DEAE-Sephacel Column Chromatography Research Articles

Effect of tartrate on the colchicine-binding activity in cultured carrot cell extract

The colchicine-binding activity in carrot-cell extract was markedly enhanced and also stabilized by the addition of tartrate. The optimum temperature for the binding reaction in the presence of tartrate shifted to 41 °C from 30 °C in its absence. The apparent affinity constant in the presence of tartrate was 1.6 × 105l/mol at 37 °C which is much higher than the value obtained in the absence of tartrate. It was confirmed from the following evidence that the binding reaction in the presence of tartrate represented the colchicinetubulin interaction. 1. The reaction was inhibited by the preincubation with proteases and not with lumicolchicine. 2. The colchicine-binding component had a similar property to porcine brain tubulin in DEAE-Sephacel column chromatography and SDSpolyacrylamide gel electrophoresis.

Amino acid sequence of human beta-factor XIIa.

Human factor XII was activated by limited proteolysis with trypsin, and the resulting beta-factor XIIa (Mr = 30,000) was isolated by DEAE-Sephacel column chromatography. The complete amino acid sequence of beta-factor XIIa was then determined on peptides produced by enzymatic digestion with either trypsin, chymotrypsin, or Staphylococcus aureus V8 protease and by chemical cleavage at methionyl and tryptophyl bonds. beta-Factor XIIa is a glycoprotein composed of a heavy chain (243 amino acid residues) and a light chain (9 amino acid residues), and these two chains are held together by a disulfide bond. The carbohydrate is attached to asparagine residue 61 in the heavy chain. The amino acid sequence of the heavy chain shows a high degree of homology to the corresponding regions of other plasma serine proteases, such as plasmin, thrombin, factor IXa and factor Xa, as well as the pancreatic digestive enzymes. These results demonstrate that factor XII is the precursor of a typical serine protease that participates in the coagulation cascade.

Simultaneous simple purification of tyrosine hydroxylase and dihydropteridine reductase

A rapid and simple simultaneous micropurification procedure of tyrosine hydroxylase (TH) and dihydropteridine reductase (DPR) was developed from soluble supernatants of 1 to 2 g of rat adrenal gland or caudate nucleus. All purification procedures for the two enzymes were complete within 3 days. The recovery of TH and DPR was reproducible and approximately 20 and 40%, respectively. Purification procedure for TH involved chromatographies with DEAE-Sephacel, Bio-Gel A-1.5 m, and heparin-Sepharose. As judged by gel filtration and sodium dodecyl sulfate-gel electrophoresis, the enzyme purified from each tissue appeared to be homogeneous and was composed of an identical subunit, each possessing a M r of 60,000. With DEAE-Sephacel column chromatography, TH was separated completely from DPR. DPR was purified by subsequent chromatographies with Sephadex G-50 and blue Sepharose to a purity of 50%. DPR in adrenals and brain was found to be a NADH-dependent type. This micropurification procedure is applicable to assessing the molecular properties of TH modified physiologically or pharmacologically in vivo, and to getting a small amount of the pure enzyme as antigen for producing its antibody.

Biosynthesis of glycogen in Neurospora crassa. Purification and properties of the branching enzyme.

A branching enzyme was extracted from the mycelia of Neurospora crassa and was purified to electrophoretic homogeneity by procedures including DEAE-Sephacel column chromatography, 6-aminohexyl-Sepharose 4B column chromatography and gel filtration on Toyopearl HW-55S. The final yield of the branching enzyme activity was 15.1%, and the final purified enzyme preparation showed a specific activity of 702 units per mg of protein. The molecular weight of this enzyme was estimated to be 80,000 by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel. The amino acid composition and the carbohydrate content of this enzyme were analyzed. The isoelectric point of this enzyme determined by polyacrylamide gel isoelectrofocusing was 5.6. The branching activity of the enzyme was confirmed by its action on amylopectin as well as by the combined action of this enzyme and N. crassa glycogen synthase. The action of this enzyme on amylopectin decreased the wavelength of the absorption maximum of the glucan-iodine complex, and increased the amount of the short unit chains of the debranched product. The product obtained by the combined action yielded beta-limit dextrin upon hydrolysis with beta-amylase. No multiplicity was found for the branching activity either by chromatography or by electrophoresis.

