Subsequent Reversed-phase HPLC Research Articles

Biochemical characterization of the major N-acetylmuramidase from Lactobacillus buchneri.

Bacterial cell wall hydrolases are essential for peptidoglycan remodelling in regard to bacterial cell growth and division. In this study, peptidoglycan hydrolases (PGHs) of different Lactobacillus buchneri strains were investigated. First, the genome sequence of L. buchneri CD034 and L. buchneri NRRL B-30929 was analysed in silico for the presence of PGHs. Of 23 putative PGHs with different predicted hydrolytic specificities, the glycosyl hydrolase family 25 domain-containing homologues LbGH25B and LbGH25N from L. buchneri CD034 and NRRL B-30929, respectively, were selected and characterized in detail. Zymogram analysis confirmed hydrolysing activity on bacterial cell walls for both enzymes. Subsequent reversed-phase HPLC and MALDI-TOF MS analysis of the peptidoglycan breakdown products from L. buchneri strains CD034 and NRRL B-30929, and from Lactobacillus rhamnosus GG, which served as a reference, revealed that LbGH25B and LbGH25N have N-acetylmuramidase activity. Both enzymes were identified as cell wall-associated proteins by means of immunofluorescence microscopy and cellular fractionation, as well as by the ability of purified recombinant LbGH25B and LbGH25N to bind to L. buchneri cell walls in vitro. Moreover, similar secondary structures mainly composed of β-sheets and nearly identical thermal stabilities with Tm values around 49 °C were found for the two N-acetylmuramidases by far-UV circular dichroism spectroscopy. The functional and structural data obtained are discussed and compared to related PGHs. In this study, a major N-acetylmuramidase from L. buchneri was characterized in detail for the first time.

A Systems Biology Approach to Restrictive Cardiomyopathy in Drosophila

Drosophila melanogaster possess a simple linear heart tube and constitute an excellent genetic model system with which to investigate the effects of cardiomyopathic mutation. The Mhc5 myosin heavy chain mutation is located in the ‘transducer’domain and elicits hypercontractile function at the molecular level characterized by high ATPase activity and enhanced in vitro motility properties. Additionally, its expression impairs diastolic relaxation of the cardiac tube reminiscent of restrictive cardiomyopathy in humans. We have investigated the effect of the Mhc5 mutation on cardiac structure/function by quantitative proteomics using isobaric tags for relative quantification (iTRAQ). Excised fly hearts from yw (control) and Mhc5 strains were digested with trypsin, reduced, alkylated and labeled with ITRAQ reagent. Peptides from each pool were mixed together prior to fractionation by strong cation exchange chromatography and subsequent reversed-phase HPLC coupled to tandem mass spectrometry. This approach identified approximately 600 proteins, of which 94 were upregulated and 86 were downregulated in Mhc5 hearts relative to yw hearts (p<0.05). Ontological cluster analysis of the genes encoding the regulated proteins revealed that myofibrillar disarray in Mhc5 hearts likely stems from overexpression of actin with concomitant reduction of myofibrillar assembly proteins such as spectrin, and other actin-binding proteins. Structural remodeling was also characterized by increased expression of extracellular matrix proteins. Upregulation of proteins involved in mitochondrial oxidative phosphorylation and fatty acid catabolism suggests further bioenergetic remodeling. The proteomic, structural and ultrastructural data are consistent with a model whereby the elevated ATPase activity caused by Mhc5 mutation increases energetic demand, thereby stimulating a concerted compensatory metabolic response to maintain energetic homeostasis. Ongoing protein-network/interactome analysis will help to further refine the model.

Solid-phase/supercritical-fluid extraction for liquid chromatography of phenolic compounds in freshwater microalgae and selected cyanobacterial species

