Components and proteolytic processing sites of arylsulfatase B from human placenta.

Previous studies have shown that mature arylsulfatase B purified from human sources is composed of two non-identical chains with apparent molecular masses of 43 kDa and 8 kDa. Arylsulfatase B purified from human placenta in the present study, however, included another 7 kDa component that could be detected only by carbohydrate staining on reducing SDS-PAGE employing the Tris-Tricine system. The 43 kDa and 7 kDa components contained a carbohydrate moiety, but the 8 kDa one did not, as demonstrated by periodic acid-Schiff staining, Con-A lectin blotting, endo-glycosidase treatment and in vitro phosphorylation by UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. The purified arylsulfatase B migrated as a single polypeptide of 58 kDa on non-reducing SDS-PAGE, indicating that the three chains are linked by disulfide bonds. In order to determine the origin of the components, N-terminal sequencing of the isolated polypeptides was performed. As a result, the 43, 7 and 8 kDa components were found to commence with Ala-41, Ala-424 and Asp-466, respectively. These results suggest that after removal of the signal peptide, human arylsulfatase B undergoes proteolytic processing on at least two sites during maturation.     

Affinity chromatography od Klebsiella arylsulfatase on tyrosyl-hexamethylenediamine-beta-1,3-glucan and immunoadsorbent.

A simple and convenient method for preparation of a highly purified arylsulfatase (EC 3.1.6.1) from Klebsiella aerogenes has been developed. Specificity of purification was achieved by using affinity chromatography on a tyrosyl-hexamethylenediamino-beta-1,3-glucan or on a solid phase immunoadsorbent. By using affinity chromatography a homogeneous enzyme was obtained with high yield. It is also proposed that the beads of curdlan type polysaccharide consisting of beta-1,3-glucan can be used as a good matrix for affinity chromatography.     

Rapid purification of annexin V from human placenta by affinity chromatography

Affinity purification of annexin V from human placenta on column with appropriate monospecific antibodies is developed. The procedure permits purification of the protein to a highly purified state by a two stage procedure. The yield of the protein is about 5 mg per 100 g of wet tissue. Because of high homologies between various annexins, it was supposed that this procedure can be also applied for purification of other annexins from other tissues.     

Purification and properties of arylsulfatase C from human placenta

Arylsulfatase C (ASC) was purified about 1,000-fold from human placenta. The major steps in the procedure included chromatography on Con A-Sepharose and Bio-Gel A-1.5 m. The purified enzyme was homogeneous by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The native enzyme has an apparent molecular weight of 238,000 resulting from three identical subunits of 78,000 daltons. The purified enzyme hydrolyzes the artificial substrate p-nitrophenyl sulfate (NPS), and the two natural substrates estronesulfate (ES) and dehydroepiandrosterone sulfate (DHEAS), the ratio of these three activities being constant throughout the purification. ES and DHEAS are powerful competitive inhibitors of the enzymatic hydrolysis of NPS. ASC, ESase and DHEASase activities show the same thermal stability. These results strongly suggest that a single enzyme is responsible for the hydrolysis of the two natural and the artificial substrates.     

Characterization of arylsulfatase C isozymes from human liver and placenta

Arylsulfatase C and steroid sulfatase were thought to be identical enzymes. However, recent evidence showed that human arylsulfatase C consists of two isozymes, s and f. In this study, the biochemical properties of the s form partially purified from human placenta were compared with those of the f form from human liver. Only the placental s form has steroid sulfatase activity and hydrolyses estrone sulfate, dehydroepiandrosterone sulfate and cholesterol sulfate. The liver f form has barely detectable activity towards these sterol sulfates. With the artificial substrate, 4-methylumbelliferyl sulfate, both forms demonstrated a similar KM but the liver enzyme has a pH optimum of 6.9 while the placental form displayed two optima at 7.3 and 5.5. The molecular weight of the native enzyme determined with gel filtration was 183,000 for the s form and 200,000 for the f form and their pI's were also similar at 6.5. However, the T50, temperature at which half of the enzyme activity was lost, was 49.5 degrees C for the f form and 56.8 degrees C for the s form. Polyclonal antibodies raised against the placental form reacted specifically against the s and not the f form. They immuno-precipitated concomitantly greater than 80% of the total placental arylsulfatase C and steroid sulfatase activities while less than 20% of the liver enzyme was immuno-precipitable. In conclusion, the two isozymes s and f of arylsulfatase C in humans purified from placenta and liver, respectively, have similar KM, pI' and native molecular weight. However, they are distinct proteins with different substrate specificity, pH optima, heat-lability and antigenic properties. Only the s form is confirmed to be steroid sulfatase.     

Centrifugal column chromatography for small sample purification

We evaluated several gel filtration materials for suitability in spun column chromatography a rapid desalting method. The materials were evaluated for resistance to column cracking and to radial shrinkage for sample recovery and for salt separation. A series of graphs can be derived from these evaluations which relates the gel heights to sample volumes for maximum recovery and salt separation while maintaining low sample dilution.     

