Reexamination of spectroscopic properties of ceruloplasmin freshly isolated with a novel very rapid single-step procedure

A novel, single-step, chromatographic procedure for the purification of ceruloplasmin is described. The procedure consists of a single chromatographic step, leading in approximately 5 hours from 2 liters of plasma to an electrophoretically homogeneous ceruloplasmin preparation which exhibits different spectroscopic properties with respect to the protein obtained with more lengthy, currently available procedures. The method is suitable for either small or large scale purification from various vertebrate plasma.     

Structural and immunochemical characterization of human urine arylsulfatase A purified by affinity chromatography

Arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was isolated from an ammonium sulfate precipitate of urinary proteins using two different affinity chromatography methods. One method involved the use of concanavalin A-Sepharose affinity chromatography at an early stage of purification, followed by preparative polyacrylamide gel electrophoresis. The other procedure employed arylsulfatase subunit affinity chromatography as the main step and resulted in a remarkably efficient purification. The enzyme had a specific activity of 63 U/mg. The final preparation of arylsulfatase A was homogeneous on the basis of polyacrylamide gel electrophoresis at pH 7.5, and by immunochemical analysis. However, when an enzyme sample obtained by either method of purification was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing or non-reducing conditions, peptide subunits, of 63.5 and 54.5 kDa, were observed. Immunological tests with 125I-labeled enzyme established the presence of a common protein component in both of the electrophoretically separable peptide subunits of human urine arylsulfatase. The amino acid analysis of homogeneous human urine arylsulfatase A showed only a few differences between it and the human liver enzyme. However, immunological cross-reactivity studies using rabbit anti-human urine arylsulfatase revealed immunological difference between the human urine and liver arylsulfatase A enzymes.     

Bovine liver thiol-protein disulphide oxidoreductases. An alternative method for differential purification and resolution of protein disulphide-isomerase and glutathione-insulin transhydrogenase.

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) activities in bovine liver were studied in parallel during purification of 'thiol-protein disulphide oxidoreductase' by the procedure of Carmichael, Morin & Dixon [(1977) J Biol. Chem. 252, 7163-7167]. The two activities showed no quantitative co-purification and were partially resolved by (NH4)SO4 precipitation, indicating that distinct enzymes are present. 2. Protein disulphide-isomerase was purified by a relatively rapid method involving a combination of the early stages of the Carmichael procedure and covalent chromatography, with a new stepwise elution procedure. Ion-exchange chromatography yields a homogeneous preparation of mol.wt. 57 000. 3. The relationship between protein disulphide-isomerase, glutathione-insulin transhydrogenase and 'thiol-protein disulphide oxidoreductase' is discussed.     

Interaction between heparin and human pregnancy-associated plasma protein A (PAPP-A): a simple purification procedure

Human pregnancy-associated plasma protein A (PAPP-A) binds to heparin-Sepharose. This affinity chromatography preceded by molecular sieve chromatography provides a simple two-step purification procedure of PAPP-A from late pregnancy plasma. One hundred percent of the applied PAPP-A was recovered, with more than 40% being electrophoretically homogeneous after the two procedures. The remaining PAPP-A could be purified by negative affinity chromatography on anti-total human serum immobilized on agarose.     

Purification and Characterization of Lipase from Wheat (Triticum aestivum L.) Germ

A method of isolation and purification of lipase (EC 3.1.1.3) from the germ of wheat (Triticum aestivumL.) is described. An electrophoretically homogeneous preparation of the enzyme (specific activity, 622.5 × 10^–3 mol/min per mg protein) was obtained after 61-fold purification. The molecular weight of the enzyme, determined by gel chromatography, was 143 ± 2 kDa. The optimal conditions for the enzyme were 37°C and pH 8.0. The homogeneous preparation of the lipase exhibited high thermal stability: over 20% of the original activity was retained after incubation of the preparation at high temperatures (60–90°C) for 1 h at pH 8.0.     

