scholarly journals Ca2+-binding protein from human kidney. Purification and properties

1984 ◽  
Vol 217 (1) ◽  
pp. 229-237 ◽  
Author(s):  
M Staun ◽  
O Norén ◽  
H Sjöström
Keyword(s):  
Specific Antibody ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Human Kidney ◽  
Fold Increase ◽  
Binding Activity ◽  
Cytosol Fraction ◽  
Crossed Immunoelectrophoresis

A Ca2+-binding protein (CaBP) from human kidney was purified by two different procedures. The first involved heat-precipitation of a kidney cytosol fraction followed by gel filtration and chromatofocusing. This resulted in a 200-fold increase in the specific Ca2+-binding activity with a yield of 10%. A specific antibody was raised against the purified CaBP, as demonstrated by one precipitate in crossed immunoelectrophoresis of a kidney cytosol fraction. The antibody was coupled to Sepharose 4B and CaBP was then purified by immunoadsorbent chromatography. Applying this technique, a 500-fold purification of CaBP with a yield of 50% was obtained. Both preparations appeared homogeneous in crossed immunoelectrophoresis against a polyvalent antiserum and migrated as a single band corresponding to a mol.wt. of 26000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In gel filtration under non-denaturing conditions CaBP was eluted corresponding to a mol.wt. of 28000. The association constant for the high-affinity Ca2+-binding sites of CaBP was estimated by gel filtration to be 0.1 × 10(6)M-1, and the protein displayed Ca2+-dependent electrophoretic mobility, with more rapid anodic migration in the presence of EDTA. The protein eluted at a position corresponding to a pI of 4.5 in chromatofocusing. Immunochemical experiments with the specific antibody showed no cross-reaction between renal and intestinal CaBP.

scholarly journals A fibronectin-binding glycoprotein from human platelet membranes

1982 ◽  
Vol 201 (3) ◽  
pp. 629-633 ◽  
Author(s):  
M S Hansen ◽  
I Clemmensen
Keyword(s):  
Human Platelet ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Relative Molecular Mass ◽  
Crossed Immunoelectrophoresis ◽  
Platelet Membranes ◽  
Fibronectin Binding ◽  
Cold Insoluble Globulin ◽  
Binding Glycoprotein

Fibronectin (‘cold-insoluble globulin’) has been suggested as a possible mediator of platelet adhesion. A fibronectin-binding protein as partially purified from washed solubilized human platelet membranes by affinity chromatography on fibronectin-Sepharose. The isolated protein migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr (relative molecular mass) of approx. 125 000 under reducing conditions. The protein migrated as a dimer in non-reduced gels. The purified protein did not react with immunoglobulins against fibrinogen or fibronectin when tested in crossed immunoelectrophoresis or electroimmunoassay. The protein and purified fibronectin formed a complex that had a significantly faster mobility in crossed immunoelectrophoresis than did native fibronectin. The presence of heparin in the binding-protein-fibronectin mixture resulted in an even faster mobility of the complex, whereas the mobility of native fibronectin was unaffected. Crossed affinoimmunoelectrophoresis of the complex using different lectins suggested that the binding protein is a glycoprotein containing N-acetylglucosamine residues. The complex, but not purified fibronectin, bound to phenyl-Sepharose on crossed hydrophobic-interaction immunoelectrophoresis. The results strongly suggest the presence of a fibronectin-binding glycoprotein in the platelet membrane.

scholarly journals Comparison of the Fibronectin-Binding Protein FNE fromStreptococcus equi Subspecies equi with FNZ from S. equi Subspecies zooepidemicus Reveals a Major and Conserved Difference

2001 ◽  
Vol 69 (5) ◽  
pp. 3159-3163 ◽  
Author(s):  
Hans Lindmark ◽  
Martin Nilsson ◽  
Bengt Guss
Keyword(s):  
Dna Sequences ◽  
Stop Codon ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Binding Activity ◽  
Base Pairs ◽  
Reading Frame ◽  
A Cell

