scholarly journals Purification and characterization of three constitutive cytochrome P-450 isoforms from bovine olfactory epithelium

1997 ◽  
Vol 323 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Vincenzo LONGO ◽  
Giada AMATO ◽  
Annalisa SANTUCCI ◽  
Pier Giovanni GERVASI
Keyword(s):  
Spin State ◽  
High Spin State ◽  
Total Oxidation ◽  
Oxidation Rates ◽  
Sds Page ◽  
Molecular Masses ◽  
Endogenous Substrates ◽  
Cytochrome P 450 ◽  
Great Portion

Three constitutive forms of cytochrome P-450 (P-450s) were isolated from olfactory microsomes of cattle. The purified P-450s, designated P-450bov1, P-450bov2 and P-450bov3, were electrophoretically nearly homogeneous by SDS/PAGE and their apparent relative molecular masses were estimated to be 50000, 53000 and 51000 respectively. As indicated by several criteria including the N-terminal sequence and absorption spectra, the three olfactory forms of P-450 were distinct from each other and from all the other P-450s currently known in cattle. P-450bov1 and P-450bov2 were purified in the low-spin state, whereas P-450bov3 was in the high-spin state. Studies to evaluate, by Western blot analysis, the reactivity of these purified P-450s with antibodies raised against rat hepatic P-450 2E1, 2B, 1A and 3A and rabbit olfactory P-450NMa and P-450NMb showed that P-450bov3 strongly cross-reacted with anti-P-450NMb IgG, and P-450bov1 moderately with anti-P-450NMa IgG. As determined by immunoblots, P-450bov1 and P-450bov3 represented a great portion of the total olfactory P-450. In a reconstituted system with NADPH:cytochrome P-450 reductase and phospholipids, P-450bov1 was more active in the metabolism of xenobiotic compounds (i.e. O-de-ethylation of ethoxycoumarin and N-demethylation of hexamethylphosphoramide) than towards endogenous substrates (testosterone and progesterone). Conversely, P-450bov3 metabolized the xenobiotics at lower rates but exhibited total oxidation rates of the above sex hormones higher than those of P-450bov1. From the comparison of the catalytic, immunochemical and structural properties, it was inferred that P-450bov1 and P-450bov3 are the bovine orthologues of P-450NMa (2A) and P-450NMb (2G1) respectively, the only two olfactory P-450s previously purified from rabbit. P-450bov2, which showed low activity toward some exogenous and endogenous compounds, represents a novel purified olfactory hemoprotein possibly belonging to the 3A subfamily. These results are consistent with a specific presence of catalytically and structurally similar P-450s, at least for the major ones, in the olfactory mucosa of mammals.

scholarly journals Purification and characterization of three extracellular (1→3)-β-d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum

1995 ◽  
Vol 308 (3) ◽  
pp. 733-741 ◽  
Author(s):  
S M Pitson ◽  
R J Seviour ◽  
B M McDougall ◽  
J R Woodward ◽  
B A Stone
Keyword(s):  
Filamentous Fungus ◽  
Gel Filtration ◽  
High Specificity ◽  
Major Product ◽  
Ph Optimum ◽  
Sds Page ◽  
Molecular Masses ◽  
Monomeric Proteins ◽  
Ph Range

Three (1-->3)-beta-D-glucanases (GNs) were isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. Homogeneity of the purified proteins was confirmed by SDS/PAGE, isoelectric focusing and N-terminal amino acid sequencing. All three GNs (GN I, II and III) are non-glycosylated, monomeric proteins with apparent molecular masses, estimated by SDS/PAGE, of 81, 85 and 89 kDa respectively. pI values for the three enzymes are 5.3, 5.1, and 4.4 respectively. The pH optimum for GN I is 6.5, and 5.0 for GN II and III. All three purified enzymes displayed stability over the pH range 4.5-10.0. Optimum activities for GN I, II and III were recorded at 65, 55 and 60 degrees C respectively, with both GN II and III having short-term stability up to 50 degrees C and GN I up to 55 degrees C. The purified GNs have high specificity for (1-->3)-beta-linkages and hydrolysed a range of (1-->3)-beta- and (1-->3)(1-->6)-beta-D-glucans, with laminarin from Laminaria digitata being the most rapidly hydrolysed substrate of those tested. K(m) values for GN I, II, and III against L. digitata laminarin were 0.1, 0.23 and 0.22 mg/ml respectively. D-Glucono-1,5-lactone does not inhibit any of the three GNs, some metals ions are mild inhibitors, and N-bromosuccinimide and KMnO4 are strong inhibitors. All three GNs acted in an exo-hydrolytic manner, determined by the release of alpha-glucose as the initial and major product of hydrolysis of (1-->3)-beta-D-glucans, and confirmed by viscometric analysis and the inability to cleave periodate-oxidized laminarin, and may be classified as (1-->3)-beta-D-glucan glucohydrolases (EC 3.2.1.58).

