Biomedical subjects

O Cascone

Publications and source records attributed to O Cascone.

At least 19 recordsLinked to original sources

PubMed2005

Rapid affinity purification processes for cyclodextrin glycosyltransferase from Bacillus circulans.

Two rapid and easy-to-scale-up methods for the purification of cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans were developed: affinity precipitation with starch and aqueous two-phase partition. The first method, optimised by a factorial design, gave an 80% CGTase adsorption at 11% starch and 1.6% ammonium sulphate, and a 65% recovery after elution with 10 mM alpha-cyclodextrin. The purification factor was 17. Aqueous two-phase partition yielded a 72% CGTase recovery in a two-step procedure; CGTase was obtained in the bottom phase with a purification factor of 37.

Bacillus↗

PubMed2004

Fractionation of the naringinase complex from Aspergillus terreus by dye affinity chromatography.

Affinity chromatography with immobilised triazine dyes was used to separate the main enzymes present in the naringinase complex produced by Aspergillus terreus CECT 2663. One alpha-L-rhamnosidase and two beta-D-glucosidases (beta G1 and beta G2) were separated by a simple two-step procedure involving chromatography with Red HE-3B immobilised on Sepharose 4B first at pH 5.5 and then at pH 4.7. Maximum activity of the beta-D-glucosidases was from pH 4 to 6 and at 65 degrees C. Both glucosidases were active on p -nitrophenol glucoside and prunin with respective Km values of 1.9 mm and 1.6 mm for beta G1 and 2.1 mm and 0.25 mm for beta G2. Only beta G1 hydrolysed cellobiose (Km = 5.7 mm).

Adsorption↗

PubMed2003

Monoclonal antibody purification by affinity chromatography with ligands derived from the screening of peptide combinatory libraries.

The peptide, Ala-Pro-Ala-Arg (APAR), was selected from the screening of a tetrapeptide combinatorial synthetic library as the ligand for affinity purification of an anti-Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) monoclonal antibody (Mab) developed in mouse ascitis. The affinity chromatographic matrix obtained by attachment of APAR to agarose, having a peptide density of 0.5 micromol ml(-1), showed a maximum capacity of 9.1 mg Mab ml(-1) and a dynamic capacity of 3.9 mg Mab ml(-1). A 95% yield of electrophoretically pure anti-GM-CSF was obtained in a single step.

Antibodies, Monoclonal↗

PubMed2000

High-speed pectic enzyme fractionation by immobilised metal ion affinity membranes.

Immobilised metal ion affinity polysulfone hollow-fibre membranes, with a high capacity for protein adsorption, were prepared and their utilisation for commercial pectic enzyme fractionation was studied. The pass-through fraction containing pectinlyase is useful for fruit-juice clarification without methanol production on account of pectinesterase being retained by the IDA-Cu2+ membrane.

Chromatography, Affinity↗

PubMed1999

Protein adsorption onto tentacle cation-exchange hollow-fiber membranes

A sulfonic group (up to 200 &mgr;mol/mL) membrane was incorporated to epoxy-activated microporous hollow fibers to obtain high-capacity convective ion exchangers. The pure water flux through the membrane decreased exponentially with sulfonic group density and protein binding capacity increased accordingly. At sulfonic group density of 70 &mgr;mol/mL, the membrane lysozyme maximum binding capacity was 84 +/- 9 mg/mL in comparison with its theoretical monolayer maximum binding capacity of 20 mg/mL, thus evidencing tentacle formation. After a cycle of adsorption in a 30 mM sodium phosphate buffer, pH 7. 0, adsorbed lysozyme could be quantitatively recovered following elution with 0.5 M NaCl in the same buffer. Dynamic capacity for lysozyme was 67% of maximum binding, and this value did not change at space velocities ranging from 10 to 40 min-1 as shown by the superimposition of the corresponding breakthrough curves. A cartridge assembled with 21 fibers showed a dynamic-to-static capacity ratio for lysozyme of 0.60 with 1 mg/mL pure lysozyme solution, and 0.42 with a particulate feed composed of 1 mg/mL lysozyme and 0.1 mg/mL yeast.