ABSTRACT The gene fnz from Streptococcus equisubspecies zooepidemicus encodes a cell surface protein that binds fibronectin (Fn). Fifty tested isolates of S. equi subspecies equi all contain DNA sequences with similarity to fnz. This work describes the cloning and sequencing of a gene, designated fne, with similarity tofnz from two S. equi subspeciesequi isolates. The DNA sequences were found to be identical in the two strains, and sequence comparison of the fne andfnz genes revealed only minor differences. However, one base deletion was found in the middle of the fne gene and eight base pairs downstream of the altered reading frame there is a stop codon. An Fn-binding protein was purified from the growth medium of a subspecies equi culture. Determination of the NH2-terminal amino acid sequence and molecular mass, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that the purified protein is the gene product of the 5′-terminal half of fne. Fn-binding activity has earlier only been found in the COOH-terminal half of FNZ. By the use of a purified recombinant protein containing the NH2 half of FNZ, we provide here evidence that this half of the protein also harbors an Fn-binding domain.

scholarly journals Purification and photoaffinity labelling of a rat cytosolic binding protein specific for 3-methylcholanthrene

1987 ◽  
Vol 242 (2) ◽  
pp. 375-381 ◽  
Author(s):  
P S Arnold ◽  
R C Garner ◽  
B Tierney
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Binding Activity ◽  
Liver Protein ◽  
Photoaffinity Labelling ◽  
High Affinity ◽  
Apparent Homogeneity ◽  
Stokes Radius

Rat hepatic cytosolic proteins which sediment at 4-5 S on sucrose gradients exhibit high-affinity saturable binding for the carcinogen 3-methylcholanthrene. A rat liver protein of Stokes' radius 3 nm, Mr by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 39,000 and with specific 3-methylcholanthrene-binding activity sedimenting at 4.5 S, has been purified 315-fold to apparent homogeneity by using affinity chromatography on a column of 1-hydroxy-3-methylcholanthrene coupled to epoxy-activated Sepharose 6B, in conjunction with two gel-filtration steps. The protein purified by this technique was shown to be associated with the observed specific 3-methylcholanthrene-binding activity by photoaffinity labelling with 1-oxo-3-methylcholanthrene.

scholarly journals Purification of two hexosaminidases from human kidney

1977 ◽  
Vol 163 (1) ◽  
pp. 133-140 ◽  
Author(s):  
D V Marinkovic ◽  
J N Marinkovic
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Human Kidney ◽  
Protein Band ◽  
Iodoacetic Acid ◽  
Homogeneous State ◽  
Molecular Weights ◽  
Hexosaminidase A

Hexosaminidase forms A and B were isolated from human kidney in a homogeneous state as demonstrated by electrophoretic and enzymic criteria. The enzymes were stable for at least 18 months when stored at -20 degrees C in 0.025 M-phosphate buffer, pH 6.5. The molecular weights of forms A and B were estimated by gel filtration to be 111 000 +/- 1500 and 114 000 +/- 1600 respectively. The molecular weights of hexosamidase A and B subunits were determined by using polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Hexosaminidase A dissociated into one subunit with mol.wt. 68 000. Hexosaminidase B dissociated into three subunits with mol. wts. 100 000, 68 000 and 37000 respectively, and one protein band of mol.wt. 140 000. After treatment of hexosaminidases A and B with iodoacetic acid, the molecular weights of the carboxymethylated polypeptide subunits were also estimated. Carboxymethylated hexosaminidase A dissociated into one major subunit of mol.wt. 18 000 and two other protein bands of mol.wts. 65 000 and 100 000. Carboxymethylated hexosaminidase B dissociated into one major subunit for mol.wt. 19 000 and an additional band of mol.wt. 37 000. The Km of the enzymes for the synthetic substrate p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside was 0.8 mM. Both enzymes were inhibited or activated by various metal ions. Double pH optima for the enzymes were found at pH 4.5 and 4.8.

scholarly journals Isolation and characterization of riboflavin-binding protein from pregnant-rat serum

1980 ◽  
Vol 187 (2) ◽  
pp. 537-540 ◽  
Author(s):  
K Muniyappa ◽  
P R Adiga
Keyword(s):  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Molar Ratio ◽  
Rat Serum ◽  
Pregnant Rat ◽  
Isolation And Characterization

A high-affinity riboflavin -binding protein was isolated and characterized for the first time from pregnant-rat sera by affinity chromatography on a lumiflavin-agarose column. The purified protein was homogeneous by the criteria of analytical polyacrylamide-gel disc electrophoresis, gel-filtration chromatography on Sephadex G-100 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It had a molecular weight of 90000+/-5000 and interacted with [14C]riboflavin with a 1:1 molar ratio with a dissociation constant (Kd) of 0.42 micron.

