Binding of bovine brain tissue factor to concanavalin A-Sepharose. Partial purification of coagulant, arylamidase, and alkaline phosphatase activities.

Tissue factor coagulant activity is adsorbed onto concanavalin A-Sepharose from sodium deoxycholate extracts of delipidated bovine brain powders. Coagulant activity is eluted with alpha-methyl-D-glucoside in sodium deoxycholate with 2--25-fold purification. This material has the same coagulant specific activity as that previously prepared in this laboratory. Alkaline phosphatase and alanyl-beta-naphthylamidase activities in the detergent extract also bind to concanavalin A-Sepharose and elute under the same conditions with 4- and 7-fold purification. In addition to these biological activities, the eluate was composed of protein (67.7%), neutral and amino sugars and sialic acid (22.3%), phospholipid (4.5%), uronic acid (3.8%) and nucleic acid (1.7%). This preparation is slightly enriched in carbohydrates compared to previous preparations. Concanavalian A-Sepharose therefore appears to be useful material for partial purification of several mammalian plasma membrane components with retention of biological function.     

Studies on membrane proteins involved in ribosome binding on the rough endoplasmic reticulum. Ribophorins have no ribosome-binding activity.

A membrane protein fraction showing affinity for ribosomes was isolated from rat liver microsomes (microsomal fractions) in association with ribosomes by treatment of the microsomes with Emulgen 913 and then solubilized from the ribosomes with sodium deoxycholate. This protein fraction was separated into two fractions, glycoproteins, including ribophorins I and II, and non-glycoproteins, virtually free from ribophorins I and II, on concanavalin A-Sepharose columns. The two fractions were each reconstituted into liposomes to determine their ribosome-binding activities. The specific binding activity of the non-glycoprotein fraction was approx. 2.3-fold higher than that of the glycoprotein fraction. The recovery of ribosome-binding capacity of the two fractions was about 85% of the total binding capacity of the material applied to a concanavalin A-Sepharose column, and about 90% of it was found in the non-glycoprotein fraction. The affinity constants of the ribosomes for the reconstituted liposomes were somewhat higher than those for stripped rough microsomes. The mode of ribosome binding to the reconstituted liposomes was very similar to that to the stripped rough microsomes, in its sensitivity to proteolytic enzymes and its strong inhibition by increasing KCl concentration. These results support the idea that ribosome binding to rat liver microsomes is not directly mediated by ribophorins I and II, but that another unidentified membrane protein(s) plays a role in ribosome binding.     

Acid sphingomyelinase from human urine: purification and characterization

Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human urine in the presence of 0.1% Nonidet P-40. The activity could be enriched 23,000-fold by sequential chromatography on octyl-Sepharose, concanavalin A-Sepharose, blue Sepharose and DEAE-cellulose. The last purification step yielded an enzyme preparation with a specific activity of about 2.5 mmol sphingomyelin cleaved/h per mg protein and with a yield of about 3%. Purified sphingomyelinase appeared to be homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 70 kDa. In the presence of 0.08% (w/v) sodium taurodeoxycholate the preparation showed phosphodiesterase activity toward sphingomyelin, phosphatidylcholine and phosphatidylglycerol. These activities co-purified during the entire purification procedure, indicating that the acid sphingomyelinase hydrolyses not only sphingomyelin but also the other two phospholipids, phosphatidylcholine and phosphatidylglycerol. Addition of 100 microM tripalmitoylglycerol to the assay system (which contains 100 microM sphingomyelin) instead of detergent, stimulated the reaction about 20-fold compared to an assay which did not contain detergents, thus offering a very sensitive and efficient system for the assay of sphingomyelinase in a system free of detergents. Sphingomyelin degradation was strongly inhibited by phosphatidylinositol 4',5'-bisphosphate, adenosine 3',5'-diphosphate and adenine-9-beta-D-arabinofuranoside 5'-monophosphate (50% inhibition at inhibitor concentrations of 1-5 microM).     

