Affinity Purification of Methyllysine Proteome by Site-Specific Covalent Conjugation.

A basic but critical step in targeted proteomics by mass spectrometry is the separation of the targeted proteins from the complex mixture of the whole proteome by affinity purification. The bait protein is usually immobilized on the surface of a solid support to enable affinity-based purification of the targeted proteome. Here, we developed a site-specific covalent immobilization of the bait protein through affinity-guided covalent coupling (AGCC) of a single cysteine residue of an SH2 domain (utilized as an affinity tag for the protein target) with an engineered ligand peptide. Site-specific covalent immobilization of a methyllysine-binding protein HP1ß chromodomain on the agarose resin was used to purify the methyllysine proteome from the whole-protein mixture. This new bait immobilization led to a notably low background in the affinity purification step, markedly outperforming the conventional (His)6 tag-nickel nitrilotriacetic acid (Ni-NTA) immobilization method. Subsequent analysis of the purified proteome identified 275 lysine methylated sites and 184 methylated proteins from 332 HP1ß CD-binding proteins, including 30 novel methylated proteins. This work demonstrates that a robust site-specific covalent protein immobilization method is well-suited for proteomic analysis of low-abundance proteins. This method also enables the identification of new methylated proteins and methylation sites in the methyllysine proteome.

Inhibition of listeria monocytogenes in minas frescal cheese by free and nanovesicle-encapsulated nisin

The effectiveness of free and nanovesicle-encapsulated nisin to control Listeria monocytogenes in Minas Frescal cheese was investigated. Commercial nisin was encapsulated into liposomes of partially purified soy lecithin. Free (0.1 mg/mL and 0.25 mg/mL) and nanovesicle-encapsulated nisin (0.25 mg/mL) were applied onto the surface of cheese samples, and L. monocytogenes was inoculated before incubation at 6-8°C for 28 days. A bactericidal effect was observed with 0.25 mg/mL free nisin; a bacteriostatic effect was observed for liposome-encapsulated nisin and 0.1 mg/mL free nisin. Free nisin was more efficient than nisin-loaded liposomes in controlling L. monocytogenes. Possible reasons for this behavior, and also the significance of nisin to soft cheeses are discussed. Nisin acted as a suitable barrier within hurdle technology, potentially extending the shelf-life and safety of fresh cheeses.

[Preparation of poly(methyl acrylate) microfluidic chips surface-modified by hyperbranched polyamide ester and their application in the separation of biomolecules].

The surface of poly (methyl acrylate) (PMMA) microfluidic chips were modified using hyperbranched polyamide ester via chemical bonding. The contact angles of the modified chips were measured. The surface morphology was observed by scanning electron microscope (SEM) and stereo microscope. The results showed that the surface of the modified chips was coated by a dense, uniform, continuous, hydrophilic layer of hyperbranched polyamide ester. The hydrophilic of the chip surface was markedly improved. The contact angle of the chips modified decreased from 89.9 degrees to 29.5 degrees. The electro osmotic flow (EOF) in the modified microchannel was lower than that in the unmodified microchannel. Adenosine and L-lysine were detected and separated via the modified PMMA microfluidic chips. Compared with unmodified chips, the modified chips successfully separated the two biomolecules. The detection peaks were clear and sharp. The separation efficiencies of adenosine and L-lysine were 8.44 x 10(4) plates/m and 9.82 x 10(4) plates/m respectively, and the resolutions (Rs) was 5.31. The column efficiencies and resolutions of the modified chips were much higher than those of the unmodified chips. It was also observed that the modified chips possessed good reproducibility of migration time. This research may provide a new and effective method to improve the hydrophilicity of the PMMA surface and the application of PMMA microfluidic chips in the determination of trace biomolecules.

A rapid and robust assay for the determination of the amino acid hypusine as a possible biomarker for a high-throughput screening of antimalarials and for the diagnosis and therapy of different diseases.

