Isolation and some properties of a new binding protein for asialoorosomucoid from rat liver

Binding proteins for asialoorosomucoid were prepared from rat liver previously labeled in vivo with [3H]leucine by affinity chromatography on asialoorosomucoid-Sepharose 4B. They were subjected again to the same affinity chromatography and eluted into two fractions successively with 10 mM Tris-HCl buffer, pH 7.8, containing 1.25 M NaCl, 1% Triton X-100 and 50 mM lactose and 20 mM ammonium acetate buffer, pH 6.0, containing 1.25 M NaCl and 1% Triton X-100, and designated as ABP-I and ABP-II (asialoorosomucoid binding proteins), respectively. ABP-I corresponds to the receptor protein specific for asialoglycoproteins which has been extensively investigated by Ashwell and collaborators (J. Biol. Chem. 254, 1038-1043, 1979). ABP-II is different from ABP-I in several properties such as molecular weight, antigenicity and solubility. The molecular weight of ABP-II was estimated to be 29,000 by SDS-PAGE. On gel filtration it behaved as a pentamer with an apparent molecular weight of 150,000. Unlike ABP-I, ABP-II showed no detectable binding activity when assayed according to the procedures of Hudgin et al. (J. Biol. Chem. 249, 5536-5543, 1974). The calcium ion was, however, essential for the binding of ABP-II to asialoorosomucoid-Sepharose 4B similar to ABP-I. ABP-II can be extracted from the total microsomes of rat liver in 1.0 M NaCl by sonication after freezing and thawing. This suggests that ABP-II is either a soluble protein or a peripheral membrane protein loosely attached to the intracisternal cavities of the microsomal membranes.     

Characterization and purification of the hyaluronan-receptor on liver endothelial cells.

In order to characterize the proteins on liver endothelial cells that bind hyaluronan (HYA), liver endothelial cells were surface-iodinated with 125I, solubilized by Triton X-100 and passed through a column containing HYA coupled to agarose. The column was washed and eluted with HYA-oligosaccharides. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted material, followed by autoradiography, showed a major band with a molecular mass of 100 kDa, that upon reduction gave major bands of 20 and 35 kDa, and minor doublet bands at 60 and 80 kDa. Two-dimensional electrophoresis of liver endothelial cell membrane proteins revealed that the 100 kDa protein has a pI of 6.6-6.8. The protein was purified by preparative SDS-PAGE of liver endothelial cell membrane proteins. The 100 kDa protein was excised from the gel and used for immunization of rabbits. Antiserum from immunized rabbits specifically recognized only the 100 kDa protein on immunoblots of liver endothelial cell membrane proteins separated by SDS-PAGE. The binding of 3H-HYA to liver endothelial cells and liver endothelial cell membranes could be specifically inhibited by Fab-fragments of the antibodies. When we tried to isolate the receptor in large scale by affinity chromatography of proteins from purified liver endothelial cell membranes, the 100 kDa protein could often not be detected on immunoblots or by silver staining following SDS-PAGE of the eluted material. Instead, proteins with molecular masses of 55 and 15 kDa were detected, but the antibodies reacted specifically with these proteins. Thus the 100 kDa protein is apparently susceptible to cleavage into distinct subcomponents.     

Membrane-bound heparin binding proteins from HL-60 cells purified in a two-step affinity chromatography differentially eluted with divalent cations

Solubilized membrane proteins from HL-60 cells were separated by two-step affinity chromatography. Proteins eluted with MgCl2 in the first heparin-gel were applied to the second heparin-gel and eluted with CaCl2. The eluted proteins were analysed and purified by electrophoresis. N-terminal amino acid sequences of eight proteins on the characteristic bands were determined. Homology search for the sequences indicated that three microsomal proteins, two nuclear proteins and a glycolytic enzyme were eluted with divalent cations, whereas a nuclear ribonucleoprotein and a membrane-cytoskelton linker protein were not dissociated with divalent cations, but with 2 M NaCl. Heparin affinity chromatography combined with differential elution with divalent cations can be a useful method for separation of membrane proteins.     