Purification of human gamma-interferon to essential homogeneity and its biochemical characterization

A multistep procedure has been developed which enables human gamma-interferon (HuIFN-gamma) to be purified to essential homogeneity. The procedure takes advantage of a modification of a previously described sequential chromatographic technique [Braude, I.A. (1983) Prep. Biochem. 13, 177-190] and the high isoelectric point of HuIFN-gamma (pH 9.5-9.8). The steps include Controlled Pore Glass adsorption chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, cation-exchange chromatography, and gel filtration chromatography. The purified HuIFN-gamma had a specific activity of 5.9 X 10(7) units/mg. This represents a purification of more than 70 000-fold and a 33% recovery. In addition, one gel filtration fraction had a specific activity of 2.5 X 10(8) units/mg. This represents a purification of greater than 300 000-fold and a recovery of greater than 17%. This fraction, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was shown to be composed of one major 26-kilodalton (kDa) species and four minor species of 74, 67, 56, and 22 kDa. Analysis of this material with anti-HuIFN-gamma monoclonal antibody immunoabsorbent columns indicates that both the 26- and the 22-kDa species are HuIFN-gamma. Thus, the final product is essentially homogeneous (90-92% HuIFN-gamma), and the specific activity of pure HuIFN-gamma is approximately (2.7-2.8) X 10(8) units/mg of protein. Finally, the 26- and 22-kDa moieties are shown to be similar, if not identical, proteins as judged by amino acid and sequence analyses.     

Studies of human kidney gamma-glutamyl transpeptidase. Purification and structural, kinetic and immunological properties.

gamma-Glutamyl transpeptidase, present in various mammalian tissues, transfers the gamma-glutamyl moiety of glutathione to a variety of acceptor amino acids and peptides. This enzyme has been purified from human kidney cortex about 740-fold to a specific activity of 200 units/mg of protein. The purification steps involved incubation of the homogenate at 37 degrees followed by centrifugation and extraction of the sediment with 0.1 M Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate; batchwise absorption on DEAE-cellulose; DEAE-cellulose (DE52) column chromatography; Sephadex G-200 gel filtration; and affinity chromatography using concanavalin A insolubilized on beaded Agarose. Detergents were used throughout the purification of the enzyme. The purified enzyme separated into three protein bands, all of which had enzyme activity, on polyacrylamide disc electrophoresis in the presence of Triton X-100. The enzyme has an apparent molecular weight of about 90,000 as shown by Sephadex G-200 gel filtration, and appears to be a tetramer with subunits of molecular weights of about 21,000. The Km for gamma-glutamyl transpeptidase using the artificial substrate, gamma-glutamyl-p-nitroanilide, with glycylglycine as the acceptor amino acid was found to be about 0.8 mM. The optimum pH for the enzyme activity is 8.2 and the isoelectric point is 4.5. Both GSH and GSSG competitively inhibited the activity of gamma-glutamyl transpeptidase when gamma-glutamyl-p-nitroanilide was used as the substrate. Treatment of the purified enzyme with papain has no effect on the enzyme activity or mobility on polyacrylamide disc electrophoresis. The purified gamma-glutamyl transpeptidase had no phosphate-independent glutaminase activity. The ratio of gamma-glutamyl transpeptidase to phosphate-independent glutaminase changed significantly through the initial steps of gamma-glutamyl transpeptidase purification. These studies indicate that the transpeptidase and phosphate-independent glutaminase activities are not exhibited by the same protein in human kidney.     

Sepharose-linked concanavalin A in the purification and characterization of glycoprotein hormones of the bovine pituitary

Affinity chromatography on concanavalin A-Sepharose is a time saving step in both large and small scale isolations of the bovine pituitary glycoprotein hormones. After ion-exchange chromatography, the final yield of purified lutropin is 40-50% of material in starting concentrates and of purified thyrotropin is approximately 20%. The final products have the same electrophoretic and immunological properties and amino acid compositions as previous preparations. Less than 3% of the immunoreactive lutropin, follitropin and thyrotropin are present as non-glycosylated forms in either crude pituitary extracts or concentrates. Thyrotropin and follitropin elute from the immobilized lectin as a single fraction, whereas lutropin separates into two glycosylated fractions. Gel filtration of both crude extracts and the glycoprotein fractions shows that less than 5% of the immunoreactivity of the hormones is present as material of apparently high molecular weight. Substantial alpha subunit immunoreactivity, however, is in three fractions (as found by others in human pituitary extracts) corresponding to "high molecular weight material" (7%), intact hormones (46%) and free subunit (47%).     

