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Biomedical subjects

G Marino

Publications and source records attributed to G Marino.

At least 19 recordsLinked to original sources
PubMed1995

Amino acid sequence and disulphide-bridge pattern of three gamma-thionins from Sorghum bicolor.

The complete primary structure of a new alpha-amylase inhibitor from Sorghum bicolor belonging to the gamma-thionin family has been determined and the amino acid sequences of two components of the family already elucidated have been corrected by combining the classical Edman degradation with advanced mass spectrometric procedures. The same integrated approach allowed us to define the pattern of the disulphide bridges in the three isoinhibitors. The arrangement of the cysteine pairing was determined as Cys3-Cys47, Cys14-Cys34, Cys20-Cys41 and Cys24-Cys43. The amino acid sequences of the alpha-amylase inhibitors share a high degree of similarity with the related plant gamma-thionins. All these proteins consist of 47 residues, contain eight cysteine residues forming four disulphide bridges, and show the presence of two clusters of basic amino acids located at both ends of the polypeptide chain. The pattern of S-S bridges determined for the isoinhibitors is identical to that inferred by NMR analysis in two related gamma-thionins, thus suggesting a highly conserved organization of the disulphide pairing. These results indicate that the structural similarities among the different gamma-thionins extend far beyond the primary structure and possibly concern the secondary structure and the general folding of the entire gamma-thionin family.

Amino Acid Sequence↗
PubMed1995

An extremely thermostable aromatic aminotransferase from the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a strictly anaerobic archaeon (formerly archaebacterium) that grows optimally at 100 degrees C by the fermentation of peptides. Cell-free extracts were found to contain two distinct aromatic aminotransferases (ArAT, EC 2.6.1.57), one of which was purified to electrophoretic homogeneity. P. furiosus ArAT is a homodimer with a subunit M(r) value of 44,000 +/- 1000. Using 2-ketoglutarate as the amino acceptor, the purified enzyme catalyzed the pyridoxal 5'-phosphate (PMP)-dependent transamination of phenylalanine, tyrosine and tryptophan with respective kcat values of 253, 72 and 62 (s-1 at 80 degrees C) under saturating conditions. The Km values for all three amino acids were between 1.1 and 2.1 mM and the optimum temperature for catalysis was above 95 degrees C. The melting point for the pure enzyme was also above 95 degrees C as determined by the change in ellipticity at 220 nm. Irreversible denaturation of the pure enzyme was not apparent after 6 h at 80 degrees C in the presence of PMP and 2-ketoglutarate and the time required for a 50% loss in activity at 95 degrees C was approx. 16 h. This decreased to approx. 12 h if cofactor and substrate were not added. In contrast, the apoenzyme (lacking PMP) lost most (70%) of its activity (measured after reconstitution) after 6 h at 80 degrees C, indicating that both PMP and 2-ketoglutarate stabilize the enzyme at extreme temperatures. Although few ArATs have been characterized to date, the molecular properties and substrate specificity of P. furiosus ArAT more resemble those of the ArAT from Escherichia coli than those of the analogous enzyme from rat liver. Moreover, the P. furiosus enzyme is by far the most thermostable aminotransferase of any type to be purified so far.

Amino Acid Sequence↗
PubMed1995

Effectiveness of cascade filtration plasmapheresis in two patients affected by familial hypercholesterolemia.

