Purification de l'alcool deshydrogénase de tomate par chromatographie d'affinité

Tomato alcohol dehydrogenase has been purified 99-fold by affinity chromatography on Blue Sepharose CL-6B with 37% yield. The enzyme so obtained is homogenous in polyacrylamide gel electrophoresis. By adding 20% glycerol to the extraction and purification buffers, an enzyme is obtained which is stable for several months at 4°. The molecular weight values determined by gel filtration (Sephadex G 200) and polyacrylamide gradient gel electrophoresis on one hand and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate on the other, show that the enzyme exists in dimeric form.     

An Analysis of the Heterogeneity of Albumin

A simple and rapid procedure for characterization of serum albumin samples and for the isolation of fractions from fresh serum or commercial sources has been developed, based on DEAE Sepharose CL-6B ion exchange chromatography. Characterization of the fractions included sulfhydryl analysis, gel electrophoresis, and immunoelectrophoresis. Both analytical and preparative procedures were used. It has been shown that the relative content of the fractions varies from individual to individual. It has also been shown that certain fractions found in commercial preparations are artifacts which should be removed before performing serious studies with serum albumin.     

Urinary protein profiling by high-performance gel permeation chromatography

We describe a new application of high-performance aqueous gel permeation chromatography for the analysis of human proteinuria. Separations of urinary proteins from normal subjects and patients with renal impairment were performed with TSK G 3000 SW columns. The effects of pH and icnic strength of the eluent on the separation of urinary proteins were investigated. Albumins were selectively separated from urine by affinity chromatography on Blue Sepharose CL-6B. According to the results of clinical investigations, urinary protein pattern derived from gel permeation chromatography revealed a good prediction of the site of renal involvement. Predominant excretion of proteins with lower molecular weight than albumin correlated with tubular damage. Albumin and higher molecular weight protein patterns were associated with glomerular disease. Absorbance measurements of the eluent at 280 nm were used for quantitative determination of total urinary protein. Gel permeation chromatography was compared to sodium dodecyl sulfate—polyacrylamide gel electrophoresis and the resulting protein patterns are in good agreement.     

Purification and properties of pyridoxal kinase from bovine brain

A 27,000-fold purification of pyridoxal kinase from bovine brain tissue has been achieved by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G-150 gel filtration, Blue Sepharose CL-6B chromatography, and Phenyl-Superose chromatography. The final chromatography step yields a homogeneous preparation of high specific activity (2105 nmol/min/mg protein). The molecular mass of the native enzyme was estimated to be approximately 80,000 on gel filtration. The subunit molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be approximately 39,500. This indicates that pyridoxal kinase is a dimeric enzyme.     

Identification of albumin as the plasma carrier for zinc absorption by perfused rat intestine.

The isolated vascularly perfused rat intestine exhibits an obligatory need for a protein carrier in order to absorb zinc. Therefore this system is ideal for use as a model to identify the plasma carrier during zinc absorption. Affinity chromatography on Blue Sepharose CL-6B was employed to separate the major serum zinc-binding proteins in the portal effluent of the perfused intestine. It was found that 94% of newly absorbed 65Zn was transported in the portal serum-containing perfusate as an albumin-65Zn complex. The identity of albumin as the plasma carrier was confirmed by polyacrylamide-slab-gel electrophoresis. This evidence suggests that albumin is the plasma protein that is involved in removal of zinc from intestinal-mucosal cells and subsequent transport of the metal in portal blood to the liver.     

A high-capacity hydrophobic adsorbent for human serum albumin

A simple method, based on salting out hydrophobic interaction chromatography, for the efficient removal of trace amounts of serum albumin from partially purified protein preparations is described. The method is also successfully applied for the purification of albumin from Cohn fraction IV, a by-product obtained from the commercial fractionation of human serum proteins by the ethanol precipitation procedure. About 70% of the adsorbed albumin can be eluted by buffer of low ionic strength and can thus be lyophilized directly, if required. The adsorbent can be used for several cycles of adsorption and desorption without affecting its selectivity or capacity. Its adsorption properties and capacity for serum albumin are compared with those of the commercially available adsorbent Blue Sepharose CL-6B.     