Purification and Characterization of Hepatic P-450Iie1 from Acetone-Treated Mice

1993 ◽  
Vol 9 (3) ◽  
pp. 539-546 ◽  
Author(s):  
Vincenzo Longo ◽  
Silvia Menicagli ◽  
Michael Minks ◽  
Annalisa Santucci ◽  
Pier Giovanni Gervasi
Keyword(s):  
Polyclonal Antibodies ◽  
High Spin State ◽  
Purification And Characterization ◽  
Turnover Number ◽  
High Turnover ◽  
Hepatic Microsomes ◽  
Aminoacid Sequence ◽  
Reconstituted System ◽  
Cytochrome P 450

A new cytochrome P-450 isozyme (Mr = 52,000) was purified to apparent electrophoretic homogeneity from hepatic microsomes of mice treated with acetone and its biochemical, spectral, and immunological properties characterized. Several criteria indicated that the purified cytochrome was distinct from the known mouse P-450 isozymes. The absolute spectrum of its oxidized form indicated that it was in the high spin state. In a reconstituted system, it showed low catalytic activities towards 7-ethoxycoumarin, aminopyrine, and coumarin, whereas it catalyzed the oxidation of aniline, acetone, dimelhylnitrosoamine with high turnover number. The mouse enzyme was immunoreactive with polyclonal antibodies against rat P-45011E1 and exhibited an NH2-terminal aminoacid sequence with a high homology to that of rat-P-450IIEI. Based upon the above catalytic, spectral, immunological and structural properties, the purified mouse P-450 appears to be the ortholog of previously described P-450IIE1 (s) of other species.

scholarly journals Characterization of MAX.3 antigen, a glycoprotein expressed on mature macrophages, dendritic cells and blood platelets: identity with CD84

2000 ◽  
Vol 346 (3) ◽  
pp. 729-736 ◽  
Author(s):  
Stefan W. KRAUSE ◽  
Michael REHLI ◽  
Sven HEINZ ◽  
Reinhard EBNER ◽  
Reinhard ANDREESEN
Keyword(s):  
Dendritic Cells ◽  
Blood Platelets ◽  
Cell Types ◽  
Variable Expression ◽  
Surface Molecules ◽  
Sds Page ◽  
Molecular Masses ◽  
Antigen A

MAX.3 is a monoclonal antibody that preferentially reacts with mature macrophages (MAC), monocyte-derived dendritic cells, megakaryocytes and platelets. In this study, we describe the characterization, purification and identification of the MAX.3 antigen. Immunoprecipitation and SDS/PAGE revealed different molecular masses of MAX.3 antigen in MAC (60-90 kDa) and platelets (58-64 kDa), whereas a similar size (45 kDa) was observed in both cell types after digestion with N-glycosidase F. Lectin affinity and sequential treatment with different glycosidases suggests complex type glycosylation of MAX.3 antigen in MAC and hybrid type glycosylation in platelets. Amino acid sequencing led to the identification of a corresponding cDNA clone and showed its identity to the sequence of the CD84 antigen, a member of the CD2 family of cell surface molecules. MAX.3/CD84 was further studied by immunohistochemistry and a variable expression was found on tissue MAC, confirming this antigen to be mainly a marker for MAC in situ.

scholarly journals Characterization of prenylated protein methyltransferase in Leishmania