Hypercholesterolemia has been recognised as a primary risk factor for coronary heart disease. Reduction of plasma levels of total and LDL cholesterol has been shown to decrease coronary atherosclerosis. Plasmapheresis represents an useful non-pharmacological tool to treat severe hypercholesterolemias. We have evaluated the effectiveness of a system of plasmapheresis using a cascade filtration method in two young male subjects (aged 16 and 26 years) with homozygous familial hypercholesterolemia. Both showed severe coronary atherosclerosis as determined by angiography. Procedures were performed at intervals of 7 days in each case. We observed a mean reduction of plasma levels of total cholesterol of 59.5% (range 31.0-75.5%); LDL-cholesterol, 61.6% (range 32.6-77.1%); triglycerides, 48.1%; HDL-cholesterol, 31.1%; apo A-I, 30.8%; and apo B, 57.6%. We also noted a reduction of other parameters, such as fibrinogen (49.9%) and Lp(a) (59.9%). At the end of each procedure about 8 g of cholesterol was removed from the total body pool. A decrease of total proteins (26.9%) and albumin (19.6%) was also observed, but this was completely restored before the next apheresis (1 week). These data show the effectiveness of the removal of LDL in a cascade filtration system, which obtains results not very different from other more selective methods. The lack of selectivity is not much of a problem, since it also reduces other risk factors such as Lp(a) and fibrinogen.

Adolescent↗
PubMed1995

Biochemical and functional identification of GABA receptors in Hydra vulgaris.

GABA binding sites labelled in vitro with [3H]GABA are present in crude membrane preparations from Hydra vulgaris. [3H]GABA binding is specific (70%), saturable and it is completely displaced by the GABA mimetic muscimol but not by bicuculline or baclofen. Scatchard analysis of saturation data indicates the presence of only one population of binding sites with a Bmax of 4, 75 pmol/mg of protein and a KD of 76 nM. In the living animal GABA and benzodiazepines increase the duration of mouth opening during the glutathione-induced feeding response. Bicuculline prevents the GABA-induced increase of the feeding response. Diazepam activity is enhanced by simultaneous GABA administration and it is suppressed by the specific antagonist flumazenil. In contrast, no [3H]-flunitrazepam binding is detected in membrane preparations. We conclude that a population of GABA receptors is present in Hydra tissues, where they are involved in the modulation of response to chemical stimulation.

Animals↗
PubMed1995

Nucleotide sequence of a cDNA coding for bovine mitochondrial aspartate aminotransferase.

Aspartate aminotransferase is a pyridoxal-phosphate dependent enzyme which plays a key role in cell metabolism. We describe the cloning and sequence analysis of the cDNA encoding bovine mitochondrial aspartate aminotransferase and compare the sequence with those of isoenzymes from other mammalian species. An adult bovine heart cDNA library constructed in lambda lambda gt11 was screened using two 32P-end labeled synthetic oligonucleotides. From the screening of the cDNA library two positive clones were isolated. A subclone in pEMBL18, 6B2, generated from the longest recombinant phage was further analyzed. This clone contains an insert of 2500 bp with an Open Reading Frame of 1287 bp that encodes a protein of 430 amino acids. The deduced amino acid sequence confirms previous results obtained by mass spectrometric sequencing. We calculated the percentage of amino acid identity for each protein pair and for each comparison the average number of amino acid substitution per site (Kaa); the lowest Kaa values were obtained from the comparison between the bovine and pig enzymes. This study shows that the rate of evolution of mammalian mitochondrial AspAT is lower and more constant than the equivalent cytosolic enzyme and adds to the growing body of knowledge on the evolution of the aspartate aminotransferase.

Amino Acid Sequence↗
PubMed1994

Indole-3-glycerol-phosphate synthase from Sulfolobus solfataricus as a model for studying thermostable TIM-barrel enzymes.

Indole-3-glycerol-phosphate synthase, a thermophilic and thermostable enzyme from the archaeon Sulfolobus solfataricus, was purified and characterized. The sequence of the thermophilic enzyme was compared to the sequence of a homologous mesophilic enzyme from Escherichia coli. The secondary structure of the thermophilic enzyme was predicted taking into account the patterns of hydropathy, chain flexibility and amphipathicity and the CD spectrum. From this analysis it turned out that indole-3-glycerol-phosphate synthase from S. solfataricus can be considered a model for studying thermostable TIM-barrel enzymes. Some peculiarities of the amino-acid sequence of indole-3-glycerol-phosphate synthase from S. solfataricus are discussed in relation to the thermostability of the enzyme.

Amino Acid Sequence↗
PubMed1994

Analysis of RNase A refolding intermediates by electrospray/mass spectrometry.

