From binding proteins to hormone receptors?

Phytohormones exert in responsive plant cells specific biochemical and physiological effects. It is a widely held view that phytohormones are first recognized by specific receptors which initiate the transduction of the hormonal signal. While hormone receptors are well studied in many eukaryotes ranging from yeast to man, we are lacking a detailed understanding of phytohormone receptors. Phytohormone binding proteins have been suspected to provide candidates for such receptors. In this review recent progress towards molecular analysis of such proteins and their genes will be summarized.     

Glutathione-S-transferases are major cytosolic thyroid hormone binding proteins

Thyroid hormone binding proteins of rat liver cytosol were characterized. Glutathione-S-transferases were identified among major cytosolic proteins adsorbed by thyroxine affinity matrices. The Ya and Yb subunits of the glutathione-S-transferases were also principal proteins of cytosol covalently labeled with 3,3',5-triiodo-L-thyronine (T3) or 3,3',5,5'-tetraiodo-L-thyronine (T4) by photoaffinity methods. T3 and T4, but not L-thyronine or iodinated tyrosines, were bound with high affinity to purified glutathione-S-transferases and were potent inhibitors of their enzymatic activities. These results suggest that glutathione-S-transferases have the potential to function in the intracellular binding and transport of thyroid hormones. The proteins provide a means for regulating the action and metabolism of thyroid hormones by acting as high capacity binding components.     

Selective actions of growth hormone on rat liver estrogen binding proteins

Levels of hepatic estrogen receptor were 9.0 ± 2.4 fmoles/mg cytosol protein in intact females compared to 3.4 ± 2.2 in hypophysectomized females. Likewise, levels of receptor were 9.8 ± 1.5 fmoles/mg cytosol protein in intact males and 2.7 ± 1.8 in hypophysectomized males. Hypophysectomy abolished the sex differences in a second class of binding sites termed higher capacity lower affinity binding sites by increasing female levels and decreasing male levels. Treatment of hypophysectomized male or female rats with growth hormone (2 units/kg body wt, two times daily) restored normal levels of hepatic estrogen receptor. Administration of growth hormone to hypophysectomized rats did not reverse the effects of hypophysectomy on higher capacity lower affinity binding sites. These studies demonstrate that growth hormone exerts selective actions on different forms of hepatic estrogen binding proteins.     

Thyroid hormone binding to the rat heart cytosol proteins

The properties of the thyroid hormone binding to rat heart cytosol were studied. Cytosol proteins were found to bind specifically T4 with high affinity (Ka approximately equal to 10(8)M-1) and rT3 with lower affinity (Ka approximately equal to 10(7)M-1), but they do not bind T3. The binding of both T4 and rT3 was pH dependent, however, while T4 binding had the highest values between pH 7.0 and 10, rT3 binding increased from pH 6.0 to 10.7. Divalent ions also stimulated T4 and rT3 binding. Sulfhydryl groups blocking agents such as N-ethylmaleimide (NEM) and iodoacetamide significantly decreased rT3 binding and had less profound effect on binding of T4 to cytosol proteins. The importance of free -SH groups remains unclear as dithiothreitol was found to diminish the binding of T4 and rT3.     

Comparison of binding of thyroid hormone analogues to hepatic organic anion binding proteins

Dv protein and ligandin are two hepatic cytosolic proteins which bind organic anions, including endogenous thyroid hormones. Binding studies were performed using the ANS displacement technique to compare the binding of a variety of thyroid hormone analogues to purified organic anion binder and ligandin. Inhibition of ANS binding by these compounds was competitive. Both proteins bound L- and D-thyroxine with comparable affinity (Kd 30-45 microM), whereas ligandin bound 3',3',5-triiodo-L-thyronine, 3',3',5-triiodo-L-thyronine and most analogues with greater affinity. Nevertheless, the order of ligand affinities for both binders was highly correlated, suggesting that the nature of the binding site on both proteins is similar. The binding affinities of these organic anion binders are 2-3 orders of magnitude lower than an hepatic cytosolic thyroid binder reported by others, suggesting that ligandin and organic anion binder may not be important in intracellular thyroid hormone transfer.     