1999 ◽  
Vol 342 (3) ◽  
pp. 513-518 ◽  
Author(s):  
Marie-Pierre HASNE ◽  
Françoise LAWRENCE
Keyword(s):  
Protein Kinase C ◽  
Leishmania Donovani ◽  
Post Translational Modification ◽  
Methyltransferase Activity ◽  
Kinase C ◽  
Protein Methyltransferase ◽  
Molecular Masses ◽  
Endogenous Substrates

Prenylated protein methyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, has been characterized in a parasitic flagellated protozoan, Leishmania donovani. The activity of this enzyme was monitored by the methylation of an artificial substrate, an S-prenylated cysteine analogue, with S-adenosyl-L-[methyl-3H]methionine as methyl donor. More than 85% of the methyltransferase activity was associated with membranes. The enzyme methylates N-acetyl-S-trans,trans-farnesyl-L-cysteine and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, but N-acetyl-S-trans,trans-geranyl-L-cysteine only very weakly. In contrast with the enzyme from mammals, the leishmanial enzyme had a greater affinity for the farnesylated substrate than for the geranylgeranylated one. Activity in vitro was not modulated by cAMP, protein kinase C activator or guanosine 5′-[γ-thio]triphosphate. An analysis of the endogenous substrates showed that the carboxymethylated proteins were also isoprenylated. The main carboxymethylated proteins have molecular masses of 95, 68, 55, 46, 34-23, 18 and less than 14 kDa. Treatment of cells with N-acetyl-S-trans,trans-farnesyl-L-cysteine decreased the carboxymethylation level, whereas treatment with guanosine 5′-[γ-thio]triphosphate increased the carboxymethylation of various proteins, particularly those of molecular masses 30-20 kDa.

scholarly journals Domains of the catalytically self-sufficient cytochrome P-450 BM-3. Genetic construction, overexpression, purification and spectroscopic characterization

1992 ◽  
Vol 288 (2) ◽  
pp. 503-509 ◽  
Author(s):  
J S Miles ◽  
A W Munro ◽  
B N Rospendowski ◽  
W E Smith ◽  
J McKnight ◽  
...  
Keyword(s):  
Spin State ◽  
Equivalent Form ◽  
High Spin State ◽  
Spectroscopic Characterization ◽  
Hydroxylase Activity ◽  
Fatty Acid Hydroxylase ◽  
X Band ◽  
Resonance Raman Spectra ◽  
Cytochrome P 450 ◽  
Reductase Domain

1. The gene CYP102 encoding cytochrome P-450 BM-3 and subgenes encoding the cytochrome P-450 and cytochrome P-450 reductase domains have been cloned in Escherichia coli. 2. The protein products of these genes have been overexpressed and purified to homogeneity. 3. The cytochrome P-450 domain is purified in the ferric low-spin state, but is readily converted into the high-spin state by addition of the substrate palmitate (Ks = 1 microM). The cytochrome P-450 reductase domain readily reduces cytochrome c. Mixing the two domains reconstitutes only about one-thousandth of the fatty acid hydroxylase activity associated with the intact cytochrome P-450 BM-3. 4. The X-band e.p.r. spectra of both the cytochrome P-450 domain and intact cytochrome P-450 BM-3 give g-values indicating low-spin ferric haem. The spectra are virtually identical with those of the equivalent form of cytochrome P-450 cam indicating that the haem ligation in cytochrome P-450 BM-3 is identical with that of cytochrome P-450 cam. 5. Resonance Raman spectra of the substrate-free and substrate-bound forms of the cytochrome P-450 domain are given. Spectral differences in comparison with cytochrome P-450 cam may reflect subtle electronic differences between the respective haem environments.