Eukaryotic initiation factor 5A (eIF5A) has recently been identified as a biomarker of prognostic significance and therapeutic potential for the treatment in hepatocellular carcinoma. This prompted us to establish a rapid and robust assay to determine deoxyhypusine and hypusine formed with the purified enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) from Plasmodium to develop a rapid screening assay for antimalarial drugs. The peptide hydrolysate obtained from hypusinylated eIF5A was analyzed by ultra performance liquid chromatography (UPLC) with retention times for deoxyhypusine of 7.44 min and for hypusine of 7.30 min, respectively. The limit of detection for both compounds was 0.144 ng/µl. Determination of the specific activity of Plasmodium DOHH resulted in a twofold higher specific activity than its human counterpart. Following the iron-complexing strategy of the ferrous iron which is present in the active site of Plasmodium DOHH, a series of iron chelating compounds was tested. 2,2'-Dipyridyl and mimosine abolished DOHH activity completely while 4-oxo-piperidine-carboxylates i.e. the nitrophenylether JK8-2 and EHW 437, the oxime ether of the piperidine aldehyde, showed no inhibition although they were highly active in in vitro cultures of Plasmodium and in vivo in a rodent mouse model. The method allows a high-throughput screening (HPTS) of antimalarial drugs and the evaluation of eIF5A as a biomarker.

Extraction and separation of a lysine-rich protein by formation of supramolecule between crown ether and protein in aqueous two-phase system.

The macrocyclic calixarenes and crown ethers have recently been found to form hydrophobic complexes with the cationic protein cytochrome c (Cyt-c), by recognizing lysine residues on the protein surface. In the present study, it was found that the distribution of cytochrome c in Li(2)SO(4)/PEG aqueous two-phase system (ATPS) can be controlled by complexation with the crown ether dicyclohexano-18-crown-6 (DCH18C6). The protein was quantitatively extracted into the PEG-rich phase in the presence of DCH18C6 and perchlorate ion. Of various crown ethers and their analogues that were investigated, only DCH18C6 was able to extract cytochrome c into the PEG-rich phase. Extraction of cytochrome c in the ATPS using DCH18C6 is complete within 5 min. Cytochrome c complexed with DCH18C6 in the PEG-rich phase was quantitatively recovered into a salt-rich phase using K(2)SO(4) by ion exchange of potassium ion and cationic protein in the cationic protein complex with DCH18C6. Selective extraction of cationic proteins was demonstrated in the ATPS. Under optimum conditions, the lysine-rich protein cytochrome c was selectively extracted over other cationic proteins using DCH18C6.

Absolute configuration and antitumor activity of myxochelin A produced by Nonomuraea pusilla TP-A0861.

In the screening of antitumor compounds from microbial secondary metabolites, myxochelin A was isolated from a culture broth of Nonomuraea pusilla TP-A0861. The absolute configuration was determined to be S by synthesizing both enantiomers from an L- or D-lysine derivative and comparing their specific rotations. Both enantiomers of myxochelin A showed remarkable inhibitory effects on the invasion of murine colon 26-L5 carcinoma cells at non-cytotoxic concentrations.

Separation, identification, and quantification of amino acids in L-lysine fermentation potato juices by gas chromatography-mass spectrometry.

The amino acid composition of L-lysine fermentation juices from potatoes and cane molasses from a green biorefinery has been determined by gas chromatography-mass spectrometry. N-Methyl-N-tert(butyldimethylsilyl)tri-fluoroacetamide (MTBSTFA) was used as derivatization reagent to prepare the t-butyldimethylsilyl derivatives of the amino acids present in the juices. The amino acids in these derivatives were identified from both their EI and CI mass spectra and their retention times in the gas chromatogram, and they were quantified employing the GC response signals relative to cycloleucine as internal standard.

Application of an anhydrotrypsin-immobilized precolumn for selective separation of peptides having arginine or lysine at their C-termini by column-switching high-performance liquid chromatography.

A column-switching high-performance liquid chromatographic (CS-HPLC) system which consisted of an anhydrotrypsin (AHT)-immobilized diol-silica precolumn and a reversed-phase analytical column was developed for the selective separation of peptides having Arg or Lys at their C-termini. Tuftsin (Thr-Lys-Pro-Arg) could be enriched almost quantitatively on the precolumn when loaded with water as a carrier solvent and the precolumn was washed with 10-30 mM acetate buffer (pH 5.0). An investigation of the affinity characteristics of 55 peptides to the AHT precolumn showed that among twelve peptides having Arg or ArgNH2 at their C-termini and more than four amino acid residues, ten were retained almost quantitatively on the precolumn, and eight out of nine peptides having Lys at their C-termini were less retained. The peptide having D-Arg at its C-termini was not retained. However, twelve out of thirty peptides having no Arg or Lys at their C-termini were also retained, but the retention was greatly decreased, in contrast to the Arg peptides, when the precolumn was washed with 20 mM calcium chloride solution. The results indicate that the CS-HPLC system equipped with an AHT precolumn offers new selectivity in the HPLC selectivity in the HPLC separation of peptides.