Electrospray/mass spectrometry (ES/MS) was extensively used to obtain information on disulphide-containing intermediates formed during refolding of bovine pancreatic ribonuclease A. The analysis showed the existence of an equilibrated population of disulphide bonded intermediates, and indicates that intermediates containing two intramolecular S-S are predominant until late stages of the refolding process. Mixed disulphides with exogenous glutathione were also detected, supporting previous evidence of conformational restrictions on the ability of RNase A to form intramolecular disulphides. The results indicate that ES/MS is a suitable technique to detect and characterize refolding intermediates.

Animals↗
PubMed1994

Post-translational modifications in aspartate aminotransferase from Sulfolobus solfataricus. Detection of N-epsilon-methyllysines by mass spectrometry.

Advanced mass spectrometric procedures have been extensively used to provide an accurate structural characterization of aspartate aminotransferase from Sulfolobus solfataricus. The amino acid sequence of this enzyme had previously been deduced from the DNA sequence. The accurate molecular mass of the protein, determined using electrospray mass spectrometry, demonstrated that the amino acid sequence deduced was correct and ruled out the possible presence of large covalent modifications which had been postulated to fit the much higher molecular mass obtained from previous SDS/PAGE experiments. The definition of the entire primary structure of aspartate aminotransferase from S. solfataricus was achieved by exploiting a new mass spectrometric mapping strategy. Initially, the molecular mass of relatively large protein fragments produced by CNBr hydrolysis was accurately determined using electrospray mass spectrometry. The protein regions where structural modifications had occurred were easily identified from their anomalous mass values. The corresponding CNBr fragments were then subdigested with suitable proteases and the resulting peptide mixtures were analysed by fast-atom-bombardment mass spectrometry. This mapping approach led to the detection of two partially modified lysine residues at positions 202 and 384, which had been converted to their N-epsilon-methyl derivatives to a substoichiometric extent.

Amino Acid Sequence↗
PubMed1994

Biological activities of CNBr fragments of a major protein secreted from the rat seminal vesicle epithelium.

Two fragments of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, were produced in vitro by protein cleavage with CNBr at level of the single methionine residue (Met-70) occurring in its polypeptide chain. After their purification by reversed-phase chromatography, SV-IV/A (1-70 fragment) and SV-IV/B (71-90 fragment) were assayed as transglutaminase substrates, and their anti-inflammatory, anti-thrombotic and immunosuppressive properties were evaluated in comparison with native SV-IV. Both fragments retained the SV-IV ability to act as transglutaminase substrates in vitro; fast atom bombardment mass spectrometry analyses of the reaction products pointed to Gln-9 and Gln-86 as acyl donor sites, and to Lys-59, -79 and -80 as acyl acceptor sites. In contrast, only SV-IV/A was shown to possess, like SV-IV, the property of inhibiting both the intensity of the carrageenin-induced rat foot edema and the platelet aggregation induced in vivo by different agents. Finally, the two protein fragments were found to be completely unable to inhibit both the mitogen-induced proliferation of human T cells and the mixed lymphocyte reaction.

Amino Acid Sequence↗
PubMed1994

Hb F-Sassari: a novel G gamma variant with a threonine residue at position gamma 75, characterized by mass spectrometric techniques.

The cord blood sample of a Caucasian newborn contained about 40% of an abnormal fetal hemoglobin. The mutated gamma chain was isolated using reversed phase high performance liquid chromatography and characterized by means of electrospray and fast atom bombardment mass spectrometric techniques as a G gamma-globin variant with an Ile-->Thr substitution at position gamma 75. The variant chain shows the same structure as the previously described Hb F-Charlotte that was demonstrated to be an A gamma variant with an Ile-->Thr substitution at position gamma 75 and an additional Ala-->Gly substitution at gamma 136.

Chromatography, High Pressure Liquid↗
PubMed1994

Toxicity associated with severe inhalational and dermal exposure to dimethylacetamide and 1,2-ethanediamine.

