scholarly journals The purification of a 50 kDa protein-actin complex from unfertilized sea-urchin (Strongylocentrotus purpuratus) eggs

1989 ◽  
Vol 257 (3) ◽  
pp. 809-815 ◽  
Author(s):  
R M Golsteyn ◽  
D M Waisman
Keyword(s):  
Sea Urchin ◽  
Binding Protein ◽  
Strongylocentrotus Purpuratus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Binding Activity ◽  
Stable Complex ◽  
Chelex 100 ◽  
Activity Peak ◽  
Sds Page

The 100,000 g supernatant from the unfertilized egg of the sea urchin Strongylocentrotus purpuratus has been fractionated on DEAE-cellulose and analysed for Ca2+-binding activity by the Chelex-100 competitive Ca2+-binding activity assay. The major peak of Ca2+-binding activity was subjected to further purification and the Ca2+-binding protein responsible for this Ca2+-binding-activity peak has been isolated and characterized. Non-denaturing polyacrylamide-gel electrophoresis (PAGE) followed by 45Ca2+ autoradiography suggested a molecular mass of 80 kDa for the Ca2+-binding protein. SDS/PAGE revealed that the 80 kDa protein consisted of a 1:1 molar complex of proteins of 50 and 42 kDa. The 42 kDa protein was identified as actin. The complex was not dissociated by extensive dialysis against an EGTA-containing buffer. The EGTA-stable complex was named ‘50K-A’.

scholarly journals Differential interactions of human kallikrein-binding protein and α1-antitrypsin with human tissue kallikrein

1990 ◽  
Vol 267 (1) ◽  
pp. 79-84 ◽  
Author(s):  
L M Chen ◽  
L Chao ◽  
R K Mayfield ◽  
J Chao
Keyword(s):  
Complex Formation ◽  
Human Serum ◽  
Human Tissue ◽  
Binding Protein ◽  
Binding Activity ◽  
Stable Complex ◽  
Tissue Kallikrein ◽  
Heat Stable ◽  
Sds Page ◽  
Alpha 1 Antitrypsin

The characteristics of a new kallikrein-binding protein in human serum and its activities were studied. Both the kallikrein-binding protein and alpha 1-antitrypsin form 92 kDa SDS-stable and heat-stable complexes with human tissue kallikrein. In non-SDS/PAGE, the mobility of these complexes differ. Complex-formation between kallikrein and the binding protein is inhibited by heparin, whereas that between kallikrein and alpha 1-antitrypsin is heparin-resistant. In normal or alpha 1-antitrypsin-deficient-serum, the amount of 92 kDa SDS-stable complex formed upon addition of kallikrein is not related to serum alpha 1-antitrypsin levels. The rate of complex-formation between kallikrein and the binding protein is 12 times higher than that between kallikrein and alpha 1-antitrypsin. Purified alpha 1-antitrypsin, which exhibits normal elastase binding, has a kallikrein-binding activity less than 5% of that of serum. Binding of tissue kallikrein in serum is not inhibited by increasing elastase concentrations, and elastase binding in serum is not inhibited by excess tissue kallikrein. A specific monoclonal antibody to human alpha 1-antitrypsin does not bind to either 92 kDa endogenous or exogenous kallikrein complexes isolated from human serum. The studies demonstrate a new tissue kallikrein-binding protein, distinct from alpha 1-antitrypsin, is present in human serum.

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.

Identification of the Poly(C) Binding Protein in the Complex Associated With the 3′ Untranslated Region of Erythropoietin Messenger RNA

Blood ◽  
1999 ◽  
Vol 93 (6) ◽  
pp. 2111-2120 ◽  
Author(s):  
Maria F. Czyzyk-Krzeska ◽  
Amy C. Bendixen
Keyword(s):  
Mrna Stability ◽  
Messenger Rna ◽  
Untranslated Region ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mobility Shift ◽  
Protein Factor ◽  
Ribonucleoprotein Complexes ◽  
Sds Page