Purification, properties and glycoprotein nature of arylsulfatase A from sheep brain.

A simple and rapid method for the purification of arylsulfatase A (EC 3.1.6.1) from sheep brain has been developed. This includes the concanavalin A-Sepharose affinity chromatography and the pH-dependent polymerization and depolymerization of the enzyme. By these methods a homogeneous enzyme was obtained and the enzyme was purified 7180-fold. Sheep brain arylsulfatase A has been shown to be a glycoprotein containing 25% neutral sugar and 0.5% sialic acid. The constituent neutral sugars were identified as glucose and mannose.     

PARTIAL PURIFICATION, PROPERTIES AND GLYCOPROTEIN NATURE OF ARYLSULPHATASE B FROM SHEEP BRAIN

Abstract— A simple method has been developed for the partial purification of arylsulphatase B from sheep brain. This includes concanavalin A-Sepharose affinity chromatography and ionic strength-dependent binding and dissociation of the enzyme with Dextran Blue; by these methods the enzyme was purified 1344-fold with 10% recovery. The partially purified enzyme was shown to be a glycoprotein and its kinetic properties were compared with that of purified arylsulphatase A from the same source.     

Preparation of homogeneous human prostatic acid phosphatase using concanavalin A-sepharose 4-B.

A simple, rapid and efficient procedure is presented for the purification of human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) to homogeneity. The method employs two steps suitable for use with large quantities of material, followed by chromatography on concanavalin A-Sepharose as its sole column step. The procedure also permits the recovery of purified enzyme in higher yields than earlier methods.     

An improved purification procedure for rat liver lysosomal phospholipase A1

A purification procedure is presented for the isolation of lysosomal acid phospholipase A1 (PLA1) from livers of non-pretreated rats, in a high yield and purity. The purification starts from a crude mitochondrial-lysosomal fraction. PLA1 is solubilised and subsequently purified by chromatography on concanavalin A-Sepharose, by chromatofocusing, and by gel filtration. After chromatofocusing, the enzyme is already purified 50200-fold with a yield of 50%, and after gel filtration 56600-fold with a yield of 7%. Purified PLA1 exhibits a specific activity of approx. 8.2 mumol phosphatidylethanolamine (preferred substrate) hydrolysed per min per mg protein, and upon chromatofocusing an apparent isoelectric point of 5.3 Gel filtration of purified PLA1 suggests a molecular mass of about 29 kDa, whereas in SDS-PAGE two proteins of 27 kDa and 55 kDa (mass ratio about 1/2) were visualised.     

Purification of the insulin receptor from human placental membranes

Insulin receptors were purified from human placental microsomal membranes by solubilisation with Triton X-100 followed by Sepharose 6B chromatography, phosphate gradient elution from hydroxyapatite and affinity chromatography on concanavalin A-Sepharose. 2000-fold purification was achieved with 63% overall recovery. The purified receptor gave a single band on 3.75% polyacrylamide (0.1% Triton X-100) gel electrophoresis. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis there was a major band at 75,000 and a minor band at 80,000 daltons. The purified receptor rechromatographed on Sepharose 6B with an apparent molecular weight of 300,000.     

I-Cell disease: isoelectric focusing, concanavalin A-Sepharose 4B binding and kinetic properties of human liver acid beta-D-galactosidases.

