Tricine-sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Research Articles

Peroxynitrite-mediated nitration of tyrosine and inactivation of the catalytic activity of cytochrome P450 2B1.

The addition of peroxynitrite to purified cytochrome P450 2B1 resulted in a concentration-dependent loss of the NADPH- and reductase-supported or tert-butylhydroperoxide-supported 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B1 with IC50 values of 39 and 210 microM, respectively. After incubation of P450 2B1 with 300 microM peroxynitrite, the heme moiety was not altered, but the apoprotein was modified as shown by HPLC and spectral analysis. Western blot analysis of peroxynitrite-treated P450 2B1 demonstrated the presence of an extensive immunoreactivite band after incubating with anti-nitrotyrosine antibody. However, the immunostaining was completely abolished after coincubation of the anti-nitrotyrosine antibody with 10 mM nitrotyrosine. These results indicated that one or more of the tyrosine residues in P450 2B1 were modified to nitrotyrosines. The decrease in the enzymatic activity correlated with the increase in the extent of tyrosine nitration. Further demonstration of tyrosine nitration was confirmed by GC/MS analysis by using 13C-labeled tyrosine and nitrotyrosine as internal standards; approximately 0.97 mol of nitrotyrosine per mole of P450 2B1 was found after treatment with peroxynitrite. The peroxynitrite-treated P450 2B1 was digested with Lys C, and the resulting peptides were separated by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amino acid sequence of the major nitrotyrosine-containing peptide corresponded to a peptide containing amino acid residues 160-225 of P450 2B1, which contains two tyrosine residues. Thus, incubation of P450 2B1 with peroxynitrite resulted in the nitration of tyrosines at either residue 190 or 203 or at both residues of P450 2B1 concomitant with a loss of 2B1-dependent activity.

Purification and Characterization of a Phagocytosis Inducing Factor: Role in Cell-Substratum Adherence

The presence of two different signaling molecules is essential to induce opsonin- independent phagocytosis of particulate activators of the human alternative complement pathway by human monocytes. In addition to the involvement of a low Mrpeptide cytokine or phagocytosis inducing factor (PIF) , we have now established that the participation of bacterial lipopolysaccharide is also required. PIF has been demonstrated to be present in human cell lines of different origins, e.g., WISH cells, Raji cells, U937 cells, HL-60 cells and M21 cells. PIF has been purified to apparent homogeneity from the U937 cell line by anion-exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sephadex G-50. On the basis of Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular mass has been estimated to be approximately 1,600 Da. PIF also plays an important role in the regulation of cell-substratum adherence.

Characterization and Antimicrobial Spectrum of Bifidocin B, a Bacteriocin Produced by Bifidobacterium bifidum NCFB 1454

Five strains of Bifidobacterium bifidum (ATCC 11863 and 29591, and NCFB 1453, 1454, and 1455) were examined for production of bacteriocins in MRS broth with 0.05% cysteine. Only strain NCFB 1454 excreted a bacteriocin into the broth; it was designated bifidocin B. Bifidocin B was sensitive to several proteolytic enzymes (protease IV, pronase E, protease XVII, proteinase K, trypsin, α-chymotrypsin, papain, and pepsin), but was resistant to catalase, peroxidase, lipase, lysozyme, cellulase, ribonuclease A, and amylases. It was also resistant to organic solvents such as ethyl alcohol, acetone, hexane, chloroform, methanol, and ether, and to heating at 90°C for 15, 30, and 60 min or at 121°C for 15 min. Bifidocin B remained active after storage at −20 or −70°C for 3 months and retained biological activity after exposure to pH values of 2 to 10. Bifidocin B was active against some food-borne pathogens and food spoilage bacteria such as Listeria, Enterococcus, Bacillus, Lactobacillus, Leuconostoc, and Pediococcus species but was not active against the other gram-positive and gram-negative bacteria tested. Bifidocin B was produced during exponential phase, reaching a maximum activity of 3,200 AU/ml at early stationary phase. Bifidocin B had a molecular mass of about 3.3 kDa as analyzed by Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Isolation and characterization of a bacteriocin (Butyrivibriocin AR10) from the ruminal anaerobe Butyrivibrio fibrisolvens AR10: evidence in support of the widespread occurrence of bacteriocin-like activity among ruminal isolates of B. fibrisolvens.