Hypoxia regulates expression of erythropoietin (EPO), a glycoprotein that stimulates erythrocytosis, at the level of transcription and also possibly at the level of messenger RNA (mRNA) stability. A pyrimidine-rich region within the EPO mRNA 3′ untranslated region was implicated in regulation of EPO mRNA stability element and shown to bind protein factors. In the present study we wished to identify the protein factor binding to the pyrimidine-rich sequence in the EPO mRNA stability element. Using mobility shift assays, ultraviolet light cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electroelution of protein factors from the gel slices corresponding to the ribonucleoprotein complexes, we found that two isoforms of a 40 kD poly(C) binding protein (PCBP, also known as CP or hnRNPE), PCBP1, and PCBP2 are present in that complex. In Hep3B or HepG2 cells hypoxia induces neither expression of PCBP nor formation of the ribonucleoprotein complex associated with EPO mRNA that involves PCBP.

scholarly journals Solubilization and purification of the retinol-binding protein receptor from human placental membranes

1994 ◽  
Vol 302 (1) ◽  
pp. 245-251 ◽  
Author(s):  
A Sivaprasadarao ◽  
M Boudjelal ◽  
J B C Findlay
Keyword(s):  
Binding Protein ◽  
Binding Activity ◽  
Retinol Binding Protein ◽  
Protein Receptor ◽  
Sds Page ◽  
Minor Protein ◽  
Membrane Bound ◽  
Placental Membranes ◽  
Poly Ethylene ◽  
Solubilized Form

The membrane receptor for retinol-binding protein (RBP) has been solubilized from human placental brush-border membranes with octyl-beta-glucoside, Nonidet P-40 and CHAPS. A method, based on the preferential precipitation of 125I-RBP-receptor complex with poly(ethylene glycol) 8000, was developed in order to measure the RBP-binding activity in the detergent extracts. The receptor was fairly stable (4 degrees C, 7 days) in octyl-beta-glucoside and Nonidet P-40, but quickly lost activity in CHAPS. The detergent-solubilized form retained all the properties characteristic of the membrane-bound protein, except for a small decrease in affinity for RBP (3- and 7-fold in Nonidet P-40 and octyl-beta-glucoside respectively). The receptor was isolated using recombinant RBP coupled to Reacti-Gel 6X affinity matrix. The purified material contained major and minor protein species of 63 and 55 kDa respectively on SDS/PAGE.

scholarly journals Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go

1987 ◽  
Vol 246 (2) ◽  
pp. 431-439 ◽  
Author(s):  
G L Waldo ◽  
T Evans ◽  
E D Fraser ◽  
J K Northup ◽  
M W Martin ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Binding Activity ◽  
Bovine Brain ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Gtp Binding ◽  
Guanine Nucleotide Binding ◽  
The Brain

A guanine-nucleotide-binding protein (G-protein) was purified from cholate extracts of bovine brain membranes by sequential DEAE-Sephacel, Ultrogel AcA-34, heptylamine-Sepharose and Sephadex G-150 chromatography. Guanosine 5′-[gamma-[35S]thio]triphosphate (GTP[35S])-binding activity copurified with a 25,000 Da peptide and a 35,000-36,000 Da protein doublet. Neither pertussis toxin nor cholera toxin catalysed the ADP-ribosylation of a protein associated with the GTP[35S]-binding activity. Photoaffinity labelling of the purified protein with 8-azido[gamma-32P]GTP indicated that the GTP-binding site resides on the 25,000 Da protein. The 35,000-36,000 Da protein doublet was electrophoretically indistinguishable from the beta-subunits of other GTP-binding proteins, and the 36,000 Da protein was recognized by antiserum to oligomeric Gt. The purified protein specifically bound 17.2 nmol of GTP[35S]/mg of protein. The Kd of the binding site for radioligand was approx. 15 nM. The brain GTP-binding protein co-migrated during SDS/polyacrylamide-gel electrophoresis with a GTP-binding protein, named Gp, purified from human placenta [Evans, Brown, Fraser & Northup (1986) J. Biol. Chem. 261, 7052-7059], and cross-reacted with antiserum raised against the placental protein, but not with antiserum raised to brain Go. SDS/polyacrylamide-gel electrophoresis of the brain and placental GTP-binding proteins in the presence of Staphylococcus aureus V8 protease yielded identical peptide maps.