Acid dissociation constant and apparent nucleophilicity of lysine-501 of the alpha-polypeptide of sodium and potassium ion activated adenosinetriphosphatase.

A combination of competitive labeling with [3H]acetic anhydride [Kaplan, H., Stevenson, K. J., & Hartley, B. S. (1971) Biochem. J. 124, 289-299] and immunoaffinity chromatography is described that permits the assignment of the acid dissociation constant and the absolute nucleophilicity of individual lysines in a native enzyme. The acid dissociation constant of lysine-501 of the alpha-polypeptide in native (Na+ + K+)-ATPase was determined. This lysine had a normal pKa of 10.4. The rate constant for the reaction of the free base of lysine-501 with acetic anhydride at 10 degrees C is 400 M-1 s-1. This value is only 30% that for a fully accessible lysine in a protein. The lower than normal apparent nucleophilicity suggests that lysine-501 is hindered from reacting with its intrinsic nucleophilicity by the tertiary structure of the enzyme and is consistent with its location within a pocket that forms the active site upon the surface of the native protein.

Myxochelin A, a new iron-chelating compound from Angiococcus disciformis (Myxobacterales). Production, isolation, physico-chemical and biological properties.

Myxochelin A, a new catechole siderophore, was isolated from the culture broth of the myxobacterium, Angiococcus disciformis strain An d30. As is the case with other iron-chelating compounds the production of myxochelin A could be markedly increased up to 44 mg/liter by fermentation at low iron concentrations (10(-7) M FeCl3). The new substance showed weak activity against some bacteria.

Glidobactins A, B and C, new antitumor antibiotics. I. Production, isolation, chemical properties and biological activity.

New antitumor antibiotic glidobactins A, B and C were isolated from the cultured broth of a gliding bacterium, Polyangium brachysporum sp. nov. No. K481-B101. They are novel molecules carrying the common cyclic tripeptide nucleus substituted with different alpha, beta, gamma, delta-unsaturated fatty acids. Glidobactins exhibit broad inhibitory activity against fungi and yeasts, and prolong the life span of mice inoculated with P388 leukemia cells.

Isolation and characterization of an 18 kDa hypusine-containing protein from cultured NB-15 mouse neuroblastoma cells.

An 18 kDa protein can be metabolically labeled by [3H]putrescine or [3H]spermidine in various mammalian cells. The labeling is due to a post-translational modification of one lysine residue to hypusine using the aminobutyl moiety derived from spermidine. In view of the lack of knowledge of the function of this spermidine-modified protein, we decided to use the radioactivity associated with the [3H]spermidine-labeled 18 kDa protein as a tracer to develop a simple procedure for purifying this protein from cultured cells. We first screened more than 15 different affinity adsorbents for their ability to bind the labeled 18 kDa protein. This approach enabled us to develop a four-step procedure to purify the labeled 18 kDa protein from NB-15 mouse neuroblastoma cells. The procedure, including a Cibacron Blue column, an omega-aminooctyl-agarose, a Sepharose G-50, and a Mono Q column, resulted in an 800-fold purification of the labeled 18 kDa protein. Two-dimensional gel analysis of fractions enriched in the labeled 18 kDa protein revealed (i) the presence of isoforms of hypusine-containing 18 kDa protein, with pI values ranging from 4.7 to 5.2, and (ii) the presence of an additional labeled protein with an apparent molecular mass of 22 kDa and a pI value of 5.0. The labeling intensity of the 22 kDa protein, however, was less than 5% of that of the 18 kDa protein. Peptide map analysis, using the V-8 proteinase digestion method, indicated that the 18 kDa hypusine-containing protein obtained from NB-15 cells was similar to eukaryotic initiation factor 4D isolated from rabbit reticulocytes.

Resolution of the enantiomers of various alpha-substituted ornithine and lysine analogs by high-performance liquid chromatography with chiral eluant and by gas chromatography on Chirasil-Val.

