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1.
Chemosphere ; 69(2): 229-39, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17544478

ABSTRACT

Dry olive mill residue (DOR) from the olive oil production by two phase centrifugation system was fractionated by a consecutive continuous solid-liquid extraction obtaining the EAF, PF, MF and WF fractions with ethyl acetate, n-propanol, methanol and water, respectively. The chemical, chromatographic and mass spectrometric analyses showed EAF, PF and MF to be mainly composed of simple phenols, phenolic acids, flavonoids and glycosilated phenols (glycosides of phenols, secoiridoids and flavonoids), whereas WF was mainly consisting of polymerin, the metal organic polymeric mixture previously identified in olive oil mill waste waters and composed of carbohydrates, melanin, proteins and metals (K, Na, Ca, Mg and Fe). The identification in DOR of oleoside, 6'-beta-glucopyranosyl-oleoside and 6'-beta-rhamnopyranosyl-oleoside, and of its organic polymeric component, known as polymerin, are reported for the first time in this paper. The inoculation of the previously mentioned fractions with saprobe fungi Coriolopsis rigida, Pycnoporus cynnabarinus or Trametes versicolor indicated these fungi to be able to metabolize both the phenols and glycosilated phenols, but not polymerin. In correspondence, EAF, PF, MF and WF, which proved to be toxic on Lepidium sativum, decreased their toxicity after incubation with the selected fungi, WF showing to be also able to stimulate the growth of the selected seeds. The phytotoxicity appeared mainly correlated to the monomeric phenols and, to a lesser extent, to the glycosilated phenols, whereas polymerin proved to be non toxic. However, the laccase activity was not associated with the decrease of phytotoxicity. The valorization of DOR as a producer of high added value substances of industrial and agricultural interest in native form and after their bioremediation for a final objective of the total DOR recycling is also discussed.


Subject(s)
Environmental Restoration and Remediation/methods , Lepidium sativum/chemistry , Plant Oils/chemistry , Chromatography, High Pressure Liquid , Olive Oil , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, Ultraviolet
2.
Chemosphere ; 66(1): 67-74, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16814842

ABSTRACT

Some saprobe fungi (Phlebia radiata, Trametes versicolor, Coriolopsis rigida, Pycnoporus cinnabarinus, Fomes sclerodermus or Pleurotus pulmonarius) were able to bioconvert the ethyl acetate fraction (DEAF) and the corresponding aqueous exhausted fraction (EAF) of dry olive mill residue (DOR), reducing their phytotoxicity on Lepidium sativum seeds. Large amount of hydroxytyrosol together with other eight monomeric phenols were found in the native DEAF fraction, which represents a good source of antioxidants. P. radiata, T. versicolor and F. sclerodermus caused an effective phytotoxicity reduction of EAF in the concentration range of 25-3 gl(-1). In particular, in the range between 12.5 and 3 gl(-1), the EAF samples inoculated with P. radiata and F. sclerodermus surprisingly stimulated the germinability of L. sativum, suggesting their use as a potential biofertilizer. This is the first report which showed the bioconversion of the above fractions in shorter time with respect to the previous findings concerning DOR. The possible implications of laccase in the decrease of DEAF and EAF phytotoxicity was also discussed.


Subject(s)
Acetates/chemistry , Basidiomycota/metabolism , Industrial Waste/prevention & control , Plant Oils/chemistry , Acetates/metabolism , Basidiomycota/growth & development , Biodegradation, Environmental , Chromatography, High Pressure Liquid , Germination/drug effects , Industrial Waste/analysis , Laccase/metabolism , Lepidium sativum/drug effects , Molecular Structure , Olive Oil , Phenols/metabolism , Plant Oils/metabolism , Seeds/drug effects , Spectrometry, Mass, Electrospray Ionization , Water Pollutants, Chemical/metabolism , Water Pollutants, Chemical/toxicity
3.
Dig Liver Dis ; 39(2): 122-9, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17092786

