Characterization of human alpha fetoprotein charge microheterogeneity during fetal development.

Aliquotes of human amniotic fluid (AF), fetal serum (FS), and cord blood (CB) were obtained as by-products of routine clinical diagnostic procedures at term or in the second trimester of pregnancy. When samples of CB were applied to a pH 5.5-4 chromatofocusing gradient, three isoforms of AFP could be resolved; a pl 4.57 form (isoform IA, 52% AFP), a pl 4.27 form (isoform IB, 43% AFP), and one species that was bound to the column but could be eluted with 1.0 M NaCl (isoform II, pl less than 4.00, 5% AFP). Term AF displayed a profile similar to that observed in term CB. When samples of 15-20-week gestation AF were chromatofocused, the immunoreactive AFP recovered was distributed between isoform IA and IB (60%) and isoform II (40%). FS and AF obtained from same pregnancy (23-26 weeks) displayed an identical chromatofocusing profile. Aliquotes of AF subjected to conA revealed 83% reactive variants compared with greater than 95% reactive variants for CB. FS displayed a conA profile identical to CB. When individual CB charge isoforms were isolated and subjected to conA analysis, greater than 97% of the AFP bound to conA. In contrast, when AFP isoform IA and IB were isolated from midgestation AF, approximately 22% of the AFP did not bind to the lectin while 100% of isolated AFP isoform II eluted as the reactive variant. These data suggest that human AFP exists as at least three charge and two lectin variants and that the charge profile may change during fetal development.     

Human alpha-fetoprotein purified from amniotic fluid enhances growth factor-mediated cell proliferation in vitro.

Using a primary monolayer culture of porcine granulosa cells (pGC) as an in vitro cell proliferation assay, we have examined the growth-promoting activity of alpha-fetoprotein (AFP) purified from term cord blood and midtrimester amniotic fluid. Increasing concentrations (2.5-20%) of crude human cord blood (CB) increased pGC proliferation, while identical concentrations of crude amniotic fluid (AF) were ineffective. When the cell system was maximally stimulated, AF dose dependently decreased cell proliferation. AFP purified from AF and CB (1.25-5.0 micrograms/ml) was not mitogenic alone, but, in the presence of epidermal growth factor (EGF) + insulin-like growth factor (IGF-I) (10 ng/ml each), AFP dose dependently increased cell proliferation to nearly double that of EGF + IGF-I alone. The response of pGC to the proliferative effects of AF-AFP and CB-AFP were identical at each dose of AFP tested. These results indicate that although crude, pooled midtrimester AF does not display the mitogenic activity seen in cord blood, AFP purified from pooled AF significantly synergizes with growth factors to increase cell proliferation markedly.     

甲胎蛋白受体的性质及其所介导的cAMP-PKA信号转导途径

研究HeLa细胞膜上甲胎蛋白 (alpha fetoprotein ,AFP)受体的存在情况及其介导的信号转导 .先用Na[12 5I]标记AFP ;标记的AFP和培养的HeLa细胞结合 ,Scatchard法和受体 配体结合法分析受体数目 ;再用放射免疫结合法分析在百日咳毒素 (pertussistoxin ,PTX)预处理前后AFP对细胞内环腺苷酸 (cAMP)浓度及细胞内蛋白激酶A(proteinkinaseA ,PKA)活性变化的影响 .在HeLa细胞膜表面存在 2种不同解离平衡常数 (Kd)的AFP受体 ,Kd1=5 2pmol L(2 10 0位点 细胞 ) ;Kd2 =2 3nmol L (114 0 0位点 细胞 ) .在AFP(2 0mg L)作用下 ,HeLa细胞内cAMP浓度变化及PKA活性的改变为与对照组比较 ,用PTX预处理前cAMP浓度升高 2 6 7% ,PKA活性增高 10 3 2 % ;用PTX预处理后升高 86 % ,PKA活性增高 2 5 3% .抗甲胎蛋白单克隆抗体可阻断AFP对细胞cAMP浓度和PKA活性的影响 .结果证明 ,在HeLa细胞膜上有 2种不同解离平衡常数的甲胎蛋白受体存在 ,受体有可能通过cAMP PKA途径介导信号转导 .     