A reversed-phase high-performance liquid chromatography method, with L-proline and copper as chiral mobile phase, is described for the enantiomeric resolution of various alpha-substituted ornithine and lysine analogs. Although ornithine gives no separation with the chiral eluant used, excellent resolutions are obtained for various alpha-alkyl-, alpha-halogenomethyl-, alpha-vinyl-, and alpha-ethynyl-substituted ornithines. Similar separations are also observed for the dehydroornithine and lysine analogs. Gas chromatography on a chiral stationary phase, Chirasil-Val, allows the resolution of the ornithine and lysine analogs after derivatization into the monofluoroacyl derivatives of their corresponding lactams. No resolution or only a poor resolution is obtained by GC on Chirasil-Val for the dehydroornithine analogs as their di-N-perfluoroacyl alkyl esters. The chiral eluant HPLC procedure is easily scaled up for the semipreparative resolution of several ornithine analogs, i.e., alpha-fluoromethylornithine, alpha-difluoromethylornithine, alpha-chlorofluoromethylornithine, and alpha-fluoromethyldehydroornithine, which are known as potent ornithine decarboxylase inhibitors in vitro and in vivo.

The NADP+-binding site of ferredoxin-NADP+ reductase. Sequence of the peptide containing the essential lysine residue.

The flavoprotein ferredoxin-NADP+ reductase is inactivated and loses its ability to bind NADP+ during covalent modification of a lysine by 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) [Zanetti, G. (1976) Biochim. Biophys. Acta 445, 14-24]. The substrate NADP+ gives almost complete protection against inactivation and modification. These observations are extended in this report by the characterization of an octapeptide containing the dansyl-lysine which was isolated by high-performance liquid chromatography from tryptic digests of protein modified with radiolabeled reagent. The amount of this peptide was severely reduced in protein modified in the presence of NADP+. The sequence of the dansyl-peptide, only partially obtained by Edman degradation, was completed by analysis of the fragments resulting from thermolysin digestion of the purified tryptic dansyl-peptide. Thus, the octapeptide containing the essential lysine residue has the following sequence: H2N-Ser-Val-Ser-Leu-Cys-Val-Lys-Arg-COOH. A comparison with corresponding sequences of other known NADP+-dependent dehydrogenases is attempted.

Proximity of reactive lysyl residue to the antigenic site in rabbit skeletal myosin against the monoclonal antibody (MF-18) generated to chicken skeletal myosin.

MF-18, one of the monoclonal antibodies generated to chicken myosin, cross-reacted with rabbit skeletal myosin subfragment-1 (S1). Utilizing an improved procedure of immuno-blotting, a decrease in reactivity of MF-18 to S1 by trinitrophenylation was observed. This indicates that the reactive lysyl residue is very close to the hapten site. This is consistent with the evidence that the hapten site resides in the 26,000 dalton tryptic fragment of S1. Use of such antibodies as labels may open the way to determining the location of specific hapten sites in the three-dimensional image of actin-S1 complex reconstructed from the electron micrographs.

Characterization of phospholipases A2 of Naja melanoleuca snake venom modified at the N-terminal region.

In phospholipase A2 from Naja melanoleuca snake venom all four lysines were converted into the epsilon-amidinated derivatives without reaction of the alpha-amino group. The amidinated phospholipase (AMPA) showed high enzymatic activity. Starting from AMPA, chemical modification reactions were carried out at the alpha-amino function. This group was blocked with a tert-butyloxycarbonyl or a phenylthiocarbamyl group. Furthermore the polypeptide chain was shortened by one residue by removing the N-terminal asparagine, resulting in the formation of des-Asn1-AMPA. The native enzyme was shortened by eight residues by cyanogen bromide cleavage at the single methionine residue. Although all modified proteins show a reduced affinity for monomeric lipids, they are easily saturated with micellar substrate analogs. Whereas the removal of the N-terminal octapeptide abolished all enzymatic activity the other modified enzymes possess a low (1%), but measurable enzymatic activity. It is concluded that chemical modifications in the N-terminal region give rise to a distortion of the active site, thus reducing the activity of the lipid-bound enzyme.

A 1H-NMR study of isolated domains from human plasminogen. Structural homology between kringles 1 and 4.