ABSTRACT

BACKGROUND: To understand the molecular changes underlying Helicobacter pylori-related gastric diseases is mandatory to prevent gastric cancer. Proteomic technology is providing a rapid expansion of the basic knowledge, particularly in the discovery of new biomarkers involved in the tumourigenesis. AIM: To characterise changes in protein expression level of the gastric mucosa in H. pylori-infected patients. METHODS: The population enrolled comprised 41 dyspeptic patients. Proteins extracted from gastric mucosal specimens were analysed by 2-dimensional electrophoresis, sequenced by MALDI-TOF and identified by Edman's degradation. RESULTS: Twenty-one out of 41 patients had H. pylori infection of whom 17 had anti-CagA IgG antibodies. Several proteins were identified, of which Rho guanosine diphosphatase dissociation inhibitor alpha and heat shock protein 27 increased and glutathione transferase and antrum mucosa protein-18 decreased in H. pylori-positive in respect to H. pylori-negative patients. Interestingly, antrum mucosa protein-18, currently referred as gastrokine-1, showed two isoforms differing in the first N-terminal amino acid residue. Both gastrokine-1 isoforms were observed in the H. pylori-negative group whereas a lower expression or even absence of the gastrokine-1 basic isoform was found in a subgroup (7/21) of H. pylori-positive patients with moderate-severe gastritis. CONCLUSION: Our study demonstrated the presence of gastrokine-1 isoforms of which the basic isoform was reduced in a subset of patients with H. pylori infection.


Subject(s)
Dyspepsia/metabolism , Endonucleases/biosynthesis , Gastric Mucosa/metabolism , Helicobacter Infections/metabolism , Helicobacter pylori , Adult , Blotting, Northern , Blotting, Western , Electrophoresis, Gel, Two-Dimensional , Female , Gene Expression Regulation , Guanine Nucleotide Dissociation Inhibitors/biosynthesis , HSP27 Heat-Shock Proteins , Heat-Shock Proteins/biosynthesis , Humans , Male , Middle Aged , Molecular Chaperones , Neoplasm Proteins/biosynthesis , Oligonucleotide Array Sequence Analysis , Peptide Hormones , Protein Isoforms/biosynthesis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , rho-Specific Guanine Nucleotide Dissociation Inhibitors
4.
Eur J Biochem ; 268(13): 3858-69, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11432754

ABSTRACT

In this study we show that SV-IV, a major immunomodulatory, anti-inflammatory, and sperm immunoprotective protein secreted from the rat seminal vesicle epithelium, acts in vitro as a substrate of protein kinase C (PKC) competing efficiently with H1 histone, a very well known PKC substrate. Electrospray mass spectrometry (ES-MS) analysis demonstrated that approximately 10% of the native SV-IV molecules were phosphorylated by PKC and that such a modification involved only a single serine residue (Ser58) out of the 22 occurring in the protein. Interestingly, this modification produced a substantial enhancement (approximately 50%) of the native SV-IV's ability to stimulate the activity of both horseradish peroxidase (POD) and selenium-dependent glutathione peroxidase (GPX), an enzyme that is known to protect the mammalian spermatozoa from oxidative stress and loss of motility in the female genital tract following ejaculation. In contrast, the phosphorylation of SV-IV on Ser58 did not produce any effect on the anti-inflammatory properties of SV-IV, as measured by its ability to inhibit the phospholipase A2.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Glutathione Peroxidase/metabolism , Horseradish Peroxidase/metabolism , Protein Kinase C/metabolism , Proteins/chemistry , Proteins/metabolism , Seminal Vesicles/metabolism , Serine , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Kinetics , Male , Molecular Sequence Data , Peptide Fragments/chemistry , Phosphorylation , Proteins/isolation & purification , Rats , Rats, Inbred F344 , Rats, Wistar , Seminal Plasma Proteins , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tetradecanoylphorbol Acetate/pharmacology
5.
Rapid Commun Mass Spectrom ; 15(8): 527-40, 2001.
Article in English | MEDLINE | ID: mdl-11312501

ABSTRACT

The structural characterisation of the adducts formed by in vitro interaction of hemoglobin (Hb) with 1,2,3,4-diepoxybutane (DEB), the most reactive 1,3-butadiene (BD) metabolite, was obtained by liquid chromatography/electrospray ionisation mass spectrometry (LC/ES-MS) analysis of modified tryptic peptides of human hemoglobin chains. The reactive sites of human hemoglobin towards DEB and its hydroxylated derivatives (trihydroxybutyl (THB)-derivatives) were identified through the characterisation of alkylated tryptic peptides by matrix-assisted laser desorption/ionisation tandem mass spectrometry (MALDI-MS/MS). Based on this characterisation, a procedure was set up to measure the Hb-adducts of THB-derivatives by isotope dilution mass spectrometry with the use of a deuterated peptide standard. The results obtained here could permit optimisation of molecular dosimetry of BD-adducts, and extension of the analysis to the biological monitoring of occupational exposure to butadiene.


