Gel filtration and sedimentation behaviour of receptor-gonadotrophin complexes from rat ovary

1981 ◽  
Vol 97 (2) ◽  
pp. 258-269 ◽  
Author(s):  
Kalle Jääskeläinen ◽  
Hannu Rajaniemi ◽  
Leo E. Reichert
Keyword(s):  
Physical Properties ◽  
Granulosa Cells ◽  
Developmental Stages ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Specific Receptor ◽  
Receptor Complexes ◽  
Specific Complex ◽  
Stokes Radius ◽  
Membrane Components

Abstract. Membrane particles of luteinized rat ovaries and immature and mature granulosa cells of rat ovarian follicles were labelled with [125I]human chorionic gonadotrophin (hCG), membranes of mature granulosa cells were also labelled with [125I]ovine follitropin (oFSH). Hormone-receptor complexes were solubilized with Triton X-100 and their physical properties were characterized by sedimentation and gel filtration. No difference in the macroscopic physical properties of the specific receptor-hCG-complex was observed between different developmental stages of granulosa cells. The Stokes radius of the specific receptor-[125I]hCG-complex was 58 Å and the sedimentation coefficient 6.7 ± 0.3 S. The physical properties of free LH/hCG-receptor of luteinized ovaries were also characterized. The Stokes radius was found to be 57–59 Å and the sedimentation coefficient 4.8–5.6 S. Both [125I]hCG and [125I]oFSH formed non-specific complexes with ovarian as well as with non-gonadal membrane components. The sedimentation coefficient of the non-specific complex was 1.3 ± 0.5 S and the Stokes radius 49 Å. These results show that the macroscopic physical properties of the receptor-hCG-complex do not change during the maturation and subsequent luteinization of the granulosa cells. The methods used were not enough discriminating to reveal the existing structural differences between receptor-oFSH-complex and receptor-hCG-complex.

scholarly journals Cytosol oestrogen receptor of lactating mammary gland. Effect of heparin on the aggregation of the receptor and interaction of the receptor with heparin-Sepharose

1978 ◽  
Vol 171 (1) ◽  
pp. 137-141 ◽  
Author(s):  
F Auricchio ◽  
A Rotondi ◽  
P Sampaolo ◽  
E Schiavone
Keyword(s):  
Mammary Gland ◽  
Oestrogen Receptor ◽  
High Speed ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Large Aggregate ◽  
Low Salt ◽  
Lactating Mammary Gland ◽  
Stokes Radius

1. An oestrogen receptor is present in low-salt cytosol of the mammary gland of lactating mice as a large aggregate; it is excluded from gel matrix when filtered on a Sephadex G-200 column and sediments at 7S in sucrose gradients. After incubation of cytosol with heparin, the receptor is dissociated. On a Sephadex G-200 column, it is included in the gel matrix and eluted as a protein with mol.wt. 260000 and a Stokes radius of 6.8nm; it sediments at 6S in sucrose gradients. 2. Dissociation of the mammary-gland cytosol oestrogen receptor seems to be the result of interaction of the oestrogen-receptor complex with heparin. This receptor interacts with heparin covalently bound to Sepharose, thereafter sedimenting at 6S. By using this interaction, the cytosol receptor was purified 200-fold compared with the homogenate, with a yield of 70%. 3. The cytosol receptor that was not incubated or was incubated with heparin was much smaller during sucrose-gradient centrifugation than during gel filtration. This discrepancy can be explained by pressure-induced dissociation during high-speed centrifugation. This possibility is supported by the decrease in the sedimentation coefficient of the receptor with increased duration of centrifugation.

scholarly journals PURIFICATION AND PHYSICAL PROPERTIES OF GROUP C STREPTOCOCCAL PHAGE-ASSOCIATED LYSIN

1971 ◽  
Vol 133 (5) ◽  
pp. 1105-1117 ◽  
Author(s):  
Vincent A. Fischetti ◽  
Emil C. Gotschlich ◽  
Alan W. Bernheimer
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Physical Properties ◽  
Total Protein ◽  
Sedimentation Coefficient ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
Purification Procedure ◽  
Sh Group ◽  
Stokes Radius

A purification procedure for the Group C phage-associated lysin is described utilizing tetrathionate to protect the enzyme's -SH group(s) from thiol-inactivating agents. A 652-fold purification has been accomplished yielding a solution in which the enzyme activity corresponds to essentially a single band on polyacrylamide gel which accounts for 70% of the total protein in the preparation. A molecular weight of 101,000 and frictional ratio of 1.526 was determined for the lysin utilizing experimentally determined values for its Stokes radius and sedimentation coefficient.