The purification and properties of extracellular glycosidases of the cellular slime mould Dictyostelium discoideum

The purification of beta-N-acetylhexosaminidase, alpha-glucosidase, alpha-mannosidase and beta-glucosidase from the spent growth medium of Dictyostelium discoideum strain Ax-2 myxamoebae is described. beta-N-Acetylhexosaminidase and alpha-glucosidase were obtained in high yield and as homogeneous preparations whereas the alpha-mannosidase preparation consisted of two electrophoretically distinct isoenzymes. The physical, chemical and kinetic properties of these enzymes are described.     

Purification of cytochrome P-450, NADPH-cytochrome P-450 reductase,and epoxide hydratase from a single preparation of rat liver microsomes

A simplified procedure is presented for the simultaneous purification of the enzymes cytochrome P-450, epoxide hydratase (EC 3.3.2.3), and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) from a single preparation of rat liver microsomes. All three enzymes can be recovered after chromatography of detergent-solubilized microsomes on a column of n-octylamino-Sepharose 4B. The major form of cytochrome P-450 (of phenobarbitaltreated rats) is purified by subsequent DEAE-cellulose chromatography, epoxide hydratase is purified by DEAE- and O-(carboxymethyl)-cellulose chromatography, and NADPH-cyto-chrome P-450 reductase is purified using 2′,5′-ADP agarose chromatography. The nonionic detergent Lubrol PX and the ionic detergents sodium cholate and deoxycholate are used in these procedures to permit utilization of uv-absorbance measurements in monitoring protein during purification. Overall yields of the three enzymes are approximately 20, 25, and 60%, respectively. All three enzymes are apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are functionally active. The same procedure can be used to obtain the major cytochrome P-450 present in liver microsomes isolated from β-naphthoflavone (5,6-benzoflavone)- or 3-methylcholanthrene-treated rats. Thus, the described procedures permit the rapid and reproducible purification of three major rat liver microsomal enzymes which can be coupled to study bioactivation and detoxification of a variety of xenobiotics in reconstituted systems.     

Purification of Squash NADH:Nitrate Reductase by Zinc Chelate Affinity Chromatography

NADH:nitrate reductase (EC 1.6.6.1) was isolated and purified from the green cotyledons of 5-day-old squash seedlings (Cucurbita maxima L.). The 10-hour purification procedure consisted of two steps: direct application of crude enzyme to blue Sepharose and specific elution with NADH followed by direct application of this effluent to a Zn²⁺ column with elution by decreasing the pH of the phosphate buffer from 7.0 to 6.2. The high specific activity (100 micromoles per minute per milligram protein) and high recovery (15-25%) of electrophoretically homogeneous nitrate reductase show that the enzyme was not damaged by exposure to the bound zinc. With this procedure, homogeneous nitrate reductase can be obtained in yields of 0.5 milligram per kilogram cotyledons.     

Purification and properties of the cytosolic and mitochondrial forms of aspartate aminotransferase and malate dehydrogenase from rat heart

The present work describes the purification from rat heart of the mitochondrial and cytosolic forms of the enzymes of the malate--aspartate shuttle, aspartate aminotransferase (EC 2.6.1.1) and malate dehydrogenase (EC 1.1.1.37), by a single procedure after the preparation of the original crude extract. In 10 purification steps, the four enzymes were obtained electrophoretically pure in yields ranging from 6 to 54% of their respective isoenzyme levels in the crude extract. Apoenzymes were formed from the aminotransferases by reacting them with cysteine sulfinate and dialyzing. Complete reconstitution was obtained after a brief incubation with pyridoxal phosphate. All four enzymes are dimers. The mitochondrial isoenzymes are of slightly lower molecular weight than their respective cytosolic forms. Michaelis constants and maximal velocities were derived by the use of primary and secondary plots. In general, the properties of the enzymes from rat heart are similar to the properties of the enzymes from other animal sources.     