Subject(s)
Epoxy Compounds/chemistry , Hemoglobins/analysis , Alkylation , Chromatography, High Pressure Liquid , Globins/analysis , Indicators and Reagents , Peptides/analysis , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin/chemistry
6.
J Mass Spectrom ; 36(1): 47-57, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11180646

ABSTRACT

The reaction products of epichlorohydrin with human alpha- and beta- globins, obtained through in vitro incubation of these compounds and red blood cells, were determined by using reversed-phase high-performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization tandem mass spectrometry. The alpha-globin was much more reactive than the beta-globin. At low incubation ratios, approximating the order of magnitude of epichlorohydrin concentration as found in workplaces, the only modified peptide still detectable was the 62-90 belonging to the alpha-chain and carrying an incremental mass of 92 u on either His72 or His89. Given that the two peptides co-eluted in a single chromatographic peak during RP-HPLC separation, they could be chosen as suitable biomarkers for quantification in the setting up of a new methodology for the biological monitoring of persons occupationally exposed, replacing currently known procedures.


Subject(s)
Biomarkers/blood , Epichlorohydrin/blood , Erythrocytes/chemistry , Mass Spectrometry , Amino Acids/analysis , Chromatography, High Pressure Liquid , Globins/metabolism , Humans , Occupational Exposure , Peptide Fragments/blood , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin/metabolism
7.
J Mass Spectrom ; 36(1): 38-46, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11180645

ABSTRACT

The primary structure of saporin-S9 and MAP-S, two type-1 ribosome-inactivating proteins isolated from the seeds of Saponaria officinalis L. and Mirabilis jalapa, respectively, was determined using a combined approach based on Edman degradation and electrospray ionization mass spectrometry (ESMS). Saporin-S9 has 253 amino acids with a calculated molecular mass of 28,492.99, which is in good agreement with that determined by ESMS (28 495 +/- 2 Da). Unlike other saporins with known primary structure, saporin-S9 contains four histidinyl residues (positions 111, 121, 216 and 248). By comparing the amino acid sequence of saporin-S9 with that of saporin-S6, we found 22 amino acid substitutions (8.7%), 13 of which are conservative and nine non-conservative. The residues known to be involved in the definition of the active site and with RNA base recognition are conserved. The four histidinyl residues and especially Lys for Gln203 contribute to the higher calculated pI value (10.17) of saporin-S9 compared with saporin-S6 (9.98). MAP-S contains 250 amino acid residues with a calculated molecular mass of 27,789.49, in good agreement with that determined by ESMS (27,789 +/- 2). Cys36 and Cys220 form a disulphide bridge and only four amino acid residues are different from the amino acid sequence of MAP, isolated from the roots of the same plant, i.e. Leu34 (Glu), Ile161 (Leu), Asp185 (Glu) and Asp191 (Glu) (in parentheses, the residues present in MAP). The reported approach can provide rapid and reliable sequence screening in the analysis of homologous proteins, including the presence of disulphide bridges.


Subject(s)
Immunotoxins , N-Glycosyl Hydrolases , Plant Proteins/chemistry , Sequence Analysis, Protein , Spectrometry, Mass, Electrospray Ionization , Amino Acid Sequence , Conserved Sequence , Cyanogen Bromide , Endopeptidases , Metalloendopeptidases , Molecular Sequence Data , Peptide Fragments/chemistry , Ribosome Inactivating Proteins, Type 1 , Saporins , Sequence Homology , Trypsin
8.
Rapid Commun Mass Spectrom ; 14(23): 2247-59, 2000.
Article in English | MEDLINE | ID: mdl-11114036