Journal Article↗

PubMed1999

Purification and characterization of an acid proteinase from mesophilic Mucor sp. solid-state cultures.

The fourth-day extract of a solid-state culture of the mesophilic Mucor sp. (M-105) strain showed a high milk-clotting activity and a clotting/proteolytic activity ratio similar to that of commercial preparations from microbial origin used in cheese manufacture. After ultrafiltration of the crude extract, the milk-clotting proteinase was purified in two steps: ion-exchange followed by size-exclusion chromatography. Enzyme homogeneity was assessed by HPLC, SDS-PAGE and N-terminal residue determination. A pI value of 4.21 was obtained and a molecular weight of 33 kDa was calculated from size-exclusion chromatography and SDS-PAGE data. The optimum pH for proteolytic activity towards dimethylcasein was in the 3.0-3.5 range. The proteinase retained 26 and 13% of its proteolytic activity after a 30-min incubation period, at pH 5.0 and 50 and 60 degrees C, respectively. This evidenced a lower heat stability than that of the thermophilic enzymes currently used in the cheese industry and also than that of bovine chymosin. The enzyme was fully inhibited by pepstatin A and no effect was observed with PMSF, p-CMPS or EDTA. The N-terminal amino acid sequence: GTGTVPVTDDGNLNEYYXTVTVGXP was compared with those from other fungal enzymes.

Amino Acid Sequence↗

PubMed1996

Foot and mouth disease virus concentration and purification by affinity chromatography.

Foot and mouth disease virus, (FMDV) from a crude cell lysate was purified in a single step by affinity chromatography with heparin as a ligand. The virus eluted from an Heparin-Ultrogel A4R column at 1M sodium chloride in 10 mM sodium phosphate buffer, pH 7.0, while most cell protein and albumin did so at lower concentrations of sodium chloride in the same buffer. Purity of the eluted fraction containing the virus was assessed by SDS-PAGE, HPLC, ultracentrifugation, and UV absorption spectrum. With this method, intact viral particles are recovered in high yield (over 90%) and specific virus purity increases nearly 1000-fold. The capacity of the chromatographic matrix for the virus was found to be 1.1 mg viral mass per mL of hydrated gel.

Aphthovirus↗

PubMed1996

Purification of a lipase from Acinetobacter calcoaceticus AAC323-1 by hydrophobic-interaction methods.

The performance of hydrophobic-interaction chromatography (HIC) for the purification of Acinetobacter calcoaceticus AAC323-1 lipase was compared with that of various aqueous two-phase systems. While a 42% lipase yield with a purification factor of 140 could be recovered by HIC, higher yields were achieved by using aqueous two-phase systems, either those formed by poly(ethylene glycol) and dextran or those based upon the use of a detergent. Triton X-114-based aqueous two-phase partition showed the best performance, with a yield of 81% and a purification factor of 68. Further detergent removal was easily achieved with an adsorbent, with no significant decrease in yields. Owing to its simplicity, the method should be easy to scale-up.

Acinetobacter calcoaceticus↗

PubMed1995

Purification and characterization of two forms of antigen 5 from polybia scutellaris venom.

Two polypeptides from the venom of Polybia scutellaris were purified to homogeneity by RP-HPLC. They differ very slightly in mol. wt (both are about 23,000) and hydrophobicity, and have isoelectric points greater than 9. Amino acid analyzes show close similarity between them and with antigen 5 of vespids from different species. The two polypeptides have an identical N-terminal sequence (18 amino acids) which shows a high degree of homology with those of other vespids. Owing to the fact that the venom of this species is non-allergenic, the data for the mol. wt, isoelectric point, amino acid composition and N-terminal sequence allow us to identify the isolated polypeptides as two forms of antigen 5. Amino acids at positions 5 and 11 in P. scutellaris antigen 5 differ from those of the previously known sequences for antigen 5, suggesting that one or other might be responsible for the lack of allergenicity of the P. scutellaris venom.