Forty-nine isolates of Butyrivibrio fibrisolvens and a single isolate of Butyrivibrio crossotus were screened for the production of inhibitors by a deferred plating procedure. Twenty-five isolates produced factors which, to various degrees, inhibited the growth of the other Butyrivibrio isolates. None of the inhibitory activity was due to bacteriophages. The inhibitory products from 18 of the producing strains were sensitive to protease digestion. Differences in the ranges of activity among the Butyrivibrio isolates and protease sensitivity profiles suggest that a number of different inhibitory compounds are produced. These findings suggest that the production of bacteriocin-like inhibitors may be a widespread characteristic throughout the genus Butyrivibrio. The bacteriocin-like activity from one isolate, B. fibrisolvens AR10, was purified and confirmed to reside in a single peptide. Crude bacteriocin extracts were prepared by ammonium sulfate and methanol precipitation of spent culture supernatants, followed by dialysis and high-speed centrifugation. The active component was isolated from the semicrude extract by reverse-phase chromatography. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that the peptide was purified to homogeneity, having an estimated molecular mass of approximately 4,000 Da. The N terminus of the peptide was blocked. A cyanogen bromide cleavage fragment of the native peptide yielded a sequence of 20 amino acids [(M)GIQLAPAXYQDIVNXVAAG]. No homology with previously reported bacteriocins was found. Butyrivibriocin AR10 represents the first bacteriocin isolated from a ruminal anaerobe.

Isolation and characterization of two fatty acid binding proteins from mouse brain.

Two fatty acid binding proteins (FABPs) were isolated from Swiss Webster mouse brains. Neither protein cross-reacted with antisera to recombinant liver L-FABP. One protein, designated brain H-FABP, migrated on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single band at 14.5 kDa with pl 4.9. Brain H-FABP bound NBD-stearic acid and cis-parinaric acid with K D values near 0.02 and 0.5 microM, respectively. Brain H-FABP cross-reacted with affinity-purified antisera to recombinant heart H-FABP. The second protein, mouse brain B-FABP, migrated on tricine SDS-PAGE gels as a doublet at 16.0 and 15.5 kDa with pl values of 4.5 and 4.7, respectively. Brain B-FABP bound NBD-stearic and cis-parinaric acid with K D values near 0.01 and 0.7 microM, respectively. The brain B-FABP doublet was immunoreactive with affinity-purified antibodies against recombinant mouse brain B-FABP, but not with affinity-purified antibodies against heart H-FABP. (3H)Oleate competition binding indicated that the two brain FABPs had distinct ligand binding specificities. Both bound fatty acids, fatty acyl CoA, and lysophosphatidic acid. Although both preferentially bound unsaturated fatty acids, twofold differences in specific saturated fatty acid binding were observed. Brain B-FABP and brain H-FABP represented 0.1% and 0.01% of brain total cytosolic protein, respectively. In summary, mouse brain contains two native fatty acid binding proteins, brain H-FABP and brain B-FABP.

Role of the leader and structural regions of prelantibiotic peptides as assessed by expressing nisin-subtilin chimeras in Bacillus subtilis 168, and characterization of their physical, chemical, and antimicrobial properties.