ABSTRACT

We report here on the structural characterization of a highly heterogeneous mixture of glucosylceramides (GlcCers) isolated from a deep-water Mediterranian dendrophylliid coral, Dendrophyllia cornigera. The neutral glycosphingolipid (GSL) components of the coral were separated into three HPLC fractions which were structurally characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS). NMR analysis revealed a beta-glucosylpyranose, a methyl branched conjugated sphingadienine and alpha-hydroxy fatty acid moieties characteristic for the species. Molecular mass distributions of the HPLC fractions were monitored using single-stage MS. At least 17 different GlcCer constituents with variable long-chain base and fatty acid residues were observed based on the molecular ion peaks in the liquid secondary ion (LSI) survey spectra. Structures of the individual components were revealed by product ion spectra of the alkali-cationized molecules ([M + Cat](+)), which resulted in two characteristic fragment ions, F(F) and F(S). Tandem MS of the same fragment ions formed in the ion source showed that F(F) carries the hydoxy fatty acid, while F(S) carries the long-chain sphingoid base, thus providing complementary structural information for the characterization of ceramide composition. Based on the tandem mass spectra of the molecular ions [M + Na](+), 26 different GlcCers of the coral were identified. The ceramide moiety showed heterogeneity in both the sphingoid portion (d18:2, d19:2, d20:2 and d20:3) and the alpha-hydroxy fatty acid chain (h19-h24, either saturated or unsaturated), forming an extremely heterogeneous mixture. The method is generally applicable to the characterization of structurally heterogeneous GlcCer mixtures.


Subject(s)
Cnidaria/chemistry , Glucosylceramides/analysis , Animals , Chromatography, High Pressure Liquid , Cnidaria/metabolism , Glucosylceramides/classification , Glucosylceramides/metabolism , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Structure , Molecular Weight
10.
J Agric Food Chem ; 48(12): 5795-801, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11141251

ABSTRACT

Electrospray ionization mass spectrometry (ESI-MS) in negative ion mode was used to monitor the possible noncovalent adduct formations between DNA analogue oligonucleotides and two Fusarium mycotoxins, fumonisin B1 and fusaproliferin. Using mild experimental ESI conditions specific noncovalent interactions were detected between both single- and double-stranded model oligonucleotides and fusaproliferin with 1:1 stoichiometry. Similar association complexes were observed for the deacetyl derivative of fusaproliferin. There were no peaks due to adduct formation present in the mass spectra of fumonisin B1, incubated with oligonucleotides in a wide concentration range, suggesting no specific interaction for this molecule. In a competitive complexation reaction, another mycotoxin, the beauvericin, forms more stable association complex with DNA than fusaproliferin. These findings can be of use in the understanding of molecular mechanisms of action during apoptosis and can be correlated with the teratogenic effect of fusaproliferin.


Subject(s)
Carboxylic Acids/chemistry , DNA, Fungal/chemistry , Fumonisins , Fusarium/metabolism , Mycotoxins/chemistry , Terpenes/chemistry , DNA Fragmentation , Fusarium/genetics , Mass Spectrometry , Oligonucleotides/chemistry
12.
J Mass Spectrom ; 34(8): 845-9, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10423565

ABSTRACT

A specific and sensitive method based on gas chromatography/tandem mass spectrometry with on-column injection was developed to quantify simultaneously cyclophosphamide and ifosphamide in urine by using trophosphamide as an internal standard. The urine samples were extracted with diethyl ether and derivatization was performed with heptafluorobutyric anhydride. The detection limits of cyclophosphamide and ifosphamide in urine samples were 0.1 and 0.5 ng ml(-1), respectively, with a signal-to-noise ratio of 3 : 1. The sensitivity, the specificity and the low cost of the instrumentation involved make this method suitable for economical analysis on a large scale, such as for the biological monitoring of occupational exposure to cyclophosphamide and ifosphamide in production plants and in hospitals during their pharmacological use.