In the present paper a new extraction technique based on the combination of solid-phase/supercritical-fluid extraction (SPE/SFE) with subsequent reversed-phase HPLC is described. The SPE/SFE extractor was originally constructed from SPE-cartridge incorporated into the SFE extraction cell. Selected groups of benzoic acid derivatives ( p-hydroxybenzoic, protocatechuic, gallic, vanillic and syringic acid), hydroxybenzaldehydes (4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde) and cinnamic acid derivatives ( o-coumaric, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acid) were extracted. Cyclic addition of binary extraction solvent system based on methanol:water (1:1, v/v) and methanol/ammonia aqueous solution was used for extraction at 40 MPa and 80 °C. The p-hydroxybenzoic, protocatechuic, vanillic, syringic, caffeic and chlorogenic acid; 4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde were identified by HPLC-electrospray mass spectrometry in SPE/SFE extracts of acid hydrolyzates of microalga ( Spongiochloris spongiosa) and cyanobacterial strains ( Spirulina platensis, Anabaena doliolum, Nostoc sp., and Cylindrospermum sp.). For the identification and quantification of the compounds the quasi-molecular ions [M−H] − and specific fragments were analysed by quadrupole mass spectrometry analyzer. Our analysis showed that the microalgae and cyanobacteria usually contained phenolic acids or aldehydes at μg levels per gram of lyophilized sample. The proposed SPE/SFE extraction method would be useful for the analysis of different plant species containing trace amount of polar fraction of phenols.

Automated synthesis of 11β-methoxy-4,16α-[16α-18F]difluoroestradiol (4F-M[18F]FES) for estrogen receptor imaging by positron emission tomography

Addition of both a 4-fluoro and 11beta-methoxy group onto 16alpha-[(18)F]fluoroestradiol ([(18)F]FES) yields 11beta-methoxy-4,16alpha-[16alpha-(18)F]difluoroestradiol (4F-M[(18)F]FES) with potential improved properties for positron emission tomography (PET) imaging of estrogen receptor densities in breast cancer patients. In order to provide 4F-M[(18)F]FES as a radiopharmaceutical for clinical trials, we developed an automated synthesis procedure using 3-O-methoxymethyl-11beta-methoxy-4-fluoro-16,17-O-sulfuryl-16-epiestriol as precursor. The radio synthesis involves stereoselective opening of the protected cyclic sulfone precursor via nucleophilic fluorination with [(18)F]fluoride in acetonitrile. After removal of the protecting ether and 17beta-sulphate groups by rapid hydrolysis in acidic ethanol and subsequent reversed-phase HPLC purification, the pure 4F-M[(18)F]FES was obtained as a sterile physiological saline solution in 45-50% radiochemical yield (decay corrected). The radiochemical purity of the final product was >98% and the effective specific activity (ESA) of 4F-M[(18)F]FES prepared under optimized conditions was >15,000 Ci/mmol. The total preparation time was 110+/-5 min and the product was shown to be stable for at least 6 h.

Determination of Amphetamine by HPLC After Acetylation

An analytical procedure has been developed for the HPLC determination of amphetamine by off-line pre-column derivatization. The proposed procedure consists of sample preparation by acetylation of amphetamine with acetic anhydride and a subsequent reversed-phase HPLC separation on an octadecyl silica stationary phase with salt-free mobile phase (tetrahydrofuran, acetonitrile, 0.1% triethylamine in water, 15:15:70 v/v) applying UV-detection. The applicability of the elaborated procedure is demonstrated with results obtained by analysis of real samples seized in the Hungarian black market.

Isolation and characterization of a new trypsin inhibitor from Crotalaria paulina seeds.

A new trypsin inhibitor (CPTI) has been isolated from Crotalaria paulina seeds. Purification of the inhibitor was carried out by gel filtration, ion-exchange chromatography, and subsequent reversed-phase HPLC. The presence of a single polypeptide chain, with a molecular mass of 20 kDa and isoelectric point 4.0, was detected. The trypsin inhibitor had a Ki value of 4.5 x 10(-8) M and was capable of acting on human, bovine, and porcine trypsin and weakly on bovine chymotrypsin. Amino acid analysis showed that CPTI has a high content of aspartate, glutamate, leucine, serine, and glycine, having 177 amino acid residues in its composition. These data suggest that the protein belongs to the Kunitz-type trypsin inhibitors.

