Purification and partial characterization of a cellular retinol-binding protein from human liver

A protein with binding specificity for retinol was purified from human liver. [³H]Retinol was added to liver extracts and the [³H]retinol-binding protein isolated by conventional chromatographic techniques including ion-exchange chromatography on DEAE-Sepharose, gel filtration on Sephadex G-75 and G-50 and preparative isoelectric focusing. The yield was 10–15% in different preparations and the degree of purification was about 3000-fold. The purified protein had a molecular weight of about 15 000 as estimated from both gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulphate and was homogeneous in several electrophoretic systems. Isoelectric focusing of the purified protein gave a doublet band. Only one fluorescent band at pH 4.70 was seen if the protein solution was incubated with excess retinol prior to isoelectric focusing. The isolated protein did not react with antiserum to the retinol-binding protein of plasma. The amino acid composition and the amino terminal amino acid sequence for the first sixteen amino acids of the purified protein differed significantly from that of the plasma retinol-binding protein.     

Purification and partial characterization of cellular retinoic acid-binding protein from human placenta

Cellular retinoic acid-binding protein (CRABP) has been purified to homogeneity from human placenta by a series of procedures, including acetone powder extraction, gel filtration on Sephadex G-50, and ion-exchange chromatography on DEAE-cellulose and on SP-Sephadex. Cellular retinol-binding protein (CRBP) was isolated concurrently. CRABP was purified 75,400-fold, based on total soluble acetone powder extract of placenta. The protein is a single polypeptide chain with a molecular mass of 14,600 Da, estimated by sodium dodecyl sulfate (SDS) gel electrophoresis or gel filtration, and has an isoelectric point of 4.78 (apo-CRABP, 4.82). On analysis of absorption and fluorescence spectra, the protein was seen to exhibit an absorption peak at 350 nm, fluorescence excitation maxima at 350 and 370 nm, and a fluorescence emission maximum at 475 nm. Human CRABP was immunologically distinct from human CRBP and serum retinol-binding protein.     

Retinoic acid-binding protein in rat tissue. Partial purification and comparison to rat tissue retinol-binding protein.

When the 100,000 X g supernatant fractions of several rat organs are incubated with all-trans-[3H]retinoic acid, a binding component for retinoic acid with a sedimentation coefficient of 2 S can be detected by sucrose gradient centrifugation. This tissue binding protein for retinoic acid is distinct from the tissue binding protein for retinol which has been previously described. The tissue retinoic acid-binding protein has been partially purified from rat testis and this partially purified protein would appear to have a molecular weight of 14,500 as determined by gel filtration and high binding specificity for all-trans-retinoic acid. Binding of [3H]retinoic acid is not diminished by a 200-fold molar excess of retinal, retinol, or oleic acid but is reduced by a 200-fold excess of unlabeled retinoic acid. Tissue retinoic acid-binding protein can be detected in extracts of brain, eye, ovary, testis, and uterus but is apparently absent in heart muscle, small intestine, kidney, liver, lung, gastrocnemious muscle, serum, and spleen. This distribution is different than that observed for the tissue retinol-binding protein. Tissue retinol-binding protein was also purified extensively from rat testis. The partially purified protein has an apparent molecular weight of 14,000 and high binding specificity for all-trans-[3H]retinol as only unlabeled all-trans-retinol but not retinal, retinoic acid, retinyl acetate, retinyl palmitate, or oleic acid could diminish binding of the 3H ligand under the conditions employed. The partially purified protein has a fluorescence excitation spectrum with lambda max at 350 nm. In contrast, the retinol-binding protein isolated from rat serum and described by others has a fluorescence excitation spectrum with lambda max at 334 nm and an apparent molecular weight of 19,000. When partially purified tissue retinol-binding protein is extracted with heptane, the heptane extract has a fluorescence excitation spectrum similar to that of all-trans-retinol.     

Purification and some properties of a deoxyribonucleic acid endonuclease endogenous to rat liver chromatin

A deoxyribonucleic acid (DNA) endonucleolytic activity has been purified from a 0.3 M KCl extract of rat liver chromatin by a combination of selective precipitation and ion-exchange and gel filtration chromatography. The purified protein has a molecular weight of 35 000 as determined by Sephadex G-200 gel filtration and sodium dodecyl sulfate-acrylamide gel electrophoresis. The nuclease activity is stimulated by the addition of Mg2+ and thus may represent the Mg2+-activated DNase endogenous to chromatin. The purified enzyme has the ability to make both single-strand nicks and double-strand cuts in DNA.     

