scholarly journals Purification by affinity chromatography and immunological characterization of a 110kDa component of the chick oviduct progesterone receptor

1984 ◽  
Vol 217 (3) ◽  
pp. 685-692 ◽  
Author(s):  
J M Renoir ◽  
J Mester ◽  
T Buchou ◽  
M G Catelli ◽  
P Tuohimaa ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Affinity Chromatography ◽  
Specific Activity ◽  
Sedimentation Coefficient ◽  
Binding Activity ◽  
B Subunit ◽  
Stokes Radius ◽  
A Subunit ◽  
Final Yield

A 110kDa component of the chick oviduct progesterone receptor (PR) has been purified to homogeneity according to electrophoretic criteria and specific activity (assuming one progestagen-binding site/110kDa). The procedure involved affinity chromatography of 0.3 M-KCl-prepared cytosol, followed by DEAE-Sephacel chromatography (elution at 0.2 M-KCl). The final yield was about 12% in terms of binding activity. Properties of the 110kDa component indicate that it is identical with the ‘B’ subunit described previously [Stokes radius approximately 6.1 nm; sedimentation coefficient, (S20, w) approximately 4S; frictional ratio approximately 1.77]. It reacted with the IgG-G3 polyclonal antibody, but not with BF4 monoclonal antibody raised against the 8S molybdate-stabilized chick oviduct PR and reacting with its 90kDa component. Another progesterone-binding component, corresponding to the ‘A’ subunit, also previously described, was eluted from the DEAE-Sephacel column at approximately 0.08 M-KCl, and contained a peptide of molecular mass approx. 75-80kDa, which had S20, w approximately 4S in a sucrose gradient. This component was also recognized by IgG-G3, but not by BF4; it was very unstable in terms of hormone-binding activity.

scholarly journals Progesterone receptor in the chick oviduct: an immunohistochemical study with antibodies to distinct receptor components.

1984 ◽  
Vol 99 (4) ◽  
pp. 1193-1201 ◽  
Author(s):  
J M Gasc ◽  
J M Renoir ◽  
C Radanyi ◽  
I Joab ◽  
P Tuohimaa ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Polyclonal Antibodies ◽  
Smooth Muscles ◽  
Immunoperoxidase Technique ◽  
Cell Nuclei ◽  
Receptor Complexes ◽  
B Subunit ◽  
Chicken Oviduct ◽  
A Subunit ◽  
Immunohistochemical Studies

We performed immunohistochemical studies of chicken oviduct after different fixation procedures, by using antibodies against the progesterone receptor: polyclonal antibodies IgG-G3 against the "8S" form (an oligomere containing progesterone-binding and nonprogesterone-binding units), polyclonal antibodies IgG-RB against the progesterone-binding B subunit, and monoclonal BF4 against the non-progesterone-binding 90,000-mol-wt protein component. Chickens were immature animals with or without estrogen priming, and with or without progesterone treatment. The antibodies were revealed by means of an immunoperoxidase technique that used the avidin-biotin-peroxidase complex, and controls were performed by presaturation of antibodies with the purified 8S-progesterone receptor, the B subunit, and 90,000-mol-wt protein. The progesterone receptor was detected not only in well-characterized target tissues, i.e., in glands and luminal epithelium, but also in stromal cells (some displayed the strongest reaction), in mesothelium, and in fibers of smooth muscles. Only in cell nuclei, whether or not the animals received an injection of progesterone was an antigen revealed corresponding to the B subunit (and/or to the A subunit, because there is immunoreactivity of IgG-RB with both hormone-binding subunits A and B). The 90,000-mol-wt protein was revealed in both cytoplasm and nuclei. These immunohistological data suggest that the concept of steroid action that necessarily involves the original formation of the hormone-receptor complexes in the cytoplasm before translocation to the nucleus, may have to be revised.

Characterization of a nonphotosynthetic NADP-malic enzyme from germinating soybean cotyledons: interaction with a 94-kDa polypeptide identified as a lipoxygenase

2005 ◽  
Vol 83 (5) ◽  
pp. 529-538
Author(s):  
María Lorena Falcone Ferreyra ◽  
María Fabiana Drincovich ◽  
Carlos S Andreo ◽  
Florencio E Podestá
Keyword(s):  
Malic Enzyme ◽  
Specific Activity ◽  
Ionization Time ◽  
Protein Protein Interaction ◽  
Sds Page ◽  
Glycine Max L ◽  
A Subunit ◽  
High Enzymatic Activity ◽  
Germinated Soybean