Subject(s)
Cyclophosphamide/urine , Ifosfamide/urine , Cyclophosphamide/analogs & derivatives , Gas Chromatography-Mass Spectrometry/methods , Humans , Monitoring, Physiologic , Occupational Exposure , Sensitivity and Specificity
13.
FEBS Lett ; 452(3): 190-4, 1999 Jun 11.
Article in English | MEDLINE | ID: mdl-10386588

ABSTRACT

The use of aspecific methylation reaction in combination with MS procedures has been employed for the characterization of the nucleophilic residues present on the molecular surface of the human 2,3-diphosphoglycerate/deoxy-hemoglobin complex. In particular, direct molecular weight determinations by ESMS allowed to control the reaction conditions, limiting the number of methyl groups introduced in the modified globin chains. A combined LCESMS-Edman degradation approach for the analysis of the tryptic peptide mixtures yielded to the exact identification of methylation sites together with the quantitative estimation of their degree of modification. The reactivities observed were directly correlated with the pKa and the relative surface accessibility of the nucleophilic residues, calculated from the X-ray crystallographic structure of the protein. The results here described indicate that this methodology can be efficiently used in aspecific modification experiments directed to the molecular characterization of the surface topology in proteins and protein complexes.


Subject(s)
2,3-Diphosphoglycerate/chemistry , Globins/chemistry , Hemoglobins/chemistry , 2,3-Diphosphoglycerate/blood , Hemoglobins/metabolism , Humans , Methylation , Models, Molecular , Protein Conformation , Spectrometry, Mass, Secondary Ion
14.
Planta ; 208(1): 125-31, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10213004

ABSTRACT

Four type-1 (single-chain) ribosome-inactivating proteins (RIPs), with isoelectric points between 9.5 and 9.7, were isolated from leaves of Phytolacca dioica L. The purification procedure furnished the four proteins with an overall yield of about 16% and separated them from a protein of 29,407 +/- 2 Da, as determined by electrospray mass spectrometry, whose N-terminal amino acid sequence differed from that of pokeweed (Phytolacca americana L.) leaf chitinase (PLC-B) by only one amino acid (R17I). The four RIPs (PD-L1 to PD-L4) inhibited protein synthesis by a rabbit reticulocyte lysate with 50% inhibition at the picomolar level, and produced the beta-fragment, diagnostic of the specific enzymatic action of RIPs, on yeast ribosomes. Comparison of their N-terminal sequences, up to residue 45, showed that PD-L1 is identical to PD-L2 [designated the isoleucine (Ile) form from the N-terminal residue] and PD-L3 is identical to PD-L4 [designated the valine (Val) form from the N-terminal residue] and that there are 35 identical residues between the two forms. Furthermore, the Val form presents the same number of identical residues as PD-S2, an RIP isolated from the seeds of the same plant. With the exception of PD-L4, the purified RIPs gave a positive reaction when stained for sugars on SDS-PAGE gels and, when analyzed by electrospray mass spectrometry, had M(r) values of 32,715 +/- 1 (PD-L1), 31,542 +/- 1 (PD-L2), 30,356 +/- 1 (PD-L3) and 29,185 +/- 1 Da (PD-L4). The 1171 kDa difference in M(r), within the same RIP form, could be due to glycosylation. Like leaf saporins and many other RIPs, the four RIPs released several adenines from poly(A), herring sperm DNA and rRNA 16S + 23S, thus acting as polynucleotide:adenosine glycosidases. This property was less pronounced in PD-L1 and PD-L3 than in PD-L2 and PD-L4, respectively. The proteins PD-L1 and PD-L4 showed 3.7% reactivity with the antiserum anti-dianthin 32 and no reactivity with antisera to PAP-R saporin-S6, momordin 1 and even PD-S2, an RIP isolated from the seeds of the same plant. Protein PD-L4 showed 12.5% cross-reactivity with anti-PD-L1, while the opposite cross-reactivity was 100%.


Subject(s)
N-Glycosyl Hydrolases/isolation & purification , Plant Proteins/isolation & purification , Ribosomes/metabolism , Trees/chemistry , Amino Acid Sequence , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , N-Glycosyl Hydrolases/chemistry , N-Glycosyl Hydrolases/metabolism , Plant Leaves/enzymology , Plant Proteins/chemistry , Plant Proteins/metabolism , Protein Conformation , Ribosome Inactivating Proteins , Sequence Homology, Amino Acid , Trees/enzymology
15.
FEBS Lett ; 462(3): 241-5, 1999 Dec 03.
Article in English | MEDLINE | ID: mdl-10622703