Biosynthesis of lantibiotics such as nisin and subtilin involves post-translational modifications, including dehydration of serines and threonines, formation of thioether cross-linkages, translocation, cleavage of a leader sequence, and release into the medium. We have studied the cellular machinery that performs the modifications by constructing and expressing nisin-subtilin chimeric prepeptides in a strain of Bacillus subtilis 168 that possesses all of the cellular machinery for making subtilin except for the presubtilin gene. The chimeras consisted of a normal subtilin leader region (SL), fused to nisin-subtilin chimeric structural regions, one of which was SL-Nis1-11-Sub12-32, in which the N-terminal portion of the structural region was derived from nisin, and the C-terminal portion derived from subtilin. This chimera was accurately and efficiently converted to the corresponding mature lantibiotic, as established by reverse phase high performance liquid chromatography profiles, proton NMR spectroscopy, mass spectral analysis, and biological activity. A succinylated form of the chimera was also produced. Another chimera was in the reverse sense, with subtilin sequence at the N terminus and nisin sequence at the C terminus of the structural region (SL-Sub1-11-Nis12-34). It was processed into a heterogeneous mixture of products, none of which had the characteristics of a correctly processed polypeptide, but did contain a minor component that was active, with a specific activity that considerably exceeded nisin itself. These results, together with results published earlier, establish that processing requires specific recognition between the prelantibiotic peptide and the processing machinery, and in order for the processing to occur correctly, there must be an appropriate combination of the N-terminal part of the leader region and the C-terminal part of the structural region of the prepeptide.

Malonyl-CoA:Acyl Carrier Protein Transacylase from Developing Seeds of Cuphea lanceolata

Malonyl-CoA:acyl carrier protein (ACP) transacylase (MAT; E.C. 2.3.1.39.) was partially purified from developing seeds of Cuphea lanceolata , a crop accumulating up to 90% decanoic acid in storage triacylglycerols. Upon treatment of a cell-free seed extract with ammonium sulfate, most of the enzyme activity was precipitated between 40–75% ammonium sulfate saturation. Further purification was accomplished in 4 steps that included gel filtration on Sephacryl S 200 HR, anion exchange chromatography on Mono Q (0.0 to 0.2M LiBr gradient), rechromatography on Mono Q (0.00 to 0.12M LiBr gradient) and gel filtration on Sephacryl S 100 HR. The MAT-preparation obtained after anion exchange chromatography was free from any β-ketoacyl-ACP synthase (KAS; E.C. 2.3.1.41.) activity and could be used in the synthesis of malonyl-ACP to study medium-chain fatty acid biosynthesis. The molecular mass of the MAT was 27.5 kDa based on gel filtration and 23.5 kDa based on Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-aminoterminal sequence of this enzyme (VAVAELQVE-FI; -,not determined), for the first time determined for a MAT from a higher plant, showed a 25% homology to the enzyme from Escherichia coli .

Identification of active-site peptides from 3H-labeled 2-ethynylnaphthalene-inactivated P450 2B1 and 2B4 using amino acid sequencing and mass spectrometry.

2-Ethynylnaphthalene (2EN) is a mechanism-based inactivator of rat cytochrome P450 (P450) 2B1 with 1.3 mol of adduct bound per mole of P450 inactivated [Roberts, E.S., Hopkins, N.E., Alworth, W.L., & Hollenberg, P.F. (1993) Chem. Res. Toxicol. 6, 470-479]. Further studies have shown that 2EN is also an efficient mechanism-based inactivator of the 7-ethoxycoumarin O-deethylase activity of rabbit P450 2B4 with 0.83 mol of adduct bound per mole of P450. Cleavage of [3H]2EN-inactivated 2B1 with cyanogen bromide, separation of the peptides by HPLC, and further purification of the radiolabeled fraction by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) led to the identification by autoradiography of a radiolabeled peptide (M(r) approximately 3000). Amino acid sequence analysis of the first 12 N-terminal residues revealed the sequence ISLLSLFFAGTE corresponding to positions 290-301 in the protein. When the radiolabeled fraction from the HPLC separation was analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), peaks at m/z 2722.5 and 2890.6 were detected. The lower mass peak corresponds to the molecular ion (average mass) of the cyanogen bromide peptide Ile290 to Met314 (theoretical 2722.2), while the higher mass peak corresponds to the same peptide with a bound 2-naphthylacetyl group (theoretical 2890.4). When [3H]2EN-inactivated 2B4 was treated with cyanogen bromide, the peptides were separated by HPLC, and the fractions were analyzed by Tricine-SDS-PAGE, two radiolabeled peptides (M(r) = 5000 and 8000) were identified by autoradiography. Amino acid sequence analysis of the first 11 residues revealed identical N-termini with the sequence EKDKSDPSSEF corresponding to positions 273-283.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism-based inactivation of cytochrome P450 2B1 by 2-ethynylnaphthalene: identification of an active-site peptide.

