Further purification and characterization of newly synthesized anionic glycoconjugates secreted by cultured UMR-106 cells: evidence that the major anionic glycoconjugate secreted by these cells is similar to bone sialoprotein II

1991 ◽  
Vol 69 (8) ◽  
pp. 523-530 ◽  
Author(s):  
Ravi K. Chopra ◽  
Tassos P. Anastassiades ◽  
David Lohnes ◽  
Glenville Jones
Keyword(s):  
Amino Acid ◽  
Cetylpyridinium Chloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Minor Component ◽  
Keratan Sulfate ◽  
Bone Sialoprotein ◽  
Terminal Amino ◽  
A Minor ◽  
Umr 106

Following incubation of UMR-106 cells for 48 h in the presence of [3H]glucosamine and [35S]sulfate, the newly synthesized anionic glycoconjugates were isolated from the culture medium by cetylpyridinium chloride/ethanol precipitation and further separated by DEAE-Sephacel chromatography into two radiolabelled fractions, a major component, UM I, and a minor component, UM II. UM I appeared to be homogeneous as shown by Sepharose CL-4B chromatography under dissociative conditions, and SDS-polyacrylamide gel electrophoresis. It showed a molecular mass of approximately 93 kDa on 4–15% gels. UM I was partially degraded by brief treatment with trypsin, releasing a small, terminal peptide that contained 47.6% of 35S but no 3H. Treatment of UM I with neuraminidase and 0.1 N H2SO4 (1 h at 80 °C), respectively, released 27% 3H and 38.4% 3H plus 41% 35S, suggesting the presence of a significant number of sialic acid residues, as shown by Sephadex G-50 chromatography of the digests. Amino acid analysis showed that the UM I glycoconjugate was rich in acidic amino acids (12.6% aspartic acid and 21.2% glutamic acid residues) and its N-terminal sequence was Phe-Ser-Met-Lys-Asn-Phe-, which is identical to the published N-terminal amino acid sequence of rat bone sialoprotein II. Keratanase treatment of UM I released 26% of the incorporated radioactivity, suggesting the presence of keratan sulfate chains. UM II contained a chondroitinase ABC-sensitive proteoglycan.Key words: UMR-106 cells, anionic glycoconjugates, bone sialoprotein II.

PURIFICATION AND PARTIAL CHEMICAL CHARACTERIZATION OF SHEEP NEUROPHYSIN

1973 ◽  
Vol 74 (2) ◽  
pp. 226-236 ◽  
Author(s):  
Michel Chrétien ◽  
Claude Gilardeau
Keyword(s):  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chemical Characterization ◽  
Terminal Amino Acid ◽  
Amino Acid Determination ◽  
Pituitary Glands ◽  
Terminal Amino ◽  
Partial Chemical ◽  
Acid Determination

ABSTRACT A protein isolated from ovine pituitary glands has been purified, and its homogeneity assessed by NH2- and COOH-terminal amino acid determination, ultracentrifugation studies, and polyacrylamide gel electrophoresis after carboxymethylation. Its chemical and immunochemical properties are closely similar to those of beef and pork neurophysins, less similar to those of human neurophysins. It contains no tryptophan (like other neurophysins) or histidine (like all except bovine neurophysin-I and human neurophysins). It has alanine at the NH2-terminus and valine at the COOH-terminus. Its amino acid composition is similar to, but not identical with those of porcine and bovine neurophysins.

Partial sequence of hemoglobin from cobra (Naja naja naja)

1987 ◽  
Vol 7 (10) ◽  
pp. 813-819 ◽  
Author(s):  
Sabira Naqvi ◽  
Iffat N. Nadvi ◽  
Zafar H. Zaidi
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Minor Component ◽  
Partial Sequence ◽  
Terminal Amino Acid ◽  
Naja Naja ◽  
Terminal Amino ◽  
A Minor ◽  
Cellulose Column ◽  
Terminal Amino Acid Sequence

Hemoglobin from the cobra snake, Naja naja naja, was isolated and its chains separated on a CM-cellulose column. The separation profile revealed an α and two β chains having the molar proportions of [α]2,[β1]1,[β2]1. The N-terminal amino acid sequence of the intact chains and of the CNBr peptides were carried out. The β2 chain was found to be heterogeneous comprising a minor component amounting to 11%. This later showed changes at two positions 9 and 14 in the first 30 residues sequenced.

scholarly journals Purification and characterization of a labile rat glutathione transferase of the Mu class

1989 ◽  
Vol 260 (3) ◽  
pp. 789-793 ◽  
Author(s):  
A Kispert ◽  
D J Meyer ◽  
E Lalor ◽  
B Coles ◽  
B Ketterer
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Glutathione Transferase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Cnbr Cleavage

