Purification of a soluble ATPase from rat liver mitochondria by AMP-Sepharose affinity chromatography.

ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity was shown in the soluble fraction of rat liver micochondria. Two molecular forms (ATPase 1 and 2) were isolated. ATPase 1 has already been studied. The present paper deals with the purification method of ATPase 2 which was achieved by the following steps: (NH4)2SO4 precipitation. DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G100 filtration and AMP-Sepharose affinity chromatography. The purified protein was characterized by bidimensional polyacrylamide gel electrophoresis. Molecular weight evaluated by SDS-polyacrylamide gel electrophoresis and Sephadex G100 gel filtration was found to be 61 500 +/- 3000.     

Isolation of neuronal parvalbumin by high-performance liquid chromatography. Characterization and comparison with muscle parvalbumin

Neuronal parvalbumin has been isolated from rat brain and purified to homogeneity by high-performance liquid chromatography (HPLC) on reverse-phase supports. This procedure includes four consecutive chromatographic steps with an overall protein recovery of 74% and a 26 400-fold purification. The concentration of parvalbumin was found to be approximately 10 mg/kg wet weight in brain tissue, which is about 100 times lower than that in rat muscle. The physical properties of brain parvalbumin are described and compared with those of the muscle counterpart. These proteins were identical in their molecular weights (12 000), isoelectric points (4.9), retention times on C-18 reverse-phase HPLC columns, Ca2+ content (two per molecule), amino acid compositions, and immunological properties. A comparison of the tryptic peptide maps of brain and muscle parvalbumin by analytical HPLC also revealed identity and showed that the isolation method described here did not alter the chemical structure of the protein.     

Purification of basic fibroblast growth factor from rat brain: identification of a Mr 22,000 immunoreactive form

A 18,000-dalton protein which stimulates plasminogen activator (PA) activity in endothelial GM 7373 cells has been purified from rat brain by using heparin affinity chromatography and ion-exchange chromatography. The purified molecule stimulates PA activity in a dose-dependent manner between 1 and 30 ng/ml. It also stimulates proliferation of GM 7373 cells and DNA synthesis in NIH 3T3 cells in a similar concentration range. The molecule has been identified as a bFGF-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with anti-human bFGF antibodies. In the final preparation of the rat brain bFGF, trace amounts (less than 5%) of a contaminant were detectable. This contaminant has a molecular weight of 22,000 and cross reacts with several anti-human placental bFGF antibodies. On the basis of its affinity for heparin-Sepharose and its immunological characteristics, this protein appears to be an high molecular weight form of bFGF.     

High-performance liquid chromatography of proteins: Purification of the acidic isozyme of adenylosuccinate synthetase from rat liver

High-performance ion-exchange liquid chromatography was utilized for the purification of the acidic isozyme of adenylosuccinate synthetase from rat liver. Initial steps in the purification included ammonium sulfate fractionation and DEAE-cellulose and agarose-GTP affinity columns. The final steps were done on a SynChropak AX-300 anion-exchange support. The enzyme was purified 3000-fold with an overall yield of 10%. The enzyme preparation exhibited only one protein band on gel electrophoresis.     

Characterization of Adenylate Cyclase Purified from Rat Brain by Hydrophobic Chromatography

Abstract. Hydrophobic chromatography of detergent-solubilized rat brain adenylate cyclase on dodecyl-Sepharose produced a species that was soluble in the absence of detergent and could be manipulated like a conventional hydrophilic protein. Sevenfold purification was achieved by this technique. Further purification could then be effected by affinity chromatography on ATP-Sepharose. The purified enzyme was no longer sensitive to fluoride or guanyl nucleotides. No interaction of brain adenylate cyclase was observed with immobilized triazinyl dyes such as Cibacron Blue 3GA nor with concanavalin A-Sepharose. The molecular weight of the fluoride-activated catalytic complex in a freeze-dried membrane preparation was estimated to be 133,000 by irradiation inactivation.     