Characterization of immunosuppressive substances in the basic protein fraction of uterine secretions from pregnant ewes

The basic protein fraction of ovine uterine secretions collected late in pregnancy (Days 125-140) contains a substance capable of inhibiting in vitro blastogenic responses of lymphocytes to phytohemagglutinin (PHA) or mixed lymphocyte reactions. In this study, the immunosuppressive substance in the basic protein fraction of uterine secretions was further defined by gel filtration. The immunosuppressive activity resided in a group of high molecular weight proteins eluting at the void volume of Sephacryl S-200 and Sepharose CL-6B columns. For example, incorporation of thymidine by PHA-stimulated lymphocytes incubated with 20, 40, 80, and 120 micrograms/ml of protein from the void volume of Sepharose CL-6B was 65, 28, 2, and 0 percent of control lymphocytes, respectively. Based on polyacrylamide-gel electrophoresis in the presence of sodium dodeylsulfate (SDS), the immunosuppressive fraction from Sepharose CL-6B chromatography contained aggregates of uterine milk proteins (UTM-proteins) and a pair of proteins running at the top of a 5% (w/v) polyacrylamide gel. Other protein peaks resolved by Sephacryl S-200 and Sepharose CL-6B contained aggregates of UTM-proteins but were not immunosuppressive. The substance inhibiting in vitro lymphocyte function was not of conceptus origin, because it was found in fluid from the ligated uterine horn of unilaterally pregnant ewes and from the uterus of an ovariectomized ewe treated for 60 days with progesterone and estrone.     

Separation of Neurospora crassa myo-inositol-1-phosphate synthase from glucose-6-phosphate dehydrogenase by affinity chromatography

The purification of Neurospora crassa myo-inositol-1-phosphate synthase (EC 5.5.1.4) was studied by affinity chromatography using the substrate (glucose-6-phosphate), the inhibitor (pyrophosphate), the coenzyme (NAD+) and the coenzyme analogues (5'AMP and Cibacron Blue F3G-A) of the enzyme as adsorbents attached to agarose gel. Myo-inositol-1-phosphate synthase could be separated completely from the contaminating substance, glucose-6-phosphate dehydrogenase (EC 1.1.1.49), on Blue Sepharose CL-6B and on pyrophosphate-Sepharose. The purified enzyme had a specific activity of 16 400 U/mg. The sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the 60 micrograms of this purified enzyme gave a homogenous band. The enzyme was found to be composed of four identical subunits having a molecular weight of 65 000.     

Separation of Neurospora Crassa Myo-Inositol-1-Phosphate Synthase from Glucose-6-Phosphate Dehydrogenase by Affinity Chromatography

The purification of Neurospora crassa myo-inositol-1-phosphate synthase (EC 5.5.1.4) was studied by affinity chromatography using the substrate (glucose-6-phosphate), the inhibitor (pyrophosphate), the coenzyme (NAD⁺) and the coenzyme analogues (5′AMP and Cibacron Blue F3G-A) of the enzyme as adsorbents attached to agarose gel. Myo-inositol-1-phosphate synthase could be separated completely from the contaminating substance, glucose-6-phosphate dehydrogenase (EC 1.1.1.49), on Blue Sepharose CL-6B and on pyrophosphate-Sepharose. The purified enzyme had a specific activity of 16 400 U/mg. The sodium dodecyl sulfate/polyacrylamide gel electrophoresis of 60 μq of this purified enzyme gave a homogenous band. The enzyme was found to be composed of four identical subunits having a molecular weight of 65 000.     

Immobilization of proteins on oxidized crosslinked Sepharose preparations by reductive amination

Mild periodate oxidation of certain commercially available crosslinked agarose beads (Sepharose CL-4B and CL-6B) results in the generation of aldehydo groups which were useful for immobilization of amino compounds by reductive amination using pyridine borane. Consumption of periodate ion and production of formaldehyde were only observed with crosslinked Sepharose preparations and were correlated with a binding capacity much greater than that of uncross-linked gels when subjected to the reductive amination reaction. Up to 50 mg (approximately 0.73 mumol) of bovine serum albumin and 30 mumol of glycylglycine were coupled per gram of moist oxidized Sepharose CL-6B. The immobilization reaction was shown to proceed at neutral pH requiring about 12 h for completion and to be relatively insensitive to temperature and pyridine borane concentration. The oxidized gel was shown to be stable for at least 2 months upon storage in 0.1 M acetic acid. This method has proven to be useful for the preparation of a variety of affinity matrices and immobilized enzymes.     

大肠杆菌半乳糖结合凝集素的提取纯化

采用琼脂糖凝胶CL-6B（Sepharose CL-6B）亲和层析以及Sephadex G-75凝胶分子筛等对大肠杆菌（Esche-richia coli,E.coli）半乳糖凝集素进行了纯化。结果显示,目标蛋白经简单的步骤即可以得到纯化,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）及凝血实验证明纯化蛋白为E.coli半乳糖凝集素,蛋白提取回收率为11.4%。研究首次从E.coli蛋白提取液中分离得到纯的半乳糖凝集素,且此方法简单快捷,优越性明显。应用此方法将有利于微生物半乳糖凝集素的深入研究。     

