Purification of Human Placental Trophoblast Interferon by Two-Dimensional High Performance Liquid Chromatography

Abstract

Human placental trophoblast challenged with Sendai virus induced IFNs mainly of the β-type (75%) and relatively low levels of the α-type (25%). A two-step high performance liquid chromatographic procedure (“two-dimensional HPLC”) has been developed for the complete purification of the placental trophoblast interferon beta (tro-IFN-β) from serum-containing culture supernatant. The method involved a combination of high performance liquid affinity chromatography (HPLAC) on Cibacron Blue 3GA immobilized on an activated pressure stable macroporous synthetic polymer, 2-hydroxyethyl methacrylate vinyl sulphone (HEMA-BIO 1000 VS), as the first dimension and reversed-phase high performance liquid chromatography (RP-HPLC) on Separon SGX C-18 as the second. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot experiments showed that the tro-IFN-β was present as a 24 kDa protein. Densitometric scanning analysis of Coomassie-stained gel revealed the purity of the final preparation to be greater than 99%. The purified tro-IFN-β had a specific activity of 1.03 × 10⁸ IU/mg of protein and the overall recovery was 81% of the total IFN-β activity in the crude preparation and 61% of the total IFN activity.     

Simplified procedure for purification of Bordetella bronchiseptica dermonecrotic toxin

Abstract Bordetella bronchiseptica dermonecrotic toxin was purified by a simplified method. The method consisted of SP Toyopearl 650M chromatography and high performance liquid chromatography on a TSK gel G3000SW column. 47.5% of the activity of the crude cell extract was recovered. The purified toxin behaved as a homogeneous protein in sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance liquid chromatography, and agar gel double diffusion tests.     

N-terminal sequence analysis of human placental protein 14, purified in high yield from decidual cytosol

Human placental protein 14 (PP14) has been purified in high yield from first trimester decidual cytosol. High-performance liquid chromatography on anion exchange, gel filtration and reverse-phase chromatography were used. The protein obtained is approximately 97% pure with an overall recovery of about 50% from the original tissue extract. The first 24 amino acids of the N-terminal were found to be Met-Asp-Ile-Pro-Gln-Thr-Lys-Gln-Asp-Leu-Glu-Leu-Pro-Lys-Leu-Ala-Gly-Thr-Glu-His - Glu-Met-Ala-Met. PP14 has been characterized in this study to be a dimeric glycoprotein of Mr 60,000, with homologous subunits having an Mr of 28,000.     

Purification of the thromboxane A2/prostaglandin H2 receptor from human blood platelets

S-145 (5Z-7-(3-endo-phenylsulfonylamino-(2.2.1.)-bicyclohept -2-exo-yl) heptenoic acid) is a potent and selective antagonist for thromboxane A2/prostaglandin H2 receptor. Using this compound as an immobilized ligand for affinity chromatography and [3H]S-145 as a radioligand, we have purified the thromboxane A2/prostaglandin H2 receptor from the membranes of human blood platelets. The purification procedures consisted of solubilization of the receptor with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), affinity chromatographies on columns of S-145 affinity gel, wheat germ agglutinin agarose and red agarose, and repeated gel filtration high performance liquid chromatography on a TSK gel G-3000SW column. On the second gel filtration high performance liquid chromatography, the [3H]S-145 binding activity was eluted as a symmetrical peak which overlapped exactly with a peak of ultraviolet absorption at 280 nm. By these procedures, the receptor was purified about 8700-fold from the solubilized extract with a recovery of 6%. The final preparation showed a broad protein band at Mr 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and maximally bound 19.2 nmol of [3H]S-145/mg protein with a Kd of 29.8 nM. The [3H]S-145 binding to the purified receptor was specifically displaced by several thromboxane A2/prostaglandin H2 analogues.     