Neuronal-derived factors regulating glial cell proliferation and maturation

We purified to homogeneity two active factors, named astroglial growth factors (AGFs: AGF2 and AGF1), from bovine brain after two and three chromatographic steps, respectively. We found that AGFs have a strong affinity for heparin. Therefore, heparin affinity chromatography was used to purify rapidly and efficiently these growth factors. The purified AGF1 is an acidic protein (pI: 5.5) with an apparent molecular weight of about 17,500 daltons; the AGF2 is a basic protein (pI: 9.5) of 18,500 daltons. The comparison of the physico-chemical properties, the aminoacid composition and the amino-terminal sequence of the AGFs with that of other growth factors isolated from the brain and affecting the proliferation of other cell types has indicated that AGF1 and AGF2 are identical to the acidic and basic fibroblast growth factor (FGF), respectively. Both factors stimulate the proliferation as well as the morphological and biochemical maturation of the astroglial cells. Both factors enhance also the multiplication of oligodendroglial cells. Polyclonal and monoclonal antibodies against AGFs have been prepared and used for immunocytochemical localization of these molecules in the rat brain and cerebellum. AGFs are found exclusively in neuronal cells.     

Soluble guanylate cyclase from rat lung exists as a heterodimer

The soluble form of guanylate cyclase (EC 4.6.1.2) from rat lung has been purified to homogeneity by a one-step immunoaffinity chromatographic procedure. The purified soluble guanylate cyclase has specific activities of 432 and 49.1 nmol of cyclic GMP formed per min/mg protein with manganese and magnesium ions as a cofactor, respectively. This represents a purification of approximately 2,000-fold with a 50% recovery. The native enzyme has a molecular weight of 150,000 and a Stokes radius of 4.8 nm as determined on Spherogel TSK-G3000SW gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with molecular weights of 82,000 and 70,000. The purified soluble guanylate cyclase was also subjected to native polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, ion exchange chromatography, and GTP-agarose affinity chromatography. These additional purification procedures confirmed the presence of a single protein peak coincident with enzyme activity. The two subunits separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were shown to have different primary structures by immunoblotting with monoclonal and polyclonal antibodies prepared against purified soluble guanylate cyclase and by peptide mapping with papain or Staphylococcus aureus V8 protease treatment. These data demonstrate that soluble guanylate cyclase purified from rat lung is a heterodimer composed of 82,000- and 70,000-dalton subunits with different primary structures.     

Purification of an active opioid-binding protein from bovine striatum

We report the purification to apparent homogeneity of an active opioid-binding protein solubilized from bovine striatal membranes. The purification was accomplished in two steps: affinity chromatography on beta-naltrexylethylenediamine (NED)-CH-Sepharose 4B followed by lectin affinity chromatography on wheat germ agglutinin-agarose. The ligand affinity-purified fraction exhibits stereospecific and saturable binding of opiates and is heat-sensitive. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the NED-purified material gave 6-8 bands by silver staining or autoradiography of radioiodinated material. Under nondenaturing conditions, the NED-purified material elutes in a molecular mass range between 300 and 350 kDa from gel exclusion chromatography on Ultrogel AcA-34. The specific activity of the affinity-purified fraction (800-1500 pmol/mg protein) is enriched 4000 to 7000-fold over that of the membrane-bound or unpurified soluble receptor. Further purification (10-20-fold) is achieved by chromatography of the NED eluate on wheat germ agglutinin-agarose. The eluted fraction shows a single protein (65 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified material is an acidic glycoprotein with a pI of 6.0-6.3 and binds opiates with a specific activity (12,000-15,000 pmol/mg) that is 65,000 to 75,000-fold greater (theoretical, 77,000-fold) than that of the membrane-bound or crude soluble receptors.     

Cellular retinoic acid-binding protein from rat testis. Purification and characterization

Cellular retinoic acid-binding protein has been purified to homogeneity from rat testes. The procedures utilized in the purification included acid precipitation, gel filtration, and chromatography on DEAE-cellulose. The binding protein was purified approximately 12,000-fold, based on total soluble testicular protein. The protein is a single polypeptide chain with a molecular weight of 14,600, determined by information from gel filtration and sodium dodecyl sulfate-polyacrylamide electrophoresis. The protein binds retinoic acid with high affinity; the apparent dissociation constant was determined by fluorometric titration to be 4.2 X 10(-9) M.     