Isoelectric focusing of the acid beta-D-galactosidases (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in normal crude liver supernatant fluids demonstrated multiple isoelectric forms in the pH range 4.58-5.15, while corresponding I-cell disease samples showed an absence of isoelectric forms in the pH range 4.99-5.15. Concanavalin A-Sepharose 4B chromatography of the I-cell disease mutant C.A. demonstrated a 31% and 37% decrease in the binding of 4-methyl-umbelliferyl-beta-D-galactosidase and GM1 beta-D-galactosidase activities, respectively, when compared to normal samples. Isoelectric focusing profiles of the concanavalin A-Sepharose 4B alpha-methyl-D-mannoside effluents containing normal and I-cell disease acid beta-D-galactosidase were generally similar, but the unadsorbed I-cell disease enzyme from concanavalin A-Sepharose 4B demonstrated more activity in the pH range 4.21-4.49 than normals. Normal and I-cell disease acid beta-D-galactosidase "A" and "B", separated by gel column chromatography were found to have similar properties with respect to apparent molecular weights pH vs. activity profiles and apparent Km values for the 4 methylumbelliferyl-beta-D-galactopyranoside, GM1-ganglioside and asialofetuin (ASF) substrates. However, the apparent V values for the ICD samples were consistently reduced when compared to the results obtained with the corresponding normal fractions. The greatest decreases in apparent V were obtained for acid beta-D-galactosidase activities in I-cell disease crude supernatant fluids, and for the separated I-cell disease "B" enzyme. The differences in the isoelectric focusing profiles, the altered binding to concanavalin A-Sepharose 4B, and the reduced V values with natural and synthetic substrates may be related to changes in carbohydrate composition of I-cell disease acid beta-D-galactosidase.     

Mouse bone collagenase inhibitor: purification and partial characterization of the inhibitor from mouse calvaria cultures

The organ culture of neonatal mouse calvaria produced both collagenase and collagenase inhibitor. The inhibitor was purified by a series of column chromatographies: DEAE-cellulose and CM-cellulose ion-exchange chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, and finally by Sephacryl S-200 gel filtration. The purified inhibitor migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular mass of 28,000. The inhibitor was purified 140-fold to a specific activity of 163 units/mg with a yield of 18% over the first step of the purification by DEAE-cellulose chromatography. The inhibitor stained positively for carbohydrate with periodic acid-Schiff's reagent indicating, in conjunction with its affinity to concanavalin A, that the inhibitor is a glycoprotein. In addition to mouse bone collagenase, this inhibitor also inhibited chick bone, rat bone, rabbit corneal, and human gingival collagenase, but did not inhibit bacterial collagenase.     

Purification and characterization of the mouse thymocyte Thy-1 glycoprotein.

The thymocyte Thy-1 glycoprotein was purified from mouse strain NMRI (Thy 1.2) thymus. Crude membranes were prepared in Tween 20 and solubilized in deoxycholate. The glycoproteins were isolated by affinity chromatography on concanavalin A-Sepharose, and Thy-1 was further purified by two gel-filtration cycles on Sephacryl S-200. The concentration of Thy-1 in fractions obtained during purification was measured by a solid-phase radioimmunoassay with pure mouse brain Thy-1 as standard. Analysis of the purified preparation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed at least five distinct bands in the apparent-Mr region 25000-30000, the polymorphism probably being due to carbohydrate heterogeneity. Amino-acid-analysis data were compatible with the previously published sequence of mouse brain Thy-1. Sugar content was determined at 31% (w/w), and the carbohydrate composition indicates the presence of 'complex-type' oligosaccharide chains. The mean Mr of mouse thymocyte Thy-1 was calculated to be 18 100.     

Characterization of human liver alpha-D-mannosidase purified by affinity chromatography.

Human liver acidic alpha-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral alpha-mannosidase did not bind to the concanavalin A-Sepharose so the two types of alpha-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was alpha-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that alpha-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of alpha-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human alpha-mannosidase. The purified enzyme completely removed the alpha-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic alpha-mannosidase.     

Isolation and characterization of the pig endometrial arylsulphatase A.