Characterization of feline conceptus proteins during pregnancy

This study characterized proteins secreted de novo by feline conceptuses collected on Days 10, 12, and 15 (n = 22, preimplantation blastocysts); Days 15, 16, 17, 19, 21, and 25 (n = 6, postimplantation zonary girdle [ZG] i.e. trophoblast and endometrium); and Days 30, 36, 39, and 50 (n = 5, postimplantation ZG and free chorioallantois [CA]) and cultured in Minimal Essential Medium. De novo secretion was shown by incorporation of 3H-leucine into proteins detected in culture media by 2D-PAGE and fluorography. Western blotting, and NH2-terminal amino acid microsequencing. Major radiolabeled proteins identified as they appeared temporally on fluorographs were as follows: feline conceptus protein 1 (fCP1), Mr = 20,000, pI 5.0-5.3; fCP2, Mr = 80,000, pI 6.5-7.2; fCP3a, Mr = 67,000, pI 6.3-6.5; fCP3b, Mr = 67,000, pI 5.9-6.3; fCP4, Mr = 56,000, pI 5.0-6.0; and fCP5, Mr = 29,000, pI 5.0-5.8. The fCP1 was produced by blastocysts on Days 10-15, ZG on Days 16-25, and CA on Day 30; on Days 39-50, CA synthesized 5 proteins, possibly fCP1 isomers. The fCP2, fCP3a and b, and fCP4 were produced by blastocysts on Day 15, ZG on Day 25, and CA on Days 30-50. The fCP5 was made by ZG on Days 16-36 and by CA on Days 30-39. Western blotting identified fCP1 as retinol-binding protein (RBP), fCP2 as alpha fetoprotein, fCP3a as albumin, and fCP3b as transferrin. Amino acid sequence homologies between fCP1 and rabbit and human plasma RBP and porcine conceptus RBP2 were 93, 96, and 100%, respectively, at the first 37 NH2-terminal amino acids. The identities of fCP4 and fCP5 have not been established. Antiviral activity detected in all media was less than 3 units/ml when tested with feline fibroblast cells infected with vesicular stomatitis virus.     

Plasma luteinizing hormone during pregnancy in the pig

Three chronically catheterized Duroc gilts were used to characterize the pattern of plasma LH in the systemic circulation during pregnancy. Blood samples were collected four times daily (08.00, 12.00, 16.00 and 20.00 h) from the second day of estrus until day 7 postpartum in one pig and to 108 and 98 days of gestation in the remaining two. The concentration of plasma LH fluctuated in a pulsatile manner throughout the studied periods of gestation in all three pigs, with decreasing amplitude towards parturition. Significant correlations between the decline of LH levels and the day of pregnancy were found, and the equations for the linear regression lines are presented. It is suggested that the level of LH in early and mid-pregnancy mimics LH concentrations in the midluteal phase of the estrous cycle.     

Urinary eCG patterns in the mare during pregnancy

Blood and urine samples collected from 12 mares at frequent intervals from 25 to 210 d of pregnancy were analyzed for equine chorionic gonadotropin (eCG). Blood and urine samples were collected daily through two consecutive ovulatory periods from five cyclic mares for comparative purposes. Separate radioimmunoassays (RIA) were developed to detect eCG in the urine and plasma. A simple and quick commercial dipstick enzyme-linked immunospecific assay (ELISA), developed for eCG in the blood, was also utilized in this study to detect eCG in the urine. In the 12 pregnant mares, eCG concentrations in both the plasma and urine as detected by RIA rose significantly on Day 40, peaked by Day 60 and slowly dropped to low levels by Day 200. The dipstick ELISA appeared more reliable for eCG in the plasma than in the urine of the five pregnant mares tested. However, on peak days (50 to 60), both the plasma and urine tested positive in all five mares. Similar eCG profiles were observed when urine samples from seven of the mares were assayed in the dipstick ELISA and RIA. The highest percentage of mares (86%) were positive for eCG by ELISA between Days 65 and 85. The highest concentration of eCG in the urine as detected by RIA was observed between Days 55 and 90. ECG-like immunoactivity was not detected by the ELISA in the urine of cyclic mares, but the RIA showed variable patterns with increases in immunoactivity that could not be correlated with physiological events. In summary, eCG in urine follows a similar profile as the eCG in plasma of mares during their first trimester of pregnancy.     

Electrophoretic patterns and thermostability of some proteins from heat-hardened wheat

Heat hardening of wheat (Triticum aesticum L.) resulted in increased heat stability in leaf extracts of fraction I protein, malate dehydrogenase and peroxidase. Polyacrylamide disk gel electrophoretic atterns of fraction 1 protein, malate dehydrogenase and peroxidase did not change after the heat hardening. The individual isozymes of malate dehydrogenase and peroxidase were found to possess the different heat stabilities. After heat hardening only some of the isozymes had an increased heat stability.     

Ethnic differences in adrenocorticotropic hormone, cortisol and corticotropin-releasing hormone during pregnancy

Significant ethnic disparities exist in reproductive outcomes. A potential contributing factor may be the functioning of the hypothalamic-pituitary-adrenal (HPA) axis and placenta during pregnancy. In the present study, levels of cortisol, ACTH and CRH were determined longitudinally from the plasma of 310 African American, Hispanic and non-Hispanic White women at 18-20, 24-26 and 30-32 weeks' gestation. During pregnancy, African American women exhibited lower levels of cortisol than non-Hispanic women and higher levels of ACTH than Hispanic women. The trajectory of CRH increase also differed by ethnicity, with African Americans exhibiting the lowest levels both early and late in pregnancy. Higher levels of cortisol at 18-20 weeks were associated with higher levels of CRH at 30-32 weeks among the African American and Hispanic women, but not among non-Hispanic women. Ethnic differences persisted when adjusting statistically for sociodemographic and biomedical factors. The findings are consistent with the possibility that ethnic disparities in adverse birth outcomes may be due, in part, to differences in HPA axis and placental function.     