We present the case of a worker who was accidentally exposed (inhalational and dermal routes) to the chemicals dimethylacetamide and ethylenediamine for 90 minutes in a confined space. Clinical effects included delirium, hallucinations, skin burns, cellulitis, bilateral conjunctivitis, hepatitis, secondary coagulopathy, rhabdomyolysis, and a grade 2 esophagitis. Urinary monomethylacetamide levels 6 days after the exposure were 61 ppm.

Accidents, Occupational↗
PubMed1992

Expression of a hyperthermophilic aspartate aminotransferase in Escherichia coli.

The gene for an archaebacterial hyperthermophilic enzyme, aspartate aminotransferase from Sulfolobus solfataricus (AspATSs), was expressed in Escherichia coli and the enzyme purified to homogeneity. A suitable expression vector and host strain were selected and culture conditions were optimized so that 6-7 mg of pure enzyme per litre of culture were obtained repeatedly. The recombinant enzyme and the authentic AspATSs are indistinguishable: in fact, they have the same molecular weight, estimated by means of SDS-PAGE and gel filtration, the same Km values for 2-oxo-glutarate and cysteine sulphinate and the same UV-visible spectra. Moreover, recombinant AspATSs is thermophilic and thermostable just as the enzyme extracted from Sulfolobus solfataricus. The protocol described may be used to produce thermostable arachaebacterial enzymes in mesophilic hosts.

Aspartate Aminotransferases↗
PubMed1992

Mass spectrometric analysis of the HMGY protein from Lewis lung carcinoma. Identification of phosphorylation sites.

The primary structure of the Lewis lung carcinoma protein HMGY belonging to the nuclear group of proteins HMGI (high mobility group I) was determined using electrospray and fast atom bombardment mass spectrometry. It was demonstrated that the sequence of the tumor protein corresponds to the amino acid sequence derived from the cDNA from cultured cells and that the N-terminal serine residue is N-acetylated. Moreover, the two high performance liquid chromatography-purified forms Y1 and Y2 of the protein HMGY were shown to differ at the level of serine phosphorylation, since they contain three phosphate and two phosphate groups, respectively, in the C-terminal region. No other modification was detected in the remaining part of the molecule.

Amino Acid Sequence↗
PubMed1992

Limited proteolysis as a probe of conformational changes in aspartate aminotransferase from Sulfolobus solfataricus.

The analysis of conformational transitions using limited proteolysis was carried out on a hyperthermophilic aspartate aminotransferase isolated from the archaebacterium Sulfolobus solfataricus, in comparison with pig cytosolic aspartate aminotransferase, a thoroughly studied mesophilic aminotransferase which shares about 15% similarity with the archaebacterial protein. Aspartate aminotransferase from S. solfataricus is cleaved at residue 28 by thermolysin and residues 32 and 33 by trypsin; analogously, pig heart cytosolic aspartate aminotransferase is cleaved at residues 19 and 25 [Iriarte, A., Hubert, E., Kraft, K. & Martinez-Carrion, M. (1984) J. Biol. Chem. 259, 723-728] by trypsin. In the case of aspartate aminotransferase from S. solfataricus, proteolytic cleavages also result in transaminase inactivation thus indicating that both enzymes, although evolutionarily distinct, possess a region involved in catalysis and well exposed to proteases which is similarly positioned in their primary structure. It has been reported that the binding of substrates induces a conformational transition in aspartate aminotransferases and protects the enzymes against proteolysis [Gehring, H. (1985) in Transaminases (Christen, P. & Metzler, D. E., eds) pp. 323-326, John Wiley & Sons, New York]. Aspartate aminotransferase from S. solfataricus is protected against proteolysis by substrates, but only at high temperatures (greater than 60 degrees C). To explain this behaviour, the kinetics of inactivation caused by thermolysin were measured in the temperature range 25-75 degrees C. The Arrhenius plot of the proteolytic kinetic constants measured in the absence of substrates is not rectilinear, while the same plot of the constants measured in the presence of substrates is a straight line. Limited proteolysis experiments suggest that aspartate aminotransferase from S. solfataricus undergoes a conformational transition induced by the binding of substrates. Another conformational transition which depends on temperature and occurs in the absence of substrates could explain the non-linear Arrhenius plot of the proteolytic kinetic constants. The latter conformational transition might also be related to the functioning of the archaebacterial aminotransferase since the Arrhenius plot of kcat is non-linear as well.