A labile GSH transferase homodimer termed 11-11 was purified from rat testis by GSH-agarose affinity chromatography followed by anion-exchange f.p.l.c. The enzyme is unstable in the absence of thiol(s) and has relatively low affinity for both 1-chloro-2,4-dinitrobenzene (Km 4.4 mM) and GSH (Km(app.) 4.4mM). Its mobility on SDS/polyacrylamide-gel electrophoresis is slightly less than that of subunits 3 and 4 and its pI is 5.2. Subunit 11 has a blocked N-terminal amino acid residue, but after CNBr cleavage fragments accounting for 113 amino acid residues were sequenced and showed 65% homology with corresponding sequences in subunit 4, indicating that it is a member of the Mu family. GSH transferase 11 is a major isoenzyme in testis, epididymis, prostate and brain and present at lower concentrations in other tissues.

scholarly journals Characterization of the d-Xylulose 5-Phosphate/d-Fructose 6-Phosphate Phosphoketolase Gene (xfp) from Bifidobacterium lactis

2001 ◽  
Vol 183 (9) ◽  
pp. 2929-2936 ◽  
Author(s):  
Leo Meile ◽  
Lukas M. Rohr ◽  
Thomas A. Geissmann ◽  
Monique Herensperger ◽  
Michael Teuber
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Guanosine Monophosphate ◽  
Bifidobacterium Lactis ◽  
Terminal Amino ◽  
Cloned Gene

ABSTRACT A d-xylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase (Xfp) from the probioticBifidobacterium lactis was purified to homogeneity. The specific activity of the purified enzyme with d-fructose 6-phosphate as a substrate is 4.28 Units per mg of enzyme.Km values for d-xylulose 5-phosphate and d-fructose 6-phosphate are 45 and 10 mM, respectively. The native enzyme has a molecular mass of 550,000 Da. The subunit size upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (90,000 Da) corresponds with the size (92,529 Da) calculated from the amino acid sequence of the isolated gene (namedxfp) encoding 825 amino acids. The xfp gene was identified on the chromosome of B. lactis with the help of degenerated nucleotide probes deduced from the common N-terminal amino acid sequence of both the native and denatured enzyme. Comparison of the deduced amino acid sequence of the cloned gene with sequences in public databases revealed high homologies with hypothetical proteins (26 to 55% identity) in 20 microbial genomes. The amino acid sequence derived from the xfp gene contains typical thiamine diphosphate (ThDP) binding sites reported for other ThDP-dependent enzymes. Two truncated putative genes, pta andguaA, were localized adjacent to xfp on theB. lactis chromosome coding for a phosphotransacetylase and a guanosine monophosphate synthetase homologous to products of genes inMycobacterium tuberculosis. However, xfp is transcribed in B. lactis as a monocistronic operon. It is the first reported and sequenced gene of a phosphoketolase.

scholarly journals Purification and characterization of acidic glutathione S-transferase 6 from human brain

1991 ◽  
Vol 274 (2) ◽  
pp. 405-408 ◽  
Author(s):  
T Suzuki ◽  
D C Shaw ◽  
P G Board
Keyword(s):  
Human Brain ◽  
Minor Component ◽  
Terminal Sequence ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
A Minor ◽  
Additional Member

An acidic glutathione S-transferase (GST) isoenzyme termed GST6 has been isolated from human brain, characterized and compared with other isoenzymes. The N-terminal amino acid sequence of GST6 was found to be identical with that of GST4 previously purified from human muscle. GST6 cross-reacted with antibody raised against GST4, but not with antisera raised against GST1, GST2 or GST3. The subunit Mr and pI of GST6 were found to be different from those of GST4. The present results indicate that GST6 is another member of the Mu evolutionary class which in man also includes GST1, GST4 and GST5. A minor component that co-purified with GST6 was shown to have an N-terminal sequence similar to, but not identical with, that of GST3. This isoenzyme may be an additional member of the Pi evolutionary class.

Functional Characterization of GABAA Receptors in Neonatal Hypothalamic Brain Slice

2002 ◽  
Vol 88 (4) ◽  
pp. 1655-1663 ◽  
Author(s):  
Ren-Qi Huang ◽  
Glenn H. Dillon
Keyword(s):  
Functional Characterization ◽  
Minor Component ◽  
Dehydroepiandrosterone Sulfate ◽  
Receptor Subtypes ◽  
Hypothalamic Neurons ◽  
Inhibitory Neurotransmitter ◽  
Old Rats ◽  
A Minor ◽  
Whole Cell Recordings