Kringles 1 and 4 from human plasminogen are polypeptide domains of Mr approximately equal to 10000 each of which can be isolated by proteolysis of the zymogen. They have been studied by 1H-NMR spectroscopy at 300 MHz and 600 MHz. The spectra, characteristic of globular structures, show striking analogies that point to a close conformational relatedness among the two kringles, consistent with their high degree of amino acid conservancy and homology. The interaction of both kringles with p-benzylaminesulfonic acid (BASA), an antifibrinolytic drug that binds to a lysine-binding site, results in better resolved, narrower lines for both spectra. Aromatic and methyl-region spectra of BASA complexes of kringles 1 and 4 were compared and the latter was studied by two-dimensional NMR spectroscopy. Analysis of the CH3 multiplets in terms of their resonance patterns, and the amino acid compositions and sequences of the two kringles, leads to the identification of most signals and to some assignments. In particular, a doublet at -1 ppm, exhibited by both kringles and also found in reported proton spectra of homologous bovine prothrombin fragments, has been assigned to Leu46, a residue that is conserved in all of the kringles studied to date by 1H-NMR. Since this resonance is somewhat more sensitive to BASA than other methyl signals, it is likely that Leu46 is proximal to the lysine-binding site. Nuclear Overhauser experiments reveal that Leu46 is surrounded by a cluster of closely interacting hydrophobic and aromatic side chains. Kringle 4 was also compared with a derivative chemically modified at Trp72 with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide. As judged from the proton spectra, the modified kringle 4 retains globularity and is perturbed mainly in the aromatic region, in analogy to that which is observed for the unmodified kringle upon BASA binding. Furthermore, although previous studies have indicated no retention of the modified kringle by lysine-Sepharose, the NMR studies point to a definite interaction between BASA and the kringle derivative. The spectroscopic data also suggest that the His31 imidazole is not significantly affected by the ligand and that the lysine-binding site is structured mostly by hydrophobic side chains, including Trp72 in the case of kringle 4, and probably Tyr72 in kringle 1.

Chemically induced gene expression. Manipulation of the transforming ability of simian virus 40 minichromatin by specific chemical hyperacetylation of histones H3 and H4.

We have developed a non-enzymatic acetylating procedure, closely resembling the situation in vivo, utilizing acetyl adenylate, an acetylating agent in vivo, that mimics the enzymatic hyperacetylation of specific histone species. Analysis of the acetylated species of calf thymus histones produced from reaction with soluble chromatin yielded the same species generated in vivo and observed during active gene transcription. Four species of histone H4 and three of histone H3 occur with no alteration in histones H2A or H2B. This procedure has been utilized to hyperacetylate simian virus 40 (SV40) minichromatin in vitro in order to study the effect of acetylated compared to non-acetylated minichromatin in cellular transformation of cultured Balb/3T3 cells. Transformed cell foci appeared only in the cultures infected with hyperacetylated SV40 minichromatin. To select for cellular transformation, foci were transferred to agar-lined culture flasks and grown in the suspension of 1% methylcellulose. The selected cells were plated on slides and analyzed for the presence of T-antigen by indirect immunofluorescence. The hyperacetylated-minichromatin-infected cells exhibited T-antigen-specific fluorescence, while non-acetylated-minichromatin-treated cells and normal cells showed no specific fluorescence. These results suggest a major role for histone hyperacetylation in the mechanism of SV40 viral transformation.

L-Lysine: 2-oxoglutarate 6-aminotransferase. Subunit structure composed of non-identical polypeptides and pyridoxal 5'-phosphate-binding subunit.

L-Lysine:2-oxoglutarate 6-aminotransferase from Flavobacterium lutescence (= Achromobacter liquidum) has been shown to be composed of one each of four non-identical subunits, A, B1, B2, and C. The subunits were isolated by gel filtration, and DEAE-cellulose chromatography in the presence of 8 M urea. Their molecular weights were determined by ultracentrifugation, gel electrophoresis and gel filtration: subunit A 24,000; B1 28,000; B2 28,000; C 45,000. These subunits were all different in amino acid composition. Of the two molecules of bound pyridoxal 5'-phosphate, the one which absorbs at 415 nm is bound to subunit B2 and participates in the catalytic action of the enzyme.