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.

scholarly journals Physical measurements of the liver glucocorticoid receptor

1978 ◽  
Vol 169 (2) ◽  
pp. 445-448 ◽  
Author(s):  
G Litwack ◽  
M H Cake ◽  
R Filler ◽  
K Taylor
Keyword(s):  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Axial Ratio ◽  
Steroid Receptor ◽  
Sucrose Density Gradient Centrifugation ◽  
Uniform Size ◽  
Receptor Complexes ◽  
Physical Measurements

Physical measurements were made on the cytosolic form of the liver [3H]dexamethasone receptor. These include a Stokes radius of 3.5 nm, determined by gel filtration, and sedimentation coefficients of 5.1 and 7-8S, by sucrose-density-gradient centrifugation. From these measurements, the following physical properties were calculated: apparent mol. wt. 78000 (the 5.1 S form); D app. 6.1 × 10(-7) cm2-S-1; f/fo 1.25; axial ratio 4.7; these indicate a globular protein. Measurements of sedimentation coefficient of cytosol steroid-receptor complexes previously subjected to various activating conditions gave different values and lead to the conclusion that the mechanism of activation in vitro enabling the steroid-receptor complex to bind to DNA is more complex than simple disaggregation to a uniform size.

scholarly journals Characterization of the molybdate-stabilized glucocorticoid receptor from rat thymus

1982 ◽  
Vol 206 (3) ◽  
pp. 633-640 ◽  
Author(s):  
P J Weatherill ◽  
P A Bell
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Glucocorticoid Receptor ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Apparent Molecular Weight ◽  
Sucrose Density Gradient ◽  
Exchange Chromatography ◽  
Receptor Complex ◽  
Stokes Radius

The untransformed glucocorticoid receptor of rat thymus cytosol was characterized in the form of its complex with [1,2,4-3H]triamcinolone acetonide by ion-exchange chromatography and by gel filtration and sucrose-density-gradient ultracentrifugation at different ionic strengths. Molybdate (10 mM) was present throughout all experimental procedures and prevented receptor inactivation and degradation as well as transformation. At low ionic strength the molybdate-stabilized steroid-receptor complex was detected as a single highly asymmetric entity with a Stokes radius of 5.85 nm, a sedimentation coefficient of 9.6 S and an apparent molecular weight of 236 000. This form was converted into a smaller, even more asymmetric, form in increasing proportion as the ionic strength was increased. In the presence of 0.4 M-KCl, the smaller form had a Stokes radius of 4.95 nm, a sedimentation coefficient of 4.6 S and an apparent molecular weight of 95 500. It is concluded that the glucocorticoid-receptor complex exists at low ionic strengths as a homodimer or as a heterodimer in which only one subunit possesses a steroid-binding site, and that the process of dissociation into subunits brought about by increasing the ionic strength is a process distinct from, but possibly preceding, the transformation phenomenon responsible for conferring DNA-binding properties on the complex.

scholarly journals Hydrodynamic and pharmacological characterization of putative α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive l-glutamate receptors solubilized from pig brain

1994 ◽  
Vol 300 (2) ◽  
pp. 365-371 ◽  
Author(s):  
T Y Wu ◽  
Y C Chang
Keyword(s):  
Binding Sites ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Glutamate Binding ◽  
Stokes Radius ◽  
Triton X

L-[3H]Glutamate binding sites with characteristics resembling that of membrane-bound alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-subtype L-glutamate receptors have been solubilized from pig brain synaptic junctions by Triton X-114. Binding of [3H]AMPA to these soluble sites in the presence of KSCN results in a curvilinear Scatchard plot that can be resolved into a high-affinity component and a low-affinity component. These Triton-X-114-solubilized sites can be further separated into two species of binding sites by gel-filtration chromatography or sucrose-density-gradient centrifugation. The pharmacological profiles of these two species of binding site are almost identical, and the rank orders of potency for glutamatergic drugs in displacing L-[3H]glutamate binding to these sites are quisqualate > 6,7-dinitroquinoxaline-2,3-dione > 6-cyano-7-nitroquinoxaline-2,3-dione > AMPA > L-glutamate > kainate >> N-methyl-D-aspartate = L-2-amino-4-phosphonobutyrate. Both sites are found to bind [3H]AMPA, and in the presence of KSCN the binding activities are significantly enhanced. Analysis of the hydrodynamic behaviour of these binding sites by sucrose-density-gradient centrifugation in H2O- and 2H2O-based solvents and gel-filtration chromatography has revealed that one of these sites (Stokes radius 8.3 nm, sedimentation coefficient 18.5 S) consists of 562 kDa protein and 281 kDa detergent, and the other site (Stokes radius 9.6 nm, sedimentation coefficient 13.4 S) consists of 352 kDa protein and 569 kDa detergent. Frictional coefficients of these sites indicate that these receptor-detergent complexes are asymmetrical in structure, consistent with large transmembrane proteins.