Conditions affecting the immunosuppressive properties of human alpha-fetoprotein.

The hypothesis that alpha-fetoprotein (AFP) is immunosuppressive in vitro was tested with immunoabsorbent purified human cord AFP in human lymphocyte cultures. Albumins were purified identically from cord plasma and pooled adult plasma. No preparations were suppressive in phytohemagglutinin-stimulated cultures. Both AFP and human cord albumin (HCA) produced 50% suppression of allogeneic lymphocyte cultures at 50 to 100 microgram/ml final concentrations, whereas adult and commercial albumins were not suppressive. When AFP was eluted from the immunoabsorbent column in 0.15 M NaCl rather than the conventional 0.5 M NaCl, activity was greatly diminished or lost, but could be restored by dialysis against 0.5 M KCl. Previously, inactive lots of HCA also demonstrated the same phenomenon. It appears, therefore, that the immunosuppressive activity of AFP (and albumin) may depend both on its source and the procedure by which it is isolated. Whether analogous conditions occur in vivo is unknown at this time.     

Ovine luteinizing hormone. III. Relationships between the charge heterogeneity of partially purified subunits and the native hormone

Extracts of anterior pituitaries from wethers were prepared by homogenization and centrifugation at 100,000 X g. When chromatofocused on pH 10.5-7.0 gradients, eight peaks of immunoreactive ovine luteinizing hormone (oLH) were observed: six exhibited apparent pIs in the range of 9.33-8.83, one eluted unbound (apparent pI greater than 9.8), and one was bound to the column (apparent pI less than or equal to 7.0). A portion of the same extracts was subjected to gel filtration on Sephadex G-100 Superfine to resolve native oLH and its uncombined subunits. oLH, oLH alpha, and oLH beta were present at concentrations of 0.907 +/- 0.127, 0.089 +/- 0.020, and 0.010 +/- 0.023 microgram/mg tissue, respectively, which translated to oLH alpha/oLH and oLH beta/oLH molar ratios of approximately equal to 0.19 and approximately equal to 0.02. Fractions containing immunoreactive oLH or uncombined subunits (oLH alpha and oLH beta) were pooled, lyophilized, and chromatofocused. Native oLH resolved from uncombined subunits by gel filtration displayed a similar pattern of isohormones to those in crude extracts. In contrast, three purified oLH preparations exhibited distinct chromatofocusing patterns. Uncombined oLH alpha in pituitary extracts resolved from native oLH by gel filtration exhibited a higher percentage (approximately equal to 37%) of acidic components when chromatofocused, while more than 97% of purified oLH alpha focused as basic forms having pIs greater than 8.9. When uncombined oLH beta in pituitary extracts was chromatofocused, more than half of the immunoreactivity was bound to the column (apparent pI less than or equal to 7.0); purified oLH beta displayed a nearly identical pattern. These results suggest that native oLH resolved from uncombined subunits by gel filtration displays a similar chromatofocusing profile to that of oLH in crude pituitary extracts.(ABSTRACT TRUNCATED AT 250 WORDS)     

The intracellular mechanism of alpha-fetoprotein promoting the proliferation of NIH 3T3 cells

AIM The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS The effect of AFP on the proliferation of NIH 3T3 cells was measured by incorporation of 3H-TdR. Receptor-binding assay of 125I-AFP was performed to detect the properties of AFP receptor in NIH 3T3 cells. The influences of AFP on the [cAMP]i and the activities of protein kinase A (PKA) were determined. Western blot was used to detect the change of K-ras P21 protein expression. RESULTS The proliferation of NIH 3T3 cells treated with 0-80 mg/L of AFP was significantly enhanced. The Scatchard analysis indicated that there were two classes of binding sites with KD of 2.722×10-9M (Bmax=12810 sites per cell) and 8.931× 10-8M (Bmax=119700 sites per cell) respectively. In the presence of AFP (20 mg/L), the content of cAMP and activities of PKA were significantly elevated . The level of K-ras P21 protein was upregulated by     