Purification and characterization of a low molecular weight zinc binding protein from human placenta

A low molecular weight, native zinc binding, cytosolic protein (LMZP) has been isolated, purified and characterized from human normal term placenta. Gel filtration of heat treated placental cytosol after sequential acetone precipitation (80% ppt) revealed a major zinc binding protein in the range of low molecular weight. This partially purified zinc binding fraction was further fractionated on DEAE-Sephadex A-25. The zinc was eluted in one of the three peak fractions. Further, the purity of zinc binding protein was confirmed on fast protein liquid chromatography (FPLC). The purified placental LMZP was homogenous on SDS-polyacrylamide gel electrophoresis with a single band. Ultraviolet (UV) spectrum of LMZP showed an absorption maximum at 257 nm which disappeared at pH 2. Molecular weight of LMZP as determined by gel chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis was 6 kDa. It was calculated that 1 g atom of zinc was bound to 1 mole of the LMZP. Unlike in classical metallothionein, the amino acid composition of placental LMZP revealed the presence of aromatic amino acids, lower content of cysteine and higher content of histidine, glutamic acid and aspartic acid (10, 9 and 5 residues/mole, respectively).     

Purification and properties of human placental NADPH-cytochrome P-450 reductase

Human placental NADPH-cytochrome P-450 reductase (EC 1.6.2.4) was purified to electrophoretic homogeneity in two chromatographic steps with a high retention of bioactivity. After solubilization with 1% sodium cholate in a protective medium containing 20% glycerol, 10 microM 4-androstene-3,17-dione, 1 mM dithiothreitol, and 0.2 mM EDTA, a 35-60% ammonium sulfate precipitate was prepared. The crude protein mixture was then applied to a 2',5'-ADP-Sepharose 4B affinity column, followed by high-performance anion-exchange chromatography (Pharmacia Mono-Q column). Two forms of the reductase were isolated. One was eluted at higher salt concentration and had a relative mass (Mr) of 79 kdaltons (kDa) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance gel permeation chromatography. A smaller size reductase with a Mr of 70 kDa, eluting at lower salt concentration, was also formed by trypsinolysis of the 79-kDa reductase. It must therefore be regarded as a proteolytic artifact. The absolute spectra in the visible region of the two reductases were identical with maxima at 376 and 452 nm, typical of a flavoprotein. They also had the same specific activity of 24.7 +/- 0.7 mumol/min per milligram protein towards cytochrome c. However, only the 79-kDa reductase showed aromatase-reconstitution activity. The homogeneity of these reductases was further confirmed by the appearance of a single peak when subjected to gradient, reversed-phase high-performance liquid chromatography. According to its amino acid composition, the 79-kDa reductase is a highly acidic and hydrophobic protein, composed of 695 residues.     

Estrogen sulfotransferase of mouse placenta: behaviour and characteristics during partial purification

Mouse placental estrogen sulfotransferase (ST) was partially purified by fast protein liquid chromatography (FPLC) gel filtration in combination with FPLC anion exchange. Owing to the highly unstable nature of the enzyme, large increases in specific activity were not obtained. Storage of the ST in the presence of thiol groups at -20 degrees C stabilized the enzyme considerably. Forty-three percent of the cytosolic ST was bound to an Affi-Gel blue column and eluted as a broad peak at approximately 0.8 M NaCl. The use of the latter procedure, in combination with FPLC gel filtration, did not increase the specific activity substantially. Larger increases in specific activity were obtained using agarose-hexane-adenosine-3',5'-diphosphate affinity chromatography. The bound ST activity was eluted under a single peak at 1 mM ADP. Increases in specific activity following use of this column averaged 54-fold but could reach 90-fold. Attempts at further purification of this material resulted in low recovery and decreased specific activity. Velocity versus substrate concentration curves show that estrone and particularly estradiol inhibit the partially purified mouse placental sulfotransferase above 0.1-0.25 microM substrate concentrations.     

Purification and biochemical characterization of alpha 2-adrenergic receptor from the rat adrenocortical carcinoma

The alpha 2-adrenergic receptor was purified from rat adrenocortical carcinoma 494 by an affinity chromatographic step using a novel para-aminoclonidine-sepharose resin followed by a gel-permeation high performance liquid chromatographic step. The iodinated receptor protein was homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by high performance liquid chromatography. Both SDS-PAGE and high performance liquid chromatographic studies revealed that Mr of the protein was 64,000, suggesting the monomeric nature of the receptor protein. The purified protein showed the typical binding characteristics of alpha 2-adrenergic receptor.     