Thyroid hormone induces the generation of a novel putative protein in piscine ovarian follicle that stimulates the conversion of pregnenolone to progesterone

1996 ◽  
Vol 134 (1) ◽  
pp. 128-135 ◽  
Author(s):  
S Bhattacharya ◽  
S Guin ◽  
A Bandyopadhyay ◽  
NR Jana ◽  
S Halder
Keyword(s):  
Thyroid Hormone ◽  
Ovarian Follicle ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fold Increase ◽  
Ovarian Follicles ◽  
Linear Increase ◽  
Putative Protein

Bhattacharya S, Guin S, Bandyopadhyay A, Jana NR, Halder S. Thyroid hormone induces the generation of a novel putative protein in piscine ovarian follicle that stimulates the conversion of pregnenolone to progesterone. Eur J Endocrinol 1996:134:128–35. ISSN 0840–4643. Ovarian follicles were collected from the perch belonging to the vitellogenic stage and incubated in vitro for 4 h in the absence (control) or presence of triiodothyronine (T3). Addition of T3 (40 ng/ml) to the follicle incubation caused a two-fold increase of [3H] pregnenolone conversion to radiolabelled progesterone (P4) as compared to the control. The increase in P4 formation in the ovarian follicle could be blocked completely by the inhibitors of protein synthesis, actinomycin D and cycloheximide (50 μg/ ml), suggesting a protein or peptide mediator of the T3 stimulatory effect. To search for this mediator, ovarian follicles from the control or T3 incubate were homogenized and ultracentrifuged and different fractions were added separately to fresh follicle incubations. Only the 100 000g supernatant from T3 incubate showed a significant (p < 0.01) increase in P4 formation, while the corresponding supernatant from control follicle incubations had no such stimulatory effect. Trypsin or heat destroyed this augmentory effect. Based on its ability to stimulate the conversion of radiolabelled pregnenolone to P4 in the ovarian follicle, the T3-induced protein (TIP) was purified to homogeneity by employing Sephadex G-75 gel filtration, FPLC Mono-Q and FPLC Superose-6 chromatography. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified TIP showed it to be a 52 K monomer protein. Addition of TIP in increasing concentrations to follicle incubations caused a linear increase in P4 formation. Experiments with radiolabelled TIP ([125 I] TIP) indicate its entry through the follicular cell membrane within the limited period of incubation. Results suggest that TIP activates ovarian 3β-hydroxysteroid dehydrogenase enzyme, thus effecting a greater conversion of pregnenolone to P4. S. Bhattacharya, Department of Zoology, Visva-Bharati University. Santiniketan 731 235, West Bengal, India

scholarly journals Purification and Characterization ofO-Methyltransferase I Involved in Conversion of Demethylsterigmatocystin to Sterigmatocystin and of Dihydrodemethylsterigmatocystin to Dihydrosterigmatocystin during Aflatoxin Biosynthesis

1998 ◽  
Vol 64 (1) ◽  
pp. 166-171 ◽  
Author(s):  
Kimiko Yabe ◽  
Ken-ichiro Matsushima ◽  
Takumi Koyama ◽  
Takashi Hamasaki
Keyword(s):  
Molecular Mass ◽  
Aspergillus Parasiticus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Substrate Inhibition ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Aflatoxin Biosynthesis ◽  
Purification And Characterization ◽  
Cytosol Fraction ◽  
Apparent Homogeneity

