorganism, tentatively assigned to the genus Arthrobacter, which produced an extracellular neuraminidase is described. The secretion of neuraminidase from washed cells in minimal medium required the presence of sialo-containing glycoproteins, whereas free N-acetyl-neuraminic asid of N-acetylmannosamine were poor inducers. No enzyme could be dected in the induction fitrated of cells, in the absence of inducer or in the culture filtrate of cells grown in a complete medium. The routine enzyme inducer was a hot-water extract of "edible bird's nest." Mild acid treatment (0.05 N H2SO4) of this extract increased enzyme activity two--to threefold and the specific activity about eightfold. Neuraminidase induction with acid-treated bird's nest was manifested at a linear rate for 6 h without increase in cell number. No other anticipated glycohydrolase or protease activities were foud. The amount of enzyme located within the cells was barely detectable as compared to that found in the induction filtrate. Experiments with chloramphenicol or chlortetracycline indicate that de novo protein synthesis was required for neuraminidase production and that this exoenzyme was not released from a preformed pool. Neuraminidase from this source has an apparent molecular weight of 87,000, a pH optimum of 5 to 6, and an apparent Km of 2.08 mg/ml for collocalia mucoid and 3.3 X 10(-3) M for N-acetylneuraminlactose and is insensitive both to Ca2+ ions and ethylenediaminetetraacetic acid. Preliminary studies indicate that the enzyme can hydrolyze alpha-2,3-, alpha-2,6-, or alph-2-8-N-acetylneuraminylglycosidic linkages. From total activity data and purification criteria, it would appear that this isolate can produce about 5 mg of enzyme per liter of induction medium.     

The role of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda) in immune responses: A first survey

For the first time, a functional study of haemocytes from the crab Carcinus aestuarii was performed in order to evaluate their involvement in immune responses. Total haemocyte count (THC), phagocytosis, haemolymph opsonisation properties, hydrolytic and oxidative enzyme activities, and production of intracellular superoxide anion were evaluated. A great variability in THC was recorded among individuals, and haemocyte mean number was 6.4 (×10⁶) cells/ml haemolymph. Although only hyalinocytes were able to phagocytose yeast cells or Zymosan, phagocytic index was low (3%) and did not increase significantly (4%) after pre-incubation of yeast and Zymosan in cell-free haemolymph, suggesting that haemolymph did not have opsonising properties. All haemocyte types produced superoxide anion, whereas only granulocytes were positive to the hydrolytic enzymes assayed. In addition, only granulocytes were positive to phenoloxidase activity. Both Petri dish and spectrophotometric assays revealed a very low lysozyme-like activity in cell-free haemolymph (CFH) and haemocyte lysate (HL), although enzyme activity was higher in CFH than in HL. Interestingly, normalisation of data as to total protein content in CFH and HL resulted in an opposite situation, lysozyme-like activity being higher in HL than in CFH. This demonstrated that haemolymph of C. aestuarii has a high quantity of total proteins, functional properties of which need to be better investigated in future studies. Overall, the results obtained in the present study indicated that C. aestuarii haemocytes are not very active phagocytic cells, but they are more active in terms of both hydrolytic and oxidative enzyme activities and superoxide anion production.     

Resistance of Escherichia coli to tetracyclines

1. A strain of Escherichia coli highly resistant to chlortetracycline and partially cross-resistant to tetracycline has been isolated. 2. The nitro-reductase system of the resistant cells was inhibited to a smaller extent by chlortetracycline than was the corresponding enzyme of sensitive cells. 3. The incorporation of leucine in vitro into the ribosomal protein of cell-free preparations from sensitive and resistant cells was equally inhibited by chlortetracycline. 4. Resistant cells accumulated much less chlortetracycline and tetracycline than did sensitive cells when both were cultured in the presence of these drugs. 5. The uptake of tetracycline by both sensitive and resistant E. coli was dependent on the presence of glucose in the medium. 6. Fractionation of cells cultured in medium containing [¹⁴C]chlortetracycline indicated that the largest proportion of radioactivity in sensitive cells was in the fraction consisting mainly of cell-wall material. There was no concentration of radioactivity in any one fraction of the resistant cells. 7. No evidence could be obtained for a specific tetracycline-excretion system in the resistant cells. 8. The significance of these results in relation to current theories of the antibiotic action of and resistance to the tetracycline drugs is discussed.     