Isolation, characterization and immunocytochemical localization of bovine trophoblast protein-1

Bovine trophoblast protein-1 (bTP-1) was isolated to 90% purity from culture medium of Day 18-20 conceptuses incubated in vitro (in the presence of L-[3H]leucine) by a combination of Sephacryl S-200 gel filtration chromatography and O-(diethylaminoethyl) (DEAE) anion-exchange high-performance liquid chromatography (DEAE-HPLC). The radiolabeled protein had an Mr of 21,200 +/- 800 by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had three isoelectric variants (pI 5.7-6.5) by two-dimensional SDS-PAGE. DEAE-HPLC-enriched bTP-1 cross-reacted with anti-o TP-1 serum on Western blots. A monospecific antiserum against bTP-1 was produced by excising the bTP-1 polypeptide band from preparative SDS-PAGE gels. Antiserum reacted with a single polypeptide with an Mr of 21,200 as determined by Western blotting of SDS-PAGE-separated conceptus medium proteins and by immunoprecipitation from L-[35S]methionine-labeled medium proteins followed by SDS-PAGE and autoradiography. Bovine trophoblast protein-1 was localized by immunocytochemistry in the cytoplasm of both mono- and binuclear trophectoderm cells of Day 20 bovine conceptuses, indicating that it is a product of the trophoblast.     

Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain

Aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp-NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp-NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated a single large protein (approximately 670 kDa), which on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi-subunit complex Asp-NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) treatment followed by size-exclusion chromatography. Asp-NAT showed an order of magnitude difference in Km between l-aspartate (l-Asp, approximately 0.5 mm) and acetyl CoA (approximately 0.05 mm). Asp-NAT showed high specificity towards l-Asp with 3% or less activity towards l-Glu, l-Asn, l-Gln and Asp-Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed.     

Estrogen-mediated induction of a vitellogenin-specific nonhistone chromatin protein in the male chicken liver

Summary Non-histone chromatin proteins prepared from the livers of estrogen-treated and nontreated male chickens were compared by reverse-phase high performance liquid chromatography (RP-HPLC), followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results revealed that the hormone-treated male liver chromatin contained a specific protein corresponding to the vitellogenin-specific protein previously purified from the liver of egg-laying hens (Nakayama 1985). The chicken protein, purified further by gel-filtration high performance liquid chromatography (GF-HPLC), showed specific binding activity to DNA fragments carrying a part of the vitellogenin gene. On the basis of similarities in the elution patterns from GF-HPLC and RP-HPLC as well as in the mobility on SDS-PAGE, we concluded that this hormone-induced protein in the male chicken liver was identical to the vitellogenin-specific protein identified in the hen liver, and assumed it to be a specific regulatory protein for the vitellogenin gene expression. The amino acid composition of this chicken protein has been determined.     

Interferon-gamma modulates protein kinase C activity in murine peritoneal macrophages

The content of Ca2+-, phospholipid-dependent protein kinase activity (protein kinase C) in murine peritoneal macrophages treated with recombinant interferon-gamma (IFN-gamma) has been investigated. Protein kinase C activity was solubilized by nonionic detergent extraction of sonicated cells and separated by high performance liquid chromatography on a TSK 4000 SW gel filtration column. The enzyme eluted from the column in a molecular weight range of 60-80 X 10(3) and was identified by virtue of Ca2+ and phospholipid requirements. Macrophages treated with recombinant IFN-gamma exhibited a substantial increase in total protein kinase activity which could be accounted for entirely by increased protein kinase C activity. This activity was enhanced as much as 5-fold over that seen in untreated macrophages and was specific for IFN-gamma in that other agents known to signal changes in macrophage function had no effect. The time required for the elevation of kinase activity was identical to that required for induction of other functions by IFN-gamma in macrophages. These observations suggest that protein kinase C may be a focus of regulatory action in IFN-gamma-mediated macrophage activation.     

Purification and partial characterization of an acid phosphatase from the body wall of sea cucumber Stichopus japonicus

A high molecular weight (HMW) acid phosphatase from the body wall of sea cucumber Stichopus japonicus was purified to homogeneity by a combination of anion exchange chromatography, gel filtration chromatography and high performance liquid chromatography (HPLC). The enzyme was purified 19.3-fold with a total yield of 1.2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of MW 147.9 kDa. The enzyme displayed maximum activity at pH 4.0 and 50 °C with p-nitrophenyl phosphate as substrate. The enzyme activity appeared to be stable over pH 2.0–5.0 and up to 40 °C. The enzyme activity was enhanced slightly by Mg²⁺, whereas inhibited strongly by Cu²⁺ and Zn²⁺. The enzyme hydrolyzes several phosphate esters, suggesting a probable non-specific nature. The amino acid sequences of three segments of the purified enzyme were analyzed by mass spectroscopy, which did not have any homology with previously described acid phosphatase.     

