Purification of mammalian arylsulfatase A enzymes by subunit affinity chromatography

Rabbit liver arylsulfatase A (arylsulfatase sulfohydrolase, EC 3.1.6.1) monomer was immobilized on cyanogen bromide-activated Sepharose-6MB and on Affi-Gel-10 under various experimental conditions in order to study the effects of variables in sulfatase monomer/oligomer subunit affinity chromatography. First, the number of reactive groups on activated Sepharose-6MB and Affi-Gel-10 was determined by a procedure involving spectrophotometric titration with L-tyrosine. After covalent coupling of sulfatase monomers to the gels, the enzyme binding capacities of the sulfatase subunit affinity gel matrixes were determined at pH 4.5. The maximum binding of free monomers from solution could be achieved when the Affi-Gel-10 protein monomer matrix was prepared at low degrees of covalent loading. The introduction of a batch technique for equilibration of the protein sample with the monomer affinity matrix also increased the efficiency of the subunit affinity gel in purification procedures. The effect of pH on the stability of the heterodimers formed between monomers of rabbit liver arylsulfatase A immobilized on Affi-Gel-10 and free monomers of arylsulfatase A enzymes from different tissues and organisms was studied using the batch technique. For all sulfatase A enzymes tested, the midpoint of the pH transition for subunit association was pH 6.2, suggesting that the amino acid residues involved in the dimerization are similar. The versatility of the Affi-Gel-10 monomer affinity matrix was further demonstrated by purifying 13 mammalian arylsulfatase A enzymes to homogeneity, as assessed by Sephacryl chromatography, native and SDS gel electrophoresis. The molecular weights of the homogeneous monomers and their peptide subunits were in the range of 110-180 KDa and 50-64 KDa, respectively. The amino acid compositions of these enzymes were also determined.     

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.     

A two-component affinity chromatography purification of Helix pomatia arylsulfatase by tyrosine vanadate

The inhibition of Helix pomatia arylsulfatase by the synergistic combination of N-acetyl-l-tyrosine ethyl ester and vanadate has been extended to affinity chromatography for purification. In the presence of vanadate, l-tyrosine ethyl ester (TEE), immobilized on CH-Sepharose 4B retained arylsulfatase from the digestive juice or lyophilized powder of H. pomatia. No enzyme was retained without vanadate or with arsenate or phosphate. Arylsulfatase was eluted from the column matrix by removing the vanadate to less than 50 microM with buffer containing EDTA to chelate the vanadate. Escherichia coli alkaline phosphatase and potato acid phosphatase, two enzymes which are inhibited by vanadate but not by the vanadate-TEE complex, were not retained by the immobilized TEE under any conditions used. The sulfatase activity was completely separated from contaminating glucuronidase activity present in the crude enzyme extracts. The Ki for the immobilized vanadate-TEE system was found to be 5.0 x 10(-7) M with a capacity of 25 mg/ml swollen gel. A purification of greater than 40-fold from the lyophilized powder of H. pomatia (Sigma Type H-5) was achieved using this technique. The Ki/Keq of other phenols with vanadate were determined in a 96-well plate format as an example of a rapid screening technique that could be extended to other phosphoryl and sulfuryl-transfer enzyme classes.     

Isolation and characterization of the two active fractions from porcine brain phosphofructokinase purified by affinity chromatography

Phosphofructokinase was purified from porcine brain by the successive affinity chromatographies of Blue-Sepharose and ATP-agarose. The purified enzyme was found to contain three subunits, namely, the L-, M- and C-type by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme separated into the two active fractions through gel filtration (FIs and FIIs). SDS-PAGE revealed that the FIs contained the L- and M-type subunits, while the FIIs contained the M- and C-type. The FIIs preparation was more sensitive to ATP inhibition, ADP activation and citrate inhibition than the FIs.     

A variant form of arylsulfatase A in human urine derived directly from the renal pelvis: kinetic and immunological characterization

Arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was examined in voided and in nephrostomic urine. A variant form of the enzyme was found in nephrostomic urine, in addition to the minor form, which is the sole component of arylsulfatase A in voided urine. The nephrostomic enzyme differed from the voided urine enzyme with respect to the kinetic parameters, the isoelectric point, heat stability and immunological reactivity. The isoelectric points of the voided urine and nephrostomic enzymes were 4.7 and 5.3, respectively. The nephrostomic enzyme was more heat-labile at 62.5 degrees C than the voided urine enzyme. Although the Km values of the two enzymes with nitrocatechol sulfate as substrate were almost the same, the V value of the nephrostomic enzyme was approx. one-hundredth that of the voided urine enzyme. The molecular weight (almost 130 000) did not differ between the voided urine and nephrostomic enzymes. It was demonstrated by various methods, using IgG antibody against the purified voided urine enzyme, that the nephrostomic enzyme was antigenically distinct from the voided urine enzyme.     