Characterization of isolated bovine preantral follicles based on morphology, diameter and cell number

Zygote ◽  
2020 ◽  
Vol 28 (2) ◽  
pp. 154-159
Author(s):  
Juliana I. Candelaria ◽  
Anna C. Denicol
Keyword(s):  
Granulosa Cells ◽  
Developmental Stages ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Culture Conditions ◽  
Preantral Follicles ◽  
Secondary Stage

SummaryPreantral follicles are a potential reservoir of oocytes to be used in assisted reproductive technologies. With the increasing interest in developing techniques to grow preantral follicles in vitro, and as the bovine emerges as an appropriate model species to understand human folliculogenesis, the establishment of an accurate classification of developmental stages is needed. Classification of bovine preantral follicles has been mostly based on histological analysis and estimation models, which may not translate well to correctly characterize preantral follicles isolated from the ovary. In this study, we classified bovine preantral follicles by morphology upon isolation, determined diameter and number of granulosa cells by direct counting, and compared our results with previous studies reporting bovine preantral follicle classification. Follicles were isolated via homogenization of ovary tissue and classified into primary, early secondary and secondary stage based on morphology and number of layers of granulosa cells. Diameter was individually measured and Hoechst 33342 was used as a nuclear stain to count granulosa cells. We found that follicles classified by morphology into primary, early secondary, and secondary had different mean diameter and cell number (P < 0.01); cell number and diameter were positively correlated, as were cell density and cell number in each developmental stage (P < 0.01). Results obtained here were mostly in agreement with previous classifications based on histological sections and on isolated follicles, with some discrepancies. The present data add accuracy to classification of bovine preantral follicles that is critical to optimize culture conditions to produce developmentally competent oocytes.

Multiple Forms of Phosphoglyceromutase from Chicken Breast Muscle

1972 ◽  
Vol 50 (10) ◽  
pp. 1132-1142 ◽  
Author(s):  
Eric James ◽  
R. O. Hurst ◽  
T. G. Flynn
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Breast Muscle ◽  
Heat Inactivation ◽  
Chicken Breast ◽  
Multiple Forms ◽  
Active Components ◽  
Net Charges

Phosphoglyceromutase (2,3-diphospho-D-glycerate: 2-phospho-D-glycerate phosphotransferase, EC 2.7.5.3) has been purified from both frozen and fresh chicken breast muscle. During purification it was found that substrate, 3-phospho-D-glycerate stabilized the enzyme against heat inactivation to almost the same extent as did the cofactor 2,3-diphospho-D-glycerate.Phosphoglyceromutase prepared from frozen chicken breast muscle separated into three peaks of activity (I, II, and III) following chromatography on DEAE-Sephadex in 0.05 μ phosphate buffer, pH 8.0, using a 0.0–0.4 M NaCl gradient. Each peak of activity was shown by polyacrylamide disc gel electrophoresis at pH 9.3 to contain two enzymically active components (isoenzymes Ia Ib, IIa IIb, and IIIa IIIb). Isoenzymes in the same peak had the same specific activity. Phosphoglyceromutase prepared from fresh chicken breast muscle yielded only one peak of activity following chromatography on DEAE-Sephadex. This peak contained two enzymically active components corresponding to isoenzymes Ia and Ib. Additional peaks of activity were not produced when phosphoglyceromutase from fresh muscle was subjected to freezing and thawing.Isoenzyme Ia and mixtures of Ia and Ib, IIa and IIb, and IIIa and IIIb were homogeneous in the ultra-centrifuge sedimenting as single peaks. The sedimentation coefficient obtained for isoenzyme Ia and for Ia and Ib combined was 4.15 S, the diffusion constant 6.62 × 10−7 cm2/s, and the molecular weight calculated from both gel filtration and sedimentation data was of the order of 59 000. These results were confirmed by charge isomer studies which also showed that the isoenzymes of phosphoglyceromutase from frozen chicken breast muscle were proteins of the same size but different net charges.