Partial purification and characterization of exoinulinase from Kluyveromyces marxianus YS-1 for preparation of high-fructose syrup

An extracellular exoinulinase (2,1-beta-D fructan fructanohydrolase, EC 3.2.1.7), which catalyzes the hydrolysis of inulin into fructose and glucose, was purified 23.5-fold by ethanol precipitation, followed by Sephadex G-100 gel permeation from a cell-free extract of Kluyveromyces marxianus YS-1. The partially purified enzyme exhibited considerable activity between pH 5 to 6, with an optimum pH of 5.5, while it remained stable (100%) for 3 h at the optimum temperature of 50 degrees C. Mn2+ and Ca2+ produced a 2.4-fold and 1.2-fold enhancement in enzyme activity, whereas Hg2+ and Ag2+ completely inhibited the inulinase. A preparation of the partially purified enzyme effectively hydrolyzed inulin, sucrose, and raffinose, yet no activity was found with starch, lactose, and maltose. The enzyme preparation was then successfully used to hydrolyze pure inulin and raw inulin from Asparagus racemosus for the preparation of a high-fructose syrup. In a batch system, the exoinulinase hydrolyzed 84.8% of the pure inulin and 86.7% of the raw Asparagus racemosus inulin, where fructose represented 43.6 mg/ml and 41.3 mg/ml, respectively.     

Induction of tryptophan oxygenase by dexamethasone in isolated hepatocytes. Dependence on composition of medium and pH.

Hepatocytes were isolated from perfused rat livers. 4 x 10-6 cells/ml were incubated at at 37 degrees C in different media in the absence and presence of a steroid hormone, dexamethasone phosphate (2 x 10-5 M). 1. Hormonal enzyme induction occurred in cells suspended in a simple salt medium, devoid of amino acids and macromolecules. This induction was completely blocked by addition of either actinomycin D (2 mu-g/ml) or cycloheximide (50 mu-g/ml). 2. Incubation of cells in media containing defatted albumin did not enhance hormonal enzyme induction, although disintegration of cells during incubation was reduced. Addition of a crude albumin fraction reduced tryptophan oxygenase induction and dextran completely blocked enzyme induction by dexamethasone. 3. An increase of dexamethasone concentration in the presence of albumin to 9 x 10-5 M was unable to raise enzyme induction further, and a still higher concentration of hormone, 3 x 10-4 M, resulted in reduced enzyme induction. 4. The hormonal induction of tryptophan oxygenase was most pronounced when the pH of the medium was between 7.0 and 7.6, with an optium at 7.3. No induction was found when the pH of the medium was either 6.6 or 7.8. The basal tryptophan oxygenase activity was much less influenced by similar pH variations. It is concluded that hepatocytes in suspension are able to carry out hormone-stimulated enzyme synthesis and that factors influencing this process may be studied under controlled conditions in such systems.     

Isolation of a highly pure preparation of cephalexin amidase from bacteria of the genus Xanthomonas

A procedure for highly purified cephalexin amidase of Xanthomonas was developed. It consists of preparation of a cell-free extract of the culture after cell disintegration, precipitation with ammonium sulfate, dissolution, concentration and elimination of ballast proteins, gel filtration on Sephadex G-25, sorption of ballast proteins on DEAE cellulose and chromatography on KM-cellulose. The enzyme yield is 45-55 per cent. The purity level is 80-90-fold.     

Improving protein array performance: focus on washing and storage conditions

For protein microarrays, maintaining protein stability during the slide processing steps of washing, drying, and storage is of major concern. Although several studies have focused on the stability of immobilized antibodies in antibody microarrays, studies on protein-protein interaction arrays and enzyme arrays are lacking. In this paper we used five bait-prey protein interaction pairs and three enzymes to optimize the washing, drying, and storage conditions for protein arrays. The protein arrays for the study were fabricated by combining HaloTag technology and cell-free protein expression. The HaloTag technology, in combination with cell-free expression, allowed rapid expression and immobilization of fusion proteins on hydrogel-coated glass slides directly from cell extracts without any prior purification. Experimental results indicate enzyme captured on glass slides undergoes significant loss of activity when washed and spin-dried using only phosphate buffer, as is typically done with antibody arrays. The impact of washing and spin-drying in phosphate buffer on protein-protein interaction arrays was minimal. However, addition of 5% glycerol to the wash buffer helps retain enzyme activity during washing and drying. We observed significant loss of enzyme activity when slides were stored dry at 4 degrees C, however immobilized enzymes remained active for 30 days when stored at -20 degrees C in 50% glycerol. We also found that cell-free extract containing HaloTag-fused enzymes could undergo multiple freeze/thaw cycles without any adverse impact on enzyme activity. The findings indicate that for large ongoing studies, proteins of interest expressed in cell-free extract can be stored at -70 degrees C and repeatedly used to print small batches of protein array slides to be used over a few weeks.     