Purification of rat chondrosarcoma xylosyltransferase

The chondroitin sulfate chain-initiating enzyme, UDP-d-xylose:core protein β-d-xylosyltransferase has been purified over 600-fold from the high-speed supernatant fraction of a rat chondrosarcoma. The purification procedure involved differential centrifugation, gel chromatography on Sephadex G-200, and affinity chromatography on a matrix consisting of core protein bound to Sepharose. The purified enzyme was homogenous by electrophoretic and immunological criteria, had a molecular weight between 95,000 and 100,000 and contained approximately 10% carbohydrate. The K_m value for UDP-xylose was 1 × 10^?5, m and for the core-protein acceptor was 330 mg/liter.     

Purification of a new acidic glutathione S-transferase, GST-Yn1Yn1, with a high leukotriene-C4 synthase activity from rat brain

A new acidic form of glutathione S-transferase (GST, pI 6.2) was purified from rat brain by S-hexylglutathione affinity chromatography followed by chromatofocusing. This form occupied 20-25% of the total activity bound to the affinity column. It had a molecular mass (subunit 26 kDa) similar to that of a major GST form of rat testis (MT or 6-6) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, it differed from the MT in isoelectric point, activity towards 1,2-dichloro-4-nitrobenzene and immunological properties. On two-dimensional gel electrophoresis the brain form gave a spot which was identical in molecular mass, isoelectric point and immunological properties to a less acidic one (Yn1) of two spots (Yn1 and Yn2) of the testis GST-MT. Therefore, the brain acidic form is a homodimer, and named GST-Yn1Yn1. The activity was inhibited by sulfasalazine, an inhibitor of leukotriene-C4 synthase. This form (GST-Yn1Yn1) showed the highest leukotriene-C4 synthase activity, 496 nmol/mg protein in 5 min, among nine cytosolic GST isoenzymes from the rat. The Km values for leukotriene A4 and glutathione were 26 microM and 3.5 mM respectively. A major GST form of rat brain, occupying about 40% of the total activity, was identical with GST-P (7-7) purified from rat liver bearing preneoplastic hyperplastic nodules and localized at astroglias. GST-P also showed the significant leukotriene-C4 synthase activity, 67.2 nmol/mg protein in 5 min, but the Km for leukotriene A4 was 100 microM, fourfold higher than that of GST-Yn1 Yn1. These results suggest that mainly GST-Yn1 Yn1 may be involved in leukotriene-C4 synthesis in rat brain.     

High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography

A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.     

人IGF-1在大肠杆菌中的可溶表达和纯化

目的:在大肠杆菌中的可溶表达和纯化人胰岛素样生长因子1(hIGF-1)。方法:根据hIGF-1的氨基酸序列和大肠杆菌密码子偏爱性,利用重叠延伸PCR的方法合成hIGF-1DNA序列,构建表达载体,在大肠杆菌OrigamiB(DE3)中与硫氧还蛋白TrxA融合表达,并通过盐析和镍柱亲合层析进行纯化。结果:SDS-PAGE分析显示,重组融合蛋白以可溶形式存在,分子量约为28kDa,占上清总蛋白的50%以上。经盐析和镍柱亲合层析进行纯化,目标蛋白纯度可达到90%左右。结论:复合干扰素在大肠杆菌中的高效可溶表达。     

Purification and characterization of an Mr 87,000 protein kinase C substrate from rat brain

We (Kligman, D., and Patel, J. (1986) J. Neurochem. 47, 298-303) and others have previously identified a major protein kinase C substrate of apparent molecular weight 87,000 (Mr 87,000). To gain insight into the function of this potentially important phosphoprotein, we have undertaken its purification and characterization from rat brain. We now report a purification scheme involving heat treatment, ammonium sulfate precipitation, anion ion-exchange and reversed-phase chromatography. This procedure gave a Mr 87,000 that was homogeneous (based on silver staining), 1,600-fold enriched relative to heat-treated material and at a yield of approximately 58 micrograms/kg wet weight. We also report the amino acid composition to be high in acidic residues and in alanine and show the protein to be phosphorylated on serine residues with a stoichiometry of 2 mol of phosphate/mol of substrate. The subcellular distribution indicates Mr 87,000 is present in two forms, membrane-bound and soluble. The membrane-bound Mr 87,000 represents 45% of the total phosphoprotein content and is enriched in microsomal and synaptic membranes. Ontogenic study has revealed this protein to be developmentally regulated, with the highest concentrations of Mr 87,000 found in prenatal animals. The availability of a purification procedure should greatly facilitate further structural characterization and elucidation of the function of Mr 87,000.     