Physicochemical, molecular and immunological characterization of camel calf rennet: a comparison with buffalo rennet

2000 ◽  
Vol 67 (1) ◽  
pp. 73-81 ◽  
Author(s):  
ELSAYED I. ELAGAMY
Keyword(s):  
Marked Decrease ◽  
Cross Reactivity ◽  
Buffalo Calf ◽  
Immunological Characterization ◽  
Milk Clotting ◽  
Electrophoretic Patterns ◽  
Sds Page ◽  
Molecular Masses ◽  
Milk Clotting Activity

Camel calf rennet (CCR) and buffalo calf rennet (BCR) were prepared from dried abomasa to study their physicochemical properties and electrophoretic behaviour and to carry out an immunological characterization of the rennet proteins. CCR was more thermostable than BCR. The milk clotting activity of both rennets increased as pH decreased. The optimum temperatures for CCR and BCR were 50 and 45 °C respectively. CCR was more sensitive to increased CaCl2 in milk than BCR. Addition of NaCl to milk in the range 0–100 g/l resulted in a marked decrease in the clotting activity of both rennets. When the rennets were treated with acetone, the activity of BCR was completely destroyed, but that of CCR was unaffected. The proteolytic activity of CCR was higher than that of BCR and pepsin towards both camel and cows' milk caseins at pH 6·0. SDS-PAGE electrophoretic patterns of CCR and BCR proteins gave two major bands with molecular masses estimated as 52 and 39 kDa for CCR and 50 and 35 kDa for BCR. Immunodiffusion and immunoelectrophoresis using anti-CCR serum demonstrated immunological cross reactivity between CCR and BCR.

scholarly journals Purification and partial characterization of a novel lectin from elder (Sambucus nigra L.) fruit

1991 ◽  
Vol 278 (3) ◽  
pp. 667-671 ◽  
Author(s):  
L Mach ◽  
W Scherf ◽  
M Ammann ◽  
J Poetsch ◽  
W Bertsch ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Amino Acid ◽  
Carbohydrate Specificity ◽  
Sambucus Nigra ◽  
Acid Glycoprotein ◽  
High Affinity ◽  
Sambucus Nigra Agglutinin ◽  
Sds Page ◽  
Molecular Masses

A previously unknown haemagglutinin, named Sambucus nigra agglutinin-III (SNA-III), has been purified from the fruit of the elder (Sambucus nigra). Whereas elder bark agglutinin I (SNA-I) is highly specific for terminal alpha 2,6-linked sialic acid residues, SNA-III displays a high affinity for oligosaccharides containing exposed N-acetylgalactosamine and galactose residues. Different N-terminal sequences and the amino acid composition distinguish the fruit lectin from elder bark agglutinin II (SNA-II), which shows a similar carbohydrate specificity. The 40-fold higher affinity of SNA-III for asialofetuin than for human asialo-alpha 1-acid glycoprotein and human asialotransferrin respectively suggests a preference for O-linked glycans. SNA-III occurs mainly as a monomeric glycoprotein, but tends to form di- and oligo-meric aggregates. This aggregation seems to mediate the multivalent interaction, leading to agglutination. SDS/PAGE revealed two major polypeptides with apparent molecular masses of 32 and 33 kDa respectively. This heterogeneity is probably a result of proteolysis in the C-terminal region. Binding to concanavalin A and susceptibility to peptide: N-glycosidase F indicated the presence of N-glycosidically linked oligosaccharides.

Purification and Partial Characterization of the Streptomyces viridochromogenes TÜ494 Phosphinothricin-N-acetyltransferase Mediating Resistance to the Herbicide Phosphinothricin in Transgenic Plants

1995 ◽  
Vol 50 (11-12) ◽  
pp. 796-805 ◽  
Author(s):  
Josef Vinnemeier ◽  
Wolfgang Dröge-Laser ◽  
Elfriede K Pistorius ◽  
Inge Broer
Keyword(s):  
Escherichia Coli ◽  
Transgenic Plants ◽  
Gene Product ◽  
Coli Strain ◽  
Bar Gene ◽  
Pat Gene ◽  
Sds Page ◽  
Purification Scheme ◽  
Molecular Masses