The pig endometrial arylsulphatase A was purified 3322-fold to a specific activity of 150 mumol/min per mg. The purification involved (NH4)2SO4 fractionation, chromatography on concanavalin A-Sepharose and DEAE-Sepharose, gel filtrations on Sephadex G-200 at pH 7.4 and 5, and a new preparative gel-electrophoresis technique. The homogeneous enzyme is a glycoprotein containing 20% carbohydrate. The purified enzyme has Mr about 120 000 and it contains subunits of Mr 63 000. The pig endometrial arylsulphatase A shows many properties in common with those of arylsulphatases A purified from other sources. The similarities include their low isoelectric points, the anomalous time-activity relationships, multi-pH optima, inhibition by SO3(2-), SO4(2-), phosphate ions, metal ions and nucleoside phosphates, pH- and ionic-strength-dependent polymerization and amino acid composition.     

Purification of soluble acetylcholinesterase from Japanese quail brain by affinity chromatography.

The purification of a soluble acetylcholinesterase from Japanese quail brain using affinity chromatography on concanavalin A-Sepharose and edrophonium-Sepharose is described. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose. Acetylcholinesterase was purified 10,416-fold with a specific activity of 2500 U/mg protein. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol gave only one band with a molecular weight of 62.5 kDa. The molecular weight of the purified acetylcholinesterase was estimated to be 245.5 kDa by gel chromatography on Sephacryl S-200 under nondenaturing conditions. Based on the molecular weight obtained by both SDS-PAGE and gel filtration the purified acetylcholinesterase was assumed to be a tetrameric form.     

Purification and N-terminal sequence of two tartrate-resistant acid phosphatases type-5 from the hairy cell leukemia spleen

Tartrate-resistant acid phosphatases types 5a and 5b were purified from human hairy cell leukemia spleen by sequential chromatography on Phenyl-Sepharose, CM-Sepharose, concanavalin A-Sepharose, FPLC Superose-12 and FPLC Mono-S. The purification over the original tissue extract was 1150- and 3300-fold, with a yield of 2.1% and 2.5%, respectively. Gel filtration indicated an Mr of about 30000 for both forms. There was a N-terminal sequence identity between the two enzymes. However, they appeared to be different as assessed by cation exchange chromatography and amino acid composition.     

Improved procedure for the purification of hepatic lipase from rat liver homogenate

A procedure is described for the purification of hepatic lipase (HL)4 from rat liver homogenate which results in a high yield (41%) of electrophoretically homogeneous enzyme. The method is based on that of Twu et al. (Biochim. Biophys. Acta 1984: 792, 330), but it is more efficient with respect to yield (about 4-fold) and purity (1.6-fold). It includes the preparation of a high-speed supernatant, chromatography in series on octyl-, heparin- and concanavalin A-Sepharose, and finally gel filtration. On SDS-PAGE analysis, the purified enzyme exhibited an apparent molecular mass of 63.6 +/- 3.2 kDa. Heterogeneity was observed, when purified HL was subjected to isoelectric focussing. The enzyme displayed a specific catalytic activity of 23,000 U* (mumol fatty acid released per h at 37 degrees C) per mg protein, when assayed with trioleoyl glycerol suspensions in arabic gum. A highly specific antiserum against rat liver HL, capable of inhibiting 817 mU* HL per microliter antiserum, was raised in rabbits.     

Purification and N-terminal amino acid sequence of the tartrate-resistant acid phosphatase from human osteoclastoma: evidence for a single structure

Tartrate-resistant acid phosphatase type-5 was purified to apparent homogeneity from human osteoclastomas by sequential chromatography on CM-Sepharose, Phenyl-Sepharose, concanavalin A-Sepharose, FPLC Superose-12, and FPLC Mono-S. The purification over the original tissue extract was 1167-fold, with a yield of 16%. An identity in the N-terminal amino acid sequence and Mr was found between this enzyme and two type-5 tartrate-resistant acid phosphatases isolated from hairy cell leukemia spleen. However, they appeared to be different as assessed by amino acid composition. In contrast to a previous report, no evidence was found for two subunits of the tartrate-resistant acid phosphatase.