Characterization of mucin isolated from rat tracheal transplants

Subcutaneous rat tracheal grafts yield several milligrams of secretions from which a homogeneous mucin fraction was isolated and purified. Histological evidence demonstrated that a normal mucociliary epithelium and mucous secretion were maintained for the 4-6 weeks of the experiment. The collected secretions were initially characterized by column chromatography on Sepharose CL-6B which separated the excluded high molecular weight mucins (unpurified mucin fraction) from most of the serum-type glycoproteins and proteins, including albumin. A reductive alkylation treatment of the unpurified mucin fraction followed by Sepharose CL-4B chromatography removed contaminating protein and most of the mannose-containing material from the mucin fraction. The void volume material from this column produced a single high molecular weight band upon sodium dodecyl sulfate agarose/acrylamide gel electrophoresis. The purified mucin fraction contained 16.5% protein and primarily galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid. This fraction also underwent beta-elimination in the presence of alkaline borohydride, demonstrating the presence of O-glycosidic linkages.     

Purification and characteristics of glucocorticoid antagonizing factor in endotoxemia

The present study involved the purification of GAF (glucocorticoid antagonizing factor) released in blood of endotoxemic mice, using the inhibition rate of tryptophan oxygenase (TO) activity in the mice liver as a parameter, to determine if this plays a role in metabolic disorders. GAF-rich serum in zymosan-primed and endotoxin-injected mice was subjected to chromatography on DEAE-Sepharose CL-6B, Blue Sepharose CL-6B and Sephadex G-200 superfine columns. Finally, GAF fractions were purified by chromatography on a DEAE-Sepharose CL-6B column. The purified GAF showed a single band in electrophoresis in sodium dodecyl sulfate (SDS) polyacrylamide gel. The molecular weight of GAF was estimated to be 90,000. The purified GAF was regarded as glycoprotein. No factor (100 micrograms) exhibited lethal action on mice. The activity of TO in cortisone treated mice after injection of purified GAF was markedly lower than that in cortisone alone treated mice. On the other hand, there were no differences in tyrosine aminotransferase activities between the GAF plus cortisone injected group and cortisone only treated group. The glucose level after injection of GAF in cortisone treated mice initially showed hyperglycemia, but declined toward hypoglycemia 2 hr after injection, and thereafter returned nearly to the normal range by 4 hr. The liver glycogen level in GAF plus cortisone-treated mice was markedly lower than that in cortisone-alone treated mice.     

Improved isolation method and some properties of soybean gamma-conglycinin

Soybean gamma-conglycinin was isolated by isoelectric precipitation and ammonium sulphate fractionation. The crude protein was purified by ion-exchange chromatography on DEAE-Sepharose CL-6B and gel filtration on Sepharose CL-6B. The purified gamma-conglycinin was homogeneous on two kinds of gel electrophoresis and an ultracentrifugal analysis. A subunit band, distinguishable from other subunit bands of beta-conglycinin and glycinin, was detected by sodium dodecyl sulphate electrophoresis. Amino acid composition was similar to those of the other storage proteins of soybean. Some physical properties were also studied.     

Purification and characterization of an aminopeptidase from Mycoplasma salivarium.

The aminopeptidase which had been shown to be present in Mycoplasma salivarium was found to be associated with the cell membranes of the organism. The enzyme was solubilized in water by papain digestion of the membranes pretreated with Triton X-100 and purified approximately 130-fold by ion-exchange chromatography on DEAE-Sephadex A-50, affinity chromatography on L-leucylglycine-AH-Sepharose 4B, and gel filtration on Sepharose CL-6B. The purified enzyme had a molecular mass of 397 kilodaltons, estimated by gel filtration through Sepharose CL-6B, and gave two bands of activity in analytical disc polyacrylamide gel electrophoresis: a dense, diffuse band and a less dense, narrow one, accounting for 90 and 5% of stained proteins in the gel, respectively. The purified protein revealed two bands with molecular masses of 50 and 46 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed selectively the cleavage of the N-terminal arginine and leucine residues of peptides; had a pH optimum at 8.5; and was inhibited remarkably by bestatin, o-phenanthroline, EDTA, and L-cysteine, but was activated nine- and twofold by MnCl2 and MgCl2, respectively. The enzyme pretreated with MnCl2 had much higher maximum velocity (Vmax) for L-leucine-p-nitroanilide than the one not treated. That is, the Michaelis constant (Km) and Vmax values of the pretreated enzyme were 10.5 mM and 12.1 microM/min, respectively, whereas those of the untreated enzyme were 5.8 mM and 1.6 microM/min, respectively.     