Adaptive changes in lipid composition of skeletal sarcoplasmic reticulum membranes associated with aging

We have undertaken a detailed examination of changes associated with aging in lipid composition and corresponding physical properties of hindlimb skeletal sarcoplasmic reticulum (SR) membranes isolated from young (5 months), middle-aged (16 months), and old (28 months) Fischer strain 344 rats. Silica gel HPLC chromatography was used to separate phospholipid headgroup species. Subsequent reversed-phase HPLC was used to resolve fatty acid chain compositions of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol species. For all three phospholipid pools, significant age-related variations are observed in the abundance of multiple molecular species, particularly those having polyunsaturated fatty acid chains. Using mass spectrometry (fast atom bombardment and tandem techniques) to distinguish ester- from ether-linked phosphatidylethanolamine species, we demonstrate that overall plasmenylethanolamine content is substantially increased with age, from 48 mol% to 62 mol%. A substantial increase is also observed in the single molecular species 18:0–20:4 phosphatidylinositol suggesting implications for signalling pathways. In addition, associated with senescence we find a significant increase in the rigidifying lipid, cholesterol. Despite these changes in lipid composition of different aged animals, the average bilayer fluidity examined at several bilayer depths with stearic acid spin labels, is not altered. Neither do we find differences in the rotational mobility of maleimide spin-labeled Ca 2+-ATPase, as determined from saturation-transfer electron paramagnetic resonance, which is sensitive to both the fluidity of lipids directly associated with the Ca 2+-ATPase and to its association with proteins.

Resolution of three Ca 2+-dependent protein kinases and endogenous substrate proteins from bitter gourd seeds

A protein that is phosphorylated by plant Ca 2+-dependent protein kinase (CDPK) was isolated from seeds of bitter gourd ( Momordica charantia). This bitter gourd protein (referred to as BGP) was purified by a procedure involving batchwise elution from carboxymethylcellulose (CM-52), gel filtration, cation exchange HPLC and reversed phase HPLC. BGP preparations exhibit three bands (11 kDa, 7 kDa and 4 kDa, respectively) on SDS-PAGE, of which the 4-kDa material copurifies on SDS-PAGE with [ 32P]phosphoBGP phosphorylated by wheat CDPK or CDPKs isolated from M. charantia seeds. The 4-kDa [ 32P]phosphoBGP can be resolved from the other components after phosphorylation using [γ- 32P]ATP and CDPK and subsequent reversed phase HPLC. Electrospray ionization mass spectrometry (ESMS) of BGP revealed a major component with an average molecular mass of 11 450 Da, minor 11 287 Da and 11 563 Da components and other minor components corresponding to K + adducts of the major component. Reversed phase HPLC of BGP after treatment with 3 M guanidine HCl and 25% (v/v) 2-mercaptoethanol resolves three very similar small proteins (BGS1, BGS2 and BGS3) having average molecular masses of 3443 Da, 3606 Da and 3720 Da, respectively, and a larger protein (BGL) having an average molecular mass of 7850 Da. The 11 287-Da, 11 450-Da and 11 563-Da BGP components correspond within experimental error to 1:1 complexes of the large subunit (BGL) with a small subunit (BGS1, BGS2 or BGS3, respectively), with all complexes involving approximately three disulphide linkages (calculated masses 11 287 ± 2 Da, 11 450 ± 1 Da and 11 563 ± 2 Da, respectively). The 3606-Da BGS2 is a poor CDPK substrate in comparison with BGS1 and BGS3. Three CDPKs (CDPKs I, II and III) were resolved from M. charantia. All three CDPKs are absolutely dependent on millimolar Mg 2+ and about 10 −7–10 −6 M free Ca 2+ for maximal activity and phosphorylate BGP, histone III-S, bovine serum albumin, casein and myosin light-chain derived synthetic peptide (MLCP) (KKRAARATSNVFA-NH 2). CDPKs I and II phosphorylate the synthetic peptide kemptide (LRRASLG) better than CDPK III. CDPKs I, II and III are inhibited by poly- l-arginine (IC 50 values 52, 70 and 43 nM, respectively) and the calmodulin antagonist calmidazolium (IC 50 values about 30 μM).