Simultaneous expression and maturation of the iron-sulfur protein ferredoxin in a cell-free system

The model iron-sulfur (Fe-S) protein ferredoxin (Fd) from Synechocystis sp. PCC 6803 has been simultaneously produced and matured in a cell-free production system. After 6 h of incubation at 37 degrees C, Fd accumulated to >450 microg/mL. Essentially all was soluble, and 85% was active. Production and maturation of the protein in the cell-free system were found to be dependent in a coupled manner on the concentration of the supplemented iron and sulfur sources, ferrous ammonium sulfate and cysteine, respectively. The recombinant expression of ISC helper proteins during cell extract preparation did not increase cell-free Fd accumulation or activity, although the efficiency of iron and cysteine utilization increased. Fd maturation was independent of protein production rate, and proceeded at a constant rate throughout the period of active translation. In addition, incubation of denatured apo Fd with cell-free reaction components resulted in recovery of Fd activity, supporting the interpretation that maturation mechanisms did not act co-translationally. Incubation at 28 degrees C increased total and active protein accumulation, but decreased the ratio of active to total Fd produced. In summary, the high product yields and folding efficiency make the cell-free system described here an attractive platform for the study of Fe-S protein production and maturation. The system enables both small-volume, high throughput investigations as well as larger scale production. To our knowledge, this is the first demonstration of directed, high-yield production and maturation of an Fe-S protein in a cell-free system.     

Evidence that Blastocladiella emersonii zoospore chitin synthetase is located at the plasma membrane

Blastocladiella emersonii zoospores are not encased by a cell wall and do not detectably synthesize or contain chitin; accompanying de novo cell wall formation during zoospore encystment, chitin rapidly accumulates and is incorporated into the cell wall. Essential for understanding this abrupt change in chitin synthesis is the location of zoospore chitin synthetase. The enzyme has previously been reported to the sequestered with distinctive cytoplasmic organelles (gamma particles) characteristic for the zoospore cell type. Using similar differential and equilibrium density centrifugation procedures to those reported previously, we have observed the vast majority of zoospore homogenate chitin synthetase activity in fractions distinct from the gamma particle-enriched fractions. Over 90% of the homogenate enzyme activity could be recovered in a sucrose buoyant density region (1.14–1.18 g/ml) containing membranous elements and well separated from the region enriched for gamma particles (1.30–1.34 g/ml). When zoospores were surface-labelled with [³H]concanavalin A prior to homogenization, the buoyant density regions of radioactivity and of chitin synthetase activity exhibited nearly complete coincidence. At least the bulk of zoospore chitin synthetase appears to be located at the plasma membrane, rather than in gamma particles.     

Hormone inhibition of adenylyl cyclase. Differences in the mechanisms for inhibition by hormones and G protein beta gamma

S49 mouse lymphoma cells contain a beta-adrenergic receptor coupled to Gs that stimulates adenylyl cyclase and a somatostatin receptor coupled to Gi that inhibits adenylyl cyclase. Membranes from these cells were used to compare the inhibitory effects of somatostatin and G protein beta gamma complex to determine under what conditions beta gamma is likely to be a mediator of somatostatin action. Somatostatin was equally effective at inhibiting basal adenylyl cyclase activity in the presence of GTP, forskolin-stimulated activity, and hormone-stimulated activity. G protein beta gamma was more effective at inhibiting basal activity than was somatostatin, and these effects were partially additive. In the presence of forskolin, the two inhibitors were equally effective and not additive. In the presence of isoproterenol, beta gamma was much less effective than somatostatin, and the two inhibitors did not have additive or synergistic effects. At very high concentrations beta gamma did inhibit isoproterenol stimulation of adenylyl cyclase, although its effects were not saturating even at 100 micrograms/ml. Under conditions where beta gamma did inhibit hormone stimulation, beta gamma was a mixed inhibitor of isoproterenol stimulation, proportionally decreasing the maximum effect of the hormone and increasing the half-maximally effective concentration. Somatostatin, on the other hand, was a simple noncompetitive inhibitor of isoproterenol stimulation. These results indicate that beta gamma and somatostatin inhibit adenylyl cyclase by different mechanisms, at least in the presence of hormones that stimulate the enzyme. It is proposed that alpha i is the primary mediator of hormone inhibition of adenylyl cyclase when Gs is activated by a hormone, but that beta gamma may have a role as a mediator of inhibition of basal activity.     

Characterization of the Bacillus subtilis WL-3 mannanase from a recombinant Escherichia coli

A mannanase was purified from a cell-free extract of the recombinant Escherichia coli carrying a Bacillus subtilis WL-3 mannanase gene. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. Optimal conditions for the purified enzyme occurred at pH 6.0 and 60 degrees C. The specific activity of the purified mannanase was 5,900 U/mg on locust bean gum (LBG) galactomannan at pH 6.0 and 50 degrees C. The activity of the enzyme was slightly inhibited by Mg(2+), Ca(2+), EDTA and SDS, and noticeably enhanced by Fe(2+). When the enzyme was incubated at 4 degrees C for one day in the presence of 3 mM Fe(2+), no residual activity of the mannanase was observed. The enzyme showed higher activity on LBG and konjac glucomannan than on guar gum galactomannan. Furthermore, it could hydrolyze xylans such as arabinoxylan, birchwood xylan and oat spelt xylan, while it did not exhibit any activities towards carboxymethylcellulose and para-nitrophenyl-beta-mannopyranoside. The predominant products resulting from the mannanase hydrolysis were mannose, mannobiose and mannotriose for LBG or mannooligosaccharides including mannotriose, mannotetraose, mannopentaose and mannohexaose. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose.