Differences in the binding of thyroid hormones and indoles by rat alpha 1-fetoprotein and serum albumin

The transport of small molecules in the blood, normally assured by serum albumin in the adult, is not well known in the fetus since the albumin concentration is low in fetal serum and inversely related to the alpha 1-fetoprotein concentration. In order to investigate whether rat alpha 1-fetoprotein might be a fetal counterpart to albumin, the binding properties of these two proteins have been compared with respect to a series of molecules of biological importance, especially during fetal development: thyroid hormones and indole analogues. Though high-affinity binding of thyroxine was found with both rat alpha 1-fetoprotein and albumin, a significant difference in the number of binding sites for this hormone was found with the two proteins. Further, while rat serum albumin strongly bound L-tryptophan and indolyl-3-acetic acid (Ka approximately equal to 10(5) M-1), rat alpha 1-fetoprotein did not bind any of the indoles tested. These results are discussed with respect to the physiological and pharmacological significance of the transport role of these proteins.     

Fluorescence studies of the interactions of serum albumin and rat alpha1-fetoprotein with aflatoxin B1. Specificity and binding parameters.

The interaction of bovine serum albumin and rat alpha1-fetoprotein with aflatoxin B1 has been followed by the fluorescence quenching of the protein in presence of the ligand. The binding parameters (n, number of sites and Kd, dissociation constant) have been determined for the bovine serum albumin-alflatoxin B1 system: n = 3.5 and Kd = 3.1 +/- 0.5 . 10(-5) M and for the alpha-fetoprotein-aflatoxin system: n = 4 and Kd = 3.7 +/-0.5 . 10(-5) M. The competition of anilino-naphthalene-sulfonate and aflatoxin B1 for the same hydrophobic sites on bovine serum albumin has been demonstrated. The fluorescence quenching of various proteins (lysozymes, egg-albumin, gamma-globulin) by aflatoxin B1 have shown that there is not a strict specificity of aflatoxin towards proteins.     

Ligand properties of diethylstilbestrol: studies with purified native and fatty acid-free rat alpha 1-fetoprotein and albumin.

We report the equilibrium binding parameters for the interactions of the estrogen analogue diethylstilbestrol (DES) with highly purified rat alpha 1-fetoprotein (AFP) and serum albumin preparations. At 25 degrees C and pH 7.4, an association constant (Ka) of about 1.5 X 10(6)M-1 and 2 sites/mole are measured with the DES-AFP system, whereas for the DES-albumin interaction, we find a Ka of approximately 2 X 10(5)M-1 and about 11 sites/mole of protein. The removal of fatty acids from pure AFP causes a reversible 3 fold increase of the number of DES binding sites; the same delipidation procedure applied to albumin slightly diminishes its DES binding parameters. We also demonstrate the capability of DES to displace competitively estradiol-17 beta (E2) from its high affinity sites on the estrophilic rat AFP. Finally, the binding behaviour of the two serum proteins towards the synthetic estrogen is compared to their interaction with the natural hormones. The physiological and pharmacological relevance of these data is discussed.     

Bovine fetus-specific serum proteins. Purification and characterization of alpha1-fetoprotein and immunological identification of alpha2- and beta-fetoproteins.

Bovine alpha1-fetoprotein was isolated from fetal calf serum by successive procedures of concanavalin A-Sepharose chromatography, DEAE-Sephadex chromatography, SP-Sephadex chromatography and preparative disc polyacrylamide gel electrophoresis. The bovine alpha1-fetoprotein preparation was considered homogeneous by several physicochemical and immunochemical criteria. Bovine alpha1-fetoprotein has a molecular weight of 68 000 with an amino acid composiotn similar to that of other mammalian alhpa1-fetoprotein. In addition, bovine alpha1-fetoprotein was shown to exist as two distinct variants on the basis of carbohydrate heterogeneity. alpha2-Fetoprotein and a new beta-fetoprotein were immunologically identified in fetal calf serum. These fetoproteins, like alpha1-fetoprotein, were not detectable in non-pregnant cow serum by immunoelectrophoresis.     

Comparison of the substrate dependence properties of the rat Na,K-ATPase alpha 1, alpha 2, and alpha 3 isoforms expressed in HeLa cells.