Purification of Human Platelet Monoamine Oxidase B by High Performance Liquid Chromatography

Abstract Human platelet monoamine oxidase (MAO B), a membrane bound enzyme was purified to homogeneity by DEAE-Sephacel column chromatography, chromatofocusing, and high performance liquid chromatography (HPLC). The crucial purification step was HPLC on a anion exchange column (SynChropak AX 300). The HPLC column was eluted initially with potassium phosphate buffer (100 mM, pH 7.4) for 10 min at a flow rate of 1.0 ml/min, followed by a gradient (0–1%) of octyl-β-D-glucopyranoside (octylglucoside) in the same buffer for 10 min, and finally with buffered octylglucoside (1%) for 40 min. The elution of pargyline-bound or active MAO was established by determining either radioactivity in each fraction when MAO B had previously been covalently labeled with [3H]-pargyline [3H(G)] or catalytic activity using [14C-methylene]-benzylamine as substrate. [3H]-pargyline-bound and active MAO B eluted from the column at approximately 34 min. The extent of homogeneity and the subunit Mr (approximately 59,000) of MAO B were determined by sodium-dodecyl sulfate polyacrylamide gel electrophoresis followed by silver staining for proteins.

Initiation of DNA synthesis in the parotid salivary gland of adult mice by a factor isolated from acellular fluid of the Ehrlich ascites carcinoma.

The rate of DNA synthesis in the parotid salivary gland of adult mice is very low. We have purified about 5 000-fold a mitogen from the acellular ascitic fluid of the Ehrlich ascites carcinoma which stimulates DNA synthesis in the parotid salivary gland in vivo. This stimulation of DNA synthesis was produced with a protein concentration of this mitogen of 3 micrograms per 25 g of body weight. The purification procedure included ammonium sulfate fractionation and DEAE Sephacel column chromatography. This potent, heat-labile mitogen is presumed to be a protein with a mol.wt. of 3.5 X 10(3) to 1.3 X 10(4). The data indicate that this new factor is quite different from epidermal growth factor and tumor growth factor. Hypophysectomy did not prevent the stimulatory effect of this mitogen on the parotid salivary gland, indicating that the pituitary gland is not involved directly in mediating the mitogenic effect.

Ｎｅｕｒｏｓｐｏｒａ ｃｒａｓｓａのＢｒａｎｃｈｉｎｇ Ｅｎｚｙｍｅ グリコーゲンの生合成における役割

A branching enzyme was extracted from the mycelia of Neurospora crassa, and was purified to electrophortic homogeneity by a procedure including DEAE-Sephacel column chromatography, 6-aminohexyl-Sepharose column chromatography and gel filtration on Toyopearl HW-55S. The subunit molecular weight of this enzyme was estimated to be 80, 000 by electrophoresis in sodium dodecyl sulfate (SDS)-polyacrylamide gel, and 84, 800 by analysis of its amino acid composition and carbohydrate content. The optimum pH of this enzyme was around 8, and the optimum temperature was 27°C. The branching activity of the enzyme was confirmed by its, action on amylopectin and other substrates as well as by the combined action of this enzyme and N. crassa glycogen synthase. Action of this enzyme on various substrates induced decrease in the wave length maximum of absorption spectrum of the glucan-iodine complex, β-amylolysis limit and in the unit chain length of the debranched product. In the combined action, the branching activity stimulated incorporation of glucose into α-glucan. The products formed by the combined action had glycogenlike form, but the unit chain profiles of synthetic products were different from that of the native glycogen on Toyopearl HW-40F.

Glutamine synthetase in alder (Alnus glutinosa) root nodules. Purification, properties and cytoimmunochemical localization

Chromatographic, kinetic and regulatory properties of glutamine synthetase (GS) were investigated in root nodules of Alnus glutinosa L. (Gaertn) grown in a culture chamber. By DEAE Sephacel column chromatography and polyacrylamide gel electrophoresis, a major form of the enzyme was identified. Molecular weight was about 360 000 for the native protein and 43 000 for the subunits. Optimum pH was about 7.3 and Km for glutamate and ATP were 0.9 mM and 0.5 mM, respectively. By immunofluorescent techniques GS was localized in the inner cortical cells but not in vesicles of alder root nodules.