Efficient removal of albumin from human serum by monosize dye-affinity beads

Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for removal of human serum albumin (HSA) from human serum. Monosize poly(GMA) beads, 1.6 microm in diameter, were produced by dispersion polymerization. Cibacron Blue F3GA loading was 1.73 mol/g. HSA adsorption experiments were performed by stirred-batch adsorption. The non-specific adsorption of HSA was low (0.8 mg/g polymer). Dye attachment onto the monosize beads significantly increased the HSA adsorption (189.8 mg/g). The maximum HSA adsorption was observed at pH 5.0. With an increase of the aqueous phase concentration of sodium chloride, the adsorption capacity decreased drastically. The equilibrium adsorption of HSA significantly decreased with increasing temperature. The elution studies were performed by adding 0.1 M Tris/HCl buffer containing 0.5 M NaSCN to the HSA solutions in which adsorption equilibria had been reached. The elution results demonstrated that the adsorption of HSA to the adsorbent was reversible. The depletion efficiencies for HSA were above 87% for all studied concentrations. To test the efficiency of HSA removal from human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gel electrophoresis. Eluted proteins include mainly albumin, and a small number of nonalbumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin and alpha1-antitrypsin were bound by the dye-affinity beads. IgA was not identified in eluted fraction.     

Immunochromatography on insolubilized antibodies of very low affinity: application to immunoadsorbence of bovine alpha-fetoprotein.

Cross-reactive antibodies were utilized to prepare immunoadsorbents possessing a very low affinity to bovine alpha-fetoprotein (AFP). A goat anti-human AFP serum cross-reactive with bovine AFP was first depleted of antibodies reactive with bovine AFP in immunodiffusion. The remaining antibodies from this serum and gamma-globulin from a sheep antiserum against rabbit AFP, without prior absorption, were coupled to Sepharose. Chromatography of fetal calf serum on these adsorbents resulted in retardation of bovine AFP relative to other proteins. A major part of the AFP eluted from the columns with phosphate-buffered saline. The rest eluted as a sharp peak with a small quantity of 4 or 6 M urea. The elution of AFP with the initial column buffer has made it possible to prepare pure AFP that has not been subjected to the chaotropic elution buffers usually employed in affinity chromatography. Elimination of the washing step and the ease of elution has allowed purification of gram amounts of AFP. The fact that immunoadsorbents prepared from antibodies with no detectable reactivity in immunodiffusion still caused delayed elution in chromatography suggests that this procedure may be useful in search of proteins cross-reactive with a known protein.     

Development of a specific radioimmunoassay for the placental folate receptor and related high-affinity folate binding proteins in human tissues

High-affinity membrane-associated and soluble folate binding proteins (FBPs) from human placenta, milk, and KB cells appear to share antigenic determinants [A. C. Antony et al. (1981) J. Biol. Chem. 256, 9684-9692 and (1985) 260, 14911-14917]. Iodination of a highly purified preparation of placental folate receptor (PFR) by various techniques resulted in significant denaturation of the PFR as evidenced by additional peaks of radioactivity on Sephacryl S-200 gel filtration in 1% Triton X-100. These denatured species had similar molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as radioiodinated and native PFR, and were also recognized, albeit with less efficiency, by specific rabbit antiserum raised against purified PFR. Since these denatured species failed to bind folate, they were specifically excluded from 125I-PFR by their inability to bind pteroylglutamate-Sepharose. This ws accomplished in a single step by iodination of PFR bound to the affinity column and elution of 125I-PFR under identical conditions that the native PFR was purified. The purified 125I-PFR comigrated with unlabeled PFR on SDS-PAGE and its elution profile on Sephacryl S-200 gel filtration was identical to radioligand bound PFR. The resulting radioimmunoassay standard curve using this affinity chromatography purified 125I-PFR, unlabeled PFR, and anti-human PFR serum had a range for measurement between 5 and 500 ng of PFR and was not affected by the concentration of folate in the sample. The practical utility of this radioimmunoassay for measuring cross-reacting material to the PFR was validated by its ability to quantitate the 40,000 and 160,000 Mr FBPs which are the two major forms of high-affinity FBPs in human tissues.     