The 7-ethoxycoumarin O-deethylase activity of rat liver cytochrome P450 2B1 reconstituted with NADPH-cytochrome P450 reductase and lipid was inactivated by 2-ethynylnaphthalene (2EN) in a time- and NADPH-dependent manner, and the loss of activity followed pseudo-first-order kinetics. The extrapolated KI and kinactivation were 0.08 microM and 0.83 min-1, respectively. The loss of 7-ethoxycoumarin O-deethylation activity displayed a number of characteristics consistent with mechanism-based inactivation, including irreversibility, saturability, protection by an alternate substrate, and the lack of an effect of exogenous nucleophiles on the inactivation. The inactivation was not accompanied by a concomitant loss of spectrally detectable cytochrome P450. HPLC analysis showed that [3H]2EN was irreversibly bound to the protein moiety of cytochrome P450 and the stoichiometry of inactivation was approximately 1.3 mol of 2EN bound per mole of cytochrome P450. Liquid chromatographic and GC-MS analyses of the organic extracts from these incubations showed that the major metabolite was 2-naphthylacetic acid, and a partition ratio of 4-5 mol of acid produced per mole of cytochrome P450 2B1 inactivated was determined. A radiolabeled peptide, approximately 6.5 kDa when analyzed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was isolated by HPLC from a tryptic digest of the [3H]2EN-inactivated cytochrome P450 and NADPH-cytochrome P450 reductase. Sequence data were obtained after cyanogen bromide cleavage of this amino-terminally blocked peptide. These results in conjunction with the results from the cleavage of the intact [3H]2EN-inactivated cytochrome P450 by cyanogen bromide and separation of the peptides either by HPLC or by Tricine-SDS-PAGE followed by transfer of the peptides to a poly(vinylidene difluoride) membrane and sequencing of the labeled peptides from both experiments, led to the identification of a 2EN-modified active-site peptide with the sequence ISLLSLFFAGTETSSTTLRYGFLLM. This corresponds to positions 290-314 in cytochrome P450 2B1. Sequence alignments of cytochrome P450 2B1 with cytochrome P450 2B1 with cytochrome P450 101 predict that this region might correspond to helix I of the bacterial protein [Poulos, T.L. (1988) Pharm. Res. 5, 67-75] that contains a highly conserved threonine residue involved in oxygen binding.

Cumene hydroperoxide-mediated inactivation of cytochrome P450 2B1. Identification of an active site heme-modified peptide.

Cumene hydroperoxide (CuOOH)-mediated inactivation of cytochromes P450 (P450) results in the degradation of their prosthetic heme to products that alkylate the apoprotein. Indirect approaches suggest that this alkylation occurs at the active site. in order to identify the specific apoprotein site(s) alkylated, purified 3H- or 14C-heme-labeled P450 2B1 was incubated with CuOOH and subjected to lysyl endopeptidase-C digestion. Two major peaks (L1 and L2) containing 3H- or 14C-labeled peptides were detected by reverse-phase high pressure liquid chromatography of the digest. L1 contained the highest specific radioactivity and after Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded 3 peptide bands (M(r) approximately 3,500 (P1), 5,000 (P2), and 7,000 (P3)). Although all 3 bands were found radiolabeled, the yield of P1 was higher than that of P2 or P3. Amino acid sequence analysis of the first 13 N-terminal residues of P1 revealed the sequence RICLGEGIARNEL, corresponding to residues 434-446 of the reported 2B1 sequence. A species with the molecular mass of 3771 +/- 1 Da was detected in preliminary electrospray mass spectrometric analysis of L1. Since the theoretical average mass of the predicted peptide (residues 434-466) is 3721.99 Da, the additional 49 +/- 1 Da are considered to be contributed by the alkylating heme fragment. This alkylated 2B1 sequence contains not only Cys436, the conserved residue that provides the SH ligand for heme, but also other highly conserved residues, and therefore corresponds to the heme-sandwiching helix L of P450cam. To our knowledge, this is the first report to localize CuOOH-induced heme alkylation of 2B1 to its active site.