Purification of glyoxysomal polypeptides

Summary A strategy for the rapid purification of proteins from glyoxysomes of castor bean (Ricinus communis cv. Hale) is described. The first step was to separate the proteins in the mixture on the basis of hydrophobicity by reversed phase high performance liquid chromatography using a gradient of increasing acetonitrile concentration. Individual protein peaks were collected and fractionated according to molecular mass by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified polypeptides were used to produce monospecific, polyclonal antibodies. One of these, an anti-catalase antibody, has been employed to assess the subcellular distribution of catalase in endosperm of maturing seeds, dry seeds and seedlings. During seed maturation 45% of the catalase activity was associated with structures sedimenting at high isopycnic densities (1.21 g/cm³). However, in dry seeds, only 6% or less of the catalase activity was associated with these dense particles. In 4-day seedlings 80% of catalase activity was associated with glyoxysomes (1.24 g/cm³). A novel catalase 59 kDa subunit was found in the cytosol of 4-day seedlings and in isolated organelles from maturing and dry seed.Abbreviations AN acetonitrile - CBBR Coomassie brilliant blue R-250 - HPLC high performance liquid chromatography - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor

A naturally occurring inducer of terminal differentiation in a murine myelomonocytic leukemia cell line (WEHI-3B) was purified to apparent homogeneity from medium conditioned by lungs from mice injected with bacterial endotoxin. The factor was purified over 400,000-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Bio-Gel P-60 in 1 M acetic acid, reverse-phase high performance liquid chromatography on a phenyl-silica column, and high performance liquid chromatography on a gel filtration column. During the first two steps, the differentiation-inducing factor was separated completely from a known proliferative regulator for normal myeloid cells, granulocyte-macrophage colony-stimulating factor, but it co-purified through all remaining steps with a distinct granulocyte-specific colony-stimulating factor. The purified factor showed a single protein band of Mr = 24,000-25,000 on sodium dodecyl sulfate-polyacrylamide gels coincident with both differentiation-inducing and granulocyte colony-stimulating activity. The granulocyte-specific colony-stimulating factor was active on WEHI-3B cells and normal granulocytic progenitor cells in vitro at the same half-maximally active concentration of 3 X 10(-12) M.     

Identification of a major GTP-binding protein in bovine aortic smooth muscle cytosol as the rhoA gene product

In bovine aortic smooth muscle, about 50% of total GTP-binding activity was present in the cytosol fraction. A major GTP-binding protein (G protein) with a Mr value of about 21,000 (21K G) in this fraction was purified to near homogeneity and characterized. 21K G bound maximally about 0.8 mol of [35S]guanosine 5'-(3-O-thio)triphosphate/mol of protein with a Kd value of about 20 nM. 21K G showed GTPase activity with a turnover number of about 0.007 min-1. 21K G was ADP-ribosylated by botulinum ADP-ribosyltransferase and about 0.4 mol of ADP-ribose was maximally incorporated into 1 mol of 21K G. 21K G and the bovine brain rhoA gene product (rhoA p21) were eluted at the same retention time on C4 reversed-phase high performance liquid chromatography and migrated at the same positions on two-dimensional gel electrophoresis. These results indicate that the major G protein in bovine aortic smooth muscle cytosol is rhoA p21.     

Interleukin 1 increases collagen type IV production by murine mammary epithelial cells

The effects of human interleukin 1 (IL 1) on collagen type IV production by normal mouse mammary epithelial cells were examined. Human IL 1 was derived from the culture media of peripheral blood monocytes or placental cells that were stimulated with silica. Although crude culture media of silica-stimulated monocytes or placental cells had no enhancing activity for type IV collagen production, IL 1-containing fractions obtained by Sephacryl S-200 gel chromatography and isoelectrofocusing from such media possessed considerable activity. To confirm the effects of IL 1 on collagen production, human monocyte-derived IL 1 was highly purified by sequential isoelectrofocusing, anion-exchange (AX 300), high-performance liquid chromatography (HPLC), and HPLC gel filtration (TSK 3000). The same HPLC gel filtration fractions contained both an activity that stimulated collagen synthesis by mammary cells and thymocyte growth-promoting activity. These activities of IL 1 differed from a number of other factors, such as epidermal growth factor and another factor produced by placental cells that stimulated type IV collagen production but not thymocyte proliferation. In fact, IL 1 induced 100-fold less collagen type IV production by mammary epithelial cells than was needed to induce thymocyte proliferation. Our data suggest that IL 1-like molecules, which reportedly are produced by many tissue cell types, may therefore play a role in promoting a basement membrane formation at stromal-epithelial boundaries.     