Allergens↗

PubMed1995

Effect of the surface charge on partitioning of chemically modified bovine serum albumin in aqueous two-phase systems.

In order to assess the influence of the protein charge on its partitioning in poly(ethyleneglycol)/salt aqueous two-phase systems, three bovine serum albumin derivatives with isoelectric points of 5.50, 6.20 and 6.85 were obtained by chemical modification of the protein with a soluble carbodiimide and glycine O-methyl ester and separation of the derivative mixture by liquid isoelectric focusing. The modification reaction was mild enough to preserve the tertiary structure of the proteins, as judged by circular dichroism and fourth derivative UV spectra. The surface hydrophobicity of the bovine serum albumin derivatives was identical, as measured by hydrophobic interaction chromatography. Partitioning of the derivatives in poly(ethyleneglycol)/phosphate and poly(ethyleneglycol)/citrate aqueous two-phase systems between pH 5.2 and 6.5 indicates that partitioning is not dependent on the protein charge in the poly(ethyleneglycol)/salt systems studied.

Amino Acid Sequence↗

PubMed1993

Oxidation of methionine residues in equine growth hormone by Chloramine-T.

1. Reactivity of methionine residues towards Chloramine-T was studied in the equine growth hormone. 2. With a 20.0-fold molar excess of reagent over methionine, full oxidation of the four residues of the protein is achieved. 3. Methionine 4 is the most reactive group, followed by methionines 72 and 178--methionine 123 being the less reactive residue. 4. As judged by circular dichroism spectra and binding assays, protein conformation and binding capacity to specific receptors remains unchanged even after full oxidation of all four methionine residues. 5. Results agree with data previously obtained with bovine growth hormone.

Amino Acid Sequence↗

PubMed1992

Purification and characterization of a milk clotting protease from Mucor bacilliformis.

An acid protease having milk clotting activity has been isolated from Mucor bacilliformis cultures. The enzyme was basically purified by ionic exchange chromatography. An average yield of 29 mg purified product was obtained from 100 mL crude extract. As purity criteria, SDS-PAGE, reverse-phase HPLC, and N-terminal analysis were performed. The protease is a protein composed of a single polypeptide chain with glycine at the N-terminus. The mol wt is approx 32,000, and its amino acid composition is very similar to those of other fungal proteases. As expected, its clotting activity was drastically inhibited by pepstatin A action. On the other hand, its instability against heat treatment and its clotting/proteolytic activity ratio indicate that it may be considered as a potential substitute for bovine chymosin.

Amino Acids↗

PubMed1992

An equine growth hormone molecular species lacking the 76-92 peptidic fragment.

A new eGH molecular species was isolated and purified by reverse phase HPLC. SDS-polyacrylamide gel electrophoresis, amino acid composition, and C- and N-terminal determinations support a primary structure identical to that described by Zakin et al. (1976), except for the lack of the 76-92 peptidic fragment and the maintaining of 30% of its biological activity.

Amino Acid Sequence↗

PubMed1991

Isolation, purification and primary structure of insulin from the turtle Chrysemys dorbigni.

Insulin A and B chains from pancreas of the turtle Chrysemys dorbigni have been purified to homogeneity, and their primary structures have been determined. The sequence of the A chain is G-I-V-E-Q-C-C-H-N-T-C-S-L-Y-Q-L-E-N-Y-C-N, and that of the B chain is A-A-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-S-P-K-A. The amino acid sequence of Chrysemys insulin is identical to that of another turtle (Pseudemys scripta), the chicken, and turkey. When compared with alligator insulin, it has three conservative substitutions in the B chain. However, there are seven substitutions when compared with the insulin of the rattlesnake.

Amino Acid Sequence↗

PubMed1991

Primary structure of alpaca growth hormone.