The fractionation of non-histone chromosomal proteins from rat kidney and liver and their DNA complexes by using the cation exchangers phosphocellulose and SE-Sephadex]

Results of the gradient chromatography of chromosomal nonhistone proteins of the rat liver and kidney and their complexes with DNA on the phosphocellulose and SE-Sephadex are presented. The profiles of elution of the preparation of the homologous tissues with NaCl linear gradient of 0.1-1.0 M are equal. In the fractions of 0.4-0.5 M NaCl, protein components are discovered only in the samples of kidney.     

Apoptosis induced in neuronal cells by C-terminal amyloid ß-fragments is correlated with their aggregation properties in phospholipid membranes

Rat brain proteins presenting high-affinity binding of S-adenosyl-L-homocysteine were solubilized and purified. Extraction of binding protein was carried out in the presence of Triton X-100 and 1 M NaCl; this solubilized fraction exhibits similar kinetic properties than the membrane proteins. Purification was performed using affinity chromatography on S-adenosyl-L-homocysteine carboxyhexyl Sepharose 48 conjugate. The analysis of the affinity gel eluate by SDS-PAGE showed high purification ratios for two proteins exhibiting 54 and 68 kDa. Three activity peaks were separated when solubilized membrane proteins were submitted to isoelectric focusing; the activity peaks corresponded to proteins of pH, 6.0, 6.5, and 7.2. SDS-PAGE separation of proteins contained in each peak showed protein aggregation; a 54-kDa subunit was present in each aggregate. Solubilized membrane proteins were labeled by photoaffinity labeling with tritiated S-adenosyl-L-homocysteine; the 54-and 68-kDa proteins were found among the specifically labeled proteins. Finally, according to the previous data from the literature, the purified S-adenosyl-L-homocysteine binding proteins do not seem to be the same as adenosine receptors or phosphatidylethanolamine-N-methyltransferase.     

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) causes increases in protein kinases particularly protein kinase C in the hepatic plasma membrane of the rat and the guinea pig

To study the cause of TCDD-evoked changes in the functions of plasma membrane constituents TCDD's effects on protein kinase activities in the liver of rats and guinea pigs were investigated. TCDD was found to cause a sharp increase in both c-AMP independent and dependent protein kinase activities in plasma membrane preparations from rat liver within 48 hours from the time of administration. Such effects reached maxima around day 20, and were quite noticeable even 40 days after a single administration of TCDD. As a result of SDS-polyacrylamide gel-electrophoresis (SDS-PAGE) analysis several substrate proteins for these increased protein kinases were observed. Among them are 170 K - 150 K bands, representing EGF receptor protein. TCDD was found to particularly stimulate protein kinase C which is known to influence many enzyme and receptor functions through protein phosphorylation. The possible significance of such an action of TCDD is discussed.     

Liver fatty acid-binding protein initiates budding of pre-chylomicron transport vesicles from intestinal endoplasmic reticulum

The rate-limiting step in the transit of absorbed dietary fat across the enterocyte is the generation of the pre-chylomicron transport vesicle (PCTV) from the endoplasmic reticulum (ER). This vesicle does not require coatomer-II (COPII) proteins for budding from the ER membrane and contains vesicle-associated membrane protein 7, found in intestinal ER, which is a unique intracellular location for this SNARE protein. We wished to identify the protein(s) responsible for budding this vesicle from ER membranes in the absence of the requirement for COPII proteins. We chromatographed rat intestinal cytosol on Sephacryl S-100 and found that PCTV budding activity appeared in the low molecular weight fractions. Additional chromatographic steps produced a single major and several minor bands on SDS-PAGE. By tandem mass spectroscopy, the bands contained both liver and intestinal fatty acid-binding proteins (L- and I-FABP) as well as four other proteins. Recombinant proteins for each of the six proteins identified were tested for PCTV budding activity; only L-FABP and I-FABP (23% the activity of L-FABP) were active. The vesicles generated by L-FABP were sealed, contained apolipoproteins B48 and AIV, were of the same size as PCTV on Sepharose CL-6B, and by electron microscopy, excluded calnexin and calreticulin but did not fuse with cis-Golgi nor did L-FABP generate COPII-dependent vesicles. Gene-disrupted L-FABP mouse cytosol had 60% the activity of wild type mouse cytosol. We conclude that L-FABP can select cargo for and bud PCTV from intestinal ER membranes.     