Evaluation by ELISA of anisakis simplex larval antigen purified by affinity chromatography

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of (1/4)00 and 1 microg/ml of antigenic concentration.     

Characterization of fragments of human albumin purified by Cibacron blue F3GA affinity chromatography.

Controlled limited proteolysis of human plasma albumin (0.3 mM; 37 degrees C; 15 min; pH 3.7) with pepsin [pepsin/albumin, 1:1000 (w/w)] in the presence of octanoic acid (4.2 mM) yields at least 14 fragments in the range of 5000--56000 Da. By utilizing a combination of conventional and affinity-chromatographic procedures, two fragments with mol. wts. 25000 and 27000 were purified to more than 99% homogeneity. The larger fragment consists of a continuous polypeptide chain and has been shown to contain the primary bilirubin-binding site. The small fragment contains an internal cleavage site. On the basis of amino acid compositions, N-terminal sequences, C-terminal sequences, molecular weights and other internal markers the locations of these fragments within the known sequence of human albumin were determined to be residues 49--308 for the 27000 Da peptide and 309--585 for the 25000 Da peptide. Peptide 309--585 contains an internal cleavage site and appears to be missing residues 408--423. These non-overlapping fragments should be useful for investigations of individual ligand-binding sites and for the determination of antigenic sites.     

Characterization of glyoxalase I purified from pig erythrocytes by affinity chromatography

The linear order of nine fragments generated by the action of endonuclease AvaI on the DNA of bacteriophage lambda was determined from the altered fragmentation patterns of bacteriophages containing known deletions and of hybrids of bacteriophages lambda and phi80. Digestion of 5'-terminally 32P-labelled bacteriophage-lambda DNA was used to identify the terminal fragments. Measurement of relative fragment lengths permitted rough mapping of the endonuclease-AvaI cleavage sites relative to the ends of the bacteriophage-lambda chromosome. The fragment order was confirmed and the map refined by analysis of the fragmentation of derivative phages containing single cleavage sites for endonuclease EcoRI.     

Human placenta estrogen synthetase (aromatase) purified by affinity chromatography

Microsomal estrogen synthetase (cytochrome P-450ES), also known as aromatase, was purified from fresh human placenta microsomes by DEAE-Trisacryl and testosterone-agarose chromatography. Estrogen synthetase assays were done with androstenedione as substrate, NADPH as electron donor, and a partially purified P-450 reductase from human placenta as the electron carrier. The specific cytochrome P-450 content of the purified P-450 was 0.67 nmol mg-1 of protein, and the preparation contained no cytochrome P-420. The absorbance maximum was 448.5 nm. The specific estrogen synthetase activity of the purified P-450ES fraction was 35 nmol min-1 nmol-1 of cytochrome P-450 or 23.3 nmol min-1 mg-1 of protein. The latter value shows a 179-fold purification with a yield greater than 1% in the two-step procedure. Kinetic constants for the reaction were measured with androstenedione as the aromatizable substrate. The Km was 1.4 nM and the Vmax was 37 nmol min-1 nmol-1 of P-450. The purified enzyme aromatized androstenedione and testosterone at identical rates; androstenedione gave only estrone, and testosterone gave only estradiol-17 beta. Dehydroepiandrosterone was not detectably aromatized or otherwise metabolized. Neither 16 alpha-hydroxytestosterone nor 16 alpha-hydroxyandrostenedione was aromatized. No hydroxysteroid dehydrogenase or reductase was detected in direct assays. No free reaction intermediates were detected in aromatization assay incubation mixtures. The purity of the product and the simplicity of the preparation recommend it for use in further studies of the enzyme.     

Trans-N-deoxyribosylase: purification by affinity chromatography and characterization.

trans-N-Deoxyribosylase (EC 2.4.2.6) is usually considered as a single protein catalyzing indifferently the transfer of the deoxyribosyl moiety to and from a purine or a pyrimidine base. Affinity chromatography of an extract from Lactobacillus helveticus with two types of ligands allowed the separation and purification of two distinct trans-N-deoxyribosylases. One catalyzes specifically the deoxyribosyl transfer to and from purine bases exclusively: trans-N-deoxyribosylase-I, the other catalyzes the transfer to and from pyrimidine and purine bases: trans-N-deoxyribosylase-II. A Tris inhibition study showed a markedly different susceptibility of the two enzymes. Preliminary results indicate that the purine-specific enzyme is a polymeric enzyme of molecular weight 86 000 (+/- 4000).     

Polymorphism and glycoprotein character of Ricinus communis lectins purified by affinity chromatography]

Two lectin fractions (S20W = 6,8 and 4,9 S) were purified from Ricinus communis seeds. The purification was carried out in four steps : ammonium sulfate fractionation, affinity chromatography on Sepharose 4 B, gel filtration on Sephadex G 150 and chromatography on CM celluloes. The purified lectins were glycoproteins whose chemical composition was determined. Amino terminal analysis of the two fractions revealed glycine and serine. Polyacrylamide gel electrophoresis of the higher molecular weight fraction allowed the separation of several components with different affinity for PAS staining.     