Behavior of antithrombin III isoforms on immobilized heparins. Evidence that the isoforms bind to different numbers of low-affinity heparin sites

Antithrombin III exists in plasma as major and minor isoforms differing in affinity for heparin. The nature of the binding of each purified isoform to immobilized heparins was investigated. Unfractionated, mixed-affinity heparin bound each isoform with both high affinity and concentration-dependent low affinity. The isoforms were resolved when filtered through low-affinity heparin (heparin repeatedly passed over immobilized antithrombin III) columns. Following chemical modification of a specific tryptophan residue required for heparin binding, each isoform failed to bind to either low-affinity or mixed-affinity heparin-agarose, but elution of the modified higher-affinity isoform was retarded on both gels. Because the modified lower-affinity isoform eluted with the similarly sized bovine serum albumin in these experiments, the difference in isoform affinity for heparin appears to be the result of a unique, secondary heparin-binding site in the higher-affinity isoform that can bind a heparin site with low affinity for antithrombin III. This interpretation was supported by the chromatographic behavior of the isoforms on mixed-affinity agarose during reverse gradient elution. Two other populations of each of the tryptophan-modified isoforms were identified. Since these isoforms bound tightly to mixed-affinity heparin-agarose but eluted at lower salt concentrations than the corresponding unmodified isoforms, both isoforms may contain additional secondary sites that interact weakly with heparin. A general model of heparin-antithrombin III interaction is proposed in which a high-affinity heparin site initially interacts with a primary site on antithrombin III. The subsequent conformational change leads to a cooperative, entropy-driven association between secondary sites on the protein and low-affinity sites on heparin, stabilizing antithrombin III in its activated form.     

Diurnal variation of leptin entry from blood to brain involving partial saturation of the transport system

The blood-brain barrier (BBB) regulates the amount of peripherally produced leptin reaching the brain. Knowing that the blood concentration of leptin has a circadian rhythm, we investigated whether the influx of leptin at the BBB followed the same pattern in three main sets of experiments. (a): The entry of 125I-leptin from blood to brain was measured in mice every 4 h, as indicated by the influx rate of 125I-leptin 1-10 min after an iv bolus injection. The blood concentration of endogenous leptin was measured at the same times. Blood leptin concentrations were higher at night and early morning (peak at 0800 h) and lower during the day (nadir at 1600 h). By contrast, the influx of 125I-leptin was fastest at 2000 h and slowest at 0400 h. Addition of unlabeled leptin (1 microg/mouse) significantly decreased the influx rate of 125I-leptin at all time points, indicating saturability of the transport system. The unlabeled leptin also abolished the diurnal variation of the influx of 125I-leptin. (b): The entry of 125I-leptin into spinal cord was faster than that into brain and showed a different diurnal pattern. The greatest influx occurred at 2400 h and the slowest at 0800 h. In spinal cord, unlike brain, unlabeled leptin (1 microg/mouse) neither inhibited the influx of 125I-leptin nor abolished the diurnal rhythm. (c): Higher concentrations of unlabeled leptin (5 microg/mouse) inhibited the uptake of 125I-leptin in spinal cord as well as in brain, but not in muscle. This experiment measured uptake 10 min after iv injection at 0600 h (beginning of the light cycle) and 1800 h (beginning of the dark cycle). Thus, influx of 125I-leptin into the CNS shows diurnal variation, indicating a circadian rhythm in the transport system at the BBB, saturation of the leptin transport system shows differences between the brain and spinal cord, and blood concentrations of leptin suggest that partial saturation of the transport system occurs at physiological concentrations of circulating leptin, contributing to the differing diurnal patterns in brain and spinal cord. Together, the results show that the BBB is actively involved in the neuroendocrine regulation of feeding behavior.     

Staphylococcal Enterotoxin B: Solid-Phase Radioimmunoassay

An immunoassay employing (125)I-labeled enterotoxin B and polystyrene tubes coated with specific antibody was used for assaying purified and crude enterotoxin. Antibody was adsorbed to untreated polystyrene tubes. Unlabeled enterotoxin competed with (125)I-labeled enterotoxin for antibody-combining sites. The uptake of (125)I-labeled toxin reflected the concentration of unlabeled toxin present. The test is sensitive to 1 to 5 ng of purified and crude enterotoxin B per ml, and cross-reactions with heterologous enterotoxins did not interfere with the specificity. This test possesses the combination of sensitivity and objectivity absent in current methods for assaying enterotoxin and provides a model for investigating other enterotoxin serotypes.     