Multiple aflatoxin B1 binding proteins exist in rat liver cytosol

The in vitro binding of aflatoxin B1 to rat liver cytosolic proteins was investigated. Aflatoxin B1 binding activity was assayed with protein purified by gel permeation chromatography, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Twenty-five percent of the total binding activity was associated with proteins eluted by 0 and 0.1 M NaCl. Over 50% of the total binding activity was associated with protein present in the 0.2 M NaCl fraction. Glutathione S-transferase activity was also monitored and found only in the low salt (less than 0.2 M NaCl) fractions. The proteins eluted by 0.2 M NaCl were further purified by hydroxylapatite column chromatography and binding was found predominantly in a single fraction. The protein purification steps resulted in a 20-fold increase in the specific binding activity over that initially observed in the cytosol. These results indicate that multiple proteins are capable of binding aflatoxin B1 in rat liver cytosol.     

Binding proteins for linear renin-inhibiting peptides in basolateral plasma membranes of rat liver.

A linear hydrophobic peptide, (Code no. EMD 55068), a synthetic renin-antagonist, competitively inhibits the uptake of taurocholate and of another linear peptide (EMD 51921) but not of oleic acid, serine or thiamin hydrochloride into isolated rat liver cells. EMD 55068 was attached to a gel matrix at a position that is not involved in the protein ligand interaction. The gel matrix used did not interact nonspecifically with solubilized proteins from rat liver. The quantity of bound ligand was determined to be 3.6 mg/ml of gel matrix. In the fraction of EDTA extracted hydrophilic membrane-associated proteins, no binding proteins were detected. Affinity chromatography of integral plasma membrane proteins resulted in four protein bands with molecular masses of 46, 49, 53 and 56 kDa in SDS-PAGE. In contrast, solubilized plasma membrane proteins from AS-30D ascites hepatoma cells, which are unable to transport bile acids and linear peptides, did not bind specifically to the affinity matrix.     

Involvement of a non-hormone-binding 90-kilodalton protein in the nontransformed 8S form of the rabbit uterus progesterone receptor

Nontransformed 8S progesterone receptor (8S-PR) was purified by hormone-specific affinity chromatography from rabbit uterine low-salt cytosol containing 20 mM molybdate. In the eluate obtained with radioactive progestin, sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) showed the presence of several bands, including three that corresponded to approximately 90-, approximately 120-, and approximately 85-kDa proteins. None of these three proteins was found in the eluate of the affinity column when the molybdate-containing cytosol was chromatographed in the presence of nonradioactive progesterone ("mock purification"). Subsequent purification of the affinity eluate by DEAE-Sephacel chromatography gave a single radioactive receptor peak at 0.15 M KCl (approximately 20% yield, 19% purity on the basis of one binding site per approximately 100 kDa) with a sedimentation coefficient of 8.5 S. Silver staining after SDS-PAGE revealed that this purified 8S-PR fraction contained mainly the 120-, 90-, and 85-kDa proteins. [3H]R5020-labeled 8S-PR purified by DEAE-Sephacel column chromatography was UV irradiated, and after SDS-PAGE the 120- and 85-kDa proteins were revealed, but the 90-kDa protein was not.(ABSTRACT TRUNCATED AT 250 WORDS)     

SDS-PAGE分析肝癌细胞和正常肝细胞膜蛋白

目的:采用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)方法获得肝癌细胞株HepG2和人正常肝细胞株L-02蛋白质表达谱,期待找到与肝癌细胞相关的差异蛋白。方法:体外培养肝癌细胞株HepG2和正常肝细胞株L-02,分离提取细胞膜蛋白,用SDS-PAGE分析细胞膜蛋白。结果:2种细胞膜蛋白的疏水相和亲水相蛋白区带位置基本相同,但肝癌细胞株HepG2膜蛋白在相对分子质量66×103处有较清晰的异常蛋白区带。结论:肝癌细胞HepG2除了具有与正常肝细胞相同的组成物质外,还有自己独特的蛋白质表达谱,这一实验可为癌症的研究提供一定的参考。     

Hyaluronate-Binding Proteins of Murine Brain

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.     