人胎肝超氧化物歧化酶的研究

采用离子交换和凝胶过滤层析法,从正常人胎肝提取、纯化铜锌超氧化物歧化酶(Cu.Zn-SOD),并对其性质作了研究,结果测得人胎肝组织Cu·Zn-SOD平均含量为6.44unit/g湿重,并发现随着胎龄的增加人胎肝Cu.Zn-SOD含量上升;纯化后的Cu·Zn-SOD在聚丙烯酰胺凝胶电泳图谱上呈现单一区带;其活性受氰化钾抑制;以原子吸收光谱测得其铜、锌含量分别为0.39％和0.1％,采用SDS-聚丙烯酰胺凝胶电泳测得其亚基分子量为16kD;氨基酸自动分析仪测得其亚基的氨基酸残基数为151.5;在紫外吸收光谱上于265nm和258nm处分别出现两个吸收高峰。本研究结果表明,人胎肝Cu.Zn-SOD与成人肝及其他组织的Cu.Zn-SOD理化性质上基本一致。     

Purification and some properties of rabbit liver cytosol thioltransferase

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.     

Purification and characterization of an extracellular H2O2-requiring diarylpropane oxygenase from the white rot basidiomycete, Phanerochaete chrysosporium

An H2O2-requiring oxygenase found in the extracellular medium of ligninolytic cultures of the white rot fungus Phanerochaete chrysosporium was purified by DEAE-Sepharose ion-exchange chromatography and gel filtration on Sephadex G-100. Sodium dodecyl sulfate (SDS)-disc gel electrophoresis indicated that the purified protein was homogeneous. The Mr of the enzyme as determined by gel filtration and SDS-polyacrylamide gel electrophoresis was 41,000. The absorption spectrum of the enzyme indicated the presence of a heme prosthetic group. The absorption maximum of the native enzyme (407 nm) shifted to 435 nm in the reduced enzyme and to 420 nm in the reduced-CO complex. The pyridine hemochrome absorption spectrum indicated that the enzyme contained one molecule of heme as iron protoporphyrin IX. Both CN- and N-3 bound readily to the native enzyme, indicating an available coordination site and that the heme iron was high spin. The purified enzyme generated ethylene from 2-keto-4-thiomethyl butyric acid, and oxidized a variety of lignin model compounds, including the diarylpropane, 1-(3'4'-diethoxyphenyl)1,3-dihydroxy-2-(4"-methoxyphenyl)propane (I); a beta-ether dimer, 1-(4'-ethoxy-3'-methoxyphenyl)glycerol-beta-guaiacyl ether (V); an olefin, 1-(4'-ethoxy-3'-methoxyphenyl)-1,2 propene (III); and a diol, 1-(4'-ethoxy-3'-methoxyphenyl)-1,2-propane diol (IV). The products found were equivalent to the metabolic products previously isolated from intact ligninolytic cultures.     

Characterization of rat liver cytosol retinyl ester lipoprotein complex

A soluble high molecular weight lipoprotein complex containing retinyl esters and unesterified retinol was isolated from rat liver cytosol. This material accounts for about 10% of the total liver retinyl compounds, and its protein moiety accounts for about 0.1% of the protein of the liver homogenate and about 0.7% of the cytosol protein. The lipoprotein was purified by gel filtration and hydroxyapatite chromatography. The lipoprotein complex gave a single band by electrophoresis on cellulose acetate as judged by both lipid- and proteinspecific stains. The lipoprotein complex did not dissociate into smaller subunits in low ionic strength buffer (1 mm sodium phosphate, pH 7.7). The retinyl ester lipoprotein complex has an absorption spectrum with peaks at 328 nm (retinyl chromophore) and 258 nm. Retinyl compounds in the carrier lipoprotein complex do not show an increase of the quantum yield of fluorescence and do not show energy transfer when excited at either 258 or 280 nm. There is no induced optical activity of the retinyl chromophore absorption band. The lipoprotein complex contains about 3% (by weight) of retinyl compounds, 96% of which are retinyl esters and 4% of which are unesterified retinol. The lipoprotein complex consists of about 66% (by weight) lipids, about 30% protein, and some 4% carbohydrate. There are at least 15 polypeptide chains ranging in size from about 2 × 10⁴ to about 2.1 × 10⁵M_r. Retinyl compounds in the lipoprotein complex are stable for at least 3 months in 0.05 m phosphate buffer, pH 7.4, at 4 °C. Stability was judged from the total amount of retinyl esters plus unesterified retinol. Retinyl compounds of the lipoprotein complex were unstable below pH 6.4 or in the presence of 1 m NaCl.     