The role of multiple isoforms for the alpha subunit of Na,K-ATPase is essentially unknown. To examine the functional properties of the three alpha subunit isoforms, we developed a system for the heterologous expression of Na,K-ATPase in which the enzymatic activity of each isoform can be independently analyzed. Ouabain-resistant forms of the rat alpha 2 and alpha 3 subunits were constructed by site-directed mutagenesis of amino acid residues at the extracellular borders of the first and second transmembrane domains (L111R and N122D for alpha 2 and Q108R and N119D for alpha 3). cDNAs encoding the rat alpha 1 subunit, which is naturally ouabain-resistant, and rat alpha 2 and alpha 3, which were mutated to ouabain resistance (designated rat alpha 2* and rat alpha 3*, respectively) were cloned into an expression vector and transfected into HeLa cells. Resistant clones were isolated and analyzed for ouabain-inhibitable ATPase activity in the presence of 1 microM ouabain, which inhibits the endogenous Na,K-ATPase present in HeLa cells (I50 approximately equal to 10 nM). The remaining activity corresponds to Na,K-ATPase molecules containing the transfected rat alpha 1, rat alpha 2*, or rat alpha 3* isoforms. Utilizing this system, we examined Na+, K+, and ATP dependence of enzyme activity. Na,K-ATPase molecules containing rat alpha 1 and rat alpha 2* exhibited a 2-3-fold higher apparent affinity for Na+ than those containing rat alpha 3* (apparent KNa+ (millimolar): rat alpha 1 = 1.15 +/- 0.13; rat alpha 2* = 1.05 +/- 0.11; rat alpha 3* = 3.08 +/- 0.06). Additionally, rat alpha 3* had a slightly higher apparent affinity for ATP (in the millimolar concentration range) compared with rat alpha 1 or rat alpha 2* (apparent K0.5 (millimolar): rat alpha 1 = 0.43 +/- 0.12; rat alpha 2* = 0.54 +/- 0.15; rat alpha 3* = 0.21 +/- 0.04) and all three isoforms has similar apparent affinities for K+ (apparent KK+: rat alpha 1 = 0.45 +/- 0.01; rat alpha 2* = 0.43 +/- 0.004; rat alpha 3* = 0.27 +/- 0.01). This study represents the first comparison of the functional properties of the three Na,K-ATPase alpha isoforms expressed in the same cell type.     

Purification of mouse alpha1-fetoprotein and preparation of specific peroxidase conjugates for its cellular localization.

Mouse alpha1-fetoprotein (AFP) was isolated from amniotic fluid by immunoadsorbent columns and preparative electrophoresis. Specific antibodies were isolated from monospecific hyperimmunsera by use of immunoadsorbents, and subsequently coupled with horseradish peroxidase. At the light microscopical level, purified antibody-peroxidase conjugates were used for the cellular localization of AFP in fetal liver by direct and indirect staining methods. Fixatives containing ethanol or aldehydes were tried for antigen staining. Prior to immunocytological reactions, endogenous peroxidases were inhibited by hydrogen peroxide.     

Mouse alpha 1-fetoprotein and albumin. A comparison of their binding properties with estrogen and fatty acid ligands

The binding of estradiol-17 beta (E2), diethylstilbestrol (DES), and polyene fatty acids, in particular arachidonate (C20:4), to alpha 1-fetoprotein (alpha-FP) and albumin purified from mouse embryo sera was studied using equilibrium dialysis and electrophoretic techniques. E2, arachidonate, and DES all bind to alpha-FP, but with decreasing strength. E2 is a high affinity, low capacity ligand (Ka approximately 0.8 X 10(8) M-1 and approximately 0.3 sites/mol of alpha-FP at 25 degrees C); arachidonate is a weaker ligand disposing of more sites (Ka approximately 0.3 X 10(7) M-1 and 4-5 sites/mol of alpha-FP); the binding of DES is of comparatively low affinity and capacity (Ka approximately 0.2 X 10(7) M-1 and n approximately 0.7/mol of alpha-FP). In spite of different structures and equilibrium parameters, E2, DES, and arachidonate are able to compete with each other for binding to the fetoprotein. The C22:4 and C22:6 fatty acids are also efficient concentration-dependent inhibitors of E2 or DES binding. Albumin binds the fatty acids and DES, but equilibrium parameters are different from those of alpha-FP. In particular, arachidonate is a better ligand for albumin, where it interacts with at least two classes of apparent sites (Ka1 approximately 0.3 X 10(8) M-1 and n1 approximately 1; Ka2 approximately 0.2 X 10(7) M-1 and n2 approximately 30). In contrast to alpha-FP, albumin virtually does not bind E2. Also, no competition could be demonstrated between DES and fatty acid ligands for binding to albumin. None of the studied interactions, with either albumin or alpha-FP, was modified even by high doses of bilirubin. The possible functions of the various binding activities present in fetal sera in the process of growth are discussed.     