Identification of the milk fat globule membrane proteins. II. Isolation of major proteins from electrophoretic gels and comparison of their amino acid compositions

The fat globule membranes of milk are derived from the apical plasma membrane of the mammary secretory cells. The nature of the membrane proteins, as isolated from cows' milk, has been studied by the use of discontinuous and continuous SDS-gel electrophoresis. Six methods of preparation of milk fat globule membrane suggested by various authors were tested; gel electrophoresis showed that five major bands were present, independent of the method of preparation. The apparent molecular masses of these proteins as determined on SDS-gels (15% T) were 167, 142, 64, 49 and 46 kDa, respectively. The 167 kDa band stained only with periodic acid-Schiff reagent, while the 142 kDa band stained only with Coomassie blue; the last three bands stained with both. Delipidated membranes were extracted stepwise with water, 0.02 M NaCl and 0.6 M NaCl. The 64 kDa band appears to be nearly insoluble, while the bands of 142, 49 and 46 kDa are fractionated by this procedure. The resolution of all of these proteins by electrophoresis was superior to that achieved by molecular sieve chromatography, and so electrophoretic extraction was used to isolate the major proteins. Dansyl chloride derived proteins were used as markers. Amino acid compositions of the recovered proteins were obtained and are compared.     

Precipitation of dilute chromatographic samples (ng/ml) containing interfering substances for SDS-PAGE

SDS-PAGE of chromatographic fractions requires prior removal of salts, detergents, denaturants, or organic solvents which may perturb the electrophoretic separation. Likewise, to successfully visualize minute amounts of protein present in chromatographic fractions, they must often be concentrated before analysis by SDS-PAGE. In this study, we used a dye precipitation procedure for simultaneous removal of interfering substances and concentration of dilute samples (ng/ml) before analysis by SDS-PAGE. Nanogram amounts of protein (143 ng) were effectively precipitated with a pyrogallol red-molybdate reagent from commonly used chromatographic buffers containing various interfering solutes or solvents. Proteins were successfully precipitated from solution in the presence of organic solvents (acetonitrile, methanol, 2-propanol), chaotropic agents (6 M urea, 6 M guanidine-HCl), a protein stabilizer (40% sucrose), metal chelators (30 mM EDTA and 30 mM EGTA), or high salt (1.0 M NaCl). Detergents, at concentrations up to twice their critical micelle concentrations, from the nonionic class (Triton X-100, Tween 20) or from the zwitterionic class (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) did not inhibit protein precipitation. Some interference was observed when proteins were precipitated in the presence of ammonium sulfate (0. 5-2.0 M). Proteins did not precipitate in the presence of ionic detergents (SDS and cetyltrimethylammonium bromide). The sensitivity of the combined pyrogallol red-molybdate precipitation/SDS-PAGE procedure is approximately 7 ng. Two other methods of precipitating proteins (trichloroacetic acid and phenol-ether) both exhibited varying degrees of effectiveness, ranging from 714 to 7 ng/ml, in the precipitation of individual proteins. In summary, the pyrogallol red-molybdate protein precipitation procedure facilitates the SDS-PAGE analysis of dilute protein samples (ng/ml) from chromatographic fractions of various compositions. The method is useful for rapid pilot-scale protein fractionation and facilitates the ongoing propensity of researchers to work with minuscule amounts of protein.     

Nuclear proteins. VI. Fractionation of chromosomal non-histone proteins using hydrophobic chromatography.

Mouse liver non-histone proteins, isolated by hydroxyapatite chromatography, were fractionated by hydrophobic chromatography using omega-amino-decyl-agarose omega-amino butyl-agarose, decyl-agarose, butyl-agarose, phenyl-Sepharose, and CPAD-Sepharose. Two column loading techniques were used. In the 0.35 M NaCl technique, the proteins were dialized into 0.35 M NaCl, applied to the column and initially eluted with 0.35 M NaCl. In the 40% (NH4)2SO4 technique, the non-histone proteins were mixed with the hydrophobic agarose, dialized against 40% (NH4)2SO4, and initially eluted with 40% (NH4)2SO4. In both cases the columns were subsequently eluted with 10 mM Tris-HCl, pH 7.5, 0.35, 1.0 and 5.0 M LiBr, and finally with 1% sodium dodecyl sulfate. The 0.35 M NaCl technique, using decyl-agarose and phenyl-Sepharose, resulted in a single step marked enrichment of the major hnRNA proteins (1 M LiBr fraction). The 40% (NH4)2SO4 technique resulted in a single step isolation of a pair of 15-20 000 dalton polypeptides.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Nuclear proteins. V. Studies of histone-binding proteins from mouse liver by affinity chromatography

