Search PubMed⌕ Search

Biomedical subjects

B J Danzo

Publications and source records attributed to B J Danzo.

At least 55 records · Page 3Linked to original sources
PubMed1982

Some physicochemical characteristics of photoaffinity-labeled rabbit androgen-binding protein.

Photoaffinity-labeled androgen-binding protein (ABP), present in cytosol prepared from the caput epididymidis of intact sexually mature rabbits, was produced using 17 beta-hydroxy-[1,2-3H]4,6-androstadien-3-one (delta 6-testosterone). Photo-labeled ABP could not be distinguished from unlabeled ABP by gel filtration chromatography, electrophoresis on nondenaturing polyacrylamide gels, or sucrose gradient ultracentrifugation. Rabbit ABP was found to have a sedimentation coefficient of 4.6S and a Stokes radium of approximately 45A. Based on these parameters, its native molecular weight was calculated to be approximately 86,300. A model of ABP as a prolate ellipsoid having an axial ratio of 10 is consistent with its frictional ratio of 1.555. When photolabeled rabbit ABP was examined on polyacrylamide gels containing sodium dodecyl sulfate, two androgen-specific peaks of approximately 52,000 and approximately 48,000 daltons were detected. Both peaks contained approximately the same amount of radioactivity. When photolabeled ABP was treated with the cross-linking reagent disuccinimidyl suberate before electrophoresis on gels containing sodium dodecyl sulfate, an additional androgen-specific peak corresponding to approximately 100,000 daltons was obtained. We interpret this peak to represent dimers of the lower molecular weight species. The 52,000- and 48,000-dalton subunits were observed regardless of whether the proteins were treated with 2-mercaptoethanol, indicating that the monomers are not linked by disulfide bonds.

Affinity Labels↗
PubMed1982

Some physicochemical characteristics of photoaffinity-labeled rabbit testosterone-binding globulin.

Photolabeled testosterone-binding globulin (TeBG), obtained from the plasma of sexually immature male rabbits, was produced using 17 beta-hydroxy-[1,2-3H]4,6-androstadien-3-one. Photolabeled TeBG could not be distinguished from unlabeled TeBG by gel filtration chromatography, electrophoresis on nondenaturing gels, or sucrose gradient ultracentrifugation. Rabbit TeBG had a Stokes radius of approximately 45 A and a sedimentation coefficient of approximately 4.6S. Based on these parameters, its native molecular weight was calculated to be approximately 78,000. The frictional ratio of rabbit TeBG was found to be approximately 1.6. When photolabeled TeBG was examined on polyacrylamide gels containing sodium dodecyl sulfate, a single androgen-specific peak of approximately 40,000 daltons was obtained. When photolabeled TeBG was treated with the cross-linking reagent disuccinimidyl suberate before electrophoresis on sodium dodecyl sulfate gels, an additional androgen-specific peak of radioactivity corresponding to approximately 100,000 daltons was obtained. We interpret this peak to represent oligomers of the TeBG subunits. The 40,000-dalton subunit was obtained regardless of whether the proteins were treated with 2-mercaptoethanol, indicating that the monomers are not linked by disulfide bonds.

Affinity Labels↗
PubMed1980

Photoaffinity labeling of rat androgen binding protein.

The photoinactivation and photoaffinity labeling of androgen binding sites present in cytosol prepared from intact sexually mature rat epididymides have been demonstrated by using unlabeled and [3H]-labeled 17 beta-hydroxy-4,6-androstadien-3-one. Both photoinactivation and photolabeling are dependent upon exposure to light. These processes are inhibited when photolysis is conducted in the presence of the photoinert compound 17 beta-hydroxy-5 alpha-androstan-3-one, suggesting that steroid-specific sites are involved in the reactions. The labeled steroid-specific product of photolysis is macromolecular with a molecular weight of 47,000 as determined by electrophoresis on polyacrylamide gels containing NaDodSO4, and proteinaceous because digestion of the cytosol with pronase before photolysis eliminates steroid-specific binding. After photolysis, the protein-steroid complex has the ability to withstand dissociation during electrophoresis under denaturing conditions, and unlabeled 17 beta-hydroxy-5 alpha-androstan-3-one fails to displace the label from the complex. Thus, the binding of [3H]17 beta-hydroxy-4,6-androstadien-3-one to the cytosolic protein is covalent. This steroid-specific product is identified as an androgen binding protein of testicular origin by its comigration with native androgen binding protein on nondenaturing polyacrylamide gels and by its molecular weight which is within the range reported for androgen binding protein subunits.

Affinity Labels↗
PubMed1979

The presence of a cytoplasmic estrogen receptor in sexually mature rabbit epididymides: comparison with the estrogen receptor in immature rabbit epididymal cytosol.

Cytosol prepared from epididymides of castrated adult rabbits contains an estrogen-specific binding component that sediments in the 4--5S region on 0.01 M KCl sucrose gradients. The estrogen-specific binding component is not detectable on sucrose gradients unless the cytosol-[3H]estradiol mixture is first extracted with charcoal to remove uncomplexed and rapidly exchangeable ligand from the sample. The macromolecular bound [3H]estradiol that remains after charcoal extraction dissociates from the receptor with a dissociation half-time of greater than 24 h and is of high affinity (Kd = 6.5 +/- 0.9 X 10(-9) M). The estrogen receptor in the adult rabbit epididymis can also be demonstrated when charcoal-extracted samples are chromatographed on Sephadex G-200. Using this system, the major estrogen-specific binder has an apparent molecular weight of less than 200,000. In contrast, the epididymal estrogen receptor in sexually immature rabbits sediments as an 8S species on low salt gradients and has an apparent molecular weight of 200,000 or greater when assessed by chromatography on Sephadex G-200. The estrogen receptor in both age groups is distributed along the length of the epididymal duct. The highest concentration of the cytoplasmic receptor in both mature and immature rabbits is in the cauda epididymidis. The age-dependent changes in the amount of the receptor suggest that it may be involved in maturational changes in the epididymis. The alterations in the physicochemical properties may reflect physiological changes in the epididymis or in the endocrine status of the animal.

Aging↗
PubMed1977

High affinity estrogen binding by rabbit liver.

Macromolecular binding components for [3H]estradiol-17beta are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [3H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4--5 S and the other had a sedimentation coefficient of 8--9 S. The two components differed from each other regarding steroid specificity and various physiocochemical parameters. [3H]estradiol binding to the 4--5 S component was not inhibited by estrogens, 5alpha-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appear to be saturable and label was rapidly stripped from it by charcoal. Estradiol binding to the 8--9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4--5 S moiety. The specific binding protein has a Kd of 3.05 . 10(-10) M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incubation of [3H]estradiol with mature male liver cytosol at 0--5 degrees C polar metabolites of estradiol are produced.

Animals↗