Purification and characterization of the extracellular α-amylase activity of the yeast Schwanniomyces alluvius

1987 ◽  
Vol 65 (10) ◽  
pp. 899-908 ◽  
Author(s):  
F. Moranelli ◽  
M. Yaguchi ◽  
G. B. Calleja ◽  
A. Nasim
Keyword(s):  
Amino Acid ◽  
Amylase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Ph Optimum ◽  
Sds Page

The extracellular α-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS–PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0–7.0. Under the conditions tested, the activity is maximal between 45 and 50 °C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.

Distinct regional localization of neurocalcin, a Ca2+-binding protein, in the bovine adrenal gland

1993 ◽  
Vol 138 (2) ◽  
pp. 283-NP ◽  
Author(s):  
A. Nakano ◽  
M. Terasawa ◽  
M. Watanabe ◽  
K. Okazaki ◽  
S. Inoue ◽  
...  
Keyword(s):  
Adrenal Gland ◽  
Dissociation Constant ◽  
Adrenal Medulla ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Physiological Role ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Sds Page

ABSTRACT Neurocalcin (molecular weight 23 000 and 24 000) is a Ca2+-binding protein with three putative Ca2+-binding domains and is present in large amounts in nervous tissues. Neurocalcin isoproteins separated by C18 reverse-phase column chromatography are insoluble in buffer solution and it is impossible to determine the dissociation constant of neurocalcin with Ca2+. To overcome this difficulty, recombinant neurocalcin was synthesized, based on one of the cDNAs of the neurocalcin isoproteins. Stoichiometric titration experiments, using recombinant neurocalcin, indicated that this protein bound 2 mol Ca2+/mol protein and that the apparent dissociation constant for Ca2+ was 2·2 μmol/l, suggesting that neurocalcin plays a physiological role in cellular function. Immunoblotting showed that neurocalcin is present in the bovine adrenal gland in addition to the nervous tissues. Neurocalcin, identified by immunoblotting, was purified from the bovine adrenal gland. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of neurocalcin from the bovine brain showed 23 kDa and 24 kDa double bands, while SDS-PAGE of neurocalcin from the adrenal gland showed a single band of apparently 24 kDa, suggesting that the expression of neurocalcin isoproteins differs from tissue to tissue. The content of neurocalcin in the adrenal gland was 10 μg protein/100 g wet tissue. Immunohistochemical analysis showed the occurrence of neurocalcin in zona glomerulosa and adrenal medulla but not in zona fasciculata or zona reticularis. The restricted localization of neurocalcin in the adrenal gland suggests that a similar Ca2+ signal pathway may be present in zona glomerulosa and the adrenal medulla. Journal of Endocrinology (1993) 138, 283–290

scholarly journals Human C4-binding protein. I. Isolation and characterization

1978 ◽  
Vol 148 (1) ◽  
pp. 207-222 ◽  
Author(s):  
J Scharfstein ◽  
A Ferreira ◽  
I Gigli ◽  
V Nussenzweig
Keyword(s):  
Complement System ◽  
Binding Protein ◽  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electrophoretic Patterns ◽  
Isolation And Characterization ◽  
Sds Page ◽  
Edta Plasma ◽  
The Complement System