Morphology and morphometry of preantral follicles, and immunolocalization of angiogenic factors in ovarian tissue from the neotropical primateSapajus apella

Zygote ◽  
2018 ◽  
Vol 26 (5) ◽  
pp. 424-429
Author(s):  
AB Brito ◽  
DCC Brito ◽  
W B Silva ◽  
APR Rodrigues ◽  
JR Figueiredo ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Developmental Stages ◽  
Ovarian Tissue ◽  
Angiogenic Factors ◽  
Oocyte Nucleus ◽  
Oocyte Diameter ◽  
Preantral Follicles ◽  
Cell Counts ◽  
Secondary Stage ◽  
Morphology And Morphometry

SummaryOvarian biopsies from five health adult monkeys were collected by exploratory laparotomy. Preantral follicles (primordial, primary, and secondary) were classified as normal or degenerated and submitted to morphometric analysis in which granulosa cell counts and the areas of follicles, oocytes, and oocyte nuclei were measured. Ovarian fragments were also immunolabelled for the quantitative analysis of VEGFA and CD31 protein expression in the ovarian tissue and in the preantral follicles. In total, 213 preantral follicles was examined for morphometry and morphological classification. From this total, 20 (9.4%) were follicles enclosing two or more oocytes, i.e. multi-oocyte follicles (MOFs). From the 193 follicles enclosing only one oocyte, 46.3% were classified as primordial, 24,1% as transition, 23.3% as primary, and 6.3% as secondary follicles. The mean number of granulosa cells surrounding primordial, transition, primary, and secondary follicles was 9.2, 12.1, 18.7, and 45.3, respectively. Increase in oocyte diameter was observed from primary to secondary follicles, while the oocyte nucleus increased only when follicles reached the secondary stage. The expression of CD31 was strong in vessels, corpus luteum, and in normal oocytes and granulosa cells from preantral follicles at all developmental stages. Likewise, VEGFA expression was observed in vessels and preantral follicles (granulosa cells, the oocyte and the oocyte nucleus). We characterized the morphology, and morphometry and expression of angiogenic factors in normal and atretic preantral follicles fromSapajus apella. This description can support the analysis of follicular quality and survival after procedures such as transplantation and cryopreservation.

scholarly journals Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose.

1994 ◽  
Vol 127 (1) ◽  
pp. 107-115 ◽  
Author(s):  
L M Machesky ◽  
S J Atkinson ◽  
C Ampe ◽  
J Vandekerckhove ◽  
T D Pollard
Keyword(s):  
Gel Filtration ◽  
Protein Sequences ◽  
Rabbit Muscle ◽  
Muscle Actin ◽  
Novel Proteins ◽  
Cell Extracts ◽  
A Value ◽  
Stokes Radius ◽  
Globular Particle ◽  
Stoichiometric Complex

We identified four polypeptides of 47, 44, 40, and 35 kD that bind to profilin-Sepharose and elute with high salt. When purified by conventional chromatography using an antibody to the 47-kD polypeptide, these four polypeptides copurified as a stoichiometric complex together with three additional polypeptides of 19, 18, and 13 kD that varied in their proportions to the other polypeptides. Partial protein sequences showed that the 47-kD polypeptide is a homologue of S. pombe act2 and the 44-kD polypeptide is a homologue of S. cerevisiae ACT2, both unconventional actins. The 40-kD polypeptide contains a sequence similar to the WD40 motif of the G beta subunit of a trimeric G-protein from Dictyostelium discoideum. From partial sequences, the 35-, 19-, and 18-kD polypeptides appear to be novel proteins. On gel filtration the complex of purified polypeptides cochromatograph with a Stokes' radius of 4.8 nm, a value consistent with a globular particle of 220 kD containing one copy of each polypeptide. Cell extracts also contain components of the complex that do not bind the profilin column. Affinity purified antibodies localize 47- and 18/19-kD polypeptides in the cortex and filopodia of Acanthamoeba. Antibodies to the 47-kD unconventional actin cross-react on immunoblots with polypeptides of similar size in Dictyostelium, rabbit muscle, and conventional preparations of rabbit muscle actin but do not react with actin.

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