Association of endodermal sinus tumour,testicular teratoma and alpha1-fetoprotein.

An endodermal sinus tumour in the retroperitoneal region was associated with the presence of alpha1-fetoprotein (AFP) in the patient''s serum. At autopsy a simple cystic teratoma of the right testicle was also found. The association of these two tumours has been reported before. The classification of these malignant germ-cell tumours and an understanding of their evolution may be aided by the discovery that AFP is often found in the patient''s serum.     

Interaction of alpha1-syntrophin with multiple isoforms of heterotrimeric G protein alpha subunits

Syntrophins are components of the dystrophin-glycoprotein complex of the plasma membrane in muscular and neuronal cells, and recruit signaling proteins such as neuronal nitric oxide synthase via their multiple protein-protein interaction motifs. In this study, we found that alpha1-syntrophin binds to various subtypes of guanine nucleotide-binding protein alpha subunits (Galpha). A pull-down analysis using full-length recombinant alpha1-syntrophin and MS analysis showed that alpha1-syntrophin was coprecipitated with several isoforms of Galpha proteins in addition to known binding partners such as dystrobrevin and neuronal nitric oxide synthase. Further analysis using recombinant Galpha isoforms showed that alpha1-syntrophin associates with at least Galphai, Galphao, Galphas and Galphaq subtypes. The region of alpha1-syntrophin required for its interaction with Galphas was determined as the N-terminal half of the first pleckstrin homology domain. In addition, the syntrophin unique domain of alpha1-syntrophin was suggested to contribute to this interaction. In COS-7 cells, downregulation of alpha1-syntrophin by RNAi resulted in enhanced cAMP production and cAMP response element-binding protein phosphorylation induced by isoproterenol treatment. These results suggest that alpha1-syntrophin provides a scaffold for the Galpha family of heterotrimeric G proteins in the brain to regulate the efficiency of signal transduction evoked by G-protein-coupled receptors.     

Mechanistic basis for kinetic differences between the rat alpha 1, alpha 2, and alpha 3 isoforms of the Na,K-ATPase.

Previous studies showed that the alpha 1, alpha 2, and alpha 3 isoforms of the catalytic subunit of the Na,K-ATPase differ in their apparent affinities for the ligands ATP, Na(+), and K(+). For the rat isoforms transfected into HeLa cells, K'(ATP) for ATP binding at its low affinity site is lower for alpha 2 and alpha 3 compared with alpha 1; relative to alpha 1 and alpha 2, alpha 3 has a higher K'(Na) and lower K'(K) (Jewell, E. A., and Lingrel, J. B (1991) J. Biol. Chem. 266, 16925--16930; Munzer, J. S., Daly, S. E., Jewell-Motz, E. A., Lingrel, J. B, and Blostein, R. (1994) J. Biol. Chem. 269, 16668--16676). The experiments described in the present study provide insight into the mechanistic basis for these differences. The results show that alpha 2 differs from alpha1 primarily by a shift in the E(1) E(2) equilibrium in favor of E(1) form(s) as evidenced by (i) a approximately 20-fold increase in IC(50) for vanadate, (ii) decreased catalytic turnover, and (iii) notable stability of Na,K-ATPase activity at acidic pH. In contrast, despite its lower K'(ATP) compared with alpha 1, the E(1) E(2) poise of alpha 3 is not shifted toward E(1). Distinct intrinsic interactions with Na(+) ions are underscored by the marked selectivity for Na(+) over Li(+) of alpha 3 compared with either alpha1 or alpha 2 and higher K'(Na) for cytoplasmic Na(+), which persists over a 100-fold range in proton concentration, independent of the presence of K(+). The kinetic analysis also suggests alpha 3-specific differences in relative rates of partial reactions, which impact this isoform's distinct apparent affinities for both Na(+) and K(+).     