Fluid phase activation of proenzymic C1r purified by affinity chromatography

1. Proenzymic C1r was purified from human plasma in a two-step technique involving indirect affinity chromatography on Sepharose Ig anti-C1s. The capacity of C1r to monomerize at pH 5.0 and to redimerize at neutral pH was used for selective elution of C1r. The yield in purified C1r was 39% from plasma; no trace of contaminating serine proteases was detected from [3H]diisopropyl phosphorofluoridate labelling of C1r. 2. C14 was able to undergo a two-way autoactivation: an intramolecular catalytic process catalysed by proenzymic C1r itself and an intermolecular reaction catalysed by activated C1r formed in the process of the reaction. DFP (5mM) and C1 Inh at a C1 Inh/C1r ratio of 1:1 were effective on the solely intermolecular activation, leading to partial inhibition of the autoactivation from proenzymic C1r: C1r formed during the activation was titrated by the inhibitors. Calcium, high ionic strength or acid pH decreased C1r activation. The pH effect was characterized by a slowed-down reaction below pH 6.0 and no net influence at values as high as 10.5. The two types of activation developed similarly as a function of pH. 3. Peripheral iodination of C1r revealed differences in label distribution between proenzymic (A chain moiety 48%, B chain moiety 52%) and activated C1r (A chain 20%, B chain 80%). Two different conformational states of C1r were also suggested by 125I-labelling at different temperatures.     

N-terminal sequence analysis of atrial granule serine proteinase purified by affinity chromatography

Atrial granule serine proteinase is considered the leading candidate endoproteolytic processing enzyme of pro-atrial natriuretic factor. Its cleavage specificity is directed toward a monobasic amino acid processing site, and as such, the atrial enzyme is distinguished from the family of prohormone convertases which act at dibasic amino acid processing sites. To delineate the molecular mechanisms which distinguish monobasic from dibasic amino acid-directed processing enzymes, pure atrial enzyme is needed for sequence determination leading to molecular cloning, and for preparation of antisera. An affinity chromatography purification scheme seemed a logical modification of our established procedures to yield suitable amounts of enzyme for further studies. Surprisingly, pseudo-peptide bond inhibitors of the atrial enzyme [Damodaran and Harris (1995),J. Protein Chem., this issue] formed ineffective affinity ligands, even though these compounds contain essential residues on either side of what would be the scissile bond in a peptide substrate. On the other hand, tripeptide aldehydes (based on the substrate recognition sequence of the atrial enzyme) linked to Sepharose formed effective affinity matrices, permitting purification of the enzyme in a single step from a subcellular fraction enriched for atrial granules and lysosomes. Hence, the enzyme was purified 2000-fold in 90% overall yield, and subjected to N-terminal sequence analysis through 26 residues. The sequence determined, XXPEAAGLPG[R, L]GNPVP[F, G]R[Q, I]XY[G, E]XR(N, A]V, indicates that the atrial enzyme is unique, showing little sequence homology to other proteins in the database.Abbreviations AGSP atrial granule serine proteinase - ANF atrial natriuretic factor - BSA bovine serum albumin - Bz benzoyl - EACA 6()-aminocaproic acid - HEPES N-2-hydroxyethylpiperazine-N'-propanesulfonic acid - HPLC high-performance liquid chromatography - PEG polyethylene glycol-3350 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Single-letter abbreviations are used to denote amino acids     

Structural and immunochemical characterization of the lipooligosaccharides expressed by Neisseria subflava 44

Neisserial lipooligosaccharides (LOSs) are a family of complex cell surface glycolipids. We used mass spectrometry techniques (electrospray ionization, collision-induced dissociation, and multiple step), combined with fluorophore-assisted carbohydrate electrophoresis monosaccharide composition analysis, to determine the structure of the two low-molecular-mass LOS molecules (LOSI and LOSII) expressed by Neisseria subflava 44. We determined that LOSI contains one glucose on both the alpha and beta chains. LOSII is structurally related to LOSI and differs from it by the addition of a hexose (either glucose or galactose) on the alpha chain. LOSI and LOSII were able to bind monoclonal antibody (MAb) 25-1-LC1 when analyzed by Western blotting experiments. We used a set of genetically defined Neisseria gonorrhoeae mutants that expressed single defined LOS epitopes and a group of Neisseria meningitidis strains that expresses chemically defined LOS components to determine the structures recognized by MAb 25-1-LC1. We found that extensions onto the beta-chain glucose of LOSI block the recognition by this MAb, as does further elongation from the LOSII alpha chain. The LOSI structure was determined to be the minimum structure that is recognized by MAb 25-1-LC1.