In situ alkylation of cysteine residues in a hydrophobic membrane protein immobilized on polyvinylidene difluoride membranes by electroblotting prior to microsequence and amino acid analysis.

For identification of cysteine residues on microsequence analysis it is crucial to derivatize the sulfhydryl groups. This reaction requires a desalting step which often represents a major obstacle, especially if the sample consists of limited amounts of a hydrophobic membrane protein. An alkylation procedure is described, allowing efficient derivatization (greater than 90%) of cysteines and cystines even in low microgram quantities, as revealed by test analyses with lysozyme and a hydrophobic membrane protein. The modified protein is recovered in high yields in a form suitable for both microsequence analysis and amino acid analysis. The method involves electrophoretic desalting by miniaturized Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in situ alkylation after electro-transfer onto polyvinylidene difluoride membranes. Precautions against NH2-terminal blocking during sample preparations are provided. The general applicability of the method is illustrated by the structural characterization of the low abundance membrane receptor for human urokinase plasminogen activator.

Isolation and characterization of a filamentous viruslike particle from Clostridium acetobutylicum NCIB 6444

A single-stranded 6.6-kb DNA molecule complexed with protein was recovered from the supernatant of Clostridium acetobutylicum NCIB 6444. Electron microscopic examination of the DNA-protein complex revealed the presence of a filamentous viruslike particle, which was designated CAK1. The possible double-stranded plasmidlike replicative form and the single-stranded prophage were also recovered from the cell culture following alkaline lysis. CAK1 was released from the C. acetobutylicum cell culture in the absence of cell lysis. Polyethylene glycol-NaCl coprecipitation of the DNA-protein complex revealed the presence of single-stranded DNA complexed with protein in a manner rendering the DNA resistant to Bal 31 exonuclease. Proteinase treatment of CsCl density gradient-purified CAK1 resulted in recovery of DNase-sensitive single-stranded DNA. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CAK1 demonstrated the presence of a 5-kDa major coat protein. Hybridization data indicated that the single-stranded DNA from CAK1 has homology with the M13 phage of Escherichia coli. An examination of various physical properties of CAK1 suggests that it is similar to the filamentous phage recovered from gram-negative microorganisms. Although infectivity or inducibility of CAK1 could not be demonstrated, to our knowledge this represents the first report of a nonlytic filamentous viruslike particle containing single-stranded DNA being recovered from a gram-positive bacterium.

Production and characterization of a monoclonal antibody specific for enterohemorrhagic Escherichia coli of serotypes O157:H7 and O26:H11

