Characterization of the corticosteroid-binding globulin messenger ribonucleic acid response in the pregnant hamster.

In this study we measured corticosteroid-binding globulin (CBG) mRNA levels in liver and various nonhepatic tissues of pregnant and nonpregnant hamsters. The N-terminal amino acid sequence (37 residues) of hamster CBG was determined and compared with published cDNA-deduced sequence information for rat and human CBG. Hamster CBG showed considerable sequence homology with both rat (70%) and human (59%) CBG. Because of the high level of homology, we were able to use a cRNA prepared from a rat CBG cDNA as a probe in Northern blot and solution hybridization analyses. Northern blots of hamster and rat liver RNA extracts revealed that the rat CBG cDNA probe hybridized to RNAs that were the same size in rats and hamsters. Further, the Northern blot showed that pregnant hamster liver contained substantially more CBG mRNA than nonpregnant hamster liver. The relative amounts of CBG mRNA in pregnant and nonpregnant hamster livers were compared using a solution hybridization assay. Slope-ratio analysis of the hybridization data revealed that pregnant hamster liver (day 14) contained 40-fold more CBG mRNA than nonpregnant hamster liver. When other tissues (kidney, spleen, small intestine, and decidual tissue) were assayed for CBG mRNA, a small amount of hybridization was detected by solution hybridization. However, Northern blot analysis of RNA extracts from nonhepatic tissues showed that the hybridizable sequences did not migrate at the same position as mature CBG mRNA. These results indicate that the observed increase in serum CBG during hamster pregnancy is largely attributable to an increase in hepatic CBG mRNA.

Species crossreactivity of human progesterone receptor monoclonal antibodies: Western blot analysis.

The crossreactivity of monoclonal antibodies (hPRa 1, 2, 3 and 6) generated against human progesterone receptor was examined in six mammalian and an avian species using the techniques of sodium-dodecylsulfate polyacrylamide gel electrophoresis and Western blot analysis. Immunoreactive bands were detected on protein blots of receptor-containing preparations from human endometrial carcinoma grown in nude mice, human T47D breast cancer cells, rabbit, cow and mouse uteri, and chick oviduct. No receptor-associated, immunoreactive bands were detected in rat, guinea pig or hamster uteri. The number and molecular weights of the receptor subunits detected varied between species, and only human progesterone receptor displayed electrophoretic microheterogeneity in its high molecular weight subunit. These data demonstrate that the human progesterone receptor antibodies recognize epitopes not common to all species.

RU486 is not an antiprogestin in the hamster.

The biological activity and progestin receptor binding activity of the synthetic steroid RU486 (RU38486; 17-beta-hydroxy-11-beta-(4-dimethylaminophenyl)-17-alpha-(1-propynl++ +)- estra-4,9-diene-3-one) were investigated in the hamster. RU486 demonstrated no antiprogestational activity in the female hamster in that it was ineffective in blocking decidualization or interrupting early pregnancy. Competitive binding assays showed RU486 did not compete from hamster uterine progestin receptor. It is concluded that hamster uterine progestin receptor has unique steroid binding specificity.

Purification and partial characterization of a corticosteroid-binding globulin from hamster serum.

Our objective was to characterize and purify the corticosteroid-binding proteins in hamster pregnancy serum. When [3H]cortisol-labeled pregnancy and proestrous serum were subjected to native polyacrylamide gel electrophoresis, a single peak of specific steroid-binding activity was detected in each, with identical electrophoretic mobility. The steroid-binding affinity (Ka = 1.07.10(8) M-1 for cortisol) is typical of corticosteroid-binding globulin from other species, but the steroid-binding specificity (cortisol greater than testosterone greater than progesterone) is not. An ultraviolet photoaffinity-labeling protocol was developed using 17 beta-hydroxy-4,6-[1,2-3H]androstadiene-3-one ([3H]androstadienolone), permitting analysis of ultraviolet photoaffinity-labeled proestrous and pregnancy serum by two-dimensional polyacrylamide gel electrophoresis and fluorography. Both sera contained the same labeled protein species. Corticosteroid-binding globulin was purified from pregnancy serum by DEAE-cellulose chromatography followed by steroid affinity chromatography on androstadienolone-17 beta-hemisuccinate-ethylenediamine-AffiGel 10. The purified protein (Mr = 62,250; pI = 3.95; n = 1; Stokes radius = 3.5; S = 4-5) was determined to be a glycoprotein. When analyzed by gel filtration and two-dimensional polyacrylamide gel electrophoresis, purified corticosteroid-binding globulin behaved the same as in unfractionated serum, and when ultraviolet photoaffinity-labeled with [3H]androstadienolone, purified corticosteroid-binding globulin produced the same fluorogram spot pattern seen in unfractionated serum. A specific corticosteroid-binding globulin antiserum was raised in rabbits, and this antiserum reacted with a single spot in Western blots of unfractionated serum. Thus, hamster pregnancy serum was determined to have one corticosteroid-binding protein. This protein is identical to the corticosteroid-binding globulin found in proestrous serum, with a higher titer in pregnancy serum. No other steroid-binding component is observed in hamster serum.