Identification and molecular characterisation of a biotin-binding protein distinct from avidin of chicken egg white and comparison with yolk biotin-binding protein

By immunological and biochemical methods a biotin-binding protein, distinct from avidin, has been shown to be present in chicken egg white. This vitamin-binding protein (Mr 67,000) bound [14C]biotin, displayed thermally induced biotin exchange reaction and exhibited gross immunological cross-reactivity with the purified yolk biotin-binding protein. In vitro labelling of soluble proteins with radioactive amino acids in the oviduct tissue explants from estrogenised chicks revealed that approx. 2% of the total radioactive proteins was immunoprecipitated with anti-yolk biotin-binding protein antibodies. The protein could be purified to homogeneity by employing ion-exchange chromatography on DEAE-cellulose and biotin-AH Sepharose affinity chromatography. The purified protein specifically bound [14C]biotin, and exhibited complete immunological homology with the yolk biotin-binding protein but not with avidin. Its electrophoretic mobility (at pH 8.3), acidic nature, biotin-binding characteristics, immunological cross-reactivity and tryptic peptide maps were very similar to that of yolk biotin-binding protein, and not avidin.     

Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen.

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.     

ISOLATION AND CHARACTERIZATION OF A SOLUBLE GLUCOSE-CONTAINING SIALOGLYCOPROTEIN FROM THE CORTICAL GREY MATTER OF CALF BRAIN

A sialoglycoprotein has been isolated from the cortical grey matter of calf brain after homogenization in 0.32 M-sucrose or in 0.15 M-NaCl. The sialoglycoprotein is present in the supernatant obtained after centrifugation at 100,000 g for 60 min. It is designated GP-350 on account of its elution with 350 mM-NaCl on a DEAE-cellulose column. From DEAE-cellulose chromatography it is evident that compounds comparable to GP-350 occur in the brain of calf and sheep, whereas they seem to be absent in calf liver and kidney. After purification, with polyacrylamide gel electrophoresis only one band can be shown both at pH 8.9 and 7.5. GP-350 consists of about 83 percent of protein and about 17 per cent of carbohydrate. The polypeptide core has an acidic character: amino acid analysis gives 26 per cent for glutamic acid plus aspartic acid and their amides, with a ratio of acidic to basic amino acids of 3.3. The carbohydrate moiety contains 2.4% sialic acid, 5.5 % hexosamine and 9.4% hexose. It is remarkable that this brain sialoglycoprotein comprises 4% glucose. Care was taken to prevent contamination with glucose-containing materials during the purification procedure of GP-350. The complete absence of other glucose-containing compounds which occur in brain, Le. glycogen and gangliosides, was demonstrated. GP-350 accounts for at least 3 per cent of the total saline-extractable protein and about 20 per cent of the total saline-extractable protein-bound sialic acid of the cortical grey matter of calf brain. These percentages correspond to 390 pg of protein and to 14 μg of sialic acid per g wet weight. GP-350 remains soluble when the pH is brought to 3.9 or when ethanol is added to 70 % (v/v).     

Purification and characterization of zeta-crystallin/quinone reductase from guinea pig liver.

zeta-Crystallin, a major lens protein of certain mammalian species, has recently been characterized as a novel and active NADPH:quinone oxidoreductase. Here we report the purification of this protein from guinea pig liver by utilizing sequentially: ammonium sulphate precipitation, Blue Sepharose affinity, cation exchange and hydrophobic chromatography steps. This four-step isolation procedure yielded 118-fold purification and a specific activity of 6 U/mg protein when assayed in the presence of 9,10-phenanthrenequinone. Kinetic, immunological and physical properties of this protein have been found to be identical with those of guinea pig lens zeta-crystallin. Western blot analysis using antibodies raised against zeta-crystallin peptides demonstrated the presence of substantial amounts of this protein in human liver homogenates.     

Purification of a special estrogen-binding protein from the liver of rats by affinity chromatography on estradiol sepharose

A procedure has been developed for purification of a special rat liver estrogen-binding protein. It includes protein precipitation by ammonium sulfate, gel filtration, ion exchange chromatography, and affinity chromatography on estradiol sepharose. The protein is purified 2260-fold with a 27% yield. Upon electrophoresis in 10% PAG in the presence of sodium dodecylsulfate the protein gives one polypeptide strip with a molecular weight of 31.200 +/- 400 dalton.