Column chromatography and electrophoresis of bovine uterine lumenal proteins collected during the estrous cycle

Uteri of 29 normally cycling Holstein and Jersey cows were non-surgically flushed with 50 ml sterile 1.5% saline, and fluids were recovered to evaluate biochemical methods for determination of qualitative changes in uterine lumenal protein at known stages of the estrous cycle. Total protein (mg) and number of red blood cells (million/ml) were 17.7 and 9.8; 7.6 and 7.1; 9.1 and 6.0; and 26.2 and 2.8 at day 0, 5, 10 and 15 of the bovine estrous cycle. Column chromatography (Sephacryl S-200) of uterine secretions revealed seven uterine specific peaks at ambient temperatures. One peak may be a hemoglobin contaminant. Five uterine specific protein peaks representing proteins greater than 160 000, ～ 25 000 and less than 13 700 mol. wt. (3) were eluted with high performance liquid chromatography. Native polyacrylamide disc gel electrophoresis fractionated uterine fluid into as many as 13 bands. There were differences in six protein bands between uterine fluid and plasma. The consistency between Sephacryl S-200 and high performance liquid chromatography is the presence of three to four low molecular weight (< 13 700) uterine specific proteins. Sephacryl S-200 chromatography resulted in elucidation of a uterine specific protein approximately 60 000 mol. wt. not found with high performance liquid chromatography. However, proteins with mol. wt. > 160 000 and approximately 25 000 were found with high performance liquid chromatography. Results indicate no differences in protein class during the estrous cycle and that red blood cell contamination must be monitored during qualitative evaluation of uterine proteins.     

Purification of tyrosine hydroxylase by high-pressure liquid chromatography

Our study of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) from rabbit adrenals has identified two major requirements which are likely to be of general application for the optimal purification and recovery of enzyme activity consequent to high-pressure liquid chromatography: (i) recovery of activity is maximized by pretreatment of the high-pressure liquid chromatography column before each use with protein to saturate high affinity, nonspecific sites exposed by the methanol used for washing, and storage of the column. (ii) Both purification and recovery are critically dependent upon the molarity of the mobile phase buffer. Examination of high-pressure liquid chromatography purified rabbit adrenal tyrosine hydroxylase by nondenaturing gel electrophoresis indicated that tyrosine hydroxylase activity was associated with one of the two protein bands in the gel. Thus, the convenient purification procedure described in this report leads to preparative amounts of tyrosine hydroxylase which is approximately 50% homogeneous.     

Purification of a multipotential colony-stimulating factor from pokeweed mitogen-stimulated mouse spleen cell conditioned medium

A factor able to stimulate the proliferation and differentiation of multipotential stem cells and progenitor cells of the granulocyte-macrophage, eosinophil, and erythroid lineages as well as being able to maintain factor-dependent cell lines in culture has been purified from pokeweed mitogen-stimulated mouse spleen cell-conditioned medium. The factor was purified over 2 million-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-Sepharose, reverse-phase high performance liquid chromatography on a phenyl-silica column, and gel permeation high performance liquid chromatography. All of the biological activities ascribed to the multipotential colony-stimulating factor co-fractionated through all steps, and the other known mouse-active hemopoietic regulator in pokeweed mitogen-stimulated mouse spleen cell-conditioned medium, granulocyte-macrophage colony-stimulating factor, was separated at the ion exchange step. Two protein species having Mr = 24,000 and 19,000 were visualized by silver-staining of sodium dodecyl sulfate-polyacrylamide gels of the purified factor. Both species migrated coincidently with the biological activities. The factor was active at a half-maximal concentration of 1 X 10(-13) M when assayed on a factor-dependent cell line.