The hypothalamus influences a number of autonomic functions. The activity of hypothalamic neurons is modulated in part by release of the inhibitory neurotransmitter GABA onto these neurons. GABAA receptors are formed from a number of distinct subunits, designated α, β, γ, δ, ε, and θ, many of which have multiple isoforms. Little data exist, however, on the functional characteristics of the GABAA receptors present on hypothalamic neurons. To gain insight into which GABAA receptor subunits are functionally expressed in the hypothalamus, we used an array of pharmacologic assessments. Whole cell recordings were made from thin hypothalamic slices obtained from 1- to 14-day-old rats. GABAA receptor-mediated currents were detected in all neurons tested and had an average EC50 of 20 ± 1.6 μM. Hypothalamic GABAA receptors were modulated by diazepam (EC50 = 0.060 μM), zolpidem (EC50 = 0.19 μM), loreclezole (EC50 = 4.4 μM), methyl-6,7-dimethoxy-4-ethyl-β-carboline (EC50= 7.7 μM), and 5α-pregnan-3α-hydroxy-20-one (3α-OH-DHP). Conversely, these receptors were inhibited by Zn2+ (IC50 = 70.5 μM), dehydroepiandrosterone sulfate (IC50 = 16.7 μM), and picrotoxin (IC50 = 2.6 μM). The α4/6-selective antagonist furosemide (10–1,000 μM) was ineffective in all hypothalamic neurons tested. The results of our pharmacological analysis suggest that hypothalamic neurons express functional GABAA receptor subtypes that incorporate α1 and/or α2 subunits, β2 and/or β3 subunits, and the γ2 subunit. Our results suggest receptors expressing α3–α6, β1, γ1, and δ, if present, represent a minor component of functional hypothalamic GABAA receptors.

scholarly journals The purification and properties of the second component of human complement

1978 ◽  
Vol 171 (1) ◽  
pp. 99-107 ◽  
Author(s):  
M A Kerr ◽  
R R Porter
Keyword(s):  
Amino Acid ◽  
Polypeptide Chain ◽  
Alternative Pathway ◽  
Polyacrylamide Gel Electrophoresis ◽  
Complement Factor ◽  
Human Complement ◽  
Terminal Amino Acid ◽  
Factor B ◽  
Antigenic Properties ◽  
Terminal Amino

The second component of human complement (C2) was purified by a combination of euglobulin precipitation, ion-exchange chromatography, (NH4)2SO4 precipitation and affinity chromatography. The final product was homogeneous by the criterion of polyacrylamide-gel electrophoresis and represents a purification of about 4000-fold from serum with 15-20% yield. Component C2 comprises a single carbohydrate-containing polypeptide chain, with an apparent mol.wt. of 102000; alanine is the N-terminal amino acid. The molecule is rapidly cleaved by activated subcomponent C1s with the loss of haemolytic activity to yield two fragments with apparent mol.wts. of 74000 and 34000. These fragments are not linked by disulphide bonds and can be easily separated. A second protein isolated during the purification of component C2 was identified by its haemolytic and antigenic properties as complement Factor B, the protein serving an analogous function to component C2 in the alternative pathway. The protein, which is also a single carbohydrate-containing polypeptide chain, has an apparent mol.wt. of 95000 and threonine as N-terminal amino acid. The amino acid analyses of component C2 and Factor B are compared.

scholarly journals Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

1998 ◽  
Vol 180 (2) ◽  
pp. 388-394 ◽  
Author(s):  
Masahiro Furutani ◽  
Toshii Iida ◽  
Shigeyuki Yamano ◽  
Kei Kamino ◽  
Tadashi Maruyama
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ppiase Activity ◽  
Methanococcus Thermolithotrophicus ◽  
Fk506 Binding Protein ◽  
Thermophilic Archaeon

ABSTRACT A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (k cat/Km ) measured at 15°C for the peptidyl substrates,N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide andN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM−1 s−1, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicusFK506-binding protein). The MTFK gene (462 bp) was cloned from anM. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.

scholarly journals Purification and partial characterization of a lectin from Caesalpinia tinctoria Domb, ex Dc fruits

2003 ◽  
Vol 15 (2) ◽  
pp. 119-122 ◽  
Author(s):  
Marli Lourdes de Oliveira ◽  
Leila Maria Beltramini ◽  
Salvatore Giovanni de Simone ◽  
Maria Helena Nasser Brumano ◽  
Rosemeire Aparecida Silva-Lucca ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Terminal Amino Acid ◽  
Saline Extract ◽  
Hydrophobic Residues ◽  
Terminal Amino ◽  
Helix Conformation ◽  
Far Ultraviolet ◽  
Terminal Amino Acid Sequence

A lectin was isolated from the pod saline extract of Caesalpinia tinctoria by dialoconcentration on Centripep-10 and affinity chromatography on chitin column. The purified lectin was partially characterized with respect to its biochemical and structural properties. It contains 8.3 % of carbohydrate and exhibited an agglutinating activity against human erythrocytes (ABO groups). Its amino acid composition was characterized by a great number of acidic and hydrophobic residues and the estimated molecular mass was 12.5 kDa. The presence of only one N-terminal amino acid sequence (D¹-V-P-A-Y-V-Y-V-H-F10-G-F-G-E-E-H-R -D-V-F20-D), showed the homogeneity of the purified lectin. The far-ultraviolet circular dichroism (CD) spectrum of lectin indicated that it contains 10 % a-helix, 38 % b-sheet, 28 % unordered form and 6 % of P II (poly-L-proline II helix conformation).

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