Peroxisomal and mitochondrial carnitine acetyltransferases in alkane-grown yeast Candida tropicalis.

Two types of carnitine acetyltransferases (EC 2.3.1.7) were first isolated from a microorganism, alkane-grown yeast Candida tropicalis. Carnitine acetyltransferase activity was induced in the alkane-grown cells, reaching about twenty times higher than that in the glucose-grown cells. Localization of the enzyme activity was demonstrated, at least, in peroxisomes (microbodies), profusely occurred in the alkane-grown cells, and in mitochondria. Peroxisomal and mitochondrial carnitine acetyltransferases could be separated using the method of DEAE-Sephacel column chromatography and both types were found to exist in the alkane-grown cells of C. tropicalis. Each carnitine acetyltransferase was purified using Sephadex G-200, Sepharose 6B, DEAE-Sephacel and Blue-Sepharose CL-6B. In DEAE-Sephacel chromatography, peroxisomal carnitine acetyltransferase was eluted below 0.15 M KCl concentration and mitochondrial carnitine acetyltransferase above 0.15 M KCl concentration. Except for the localization, little difference was observed in their kinetic properties, substrate specificity and so on. These two carnitine acetyltransferase preparations were only specific to acetyl and propionyl groups, the substrate specificity not being so broad as that of carnitine acetyltransferase obtained from mammalian tissues. Roles of these carnitine acetyltransferases in alkane metabolism in yeast are also discussed.

Studies on heterogeneity of Taka-amylase A: isolation of an amylase having one N-acetylglucosamine residue as the sugar chain.

Crystalline Taka-amylase A, prepared from Takadiastase, was fractionated into four fractions by DEAE-Sephacel and Concanavalin A-Sepharose column chromatography. The relative weight ratio of the fractions was 90 : 4 : 4 : 2. They had similar molecular weights (51,000), amino acid compositions, and hydrolytic activity against soluble starch, but different phenyl maltosidase activities and electrophoretic mobilities on polyacrylamide gel electrophoresis. Three of the fractions mainly had the high mannose type sugar chain with the sugar composition of Man5-GlcNAc2, but the other fraction had only one N-acetylglucosamine residue as the sugar chain. These results suggested that Taka-amylase A was heterogeneous both in the sugar portions and in the polypeptide portions.

Glutamine Synthetase in Rice

Chromatographic, kinetic, and regulatory properties of glutamine synthetase in rice were investigated. By DEAE-Sephacel column chromatography, two forms (glutamine synthetase 1 and glutamine synthetase 2) were identified in leaves and one form (glutamine synthetase R) was identified in roots. Purification on hydroxyapatite and gel electrophoresis showed that glutamine synthetase R was distinct from the leaf enzymes. The three isoforms were purified to similar specific activities and their properties were studied. Heat lability, pH optimum about 8, K(m) for l-glutamate of 20 millimolar, and inhibition by glucosamine 6-phosphate were the main characteristics of glutamine synthetase 2. Heat stability, pH optimum about 7.5, K(m) for l-glutamate of 2 millimolar, and no effect of glucosamine 6-phosphate differentiated glutamine synthetase 1 from glutamine synthetase 2. Glutamine synthetase R was also a labile protein but its kinetic and regulatory properties were quite similar to those of glutamine synthetase 1. These results clearly demonstrate the existence of three isoforms of glutamine synthetase in rice, two of which are located in the leaves and the third in the roots.

Deacetylation of PS-5, a new beta-lactam compound. II. Separation and purification of L- and D-amino acid acylases from Pseudomonas sp. 1158.

L-Amino acid acylase and D-amino acid acylase of Pseudomonas sp. 1158 which converted PS-5 to NS-5 (deacetylated PS-5) were separated and purified by sonication, streptomycin and ammonium sulfate fractionations, DEAE-Sephacel column chromatography and gel filtration. Molecular weight and the isoelectric point were estimated to be 75,000 and pI 5.45 for L-amino acid acylase and 100,000 and pI 4.95 for D-amino acid acylase.