Reduced and carbamidomethylated alpaca growth hormone was submitted to tryptic digestion. Peptides in the mixture were purified by reverse phase HPLC and N-terminal determination and an amino acid analysis of each was performed. Data obtained and the already known primary structure of the equine growth hormone allowed the assembly-by homology-of a definite sequence of amino acids for the polypeptide chain of the protein. Present data provide further information about the relationship between growth factors.

Amino Acid Sequence↗

PubMed1990

Conformational comparison in the growth hormone family.

1. The method of Kubota et al. [Biochim. biophys. Acta 701, 242-252 (1982)] was applied to several members of the growth hormone family in order to examine their conformational homology. 2. The method neither detects differences between rat, cow, sheep, horse and alpaca hormones, nor between monkey and human hormones. 3. Lack of homology between primate and non-primate growth hormones was found in segments 42-49 and 184-191. The first fragment could be linked to species-specificity.

Animals↗

PubMed1990

Role of histidine residues in the binding mechanism of the growth hormone family.

1. Reactivity of the hGH histidine residues were studied by reaction with ethoxyformic anhydride. Localization in the molecule of three kinetically distinguishable classes, each including only one residue, was achieved. 2. The first was composed of residue 151, with an apparent velocity constant k = 0.735/min, (similar to that of histidines 19 and 21 in bGH and eGH). The second histidine, 18, with a velocity constant k = 0.135/min, (similar to that of histidine 169 in the above hormones), and a third, histidine 21, which does not react at all. 3. Neither histidine 151 nor 18 seem to be involved, at least not directly, in bGH binding to specific rat liver sites, since the decrease in this capacity was only 47% after modification of the former by 77 and 65% after total modification of the latter. 4. These results, and those previously obtained with bGH and eGH, suggest that either histidine 21 is the only indispensable histidine for the binding of growth hormones to specific rat liver sites, or that histidine 21 and/or 18 (19 in bGH and eGH), are located within the growth hormone binding site interaction area.

Animals↗

PubMed1989

Molecular differences between rat-liver and rat-kidney biliverdin reductase. Implications for their in vivo regulation.

Rat-liver biliverdin reductase exists in two molecular forms. The major form 1 has a molecular mass of 34 kDa, while the minor form 2 has a molecular mass of 56 kDa. Form 1 was converted into a second major form (form 3) with a molecular mass of 68 kDa by a NAD+-dependent peroxisomal dehydrogenase which was induced under conditions of oxidative stress [Frydman, R. B., Tomaro, M. L., Awruch, J. & Frydman, B. (1984) Biochem. Biophys. Res. Commun. 121, 249]. Molecular form 1 from rat kidney was not affected by the dehydrogenase, and a structural explanation for this difference was therefore sought. Both form 1 biliverdin reductases, isolated from rat liver and kidney, were purified to homogeneity using affinity chromatography, FPLC and HPLC techniques. The homogeneous enzymes were found to be identical when compared by their HPLC retention times, amino acid compositions and electrophoretic behaviour on polyacrylamide gels under non-denaturing conditions and on SDS/polyacrylamide gels. On HPLC analysis the peptides resulting from the CNBr cleavage were found to be the same for both enzymes, when either the native enzymes or their thioethylpyridine derivatives were compared. When the HPLC fingerprints of the tryptic digests were compared, they were found to be very similar, except for a peptide eluting at 31.60 min in the liver digest and at 23.60 min in the kidney digest. When the enzyme from both origins was alkylated with 4-dimethylaminoazobenzene-4'-iodoacetamide and then digested with trypsin, the HPLC fingerprints of the alkylated cysteine-carrying peptides were almost identical, except for a peptide with a retention time of 19.03 min in the liver digest and of 18.19 min in the kidney digest. The liver reductase was not amenable to Edman degradation suggesting a block at the NH2-terminus; in the kidney enzyme, however, it was free and an NH2-terminal sequence of 12 amino acids could be determined. The liver enzyme was found to be more sensitive toward p-hydroxymercuriphenyl sulfonate than the kidney enzyme.

Alkylation↗