Comparison of arylsulfatase C and steroid sulfatase from human placenta and liver

Human placental and hepatic arylsulfatase C (ASC) were purified to homogeneity and about 1,000-fold, respectively. Placental ASC hydrolyzed sterol sulfates at the same active site, whereas the major hepatic ASC did not. This major hepatic ASC isozyme was more thermolabile than placental ASC and steroid sulfatase from both placenta and liver. It was not precipitated by anti-bovine ASC IgG which quantitatively precipitated both placental ASC and steroid sulfatase activities from placenta and liver. A minor hepatic ASC isozyme with similar electrophoretic mobility to the placental enzyme copurified with the major hepatic ASC and is likely responsible for the steroid sulfatase activity in this organ. Hence, placental ASC and steroid sulfatase are biochemically and antigenically identical to hepatic steroid sulfatase. In contrast, the major hepatic ASC is a distinct protein whose catalytic and structural properties differ from all the above enzymes.     

Use of the enzyme-linked immunoadsorbent assay to monitor the purification of glycosphingolipid antigens by high-performance liquid chromatography

An enzyme-linked immunoadsorbent assay (ELISA) technique has been applied to the analysis of glycosphingolipid fractions separated by high-performance liquid chromatography. Nanogram amounts of selected fractions were placed in microtiter wells and analyzed for glycosphingolipids carrying carbohydrate epitopes recognized by monoclonal antibodies using an avidin-biotin enzyme system (ABC reagents). A large number of fractions (more than 100) can be conveniently evaluated for the presence of glycosphingolipids recognized by one or more monoclonal antibodies in a single analysis. This method is a rapid and sensitive procedure for monitoring the purification of glycosphingolipid antigens and can be used in conjunction with immunostaining of glycosphingolipids separated by thin-layer chromatography.     

S-Adenosylhomocysteine hydrolase from human placenta. Affinity purification and characterization.

S-Adenosylhomocysteine hydrolase (EC 3.3.1.1) was purified to homogeneity from human placenta by using S-adenosylhomocysteine-agarose affinity chromatography. The enzyme is a tetramer with a native Mr of 189 000 and subunit Mr of 47 000-48 000; there were nine cysteine residues per subunit and no disulphide bonds. The pI was 5.7. H.p.l.c. analysis revealed that the enzyme contained four molecules of tightly bound cofactor (NAD) per tetramer, of which 10-50% was in the reduced form. The enzyme had four binding sites per tetramer for adenosine, of which 10-35% were found to be occupied. Two types of adenosine-binding sites could be distinguished on the basis of differences in rates of dissociation of the enzyme-adenosine complex, and by examining binding of adenosine at 0 degree C and 37 degrees C. The enzyme catalysed the interconversion of adenosine and 4',5'-dehydroadenosine; the equilibrium constant for this reaction was 2.1 and favoured 4',5'-dehydroadenosine formation. Variability in the specific activity of preparations of S-adenosylhomocysteine hydrolase was related to the NAD+/NADH ratio of the preparation. The capacity to bind radioactively labelled adenosine depended on the adenosine content of the purified enzyme. The rate of adenosine binding and the sensitivity of S-adenosylhomocysteine hydrolase to inactivation by adenosine were both diminished in the absence of dithiothreitol.     

Affinity purification of alkylglycerol monooxygenase from rat liver microsomes by chimyl alcohol-Sepharose 4B column chromatography

Alkylglycerol monooxygenase of rat liver microsomes was purified approximately to 97-fold with a 30% yield by procedures including affinity chromatography on chimyl alcohol-Sepharose 4B. Chimyl alcohol (1-O-hexadecylglycerol) was converted to the p-aminobenzylidene derivative and then coupled to 6-carboxyhexyl-Sepharose. The final enzyme preparation was in nearly a homogeneous state, judging from the results of sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, and it migrated to a position corresponding to an apparent molecular weight of 45,000. The results revealed that the native form of the enzyme (estimated to have a molecular weight of 400,000 as judged by Sepharose 6B column chromatography in a previous report, Ishibashi, T., and Y. Imai. 1983. Eur. J. Biochem. 132: 23-27) will polymerize to large aggregates.     

Ribonuclease inhibitor from human placenta: rapid purification and assay.

The ribonuclease inhibitor from human placenta may be isolated in 65% yield (2.5 mg per placenta) in 2 days. The performance of the affinity chromatography on Sepharose-RNase A has been expedited through adaption of the spectrophotometric assay of ribonuclease toward 2',3'-cyclic cytidine monophosphate to determination of the inhibitor activity. The result of these improvements in procedure is increased availability of the ribonuclease inhibitor for study of its chemical properties (Blackburn, P., and Jailkhani, B.L. (1979) J. Biol. Chem. 254, 12488-12493) and for its inclusion into in vitro systems in which inhibition of mammalian neutral ribonucleases is desired.     