Decidual cell function: evidence for a role in the regulation of serum CBG and a 60 kDa protein during early pregnancy in the hamster.

Several serum proteins increase in titer during pregnancy. We tested the hypothesis that decidual cells may signal the production of certain serum proteins in the hamster. Measurement of serum CBG by equilibrium binding using either [3H]-progesterone or [3H]-cortisol in conjection with ion exchange chromatography showed that decidualization increased CBG levels. Two-dimensional gel electrophoresis revealed that a 60 kDa++ protein increases markedly in the serum of the hormonally pseudopregnant (PSP) animal soon after artificial induction of decidualization on PSP day 4. The 60 kDa serum protein remains low in the nondecidualized PSP animal, but it increases in the pregnant animal. A photoaffinity labeling procedure was used to covalently bind [3H]-androstadienolone to CBG. Fluorography of 2D gels run under denaturing conditions established that the 60 kDa protein did not bind steroid as did CBG (69 kDa). To determine whether decidual cells could induce the 60 kDa and CBG proteins, different numbers of decidual cells were injected IP into PSP recipients. A single injection of 50 x 10(6) decidual cells induced both serum proteins within 48h, whereas the same number of hamster fetal cells was ineffective. Thus, these results demonstrate that hamster decidual cells induce a 60 kDa protein of unknown function and serum CBG. Since the decidual cell itself does not appear to be the source of either protein, it follows that the decidual cell signals the synthesis and secretion of these proteins elsewhere in the body, most likely in the liver. To our knowledge, this is the first demonstration that the decidual cell regulates serum CBG and other proteins in this manner.

Partial purification and characterization of two soluble c-type cytochromes from Chromatium vinosum.

Two c-type cytochromes from Chromatium vinosum have been partially purified and characterized. Cytochrome c550, which appears to function as an electron carrier in the cyclic electron transport chain of this photosynthetic purple sulfur bacterium, has a molecular weight of approximately 15,000 and an oxidation-reduction midpoint potential (Em) of +240 mV at pH 7.4. It has (in the reduced form) an alpha band at 550 nm and a beta band at 520 nm. Cytochrome c551 is characterized by absorbance maxima at 354 and 409 nm in the oxidized form and 418, 523, and 551 nm in the reduced form. The reduced cytochrome reacts with CO. Cytochrome c551 is a monomeric protein with a molecular weight of 18,800 +/- 700 and Em = -299 +/- 5 mV (pH independent between pH 6.3 and 8.0). It appears to lack a methionine axial ligand as indicated by the absence of an absorbance band at 695 nm in the oxidized form.

Reactivity of cuprous stellacyanin as a quinone and semiquinone reductase.

The reactivity of cuprous stellacyanin as a quinone and semiquinone reductase has been examined. Rate constants (25.0 degrees C) measured for the oxidation of stellacyanin by 1,4-benzoquinone and benzosemiquinone are 2.3 X 10(4) M-1 s-1 (delta H not equal to = 4.4 kcal/mol, delta S not equal to = -24 eu) and 5.1 X 10(6) M-1 s-1, respectively [pH 7.0, I = 0.1 M (phosphate)]. The agreement of these rate constants with those calculated on the basis of relative Marcus theory is discussed. Stellacyanin is more effective than laccase in quenching benzosemiquinone, suggesting that the physiological role of this metalloprotein is to regulate the concentration of free radicals generated through the laccase-catalyzed oxidation of phenols.