Amino Acid Sequence↗
PubMed1992

Characterization by mass spectrometry of a recombinant hepatitis delta virus antigen and its proteolytic products.

The recombinant hepatitis delta virus antigen was obtained as a chimaeric protein fused to the C-terminus of the phage MS2 RNA polymerase. Following induction of the temperature-sensitive promoter, two major polypeptides of about 34 kDa and 29 kDa, and two minor peptides about 21 kDa and 18 kDa, were obtained on PAGE. The 34-kDa protein was identified as the expected recombinant protein by confirming 82% of the primary structure using fast-atom-bombardment mass spectrometry. The most represented degradation product, i.e. the 29-kDa polypeptide, was also characterized by means of mass spectrometry and found to be produced by cleavage between amino acids 261 and 265. The presence of two main protein bands, with a similar difference in size, is also a typical feature of delta antigens, both extracted and recombinant, and it is considered to be derived either from heterogeneity of viral sequences, which can encode hepatitis delta antigen proteins of 195 and 214 amino acids, or from proteolysis of a single precursor. Since the data were obtained with a single viral sequence coding for 195 amino acids fused to 106 residues from MS2 polymerase, there is direct evidence that intrinsic structural properties of the protein sequence are able to cause a specific proteolysis resulting in the presence of two major forms, of which the smaller is 35-40 amino acids at the C-terminus. The recombinant protein can be used as an antigenic substitute of viral antigens both for immunoassays and for the preparation of anti-(hepatitis delta virus) antisera.

Amino Acid Sequence↗
PubMed1992

Factors affecting the fast atom bombardment mass spectrometric analysis of proteolytic digests of proteins.

The fast atom bombardment (FAB) mass spectrometric analysis of proteolytic digests of proteins is currently used in protein structural characterization. The major current limitation of this procedure is that not all the peptides generated by enzyme digestion can be observed in the spectra. Previous studies showed that in a mixture the more hydrophilic peptides are suppressed. Several enzymatic digests of 18 different proteins ranging from 10 kDa to 67 kDa in molecular weight were examined using FAB mass spectrometry. It was observed that, even though the hydrophobicity of peptides is a factor in determining their presence or absence in the spectra, the predictions of whether or not a peptide would be detected based on this criterion varies in a wide range of values. Moreover, present results seem to indicate that the presence of particular amino acid side chains within a peptide sequence capable of forming hydrogen bonds with the matrix heavily affects the behaviour of that peptide in the mixture, despite the overall hydrophobicity of the peptide itself.

Amino Acids↗
PubMed1992

[Total cholesterol, LDL-cholesterol and apoprotein B in umbilical cord blood: cross-sectional study].

The aim of the present study was to evaluate, in a large series of term newborns, the prevalence of hypercholesterolemia in cord blood, using as parameters, levels of plasma cholesterol, LDL-cholesterol and apo B higher than mean + 2 standard deviations, alone or in association. Data reported here represent the results of a transversal study. 1275 term newborns were studied. In everybody, in cord blood samples, at birth, cholesterol, triglycerides, HDL-cholesterol, apoprotein B and LDL-cholesterol have been assayed. In 3.13% of them (40 subjects), cholesterol levels > mean + 2 standard deviations were present. Moreover, only 6 subjects (0.47%) showed all three parameter levels higher than mean + 2 standard deviations, and 4 subjects (0.31%), in spite of normal cholesterol levels, presented high LDL-cholesterol and apo B levels. Integrated utilization of these parameters in the neonatal screening of familial hypercholesterolemia will have to be confirmed by follow-up studies.

Apolipoproteins B↗