Molecular mass of inhibitor-2 from rabbit liver

Inhibitor-2 was partially purified from rabbit liver by fractionation with ammonium sulphate, heat treatment at 100°C, precipitation with trichloroacetic acid, chromatography on DEAE-cellulose at pH 8.5 and 5.0 and gel filtration on Sephadex G-100 (Stokes radius, 3.4 nm). The protein behaved as a single component at each step and migrated on SDS-polyacrylamide gels as a 31 kDa protein. Its properties were indistinguishable from those of skeletal muscle inhibitor-2. The results disagree with the report of Khandelwal and Zinman (J. Biol. Chem. (1978) 253, 560–565) that hepatic inhibitor-2 is a 14 kDa protein.     

小鼠肝脏免疫抑制蛋白质(LISP)的分离纯化和鉴定

用硫酸铵分段盐析及DEAE-Sephadex A-50、羟磷灰石和CM纤维素等多种柱层析方法,从正常小鼠肝浸液中分离纯化出一种免疫抑制蛋白质(LISP)。在体外用微量该蛋白质就能强烈抑制小鼠T、B淋巴细胞对促有丝分裂原和同种异型抗原的增生反应。纯化的蛋白质在聚丙烯酰胺凝胶电泳(PACE)和等电聚焦(IEF)鉴定时均显示为一条区带,其等电点(pI)值在7.5—7.8范围。沉降系数利S_(20),w为5.39。Sephadex G-100凝胶层析测得LISP的分子量为78,000道尔顿。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)提示LISP是由二个相同的亚基组成,亚基分子量为38,500道尔顿。LISP是一种既非糖蛋白又非脂蛋白的碱性蛋白质,对它的氨基酸组成也作了分析。     

A carotenoprotein from chromatophoreses of Rhodospirillum rubrum

A carotenoprotein has been obtained by SDS-solubilization of $\text{Rhodospirillum rubrum}$ chromatophores. It was then purified by (NH₄)₂SO₄ precipitation and Sephadex G-200 filtration. SDS-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of about 12,000. The absorption spectrum of the complex is entirely different from the usual three peaked carotenoid spectrum, it has only a major peak at 370 nm. However, after acetone extraction the spectrum of spirilloxanthin reappears. The fact that the carotenoid associates with a specific protein provides strong evidence that the complex originates from the chromatophores and is not a preparative artefact.     

Purification and physical-chemical properties of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver

Acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from pigeon liver was purified by protamine sulfate precipitation, ion exchange chromatography on DEAE-A-25 Sephadex, gel filtration on Sephadex G-75, amethopterin-AH-Sepharose 4B affinity chromatography, and finally, gel filtration on Sephadex G-100. The enzyme preparation was homogeneous as judged by ultracentrifugation studies, SDS-polyacrylamide gel electrophoresis and gel filtration. The N-terminal amino acid was detected to be histidine and the complete amino acid composition is reported. The enzyme contains one disulfide bridge and two cysteine residues/mol monomer. The isoelectric point was estimated to be 4.8. The molecular weight was determined to be 32900 by high-speed sedimentation equilibrium analysis, 33000 by Sephadex G-100 gel filtration and 31600 by SDS-disc gel electrophoresis. The sedimentation coefficient from conventional sedimentation velocity runs was 3.1 S observed by ultraviolet optics. 'Active enzyme centrifugation' showed a sedimentation constant of 5.0 and 4.8 S for the purified enzyme and crude extract from pigeon liver, respectively, indicating that the enzyme forms a dimer under conditions of catalysis. It could be demonstrated that the inhibitor amethopterin was noncompetitive with respect to the acetyl donor and the acetyl acceptor. Acetyl-CoA:arylamine N-acetyltransferase was examined in different organs of pigeon. The enzyme was not inducible by 1,3-phenylenediamine and hexobarbital in vivo.     