Human mammary tumor cell proliferation: primary role of platelet-derived growth factor and possible synergism with human alpha-fetoprotein.

Human mammary medullary carcinoma cells (passages 16 to 21) were cultured for 2 days to allow for attachment, followed by 6 days of culture in either fetal calf serum, human cord blood, human amniotic fluid, or growth factors in the presence or absence of purified human alpha-fetoprotein (AFP). When growth factors were tested alone, only platelet-derived growth factor produced a significant increase in cell proliferation. Although up to 40% amniotic fluid had no effect on cell proliferation, human cord blood was two-fold more potent than fetal calf serum at similar concentrations. The addition of 10 ng/ml of platelet-derived growth factor increased the proliferative activity of human cord blood 1.5- to 2.5-fold. Ablation of endogenous AFP by affinity chromatography reduced the proliferative activity of cord blood by 75%. Similarly, the mitogenic activity of cord blood plus platelet-derived growth factor was reduced by 56% when AFP was removed. Purified AFP dose-dependently enhanced the proliferative activity of platelet-derived growth factor. This synergistic effect was specific for platelet-derived growth factor. We conclude that platelet-derived growth factor is a major growth factor controlling the proliferation of these tumor cells and that AFP may enhance growth factor proliferative activity and human mammary tumor growth.     

Investigation of the oligomeric status of the peroxisomal isoform of human 3-hydroxy-3-methylglutaryl-CoA lyase

Unprocessed 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, retaining the mitochondrial signal sequence, has been proposed to correspond to a peroxisomal isoform. Using a modified expression plasmid and purification protocol, it is now possible to isolate substantial amounts (>10mg) of highly purified peroxisomal HMG-CoA lyase. These improvements facilitate more detailed protein chemistry approaches for characterization of the enzyme, which exhibits substantial (eightfold) dithiothreitol (DTT) stimulation of activity. The C323S mutant shows little DTT activation. Superose gel filtration chromatography data have prompted other investigators to hypothesize that the peroxisomal isoform is a monomer. This study confirms the elution properties presented in that earlier report, but also demonstrates anomalous elution up on Superose chromatography. Elution properties observed using a polyacrylamide resin (Bio-Gel P100) suggest a dimeric, rather than monomeric, enzyme. This observation has been further tested by protein chemistry experiments. The peroxisomal enzyme forms a covalently linked dimeric species upon crosslinking with dibromopropanone or o-phenylenedimaleimide or upon disulfide formation as a result of incubation with diamide. Cysteine-323 is required for intersubunit covalent crosslinking. Crosslinking efficiency is not dependent on HMG-CoA lyase protein concentration nor is it influenced by the presence of varying concentrations of an unrelated protein, such as ovalbumin. Sedimentation equilibrium analyses do not indicate a monomeric form of either human mitochondrial or human peroxisomal HMG-CoA lyase; the results suggest that these proteins are predominantly dimers. The retention of the basic N-terminal mitochondrial signal sequence in the peroxisomal HMG-CoA lyase isoform may influence elution from Superose gel filtration media but does not alter the oligomeric status of the enzyme.     

以聚乙二醇为层析伴侣同时制备SOD、过氧化氢酶和血红蛋白

发展了一条从红细胞裂解液中同时制备超氧化物歧化酶(SOD)、过氧化氢酶和血红蛋白的新工艺。采用0 75 %的聚乙二醇600作为层析伴侣,使血红蛋白直接流过阴离子交换层析柱,同时吸附SOD和过氧化氢酶。经过梯度洗脱获得SOD和过氧化氢酶组分,再经过疏水性相互作用层析与凝胶过滤层析相串联,使SOD和过氧化氢酶得到纯化。纯化后的SOD和过氧化氢酶的比活力分别达到15932u/mg和65918u/mg ,血红蛋白的纯度达到99.9%以上。总回收率为:SOD ,47.4% ;过氧化氢酶,29.6% ;血红蛋白,88.7%。     