Polyanions inhibit the binding of autoantibodies with some cellular proteins

Effect of polyanions on reaction of the Ig from sera of patients with systemic lupus erythematosus and scleroderma with cellular proteins was studied by immunoblotting. It has been shown that dextran sulfate, heparin, denatured DNA and poly I inhibited the binding of autoantibodies with some polypeptides. The molecular weight of these antigens was determined. The molecular mechanism of immunological reactions studied and it's role in pathogenesis of autoimmune diseases is discussed.     

Maternal corticotropin-releasing hormone levels during pregnancy and offspring adiposity

Objective: Animal models suggest that fetal exposure to glucocorticoids can program adiposity, especially central adiposity, later in life. We examined associations of maternal corticotropin‐releasing hormone (CRH) levels in the late 2nd trimester of pregnancy, a marker of fetal glucocorticoid exposure, with child adiposity at age 3 years. Research Methods and Procedures: We analyzed data from 199 participants in Project Viva, a prospective cohort study of pregnant women and their children, At age 3 years, the main outcomes were age‐sex‐specific BMI z score and the sum of subscapular (SS) and triceps (TR) skinfold thicknesses to represent overall adiposity, and ratio of SS to TR (SS:TR) to represent central adiposity. Results: Mean (standard deviation) maternal 2nd trimester log CRH was 4.94 (0.56) pg/mL. At age 3, mean (standard deviation) for BMI z score was 0.52 (1.02); for SS + TR, 16.51 (3.94) mm; and for SS:TR, 0.67 (0.17). Log CRH was mildly inversely correlated with birth weight (r = ?0.08), chiefly because of its association with length of gestation (r = ?0.21) rather than fetal growth (r = ?0.004). After adjustment for sociodemographic factors, maternal smoking, BMI, and gestational weight gain, fetal growth, length of gestation, breastfeeding duration, and (for SS:TR only) child's 3‐year BMI, each increment of 1 unit of log CRH was associated with a reduction in BMI z score [?0.43; 95% confidence interval (CI), ?0.73, ?0.14; p = 0.004] and possible reduction in SS + TR (?1.10; 95% CI, ?2.33, 0.14; p = 0.08). In contrast, log CRH was associated with higher SS:TR (0.07; 95% CI, 0.02, 0.13; p = 0.007). Discussion: Fetal exposure to glucocorticoids, although associated with an overall decrease in body size, may cause an increase in central adiposity.     

Homologies and anomalies in primary structural patterns of nucleotide binding proteins

In searches for homology among nucleotide binding proteins, recent reports have described primary structure alignments for stretches of 30 or so amino acid residues among a variety of proteins including the ras and src oncogene products. The significance of these sequence matches has been tested by searching in available data banks for certain conserved residue patterns resulting from the alignments. The tests suggest that alignments over these limited stretches are not necessarily justifiable and any implications for residues involved in nucleotide binding must be viewed with caution.     

Insulin-like growth factors and their binding proteins define specific phases of myometrial differentiation during pregnancy in the rat

While the insulin-like growth factor (IGF) system is known to regulate uterine function during the estrous cycle, there are limited data on its role in myometrial growth and development during pregnancy. To address this issue, we defined the expression of the Igf hormones (1 and 2), their binding proteins (Igfbp 1-6), and Igf1r receptor genes in pregnant, laboring, and postpartum rat myometrium by real-time PCR. IGF family genes were differentially expressed throughout gestation. Igf1 and Igfbp1 mRNA levels were upregulated during proliferative phase (Days 6-12) of rat gestation. Igfbp3 gene expression also was elevated in proliferating smooth muscle cells (SMCs) and was highest at the time of transition between proliferative and synthetic phases (Days 12-15). Igfbp6 gene expression profile paralleled plasma progesterone (P4) concentrations, peaking during the synthetic phase (Days 17-19) and decreasing thereafter. Administration of P4 at late pregnancy (starting from Day 20) to maintain elevated plasma P4 concentrations blocked the onset of labor and prevented the fall in Igfbp6 mRNA levels. In contrast, the treatment of pregnant rats with the P4 receptor antagonist RU486 on Day 19 induced preterm labor and the premature decrease of Igfbp6 gene expression. Igfbp2 gene expression was transiently upregulated during the contractile phase of gestation (Days 21-23) solely in the gravid horn of unilaterally pregnant rats, but it was not affected in P4- or RU486-treated animals, supporting a role for mechanical stretch imposed by the growing fetuses. Igfbp5 gene was induced during postpartum involution. Our results suggest the importance of the IGF system in phenotypic and functional changes of myometrial SMCs throughout gestation in preparation for labor.     

Protein patterns during final differentiation of some rat organs

Development Genes and Evolution -