Purification of xylanase from the wood-rotting fungusTrametes hirsuta

Xylanase (EC 3.2.1.8) was purified from a crude extracellular enzyme of the wood-rotting fungusTrametes hirsuta by a four-step procedure involving precipitation with ammonium sulphate, gel filtration on Bio-Gel P-10, column chromatography on DEAE-Sephadex A-50 and preparative electrophoresis on polyacrylamide gel. The isolated enzyme, electrophoretically homogeneous, was separated from β-xylosidase and other hydrolytic enzymes studied. The analysis of the degradation products of water-soluble (4-0-methylglucurono)-D-xylan from willow by the purified xylanase showed it to be endoxylanase (β-1,4-xylan xylanohydrolase). A sequel to Kubačkováet al., Folia Microbiol.20, 29 (1975).     

Purification of pigeon liver malic enzyme by affinity chromatography

A simple and rapid method for the purification of malic enzyme (EC 1.1.1.40) from pigeon liver is described. Malic enzyme in the crude tissue extract was partially purified by heat treatment, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Final purification was achieved by affinity chromatography on immobilized N⁶-(6-aminohexyl)-adenosine 2′,5′-bisphosphate. Apparently homogeneous enzyme was obtained in 2 days with 54% yield.     

New cyclomaltodextrin-glucanotransferases, produced by Bacillus macerans

For the first time, microorganisms producing cyclomaltodextrin glucan transferases (CGT, EC 2.4.1.19) were isolated from soil samples of various ecogeographical regions. These microorganisms were identified as Bacillus macerans. The enzymes were purified by affinity chromatography on a alpha-cyclodextrin polymer and gel filtration on Biogel P-450 and proved to be electrophoretically homogeneous. Some of their physicochemical and biochemical properties are reported.     

Purification and characterization of thiamine triphosphatase from bovine brain.

Soluble thiamine triphosphatase (EC 3.6.1.28) of bovine brain has been purified 68,000-fold to an electrophoretically homogeneous state with an overall recovery of 5.5% by hydrophobic chromatography on Toyopearl HW-60, Sephadex G-75 gel filtration, DEAE-Toyopearl 650M chromatography and Blue Sepharose CL-4B chromatography. The enzyme has an absolute specificity among thiamine and nucleoside phosphate esters for thiamine triphosphate and shows no nonspecific phosphatase activities. Thiamine triphosphatase is composed of a single polypeptide chain with molecular mass of 33,900 kDa as estimated by Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 8.7 and is dependent on divalent metal ions. Mg2+ has been found to be the most effective among cations tested. A study of the reaction kinetics over a wide range of thiamine triphosphate concentrations has revealed a biphasic saturation curve being described by higher-degree rational polynomials.     

Components of human complement system. Testing and isolation of C4

A modified reagent for testing the hemolytic activity of human complement component C4 has been obtained. Reagent R4 was obtained by treatment of human blood serum pools with 0.075 M solution of hydrazine hydrate. This reagent was found to be rich in the serum fraction obtained by chromatography on DEAE-cellulose DE-52 and containing an active complement C3 component. To test the sensitivity and specificity of the reagent, component C4 was subjected to purification. This procedure resulted in a hemolytically active, electrophoretically and immunoelectrophoretically homogeneous component C4. DEAE-cellulose DE-52, DEAE-Sephacel, Ultragel AcA-34 and, again, DEAE-Sephacel were used consecutively as purification agents. The activity yield of component C4 with regard to the initial serum level was 20%.     

New cyclomaltodextrin glucan transferases produced by bacillus macerans

—For the first time, microorganisms producing cyclomaltodextrin glucantransferaseglucan transferases (CGT, EC 2.4.1.19) were isolated from soil samples of various ecogeographical regions. These microorganisms were identified asBacillus macerans. The enzymes were purified by affinity chromatography on an α-cyclodextrin polymer and gel filtration on Biogel P-150 and proved to be electrophoretically homogeneous. Some of their physicochemical and biochemical properties are reported.     