C4-binding protein (C4-bp), a new component of the complement system, was isolated from human plasma by precipitation with polyethyleneglycol, followed by chromatography on ion exchangers. C4-bp was identified on sodium dodecyl- sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by two independent criteria: its ability to bind to C4b, and immunoprecipitation with a monospecificantiserum. Purified C4-bp is a 10.7 s glycoprotein. It consists of several disulfide bonded subunits of mol wt 70,000 daltons. Under nonreducing conditions, its mol wt has been estimated on SDS-PAGE as 540- 590,000 daltons. C4-bp moves as a slow B-globulin at pH 8.6 in the absence of free divalent cations, but when the buffers contain Ca(++)-lactate, C4-bp is a gamma globulin. Purified C4-bp binds to purified C4b. The reaction proceeds in the presence or absence of divalent cations and is not inhibited by diisopropylfluorophosphate. The C4b/C4-bp complexes have sedimentation coefficients between 15 and 17 s on sucrose gradient ultracentrifugation, and can be readily identified by crossed immunoelectrophoresis (CIE). The complexes move faster toward the anode than either protein. C4-bp is multivalent. Saturation is reached at molecular ratios of C4b/C4- bp of between 4 and 5. The interaction between C4b and C4-bp may complicate the electrophoretic patterns of these proteins in normal human serum, if the complement system is activated before or during the run. However, in EDTA-plasma, native C4 and C4-bp do not form stable complexes and can be identified in separate peaks after CIE.

scholarly journals Colchicine-binding activity distinguishes sea urchin egg and outer doublet tubulins.

1984 ◽  
Vol 99 (1) ◽  
pp. 37-41 ◽  
Author(s):  
L Wilson ◽  
H P Miller ◽  
T A Pfeffer ◽  
K F Sullivan ◽  
H W Detrich
Keyword(s):  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Binding Activity ◽  
Binding Affinities ◽  
Regulatory Site ◽  
Colchicine Binding Site ◽  
Sea Urchin Egg ◽  
Enthalpy And Entropy Changes ◽  
Enthalpy And Entropy ◽  
Significant Chemical

The colchicine-binding activity of tubulin has been utilized to distinguish the tubulins from two distinct microtubule systems of the same species, the sea urchin Strongylocentrotus purpuratus. We have analyzed the colchicine-binding affinities of highly purified tubulins from the unfertilized eggs and from the flagellar outer doublet microtubules by van't Hoff analysis, and have found significant differences in the free energy, enthalpy, and entropy changes characterizing the binding of colchicine to the two tubulins. The data indicate that significant chemical differences in the tubulins from the two functionally distinct microtubule systems exist, and that the differences are expressed in the native forms of the tubulins. Our findings are discussed in terms of the possibility that the colchicine-binding site may be an important regulatory site on the tubulin molecule.

scholarly journals Purification and characterization of biotin-binding protein II from chicken oocytes

1988 ◽  
Vol 256 (3) ◽  
pp. 797-805 ◽  
Author(s):  
L Bush ◽  
T J McGahan ◽  
H B White
Keyword(s):  
Amino Acid ◽  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Chicken Egg ◽  
Biotin Binding ◽  
Cellulose Column

BBP-II, the major biotin-binding protein from chicken oocytes, was purified 12,000-fold with a 22% yield. The purification procedure includes butan-1-ol extraction of yolk lipids, phosphocellulose chromatography of the water-soluble proteins, DEAE-cellulose chromatography at pH 7.4 and hydroxyapatite column chromatography. Final purification was obtained by using a second DEAE-cellulose column chromatography at pH 6.0. BBP-I activity separated from BBP-II activity during elution from the first DEAE-cellulose column. Purified BBP-II was homogeneous on both polyacrylamide-gel electrophoresis and SDS/polyacrylamide-gel electrophoresis under conditions that would detect a 1% impurity. The subunit Mr determined from SDS/polyacrylamide-gel electrophoresis was 18,200 (72,600 for tetramer), which compares favourably with an Mr value of 17,300 (69,100) calculated from the amino acid analysis. A single precipitin line formed when rabbit antiserum to the protein was directed against a crude chicken egg-yolk sample. BBP-II purified by this procedure lacked carbohydrate and phosphate, was stable indefinitely when frozen, and was quite stable at room temperature. The N-terminal amino acid sequence showed polymorphism at three positions in the first 23 residues and was about 45% identical with the N-terminal 22 residues of avidin. Antiserum to BBP-II cross-reacted with BBP-I and similar proteins in the yolk of eggs from various birds and alligator as judged by immunodiffusion and enzyme-linked immunosorbent assays. No cross-reaction was observed with chicken egg-white by either of these methods.

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