The major NADP-malic enzyme (NADP-ME) isoform expressed in Glycine max L. cotyledons, leaves, and roots had a subunit molecular mass of 65 kDa and a pI of 6.9. The developmental profile of NADP-ME activity in cotyledons revealed a peak around day 7 post-imbibition (1.53 U·g–1 fresh mass, 33-fold that of day 1), declining markedly thereafter. NADP-ME was partially purified (33-fold) from 7-d germinated soybean cotyledons with a yield of 41% and a final specific activity of 2.8 U·mg–1. NADP-ME had an optimal pH of 7.3 and the Km values for NADP and L-malate were 33 µmol·L–1 and 0.78 mmol·L–1, respectively. The high enzymatic activity found in soybean cotyledons suggests that it plays an important role prior to the establishment of photosynthesis. NADP-ME co-purified with a 94-kDa polypeptide (p94) through various chromatographic steps. Non-denaturing electrophoresis followed by SDS-PAGE and immunoprecipitation assays indicated that NADP-ME is physically associated with p94. Matrix-assisted laser desorption and ionization – time of flight MS of p94 tryptic peptides identified p94 as a lipoxygenase. Although the association was verified by different approaches, the functional role for such interaction is still speculative. Possible hypothesis are discussed.Key words: germinating cotyledon, isozyme, lipoxygenase, NADP-malic enzyme, protein–protein interaction, soybean.

scholarly journals Purification and characterization of the 27,000 Da calcium-binding protein of bovine brain

1987 ◽  
Vol 244 (2) ◽  
pp. 401-408 ◽  
Author(s):  
M Tokuda ◽  
N C Khanna ◽  
D M Waisman
Keyword(s):  
Calcium Binding ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sedimentation Coefficient ◽  
Bovine Brain ◽  
Stokes Radius ◽  
Acidic Nature ◽  
Bovine Pancreas

A Ca2+-binding protein named CAB-27 was purified from bovine brain 100,000 g supernatant. The protein has a molecular mass of 27,000 Da as determined by SDS/polyacrylamide-gel electrophoresis and 35,500 Da by sedimentation-coefficient and Stokes-radius analysis. The protein contains about 26% Glx and Asx and 13% basic residues. The acidic nature of the molecule is confirmed by its pI of 4.80. In the presence of 3 mM-MgCl2 and 150 mM-KCl, CAB-27 binds 2.0 mol of Ca2+/mol of protein, with an apparent Kd of 0.2 microM. Ca2+-binding is unaffected by prior incubation of the protein at 80 degrees C for 2 min. Brain contains about 130 mg of CAB-27/kg. Immunoblotting identified CAB-27 in several bovine tissues; it appears to be particularly rich in brain and kidney. In addition, CAB-27 is identified as an inhibitor of bovine pancreas phospholipase A2 in vitro. The inhibitory activity of CAB-27 was 20-fold less potent than lipocortin. On the basis of the Ca2+-binding properties, intracellular concentration and tissue distribution of this protein, we suggest that CAB-27 may be an important intracellular Ca2+ receptor.

scholarly journals Properties of smooth muscle meta-vinculin.

1987 ◽  
Vol 104 (3) ◽  
pp. 473-482 ◽  
Author(s):  
J D Siliciano ◽  
S W Craig
Keyword(s):  
Smooth Muscle ◽  
Specific Activity ◽  
Sedimentation Coefficient ◽  
High Salt ◽  
Calculated Molecular Weight ◽  
Tissue Homogenates ◽  
Stokes Radius ◽  
Triton X ◽  
Peptide Maps

Quantitative studies show that meta-vinculin is ninefold more soluble in 0.6 M salt than in the 0.01 M salt buffers used to extract vinculin. Based on this finding, we have developed a protocol for the purification of meta-vinculin in 43% yield and 98% purity from a high salt extract of gizzard smooth muscle. In contrast to our earlier extraction studies, which were done on unfixed cryostat sections (30), the present studies done on tissue homogenates show that nonionic detergents are not required for solubilization of meta-vinculin. Furthermore, neither purified nor partially purified meta-vinculin binds to Triton X-114 micelles. Purified meta-vinculin is a monomeric, asymmetric molecule with a Stokes radius of 50.9 A, a sedimentation coefficient of 6.35S, and a frictional ratio of 1.46. The calculated molecular weight of meta-vinculin is 145,000. Meta-vinculin has two isoforms of pI 5.9 and 6.2, and is phosphorylated in vivo to eightfold greater specific activity than vinculin. On immunoblots of smooth muscle proteins, [125I]meta-vinculin binds specifically to talin and also to unidentified polypeptides of 180, 150, 95, 70, 68, and 45 kD. On two-dimensional peptide maps, iodinated vinculin and meta-vinculin have at least 95% of their major chymotryptic peptides in common, but each protein also has at least one highly labeled peptide that appears to be unique. Comparative peptide maps of high salt soluble meta-vinculin and the low salt soluble 152-kD protein (described by Feramisco, J.R., J.E. Smart, K. Burridge, D. Helfman, and G.P. Thomas, 1982, J. Biol. Chem., 257:11024-11031) demonstrate extensive similarities among the vinculin-like proteins but suggest a lack of complete identity. In vivo pulse-chase experiments show that meta-vinculin and vinculin do not have a precursor-product relationship. The biochemical and structural differences found between vinculin and meta-vinculin suggest that there is a unique function for meta-vinculin in smooth muscle.