ABSTRACT O-Methyltransferase I, which catalyzes conversions both of demethylsterigmatocystin (DMST) to sterigmatocystin (ST) and of dihydrodemethylsterigmatocystin (DHDMST) to dihydrosterigmatocystin (DHST) during aflatoxin biosynthesis, was purified to apparent homogeneity from the cytosol fraction of the mycelia ofAspergillus parasiticus NIAH-26 through the following chromatography series: phenyl-Sepharose, DEAE-Sepharose, phenyl-Sepharose, Sephacryl S-300, and Matrex gel Green A. The apparent molecular mass was estimated at 150 kDa based on Sephacryl S-300 gel filtration chromatography, and the denaturing molecular mass was 43 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI of the enzyme was 4.4, and the optimal pH for activity was broad, from 6.5 to 9.0. In competition experiments using the purified enzyme, the formation of ST from DMST was suppressed when DHDMST was added to the reaction mixture and DHST was newly formed. These results indicate that DMST and DHDMST commonly serve as substrates for the enzyme. TheKm of the enzyme for DMST was 0.94 μM, and that for DHDMST was 2.5 μM. Interestingly, MT-I kinetics deviated substantially from standard Michaelis-Menten kinetics, demonstrating substrate inhibition at a higher substrate concentration.

Inhibition of Urokinase by Complex Formation with Human Antithrombin III in Absence and Presence of Heparin

1978 ◽  
Vol 39 (03) ◽  
pp. 616-563 ◽  
Author(s):  
Inge Clemmensen
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Exchange Chromatography ◽  
Dependent Manner ◽  
Crossed Immunoelectrophoresis

SummaryHuman antithrombin III was purified from fresh human plasma by affinity chromatography on heparin-Sepharose®, affinity chromatography on concanavalin A Sepharose®, gel filtration on Ultrogel® AcA 34, ion exchange chromatography on DEAE A-50 Sephadex® and preparative agarose gel electrophoresis. The hydrolytic activity of urokinase (plasminogen activator from urine) on acetyl-glycyl-L-lysine methyl ester acetate (Ac-gly-lys-OMe Ac) was inhibited by antithrombin III in a slow time-dependent manner. Heparin accelerated the reaction between activator and inhibitor. Inhibition of catalytic activity was associated with the formation of an 1:1 molar complex between activator and inhibitor as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The complex was also demonstrated by crossed Immunoelectrophoresis against anti-antithrombin III.

scholarly journals Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

1981 ◽  
Vol 197 (3) ◽  
pp. 629-636 ◽  
Author(s):  
J L McKenzie ◽  
A K Allen ◽  
J W Fabre
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Human Brain ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Antibody Affinity ◽  
Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

scholarly journals Androgen-sensitive spermine-binding protein of rat ventral prostate. Purification of the protein and characterization of the hormonal effect

1979 ◽  
Vol 184 (2) ◽  
pp. 431-440 ◽  
Author(s):  
G Mezzetti ◽  
R Loor ◽  
S Liao
Keyword(s):  
Binding Protein ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Binding Activity ◽  
Ventral Prostate ◽  
Hormonal Effect ◽  
Rat Ventral Prostate ◽  
Acidic Fraction ◽  
Androgen Effect

The rat ventral prostate contains a cytosol protein that can non-covalently bind spermine much more tightly than spermidine or other natural diamines. The protein has been purified to homogeneity, as judged by electrophoresis in urea- and sodium dodecyl sulphate-containing polyacrylamide gels. The protein, with or without spermine bound to it, sediments at 3 S in a sucrose gradient with or without 0.4 M-KCl. The molecular weight of the protein is about 30 000. Each molecule of the binding protein can bind one molecule of spermine. In the prostate of rats injected with cycloheximide, the protein appears to have a half-life of about 3.5 h. The spermine-binding activity of an acidic fraction obtained by DEAE-cellulose chromatography of the prostate cytosol proteins is reduced by about 40–60% within 20–40 h after castration. This effect is reversed very rapidly within 15–30 min by intraperitoneal injection of 5 alpha-dihydrotestosterone. The hormonal effect is androgen-specific and is not mimicked by dexamethasone or oestradiol-17 beta. The androgen effect was reduced significantly when rats were injected with cycloheximide or actinomycin D, suggesting that the acidic protein may be one of the earliest proteins induced by androgen in the rat ventral prostate.

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