Purification and properties of pig liver kynureninase.

Kynureninase [L-kynurenine hydrolase, EC 3.7.1.3] was purified from pig liver by a procedure including DEAE-cellulose chromatography, hydroxyapatite chromatography, ammonium sulfate fractionation, DEAE-Bio Gel chromatography, Sephacryl S-200 gel filtration, kynurenine-Sepharose affinity chromatography, and Sephadex G-200 gel filtration. The enzyme was found to be homogeneous by the criterion of disc-gel electrophoresis. The enzyme has a molecular weight of about 100,000 and exhibits absorption maxima at 280 and 420 nm. The optimum pH and the isoelectric point of the enzyme are 8.5 and 5.0, respectively. The Michaelis constants were determined to be as follows: L-kynurenine, 7.7 X 10(-4) M; L-3-hydroxykynurenine, 1.3 X 10(-5) M; and pyridoxal 5'-phosphate, 1.8 X 10(-6) M. L-3-Hydroxykynurenine is hydrolyzed more rapidly than L-kynurenine; the liver enzyme can be regarded as a 3-hydroxy-kynureninase.     

黄精凝集素Ⅱ的纯化及部分性质研究

囊丝黄精（ＰｏｌｙｇｏｎａｔｕｍｃｙｒｔｏｎｅｍａＨｕａ．）的根状茎,经浸取、用硫酸铵分级沉淀、猪甲状腺球蛋白－Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和层析、ＣＭ－Ｓｅｐｈａｒｏｓｅ柱离子交换层析和ＳｅｐｈａｄｅｘＧ－１００凝胶过滤,可以分离纯化出黄精凝集素Ⅱ（ＰＣＬⅡ）．纯化的ＰＣＬⅡ在聚丙烯酰胺凝胶电泳中显示单一蛋白染色带;在快速高效液相色谱中亦为单一蛋白峰,经分子筛层析测得分子量为１５．９ｋＤ,最大紫外吸收值在２７８ｎｍ,ＰＣＬⅡ只凝集兔红细胞,当浓度为０．２５μｇ／ｍｌ时,即可发生凝集反应,此凝集兔红细胞的能力可被Ｄ－甘露糖和猪甲状腺球蛋白所抑制．氨基酸组成分析表明ＰＣＬⅡ分子中富含酸性氨基酸,Ｎ末端为丙氨酸．经测定ＰＣＬⅡ分子中含有３个色氨酸和２．４％的中性糖．原子发射光谱分析表明,该凝集素分子中含有Ｍｇ和Ｃａ两种金属元素．     

Purification and properties of heme oxygenase from rat liver microsomes.

Heme oxygenase was purified to apparent homogeneity from liver microsomes of rats which had been treated with either cobaltous chloride or hemin to induce heme oxygenase in the liver and the purified preparations from either rats showed an apparent molecular weight of about 200,000 when estimated by gel filtration on a column of Sephadex G-200, and gave a minimum molecular weight of about 32,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hepatic heme oxygenase could bind heme to form a heme . heme oxygenase complex showing an absorption peak at 405 nm, and the extinction coefficient at 405 nm of the heme . heme oxygenase complex was 140 mM-1 cm-1. The heme bound to the hepatic heme oxygenase protein was easily converted to biliverdin when the complex was incubated with the NADPH-cytochrome c reductase system in air. The hepatic heme oxygenase appears to have characteristics essentially similar to those of the splenic heme oxygenase (Yoshida, T., and Kikuchi, G. (1978) J. Biol. Chem. 253, 4224 and 4230). The heme oxygenase preparation which was purified from the cobalt-treated rats contained a small amount of cobaltic protoporphyrin, indicating that cobalt protoporphyrin was synthesized in these rats.     