scholarly journals Production and Characterization of Protective Human Antibodies against Shiga Toxin 1

2002 ◽  
Vol 70 (10) ◽  
pp. 5896-5899 ◽  
Author(s):  
Jean Mukherjee ◽  
Kerry Chios ◽  
Dianne Fishwild ◽  
Deborah Hudson ◽  
Susan O'Donnell ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Immunoglobulin M ◽  
B Subunit ◽  
Significant Prolongation ◽  
Uremic Syndrome ◽  
A Subunit ◽  
Light Chain Immunoglobulin ◽  
Subunit B

ABSTRACT Hemolytic-uremic syndrome (HUS) is a serious complication which is predominantly associated in children with infection by Shiga toxin-producing Escherichia coli (STEC). By using HuMAb-Mouse (Medarex) animals, human monoclonal antibodies (Hu-MAbs) were developed against Shiga toxin 1 (Stx1) for passive immunotherapy of HUS. Ten stable hybridomas comprised of fully human heavy- and light-chain immunoglobulin elements and secreting Stx1-specific Hu-MAbs (seven immunoglobulin M(κ) [IgM(κ)] elements [one specific for the A subunit and six specific for the B subunit] and three IgG1(κ) elements specific for subunit B) were isolated. Two IgM(κ) Hu-MAbs (2D9 and 15G9) and three IgG1(κ) Hu-MAbs (5A4, 10F4, and 15G2), all specific for subunit B, demonstrated marked neutralization of Stx1 in vitro and significant prolongation of survival in a murine model of Stx1 toxicosis.

scholarly journals Pathogenesis of Shigella diarrhea. IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies.

1984 ◽  
Vol 160 (6) ◽  
pp. 1767-1781 ◽  
Author(s):  
A Donohue-Rolfe ◽  
G T Keusch ◽  
C Edson ◽  
D Thorley-Lawson ◽  
M Jacewicz
Keyword(s):  
Cell Surface ◽  
Hela Cell ◽  
Specific Activity ◽  
Polyclonal Antibodies ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
High Yield ◽  
B Subunit ◽  
A Subunit ◽  
High Yields

A simple purification scheme for shigella cytotoxin was devised, resulting in high yields (approximately 50%) and a 1,300-fold increase in specific activity compared with the initial crude bacterial cell lysate. The purified toxin was enterotoxic in ligated rabbit ileal loops and neurotoxic when injected into the peritoneal cavity of mice. Measurement of specific activity of cytotoxin and enterotoxin demonstrated that these two toxicities copurify during the fractionation procedure. On sodium dodecyl sulfate gel electrophoresis, the toxin migrated as two polypeptide subunits, an A subunit of 32,000 mol wt and a B subunit of 6,500 mol wt. Chemical cross-linking experiments demonstrate that the toxin is a complex consisting of one A and five B subunits with a molecular weight of 64,000. Polyclonal rabbit anti-toxin and anti-subunit B antisera were produced as well as subunit-specific mouse monoclonal antibodies. All antibodies preincubated with toxin neutralized cytotoxic effects in HeLa cell monolayers. In contrast, only A subunit-specific antibodies were able to neutralize toxin prebound to the HeLa cell surface. Antibody to the B subunit also inhibited binding of 125I-labeled toxin to these cells by 94% or more. These data demonstrate that the B subunit is involved in shigella toxin binding to the cell surface.

Multiple Molecular Forms of Avian Aldolases. V. Purification and Molecular Properties of Chicken (Gallus domesticus) Liver Aldolase

1971 ◽  
Vol 49 (6) ◽  
pp. 647-657 ◽  
Author(s):  
Ronald R. Marquardt
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Sedimentation Velocity ◽  
Gallus Domesticus ◽  
Molecular Forms ◽  
Velocity Analysis ◽  
Multiple Molecular Forms ◽  
Stokes Radius

Aldolase (fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) was purified from chicken liver. The enzyme was shown to be homogeneous according to the following criteria: purification to a constant specific activity following sequential chromatography on carboxymethyl-Sephadex and Sephadex G-200, electrophoresis on cellulose acetate strips, sedimentation velocity analysis, absence of 10 other glycolytic enzymes, and immunodiffusion in agar.The sedimentation coefficient (s°20w 8.0), Stokes radius (47 Å), diffusion constant (D°20w 4.0 × 10−7 cm2/s), and molecular weight (160 000) are similar to those of rabbit liver aldolase and the muscle and brain enzymes from both chickens and rabbits.