A simplified method for purification of annexin V from human placenta

A simplified procedure for purification of annexin V from human placenta was developed. At first, the protein was separated from other proteins in membrane bound form in the presence of Ca2+, then was extracted with EDTA and purified by affinity chromatography on PAAG-immobilized phosphatidylserine. The purified protein gave a single band with a molecular weight of 35,000 in SDS-PAGE.     

Large-scale purification of angiotensin I-converting enzyme from human plasma utilizing an immunoadsorbent affinity gel

Angiotensin I-converting enzyme was purified 1500-fold from human plasma utilizing an immunoadsorbent affinity gel prepared by coupling antibody to baboon lung angiotensin I-converting enzyme to CNBr-activated Sepharose 4B. The enzyme was eluted from the gel using 2 m magnesium chloride, pH 5.8. Subsequent hydroxylapatite and Sephadex G-200 chromatography yielded 2.6 mg of homogenous enzyme with a specific activity of 40 units/mg with hippuryl-l-histidyl-l-leucine as substrate from 48 liters of plasma. Use of the immunoadsorbent allowed the 48 liters of plasma to be processed in one-half the time it previously took to process 2 liters of plasma by other methods. This protocol enables us to obtain sufficient amounts of enzyme for structural studies that were previously impossible because of insufficient amounts of enzyme.     

Partial purification and characterization of a peroxidase activity from human placenta.

The aim of this work was to use preparations from germinating seeds of Pisum sativum to determine the apparent equilibrium constant of the reaction catalysed by sucrose-phosphate synthase (EC 2.4.1.14) and to compare this with the mass-action ratio of the reaction in the seeds. The apparent equilibrium constant ranged from 5.3 at 0.25 mM-MgCl2, pH 7.0, to 62 at 10 mM-MgCl2, pH 7.5. The sucrose phosphate content of the seeds, 23 nmol/g fresh wt., was determined by separating sucrose phosphate from sucrose by ion-exchange chromatography and then measuring the sucrose released by alkaline phosphatase. Comparison of equilibrium constants and mass-action ratios in the cotyledons of 38 h-germinated seeds showed that the reactions catalysed by glucose-6-phosphate isomerase, phosphoglucomutase and UDP-glucose pyrophosphorylase are close to equilibrium, and those catalysed by sucrose-phosphate synthase and sucrose phosphatase are considerably displaced from equilibrium in vivo.     

The purification and identification of calmodulin from human placenta

A protein which showed similarity to bovine brain calmodulin in electrophoretic mobilities on polyacrylamide gels in the presence of 40% glycerol (pH 8.6) and 0.1% sodium dodecyl sulfate (pH 7.2) was isolated from human placenta. Its final yield was approx. 4 mg per kg human placenta. The placenta protein was similar to bovine brain calmodulin in stimulating bovine brain calmodulin-deficient cyclic nucleotide phosphodiesterase in the presence of calcium. However, its stimulating activity was eliminated by ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) or trifluoperazine. In addition, there is a close resemblance in amino acid composition between the placental protein and bovine brain calmodulin. These results indicate that calmodulin is present in human placenta.     

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.     

Efficient and rapid purification of recombinant human alpha-galactosidase A by affinity column chromatography

The lysosomal enzyme alpha-galactosidase A (alpha-Gal A) metabolizes neutral glycosphingolipids that possess alpha-galactoside residues at the non-reducing terminus, and inherited defects in the activity of alpha-Gal A lead to Fabry disease. We describe here an efficient and rapid purification procedure for recombinant alpha-Gal A by sequential Concanavalin A (Con A)-Sepharose and immobilized thio-alpha-galactoside (thio-Gal) agarose column chromatography. Optimal elution conditions for both columns were obtained using overexpressed human alpha-Gal A. We recommend the use of a mixture of 0.9 M methyl alpha-mannoside and 0.9 M methyl alpha-glucoside in 0.1 M acetate buffer (pH 6.0) with 0.1 M NaCl for the maximum recovery of glycoproteins with multiple high-mannose type sugar chains from Con A column chromatography, and that the Con A column should not be reused for the purification of glycoproteins that are used for structural studies. Binding of the enzyme to the thio-Gal column requires acidic condition at pH 4.8. A galactose-containing buffer (25 mM citrate-phosphate buffer, pH 5.5, with 0.1 M galactose, and 0.1 M NaCl) was used to elute alpha-Gal A. This procedure is especially useful for the purification of mutant forms of alpha-Gal A, which are not stable under conventional purification techniques. A protocol that purifies an intracellular mutant alpha-Gal A (M279I) expressed in COS-7 cells within 6h at 62% overall yield is presented.