PURIFICATION AND CHARACTERISTICS OF RNase INHIBITOR FROM PIG CEREBRAL CORTEX

Abstract— An RNase inhibitor has been purified from pig cerebral cortex by DEAE-cellulose and hydroxylapatite chromatography and Sephadex G-100 gel filtration. The purified RNase inhibitor could be resolved into a major band (about 80–85 per cent of total protein) and several minor components by polyacrylamide gel electrophoresis.
The ultraviolet absorption curve of the purified RNase inhibitor indicated a typical protein spectrum. The inhibitor was inactivated by digestion with trypsin or prozyme, and by heating at 70ºC for 5 min. The inhibitor was also inactivated by an SH reagent such as p -chloromercuribenzoate. The inhibitor did not affect RNase T₁. It has been suggested that the inhibitor is an acidic protein and also a SH-protein. The molecular weight of the RNase inhibitor was estimated to be about 60,000.     

Isolation and some properties of NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii.

NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii was isolated and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. This enzyme is an FAD-containing flavoprotein and has absorption maxima at 485 (shoulder0 452, 411, and 385 nm (the 411 nm band is due to cytochrome). The molecular weight of the enzyme as determined by gel filtration using Sephadex G-200 is 119,000. The enzyme catalyzes the reduction of NAD+ and NADP+ by photoreduced spinach ferredoxin or reduced benzyl viologen...     

Purification and subunit structure of mouse liver cystathionase

Cystathionase has been purified from mouse liver by ammonium sulfate precipitation, ethanol precipitation, column chromatography on DEAE-cellulose and on hydrox-ylapatite, as well as Sephadex G-200 gel filtration. These procedures yielded a chromatographically homogeneous enzyme which was purified more than 1000-fold relative to whole liver extract. Overall recovery was approximately 4%. The purified enzyme does not contain detectable carbohydrate and migrates as a single protein component on analytical disc gel electrophoresis. A sedimentation coefficient of 8.3 S has been determined for the active enzyme by rate zonal centrifugation in glycerol gradients. This value suggests a molecular weight for the native enzyme of approximately 160,000 g/mol, a value similar to that estimated by gel filtration. Following sodium dodecyl sulfate gel electrophoresis in the presence of reducing agent and at different gel concentrations, a single protein component with a molecular weight of 40,000 g/mol was obtained. Thus, the enzyme appears to consist of four subunits of equal size. The K_m value for cystathionine at pH 8.1, 37 °C, and in the presence of 1 mm dithioerythritol is approximately 1 mm.     

Isolation and various properties of thiamine-binding protein from synaptosomes in the rat brain

A thiamine-binding protein (ThBP) with a specific activity of 8.21 nmoles/mg protein was isolated from rat brain synaptosomes by affinity chromatography and gel filtration on Sephadex G-200. The protein was purified 746-fold with a 40.5% yield. ThBP was homogeneous during sodium dodecyl sulfate gel electrophoresis; its molecular mass was determined by gel filtration on Sephadex G-200 and by sodium dodecyl sulfate gel electrophoresis and was equal to 107 and 103 kD, respectively. The pH optimum for the binding is 8.35. When the ability of ThBP to bind thiamine phosphates was tested, the latter decreased in the following order: thiamine monophosphate greater than thiamine triphosphate greater than greater than thiamine diphosphate.     

Purification of rat liver nuclear protein kinase NI.

Rat liver nuclear protein kinase NI, which appears in the flowthrough of DEAE-Sephadex columns, has been purified approximately 15,000-fold from soluble nuclear protein with yields of up to 10%. The method of purification involved chromatography of the DEAE-flowthrough protein successively on phosvitin-Sepharose and casein-Sepharose followed by rechromatography on phosvitin-Sepharose. The purified enzyme has an s20,w and molecular weight of 3.7 and 47,000, respectively, as determined by sucrose density gradient centrifugation in 0.4 M NaCl. A similar molecular weight of 42,000 was determined by gel filtration using Sephadex G-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single polypeptide with a molecular weight of 25,000. Protein kinase NI therefore consists of a dimer of two identical subunits. Protein kinase NI exhibits maximal activity on casein substrate and is not stimulated by 10(-5) to 10(-4) M cAMP or cGMP when either casein or histone H2b is used as a substrate.