Purification and characterization of adenylate kinase isozymes from rat muscle and liver

Isozymes of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) were purified from skeletal muscle and liver of rats to essentially homogeneous states by acrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. The isozyme from muscle was purified by acidification to pH 5.0, and column chromatography on phosphocellulose, Sephadex G-75 and Blue Sepharose CL-6B, while that from liver was purified by column chromatography on Blue Sepharose CL-6B, Sephadex G-75 and carboxymethyl cellulose. By these procedures the muscle isozyme was purified about 530-fold in 29% yield, and the liver isozyme about 3600-fold in 27% yield from the respective tissue extracts. The molecular weights of the muscle and liver isozymes were estimated as about 23 500 and 30 500, respectively, by both sodium dodecyl sulfate gel electrophoresis and molecular sieve chromatography, and no subunit of either isozyme was detected. The isoelectric points of the muscle and liver isozymes were 7.0 and 8.1, respectively. The Km values of the respective enzymes for ATP and ADP were similar, but the Km(AMP) of the liver isozyme was about one-fifth of that of the muscle isozyme. Immunological studies with rabbit antiserum against the rat muscle isozyme showed that the muscle isozyme was abundant in muscle, heart and brain, while the liver isozyme was abundant in liver and kidney.     

Two methods for the separation of human alpha-fetoprotein and albumin. (A) Affinity chromatography using Blue Sepharose CL-6B and (B) ampholyte displacement chromatography.

Two methods for the separation of α-fetoprotein (AFB) and albumin using (A) affinity chromatography on Blue Sepharose CL-6B and (B) ampholyte displacement chromatography are described. The heterogeneity of immunoreactive human AFP is demonstrated by ampholyte displacement chromatography; possibly seven variants of AFP can be distinguished by this method.     

Isolation of human pituitary prolactin

A process developed earlier for the extraction of human follitropin, lutropin, thyrotropin and growth hormone from homogenized frozen pituitaries provided a residue utilized for the isolation of prolactin. The isolation procedure involved extraction at pH 9.8, molecular sieve chromatography on Sepharose CL-6B, hydrophobic interaction chromatography on phenyl-Sepharose CL-4B, molecular sieve chromatography on Sephadex G-100 Superfine, and ion-exchange chromatography on DEAE-Sepharose CL-6B using a convex gradient. The progressive purification was guided by radioimmunoassays. The final product was obtained in yields of 31 microgram/gland, and was equipotent with a pituitary preparation (VLS-3) supplied by the National Pituitary Agency (NIH, Bethesda, U.S.A.). Contamination hormones negligible (less than 0.05%). No heterogeneity of the isolated prolactin was observed by sedimentation-equilibrium analysis in the ultracentrifuge, by SDS electrophoresis in polyacrylamide gel or by molecular sieve chromatography in 6 M guanidine hydrochloride. These different techniques gave values in the range of 21 000-23 000 for the molecular weight of prolactin. In free zone electrophoresis, and also in polyacrylamide gel electrophoresis the prolactin preparation was, however, heterogeneous and resolved at alkaline pH into three distinct components. The former technique permitted isolation and assay of the components, indicating that they were all fully active.     

The use of a monoclonal antibody for the rapid purification of kidney neutral endopeptidase ("enkephalinase") solubilized in octyl glucoside

The neutral endopeptidase ("enkephalinase") of the rabbit brush border membrane has been purified to homogeneity by a rapid immunoaffinity method using a monoclonal antibody. In contrast with other methods used so far, a complete extraction of enkephalinase from the brush border membrane can be achieved with octyl glucoside, without loss of activity. The solubilized enzyme can be selectively separated from the other proteins in a single step using an immunoaffinity column consisting of the monoclonal antibody covalently linked to Sepharose CL-4B. It is demonstrated that enkephalinase can then be recovered in an active form by elution at low pH. The purified enzyme obtained by this method is completely inhibited by thiorphan and appears as a single 94,000 dalton protein after polyacrylamide gel electrophoresis under denaturing and reducing conditions.     

Characterization of mucin isolated from rat tracheal transplants

Subcutaneous rat tracheal grafts yield several milligrams of secretions from which a homogeneous mucin fraction was isolated and purified. Histological evidence demonstrated that a normal mucociliary epithelium and mucous secretion were maintained for the 4–6 weeks of the experiment. The collected secretions were initially characterized by column chromatography on Sepharose CL-6B which separated the excluded high molecular weight mucins (unpurified mucin fraction) from most of the serum-type glycoproteins and proteins, including albumin. A reductive alkylation treatment of the unpurified mucin fraction followed by Sepharose CL-4B chromatography removed contaminating protein and most of the mannose-containing material from the mucin fraction. The void volume material from this column produced a single high molecular weight band upon sodium dodecyl sulfate agarose/acrylamide gel electrophoresis. The purified mucin fraction contained 16.5% protein and primarily galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid. This fraction also underwent β-elimination in the presence of alkaline borohydride, demonstrating the presence of O-glycosidic linkages.