Comprehensive glyco-proteomic analysis of human alpha1-antitrypsin and its charge isoforms

Human alpha1-antitrypsin (A1PI) is a well-known glycoprotein in human plasma important for the protection of tissues from proteolytic enzymes. The three N-glycosylation sites of A1PI contain diantennary N-glycans but also triantennary and even traces of tetraantennary structures leading to the typical IEF pattern observed for A1PI. Here we present an approach to characterize A1PI isoforms from human plasma and its PTMs by LC-ESI-MS and LC-ESI-MS/MS of peptides obtained by proteolytic digestion. The single cysteine residue of A1PI formed a disulfide bridge with free cysteine. The variability of the number of antennae and hence sialic acids on glycosylation site N107, which even contained minute amounts of tetraantennary structures, emerged as a major cause for the IEF pattern of A1PI. Only negligible amounts of triantennary structures were identified attached to N70, and exclusively diantennary structures were present on site N271 in each of the isoforms analyzed. Exoglycosidase digests revealed alpha2,6-linked neuraminic acids on diantennary N-glycans, and triantennary contained additionally one single alpha2,3-neuraminic acid per N-glycan, which, together with a fucose, formed a sialyl Lewis X determinant on the beta1,4-linked N-acetylglucosamine, as shown by 2-D-HPLC of pyridylaminated asialoglycans. Fucosylation of diantennary structures was marginal and of the core alpha1,6 type.     

Isolation and quantification of the heat shock protein 90 alpha and beta isoforms from rat liver

Summary Heat shock protein 90 (Hsp90) is an abundant cytosolic protein. In higher eukaryotes two isoforms of Hsp90 exist, Hsp90 and Hsp90. Hsp90 was purified from rat liver and after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a double band at about 90 kDa. The two bands were separated and identified as the Hsp90 and Hsp90 isoforms. There was no entry in the protein databases for the Hsp90 isoform from rat. Furthermore, the ratio of the two Hsp90 isoforms was determined.     

The alpha macroglobulins of rat serum.

Cortex of rat kidney was homogenized and fractions enriched in plasma membrane, endoplasmic reticulum or brush border were prepared by several techniques of differential centrifugation. The identity and homogeneity of the membrane fragments were investigated by assaying marker enzymes and by transmission and scanning electron microscopy. Kallikrein was present in both plasma-membrane- and endoplasmic-reticulum-enriched fractions isolated by two fractionation procedures. Kallikrein was highly concentrated in a plasma-membrane fraction but was absent from the brush-border membrane of proximal tubular cells. Cells of transplanted renal tumours of the rat, originating from the proximal tubule, had no kallikrein activity. Kininase activity, angiotensin I-converting enzyme (kininase II) and angiotensinase were found in a plasma-membrane-enriched fraction and especially in the fraction containing isolated brush border. It is suggested that after renal kallikrein is synthesized on endoplasmic reticulum, it is subsequently reoriented to a surface membrane for activation and release. Renal kallikrein may enter the tubular filtrate distal to the proximal tubules. The brush-border membrane of proximal tubule is the major site of inactivation of kinins and angiotensin II..     

Suppression of the primary cell-mediated immune response by human alpha1-fetoprotein in vitro.

A possible immunoregulatory role of human α₁-fetoprotein (HAFP) was investigated. HAFP-enriched fractions as well as pure HAFP were obtained by means of two different procedures, as follows. After passage of HAFP-containing ascites of patients with primary liver carcinoma (PLC) on an anti-HAFP immunosorbent column, the retained proteins were eluted first by a glycine-NaOH buffer, pH 10.0 (resulting in HAFP I), and second by NaSCN (HAFP II). HAFP was further purified by passage of the HAFP-containing fractions an on anti-human whole serum (anti-HWS) immunosorbent column. This resulted in semipurified HAFP I and II. HAFP, pure by means of SDS-disc gel electrophoresis, Ouchterlony gel diffusion, and immunoelectrophoresis, was obtained by recycling on the anti-HWS immunosorbent column, as well as by a final Sephadex G-100 gel filtration. A possible immunoregulatory activity was assessed by testing the influence of semipurified as well as pure HAFP I and II on the uptake of tritiated thymidine by human lymphocytes stimulated by allogeneic lymphocytes in vitro. Only HAFP I, semipurified as well as pure, consistently exerted a profound suppressive effect on this primary cell-mediated immune response at concentrations of 150–200 μg/ml. In contrast, HAFP II did not show a comparable immunoregulatory effect either because there are two biologically different HAFPs or because of a loss of biological activity from HAFP II due to the use of the sodium thiocyanate elution technique.