ABSTRACT

The present study demonstrates that NO produced in vitro by inducible nitric oxide synthase in red cells can convert hemoglobin contained in the red cells to S-nitrosohemoglobin. Experiments carried out either in the absence or in the presence of a low molecular weight thiol, such as cysteine, showed that in the first case the target of NO is heme-Fe2+. On the contrary, in the presence of cysteine, the first step is the formation of S-nitrosocysteine, followed by transfer of the NO group to a particular cysteine residue of beta-globin, cysteine 93. These results confirm previous data indicating the preferential formation of S-nitrosohemoglobin at that site by chemical methods [Ferranti et al. (1997) FEBS Lett. 400, 17-24], and the existence of a physiological mechanism of inactivation for NO circulating in blood. The analysis of S-nitrosohemoglobin can also allow the quantification of the NO levels in blood to be applied for in vitro and in vivo measurements.


Subject(s)
Erythrocytes/metabolism , Hemoglobins/biosynthesis , Mercaptoethanol , Nitric Oxide Synthase/metabolism , S-Nitrosothiols , Chromatography, High Pressure Liquid , Erythrocytes/enzymology , Hemoglobins/chemistry , Hemoglobins/metabolism , Humans , Mass Spectrometry , Nitric Oxide Synthase/chemistry , Nitric Oxide Synthase Type II , Nitroso Compounds/chemistry , Nitroso Compounds/metabolism , Peptide Mapping
16.
Rapid Commun Mass Spectrom ; 12(22): 1783-92, 1998.
Article in English | MEDLINE | ID: mdl-9853387

ABSTRACT

The present study reports a procedure developed for the identification and quantitative analysis of the adducts formed by interaction of methyl bromide with human hemoglobin, based on combined analysis by electrospray mass spectrometry and automated Edman degradation of either intact globin chains or tryptic peptides of globin chains. The procedure has allowed identification of the reactive sites in human hemoglobin, and has been applied to the analysis of samples modified in vitro by methyl bromide. The results obtained represent the basis for the complete structural characterization of the modified hemoglobin and demonstrate the usefulness of the proposed analytical approach for the evaluation of the degree of alkylation and the identification of modified amino acids in proteins.


Subject(s)
Hemoglobins/chemistry , Hydrocarbons, Brominated/chemistry , Amino Acid Sequence , Amino Acids/analysis , Chromatography, High Pressure Liquid , Globins/chemistry , Globins/isolation & purification , Humans , Hydrolysis , Indicators and Reagents , Mass Spectrometry , Molecular Sequence Data , Peptides/chemistry , Trypsin
17.
Biochimie ; 80(4): 333-8, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9672754

ABSTRACT

The present work describes the biochemical procedures used to identify the cause of a quantitative and qualitative hemoglobin polymorphism found in Podolian cattle. First, to analyze the different phenotypes, isoelectric focusing (IEF) of hemoglobins and RP-HPLC of globin chains was carried out; secondly, to determine accurately the globin molecular masses, electrospray mass spectrometry was performed and finally to check the entire amino acid sequences of the proteins, several enzymatic digests were analyzed by fast atom bombardment mass spectrometry (FAB-MS) and Edman degradation procedure. As to the qualitative polymorphism, the results of RP-HPLC show the presence of two alpha-globin variants to which the extensive mass spectrometric analysis attributed a molecular mass of 15,026.47 +/- 0.44 Da and 15,079.86 +/- 0.66 Da and whose respective primary structure differed from that of the common alpha-globin chain in the amino acid substitution Asn-->Ser at position 131 and the other in the replacement of the histidine residue at position 89 with tyrosine. As to the quantitative polymorphism, on the basis of the expression gradient found out in the duplicated alpha genes of several mammals, we conceive that the alpha 89 His-->Tyr is an allelic form of the I alpha gene while the alpha 131Asn-->Ser is an allelic form of the II alpha gene.