We examined four extracts of mouse liver for histone-binding proteins using histone affinity chromatography and positively charged resins. The extracts used were cytoplasm and washes from isolated nuclei with buffers containing 0.05 M Tris, 0.15 M NaCl or 0.35 M NaCl. Proteins from the nuclear washes showed greater binding to the columns than proteins from the cytoplasm. The binding fractions were heterogeneous in gel electrophoresis systems. Proteins bound to affinity columns of individual histones were similar to those bound to columns of whole histone, polylysine and DEAE. A 25,000 dalton polypeptide (J2), found only in nuclear washes was a prominent histone-binding protein. It could be competitively eluted from DEAE with histones, suggesting polypeptide J2 may show a specific affinity for histones. Polypeptide J2 has an acidic to basic amino acid ratio of 1.58, and its amino acid composition is not similar to that of the high mobility group 1 protein. Polypeptide J2 binds to hydrophobic columns and may play a role in modifying histone-histone and histone-DNA interactions.     

Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH

While phosphoproteins have attracted great interest toward the post-genome research (e.g. clinical diagnosis and drug design), there have been few procedures for the specific enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a phosphate-binding tag molecule (i.e. a dinuclear zinc(II) complex) attached on a highly cross-linked agarose. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 1.0 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate-affinity chromatography using an epidermal growth factor-stimulated A431 cell lysate. The total time for the column chromatography (1 mL gel scale) was less than 1 h. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS-PAGE followed by Western blotting analysis.     

中度噬盐菌Bacillus sp.Ⅰ121的质膜蛋白质组学分析

膜蛋白是连接细胞与外界环境的重要载体,发挥着物质转运、信号传导等作用。通过对一种新分离出来的中度噬盐菌Bacillussp.Ⅰ121进行了质膜蛋白质组学分析,盐胁迫下该菌株主要靠积累脯氨酸来维持细胞渗透压。在对Bacillussp.Ⅰ121在不同盐浓度下的生长情况进行测定的基础上,我们选取了1%,5%,10%,15%（w/v）NaCl4个盐浓度进行细胞培养,并分别提取细菌的质膜,通过5%～20%的梯度凝胶SDS-PAGE电泳将疏水的膜蛋白进行分离,随后对盐胁迫响应的8条不同的蛋白条带进行了ESI-LC-MS/MS质谱分析。将所得结果在NCBI数据库中进行比对,最终成功鉴定出10种蛋白。这些蛋白涉及物质跨膜转运,膜的生物合成以及胁迫信号转导等功能。为膜蛋白在盐胁迫响应过程中的作用方面的研究开辟了新思路。     

A novel trypsin inhibitor from Peltophorum dubium seeds,with lectin-like properties,triggers rat lymphoma cell apoptosis

A trypsin inhibitor (PDTI) was isolated from Peltophorum dubium seeds by affinity chromatography on a thyroglobulin-agarose or a trypsin-agarose column. In both cases, SDS-PAGE showed two bands of M(r) 20,000 and 22,000, which could not be resolved. Their amino-terminal sequences were identical and similar to that of Kunitz-type soybean trypsin inhibitor (SBTI). Mass spectrometry analysis of tryptic digests of both bands showed 16 coincident peaks, suggesting that they are closely related proteins. The K(i)s for trypsin and chymotrypsin inhibitory activity of PDTI were 1.6 x 10(-7) and 1.3 x 10(-5)M, respectively. Lectin-like activity of PDTI and SBTI, detected by hemagglutination of rabbit erythrocytes, was inhibited by sialic acid-containing compounds. PDTI and SBTI caused apoptosis of Nb2 rat lymphoma cells, demonstrated by decrease of viability, DNA hypodiploidy, DNA fragmentation, and caspase-3-like activity. They had no effect on normal mouse splenocytes or lymphocytes, whereas they caused apoptosis of concanavalin A-stimulated mouse lymphocytes.