scholarly journals Oestrogen receptor of mammary gland. Inhibition of aggregation and characterization of receptor from lactating gland in the presence of sodium bromide

1978 ◽  
Vol 169 (3) ◽  
pp. 481-488 ◽  
Author(s):  
Ferdinando Auricchio ◽  
Andrea Rotondi ◽  
Ettore Schiavone ◽  
Francesco Bresciani
Keyword(s):  
Oestrogen Receptor ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Complete Disappearance ◽  
Number Of Binding Sites ◽  
Stokes Radius

1. When NaBr, a chaotropic salt, is added, in concentrations ranging from 0.5m to 2m, to low-salt mammary cytosol, (i) age-dependent aggregation of oestrogen receptor is inhibited, (ii) the receptor sediments as a sharp peak at 4.2S on sucrose-gradient centrifugation, with complete disappearance of heavier forms, and (iii) on gel filtration with Sephadex G-200, the receptor is included in the gel matrix. On a calibrated column, the receptor has a Stokes radius of 3.7nm (±6%). 2. Because NaBr inhibits interaction of receptor with other components of cytosol, the values of the sedimentation coefficient, measured by sucrose-gradient sedimentation, and of the Stokes radius, measured by gel filtration, can be accepted with confidence. From these values, it can be computed that the oestrogen-receptor form in NaBr has a mol.wt. of 64000, with a frictional ratio of 1.4. 3. Also, inhibition of aggregation by NaBr allows a 30–90-fold purification of oestrogen receptor. Analysis of this partially purified receptor by sucrose-gradient sedimentation and gel filtration in NaBr gives the same results as for receptor in crude cytosol. On electrofocusing on a pH5–8 gradient, the partially purified oestrogen receptor focuses at pH6.2. On removal of NaBr, receptor aggregates even in this partially purified state. It seems likely that at the protein and ionic concentrations of cytoplasm in vivo, the 64000-mol.wt. receptor form is part of higher states of self- and/or hetero-association with other cytoplasmic components. 4. NaBr up to a concentration of 2m does not inhibit binding of oestrogen by receptor, nor does it decrease the affinity of the interaction (KD≃8.9×10−10m). The total number of binding sites in cytosol, however, decreases by approx. 10%, but this decrease may actually be the result of elimination of lower-affinity binding by non-receptor components of cytosol. 5. NaSCN, another chaotropic salt, was also tested but gave less satisfactory results with the mammary cytosol than with uterine cytosol. EDTA was omitted from the buffers because it favours aggregation of mammary oestrogen receptor. KCl (0.4m), sucrose (15%) and ZnSO4 (3mm) did not prevent aggregation of receptor.

Purine catabolism in man: characterization of placental microsomal 5′-nucleotidase

1976 ◽  
Vol 54 (5) ◽  
pp. 462-469 ◽  
Author(s):  
Irving H. Fox ◽  
Pamela J. Marchant
Keyword(s):  
Specific Activity ◽  
Ph Optimum ◽  
Electrophoretic Variants ◽  
Molecular Weights ◽  
Wide Range ◽  
Stokes Radius ◽  
Michaelis Constants ◽  
Light Form ◽  
Molecular Weight Form

Human placental microsomal 5′-nucleotidase (EC 3.1.3.5) was prepared free of alkaline phosphatase by isoelectric focusing. A total of seven electrophoretic variants were isolated during the preparation of six placentas. Only three to six variants were found in a single placenta. The isoelectric pH's were 6.70, 6.44, 6.23, 6.02, 5.76, 5.63 and 5.44. These were found to be composed of variable quantities of a large, medium and low molecular weight form. The apparent molecular weights of the medium and light form of the enzyme were 86 500 and 43 500, respectively, as estimated from Stokes radius and sedimentation velocity determinations. The electrophoretic variants were not distinguishable with respect to specific activity and Michaelis constants for AMP, GMP or CMP or inhibition by ATP, CTP or adenosine. These electrophoretic variants appeared to be pseudoisozymes based upon different states of aggregation of a common primary sequence.There was a wide range of substrate specificity among nucleoside 5′-monophosphates which included 2-deoxyribose compounds. With AMP as 100, substrate activity was: CMP, 122; NMN, 74; GMP, 68; IMP, 63; XMP, 28 and UDP–glucose, 68. The Michaelis constants for AMP, GMP and CMP ranged from 12–18 μM, from 33–67 μM and from 170–250 μM, respectively. Although 5′-nucleotidase was active in the absence of divalent cation, 5 mM MgCl2 stimulated the enzyme activity to 234% of control and shifted the pH optimum of 9.8 to a plateau from pH 7.4–9.8.

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