Subject(s)
Cattle/genetics , Genetic Variation , Globins/genetics , Hemoglobins/genetics , Polymorphism, Genetic , Amino Acid Sequence , Animals , Bison/genetics , Chromatography, High Pressure Liquid , Globins/chemistry , Hemoglobins/chemistry , Macromolecular Substances , Mass Spectrometry , Molecular Sequence Data , Ruminants/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity
18.
J Protein Chem ; 17(8): 835-44, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9988530

ABSTRACT

The primary structure of water buffalo alpha(s1)-casein and of beta-casein A and B variants has been determined using a combination of mass spectrometry and Edman degradation procedures. The phosphorylated residues were localized on the tryptic phosphopeptides after performing a beta-elimination/thiol derivatization. Water buffalo alpha(s1)-casein, resolved in three discrete bands by isoelectric focusing, was found to consist of a single protein containing eight, seven, or six phosphate groups. Compared to bovine alpha(s1)-casein C variant, the water buffalo alpha(s1)-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine betaA2-casein variant, the two water buffalo beta-casein variants A and B presented four and five amino acid substitutions, respectively. In addition to the phosphoserines, a phosphothreonine residue was identified in variant A. From the phylogenetic point of view, both water buffalo beta-casein variants seem to be homologous to bovine betaA2-casein.


Subject(s)
Buffaloes , Caseins/chemistry , Caseins/metabolism , Amino Acid Sequence , Animals , Caseins/isolation & purification , Cattle , Isoelectric Focusing , Milk Proteins/chemistry , Milk Proteins/isolation & purification , Milk Proteins/metabolism , Molecular Sequence Data , Phosphorylation , Sequence Homology, Amino Acid , Species Specificity
19.
J Agric Food Chem ; 46(2): 411-416, 1998 Feb 16.
Article in English | MEDLINE | ID: mdl-10554255

ABSTRACT

Multiple forms of mature alpha(s1)-casein have been characterized in ovine variants A and D using a combination of mass spectrometry and automated Edman degradation. Mature ovine alpha(s1)-casein was found to be a heterogeneous mixture of at least seven molecular species. The main component, representing about 50% total alpha(s1)-casein, corresponded to the full-length (199 residues long) protein. The other components were alpha(s1)-casein of different lengths: 198 (less Gln78), 191 (less peptide 110-117), 191 residues (less peptide 140-148), 190 (less peptide 110-117 and Gln78), 190 (less peptide 140-148 and Gln78), and 183 (less peptides 110-117 and 140-148) residues long alpha(s1)-casein. Each of the alpha(s1)-casein multiple forms occurred at three different phosphorylation levels, due to the partial phosphorylation of both Ser115 (at about 50%) and Ser41 (at about 20%). In the case of deleted peptide 110-117, the protein heterogeneity linked to the partially phosphorylated Ser115 was abolished, and only two levels of phosphorylation were observed. These multiple forms differing in molecular weight and degree of phosphorylation may have been developed from an exon skipping during mRNA splicing in ovine alpha(s1)-casein, similar to that recently described in the case of its caprine counterpart.

20.
Int J Cardiol ; 62(1): 77-85, 1997 Oct 31.
Article in English | MEDLINE | ID: mdl-9363507

ABSTRACT

Oxidative modification of low density lipoprotein (LDL) and its byproducts may play a fundamental role in atherosclerosis. We report an in vitro analysis of LDL peroxidative compounds in an homozygous familial hypercholesterolemic (HFH) patient who subsequently died. During the autopsy, we analyzed lipids extracted directly from different atherosclerotic plaques, and we also provided an immunocytochemical analysis using the specific monoclonal antibody MDA2 (directed against malondialdeyde-lysine epitopes of oxidized LDL). The results showed that the same species of peroxidative compounds were present both in LDL in vitro and in lipids extracted directly from atherosclerotic lesions. Moreover, the immunocytochemistry analysis revealed a positive staining of atherosclerotic plaques, confirming the presence of LDL oxidation-specific epitopes. Although observation of a single case is necessarily limited, our findings are consistent with the hypothesis that oxidative modification of LDL is involved in human atherogenesis.


Subject(s)
Arteriosclerosis/metabolism , Cholesterol, LDL/blood , Hyperlipoproteinemia Type II/metabolism , Lipid Peroxidation , Adult , Arteriosclerosis/pathology , Autopsy , Culture Techniques , Fatal Outcome , Female , Free Radicals/metabolism , Humans , Hyperlipoproteinemia Type II/pathology , Immunohistochemistry
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