A monoclonal antibody (MAb 4E8C12) specific for Escherichia coli O157:H7 and O26:H11 was produced by immunizing BALB/c mice with a rough strain of E. coli O157:H7. The antibody reacted strongly by a direct enzyme-linked immunosorbent assay with each of 36 strains of E. coli O157:H7. No cross-reactivity was observed with strains of Salmonella spp., Yersinia enterocolitica, Shigella dysenteriae, Proteus spp., Escherichia hermanii, Klebsiella pneumoniae, Campylobacter jejuni, Serratia marcescens, Citrobacter spp., Enterobacter cloacae, Hafnia alvei, Aeromonas hydrophila, and all except five strains of E. coli other than serotype O157:H7 (including strains of serotype O157 but not H7). The E. coli strains (all of serotype O26:H11) that reacted with the antibody were enterohemorrhagic E. coli (EHEC) that were isolated from patients with hemolytic uremic syndrome or hemorrhagic colitis and produced verotoxin similar to that of E. coli O157:H7. MAb 4E8C12 belongs to the subclass immunoglobulin G2a and has a kappa light chain. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of E. coli of different serotypes followed by Western immunoblot analysis revealed that MAb 4E8C12 reacted specifically with two proteins of EHEC strains of serotypes O157:H7 and O26:H11 with apparent molecular weights of 5,000 to 6,000. These proteins appeared to be markers specific for EHEC strains of serotypes O157:H7 and O26:H11. This MAb, because of its specificity, may be a useful reagent of an immunoassay for the rapid detection of these types of EHEC isolates in clinical and food specimens.

Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis

We utilized the recently described tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (TSDS-PAGE) system to study the lipooligosaccharides (LOS) and lipopolysaccharides (LPS) of gram negative bacteria. TSDS-PAGE resulted in a high degree of resolution of LOS and LPS in the ‘mini-gel’ format. TSDS-PAGE resulted in the LOS and LPS migrating as a function of their M r during electrophoresis and allowed estimation of M r from a protein standard. Several species of LOS were analyzed. The newly described procedure allowed a more rapid and accurate analysis of LOS and the core region of LPS.

Determination of amino acid compositions and NH 2-terminal sequences of peptides electroblotted onto PVDF membranes from Tricine-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis: Application to peptide mapping of human complement component C3

The combination of high-resolution Tricine-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (H. Schägger and G. von Jagow (1987) Anal. Biochem. 166, 368–379) and electroblotting onto polyvinylidene difluoride (PVDF) membranes represents a powerful technique for the isolation of small amounts of peptides and protein fragments ( M r 1000–20,000) in a suitable form for amino acid sequencing, directly on the blotting membrane. Conditions for electrophoresis and electroblotting were optimized with respect to high transfer yield and suitability for both amino acid analysis and sequence determination of stained PVDF-bound peptides. Transfer yields were 50–80%, amino acid compositions including Cys were correct, and picomole quantities were sequenced with initial and repetitive yields as high as those we normally obtain for peptides in solution. The method was used for peptide mapping of polymorphic forms of human complement component C3.

Isolation and Characterization of a New Immunomodulatory Protein, Ling Zhi-8 (LZ-8), from Ganoderma lucidium

A novel protein with mitogenic activity in vitro and immunomodulating activity in vivo has been isolated from the mycelial extract of an Oriental medicinal fungus, ling zhi (Ganoderma lucidium). This protein was named ling zhi-8 (LZ-8) and its biochemical and immunological properties are described. LZ-8 was purified by two chromatographic systems, gel filtration and followed by ion-exchange, using an in vitro bioassay measuring blast-formation stimulatory activity toward mouse spleen lymphocytes to monitor purification. Analysis by several types of electrophoresis revealed a single band, with the molecular weight differing slightly depending on the system employed. Under reduced conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the method of Laemmli, U.K. ((1970) Nature 227, 680-685) indicated an apparent Mr = 17,100, while under nonreduced conditions an apparent Mr = 17,500 was found; and, using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a value of apparent Mr = 13,100 was obtained. LZ-8 has an isoelectric point of 4.4, and sugar analysis indicated a low carbohydrate content (1.3%). Half-cysteine, histidine, and methionine were not detected from the analysis of amino acid composition after further purification of LZ-8 by reversed-phase high performance liquid chromatography. LZ-8 was capable of hemagglutinating sheep red blood cells, but no such activity was observed toward human red blood cells (A, B, AB, and O types). In vivo, LZ-8 prevents the production of systemic anaphylaxis reaction in mice if it has been administered repeatedly, and reduction of antibody production is the suggested mechanism. The mechanisms of hemagglutination of sheep red blood cells and of blast-formation stimulation of mouse spleen cells are also discussed.