Purification of [125I]-vasoactive intestinal peptide by reverse-phase HPLC

VIP was labeled with sodium ¹²⁵iodide, and ¹²⁵I-VIP was purified by reverse-phase high performance liquid chromatography. Optimal separations of ¹²⁵I-VIP and unlabeled VIP were obtained using two C₁₈-Novapak columns in series and a gradient of acetonitrile in triethylamine phosphate for elution. The specific activity of the ¹²⁵I-VIP was 1.99±0.21 Ci/μmole, approaching the maximum specific activity of monoiodinated VIP (2.26 Ci/μmole). Radioimmunoassay and radioreceptorassay for VIP were more sensitive (2.6-fold, and 2.5-fold, respectively) using ¹²⁵I-VIP purified by HPLC compared to ¹²⁵I-VIP obtained from an open-end cellulose column. These results demonstrate the advantage of preparing purified ¹²⁵I-VIP by HPLC for the accurate assay of VIP and VIP-receptors in tissues and biological fluids.     

A simple two-step purification of human and monkey alpha fetoprotein from amniotic fluid and serum.

We developed a two-step purification system to characterize alpha fetoprotein (AFP) in early gestation amniotic fluid and late gestation fetal serum or cord blood from monkey and human. It involves only two chromatographic steps, allows preparative purification using up to 12 ml of starting sample, can purify up to 350 micrograms of AFP at one time, and can be used to purify both fetal serum or amniotic fluid AFP from two different species. This procedure will allow detailed biochemical analysis of purified AFP from different stages of fetal development.     

Solubilization and Characterization of Calcitonin Gene-Related Peptide Binding Site from Porcine Spinal Cord

The binding site for calcitonin gene-related peptide (CGRP) was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in an active form from porcine spinal cord. 125I-labeled human alpha-CGRP (125I-CGRP) binding to the solubilized protein was determined by filtration using a GF/B glass filter. The maximal binding activity (approximately 60% of the crude membrane fraction) was obtained with 5 mM CHAPS. 125I-CGRP binding to the solubilized protein was of high affinity, saturability, and high specificity, having KD and Bmax values of 3.69 pM and 338 fmol/mg of protein, respectively. The binding activity was eluted in a single peak with a molecular mass of 400,000 daltons by gel filtration on TSK gel G4000SW. These results suggest that the solubilized protein may be responsible for the specific binding site.     

Purification of the type II insulin-like growth factor receptor from rat placenta

The membrane receptor for insulin-like growth factor II (IGF II) has been purified to near homogeneity from rat placenta by chromatography of crude plasma membranes solubilized in Triton X-100 on agarose-immobilized IGF II. Elution of the IGF II receptor from the matrix at pH 5.0 in the presence of 1.5 M NaCl resulted in a receptor purification of 1100-fold from isolated plasma membranes, or 340-fold from the Triton extract with an average yield of about 50% in five separate purifications. Analysis of 125I-IGF II binding to the solubilized receptor in the Triton extract and in purified form by the method of Scatchard demonstrated no change in receptor affinity (Kd = 0.72 nM). Sodium dodecyl sulfate electrophoresis of the purified receptor showed one major band at Mr = 250,000 with only minor contamination. Affinity labeling of the receptor in isolated placenta membranes and in purified form using 125I-IGF II and the cross-linking agent disuccinimidyl suberate resulted in covalent labeling of only the Mr = 250,000 band. Such labeling was abolished by unlabeled IGF II but was unaffected by insulin, consistent with the previously reported specificity of IGF II receptor (Massague, J., and Czech, M.P. (1982) J. Biol. Chem. 257, 5038-5045). These results establish a one step affinity method for the purification of the type II IGF receptor that is rapid and highly efficient.     

枯草芽孢杆菌JA-206对皮肤癣菌抑制作用的研究

低能离子诱变筛选获得的枯草芽孢杆菌JA-206的发酵粗提液对红色毛癣菌和须毛癣菌有较强的抑菌或杀菌作用,粗提液经高温、蛋白酶处理后仍然有较高的稳定性,将JA-206粗提液经多步分离纯化,得到三种单一纯化产物抗菌肽AFP1、AFP2和AFP3,其中AFP1和AFP3对两种皮肤癣菌显示出明显的抑菌或杀菌作用。