Abstract A purification scheme for the enzyme phosphinothricin-N-acetyltransferase (PAT) originating from Streptomyces viridochromogenes {pat-gene product from Streptomyces virido­chromogenes) and mediating herbicide resistance to transgenic plants was developed. The enzyme was isolated from a transformed and overproducing Escherichia coli strain. With a combination of ammonium sulfate fractionation, chromatography on DEAE-Sephadex A50-, Phenylsepharose-, Hydroxylapatite-and FPLC-Superose 12-columns it was possible to obtain PAT which was at least 90 % homogeneous on the basis of SDS-PAGE. The properties of the isolated PAT were compared with the properties of PAT from S. hygroscopicus (bar-gene product from S. hygroscopicus) previously isolated and characterisized by Botterman, J., Gossele, V., Thoen, C., Lauwereys, M. (1991), Gene 102, 33-37. Differences were observed in the molecular masses of the two native enzymes (PAT from S. viridochrogenes being a dimer of 40 kD and PAT from S. hygroscopicus being a monomer of 21 kD), and in the temperature sensitivity of the two enzymes (the PAT from S. viridochromogenes being slightly more temperature stable than PAT from S. hygroscopicus). However, since the pat and the bar-gene are to 85 % homologous, substantial similarities exist between the two enzymes especially in the kinetic values and the substrate specificity. The isolated S. virido­chromogenes PAT did not acetylate putative substrates present in the plant cell. Antibodies were raised against the isolated protein. This antiserum was able to detect PAT in transgenic plants and therefore is suitable to analyse the fate of the protein in such plants under various stress conditions.

scholarly journals Purification, crystallization and characterization of N-acetylneuraminate lyase from Escherichia coli

1991 ◽  
Vol 276 (2) ◽  
pp. 541-546 ◽  
Author(s):  
K Aisaka ◽  
A Igarashi ◽  
K Yamaguchi ◽  
T Uwajima
Keyword(s):  
Escherichia Coli ◽  
Gel Filtration ◽  
Genetically Engineered ◽  
E Coli ◽  
Sds Page ◽  
Optimum Ph ◽  
Molecular Masses ◽  
Related Compounds ◽  
Optimum Ph And Temperature

N-Acetylneuraminate lyase produced by Escherichia coli was purified and crystallized from a genetically engineered strain (E. coli SF8/pNAL1). The enzyme showed apparent molecular masses of 105,000 Da on gel filtration and 35,000 Da on SDS/PAGE, suggesting that the enzyme is a trimer. The apparent optimum pH and temperature were found to be 6.5-7.0 and 80 degrees C respectively. The Km values for N-acetylneuraminate and N-glycollylneuraminate were 3.3 and 3.3 mM respectively. The enzyme was inhibited by reduction with NaBH4 in the presence of the substrate, indicating that the enzyme belongs to the Schiff-base-forming Class I aldolases. The enzyme was strongly inhibited by Cu2+ ions, p-chloromercuribenzoate and N-bromosuccinimide, and also inhibited competitively by the reaction product, pyruvate, and its structurally related compounds, dihydroxyacetone and DL-glyceraldehyde.

scholarly journals Isolation and characterization of galectin-1 binding proteins from human placenta

2000 ◽  
Vol 65 (2) ◽  
pp. 131-140
Author(s):  
Miroslava Jankovic
Keyword(s):  
Human Placenta ◽  
Binding Proteins ◽  
Solid Phase ◽  
Ricinus Communis ◽  
Gel Filtration ◽  
Isolation And Characterization ◽  
Sds Page ◽  
Molecular Masses ◽  
Solid Phase Binding

Galectin-1 binding proteins were isolated from human placenta by affinity chromatography on a column with immobilized endogenous lectin. The molecular masses of the isolated proteins of 170, 67 and 56 kDa were estimated by gel filtration and SDS-PAGE. These proteins were characterized as galactose-containing glycoproteins, based on their reactivity with Ricinus communis agglutinin. In addition, sialylated- lacto-N-fucopentaose II was detected in the 170 kDa protein, using anti CA 19-9 monoclonal antibodies. The interaction of the isolated proteins with human placental galectin-1 was investigated by a solid phase binding assay using asialofetuin as the glycoprotein ligand. The 67 kDa and 56 kDa proteins were found to inhibit galectin-1 binding of asialofetuin, whereas the 170 kDa protein had the opposite effect. It caused an increase in the binding of asialofetuin, suggesting a positive cooperative binding.

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