Species-specific heterogeneity for molecular weight estimates of serum extracellular superoxide dismutase activities.

1. Several apparent molecular weights (mol. wt) are reported for plasma or serum extracellular superoxide dismutase (EC SOD) activity. This study found species-dependent heterogeneity for apparent mol. wt using gel filtration with Sephadex G-150. 2. EC SOD activity in rabbit and guinea-pig serum, measured by a modified pyrogallol assay, eluted just before ceruloplasmin activity, but rat and bovine serum activity eluted after ceruloplasmin (apparent mol. wt of 142,000 and 73,000, respectively). 3. The heterogeneity between rat and rabbit serum was not eliminated by substituting a cytochrome-c-based SOD assay for the pyrogallol method, by substituting lung extracts for serum, by analysing a mixture of rat and rabbit serums, nor by analysing hemolysed serum. The apparent mol. wt of bovine serum EC SOD activity was not duplicated by gel filtration analysis of a mixture of bovine cytosolic SOD and albumin. 4. In conclusion, species-specific variation in apparent mol. wt for serum EC SOD activity was demonstrated under several circumstances.     

Purification of multiple forms of adenosine deaminase from rabbit intestine.

Two forms of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), differing in molecular size, have been purified and obtained in homogeneous form from rabbit intestine. The purification procedures involved extraction with acetate buffer, pH 5.5, precipitation and fractional reextraction with (NH4)2SO4, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-75 and Sephadex G-200. Gel filtrations analysis gave molecular weight estimates of 265 000 and 32 000 for the large and small deaminases respectively. The two enzymes forms had similar pH optima and pH stability ranges.     

Oxaloacetate keto-enol tautomerase from bovine heart mitochondrial matrix

Bovine heart mitochondrial matrix contains two proteins possessing the oxaloacetate keto-enol tautomerase (EC 5.3.2.2) activity. A procedure for the isolation and purification of the enzymes to an electrophoretically homogeneous state has been developed. The purified proteins have molecular masses of 37 kD and 80 kD and catalyze the keto-enol oxaloacetate tautomerization reaction with the turnover numbers of approximately 3000 and approximately 2000 min-1. The both enzymes were found to differ significantly in all their physicochemical and kinetic properties. Fractionation of rat liver mitochondria revealed that the oxaloacetate keto-enol tautomerase activity is predominantly localized in the mitochondrial matrix. The essential role of oxaloacetate keto-enol tautomerase in the operation of the Krebs cycle is discussed.     

A simplified procedure for the purification of large quantities of fetal bovine serum acetylcholinesterase

A simple procedure has been developed for the large scale purification of fetal bovine serum acetylcholinesterase (AChE) (EC 3.1.1.7). The procedure involves two steps: batch adsorption of the AChE from 250 L of serum onto a procainamide affinity Sepharose 4B gel; and analytical procainamide affinity chromatography of the step-1 product. Over 100 mg of AChE was purified in 10 days to apparent homogeneity with this procedure.     

Lactoperoxidase from human colostrum.

The present study has confirmed that human colostrum contains a lactoperoxidase (EC 1.11.1.7) [Langbakk & Flatmark (1984) FEBS Lett. 174, 300-303], which represents about 0.004% of the total protein in crude colostrum. An apparent 32-fold purification of the enzyme was obtained by a multistep procedure, as modified from that of the bovine enzyme, with a recovery of about 7%. By use of chromatography on an immunoaffinity column (directed against bovine lactoperoxidase B), an apparent 1450-fold purification was obtained in a single step, with a recovery of 21%. The enzyme behaved as a glycoprotein (binding to concanavalin A-Sepharose), and revealed spectral properties (Soret peak at 412 nm) and an Mr (80,000) similar to those of the bovine enzyme.