Isolation of carboxyl-termini and blocked amino-termini of viral proteins by high-performance cation-exchange chromatography

The strong cation-exchanger, PolySulfoethyl Aspartamide, has been assessed as a medium for isolation of carboxyl-terminal and blocked amino-terminal peptides from tryptic digests of small quantities of viral proteins. Peptides with a single positive charge, the blocked amino-terminal peptides of ovalbumin and the Newcastle disease virus (NDV) matrix protein and carboxyl-terminal peptides of ovalbumin and the NDV nucleocapsid protein, eluted in early ion-exchange fractions and were readily isolated in homogeneous form by subsequent reversed-phase HPLC. Some early ion-exchange fractions also contained singly charged peptides derived by “chymotryptic-like” cleavage, whilst other peptides eluted in these fractions due to their highly acidic character. Terminal sequences with additional basic residues were isolated from later eluting ion-exchange fractions. Peptides with this property included the blocked amino-terminus of the NDV nucleocapsid protein and a portion of the carboxyl-terminus of the NDV matrix protein. Hitherto undescribed polymorphism in the amino-terminal region of ovalbumin was revealed in this study. Truncated peptides from the carboxyl-terminus of the NDV matrix protein were also detected. The presence of these peptides could be a reflection of carboxyl-terminal processing of the matrix protein. The strategy described herein should be of general utility for selective microisolation of carboxyl-terminal peptides and blocked amino-terminal peptides from tryptic digests of proteins.

ANALYSIS OF γ-CARBOXYGLUTAMIC ACID IN MARINE INVERTEBRATE SHELL MATRICES

A method is presented for analysis of γ-carboxyglutamic acid (Gla) in marine invertebrate shell matrices. It is based on the enrichment of Gla by BaSO 4 adsorption of the alkaline hydrolysate, its precolumn derivatization with 4-dimethylamino-4'-azobenzene sulfonyl chloride (DABS-Cl) and subsequent reversed-phase HPLC analysis

A trace-enrichment technique for the loading of gel-permeation high-performance liquid chromatography columns

Gel-permeation high-performance liquid chromatography (GP-HPLC) columns provide rapid high-resolution separations but are frequently limited to analytical tasks because the injection volumes must be small. The reduction of volume required for the loading of solutes can often be impractical and lead to poor recoveries. We have developed a trace-enrichment technique to circumvent this problem. By placing a Waters Guard Pak within the loop of a Valco injector and connecting a pump to the injection port it is possible to concentrate proteins and peptides onto the guard column from relatively large volumes. Enrichment onto a reversed-phase guard column insert is achieved by loading solutes in an aqueous solution or one of low organic solvent concentration. Provided that the GP-HPLC is mean-while equilibrated with a solvent system of sufficiently high organic solvent concentration (i.e., 40% acetonitrile containing 0.1% trifluoroacetic acid) it is possible to elute material that has been loaded in this manner by simply placing the injection loop in line with the column. The solvent strength abruptly increases and the peptide or protein sample is loaded onto the column in a very small volume. We have applied this loading principle to both analytical and semipreparative problems. The amino-terminal fragment of pro-opiomelanocortin (POMC) has been extracted from a single human fetal pituitary (18 weeks gestation) and characterized in terms of its molecular weight. This study indicated that no proteolytic processing of the amino-terminal fragment of POMC takes place at this stage in development. In a larger scale application the amino-terminal fragment of POMC was purified from bovine anterior pituitaries. The trace-enrichment GP-HPLC loading technique was used as an initial fractionation procedure. The isolation of the polypeptide by subsequent reversed-phase HPLC was greatly facilitated by the initial removal of high- and low-molecular-weight contaminants.

Determination of Folic Acid in Commercial Diets by Anion-Exchange Solid-Phase Extraction and Subsequent Reversed-Phase HPLC

Abstract Folic acid at the μ/g level is determined in total nutritional diets by concentration on disposable commercial anion exchange columns followed by elution with a concentrated salt solution and subsequent reversed-phase HPLC with absorbance detection at 365 nm. The method is specific for folic acid with respect to some known degradants and electrochemically generated oxidation products.

Hypothalamic somatostatin-14 and -28 biosynthesis: effects of anesthetics and hypophysectomy

Somatostatin-14 and -28 biosynthesis in rat hypothalami was assessed following intracerebroventricular injection of [ 35S]cysteine, and subsequent reversed-phase HPLC separation of the labeled peptides. Identities of somatostatin-14 and -28 were verified using 3 HPLC buffer systems. The anesthetic employed during label administration itself influenced [ 35S]cysteine incorporation into both somatostatins. Hypophysectomy diminished label incorporation into both peptides, suggesting that somatostatin has an inhibitory role in the control of growth hormone secretion.