Purification of human placental alkaline phosphatase. Salt effects in affinity chromatography

Human placental alkaline phosphatase was chromatographed on Sepharose derivatives of d- and l-phenylalanine, l-leucine, glycine, aniline and p-aminobenzoic acid in high concentrations of (NH(4))(2)SO(4). Retention on these columns was greatest at the highest concentrations of (NH(4))(2)SO(4). By using decreasing concentrations and changing the types of salts, elution was effected from each of the columns. The (NH(4))(2)SO(4)-mediated retention appeared to be related to the hydrophobic character of the substituted Sepharose, rather than to any specific binding site of the enzyme. It is suggested that this provides a way of controlling hydrophobic affinity chromatography. By use of chromatography on l-phenylalanine-Sepharose and of DEAE-Sephadex chromatography in the presence of Triton X-100 detergent, a preparation of highly purified (1000-fold) human placental alkaline phosphatase was obtained in 22% yield.     

Alkaline phosphatase: affinity chromatography and inhibition by phosphonic acids.

Five phosphonic acid derivatives were synthesized, coupled to agarose, and tested for affinity chromatographic binding of alkaline phosphatase from bovine intestine. Agarose coupled to L-histidyldiazobenzylphosphonic acid was found to be a highly effective adsorbent. In order to understand the large differences in binding capacity observed with derivatized agaroses, inhibition of alkaline phosphatase by phosphonic acid ligands, and related phosphonic acids, was measured. The results of affinity chromatography and inhibition studies were in good agreement, demonstrating that phosphonic acids with large aromatic/hydrophobic, carboxylate substituents bind strongly and competitively to the enzyme active site.     

Efficient elution of functional proteins in affinity chromatography.

Many elution buffers are in use for the retrieval of proteins from affinity columns. While the aim of these buffers is to dissociate the various chemical bonds that make up protein-protein interactions and return the target protein to the mobile phase in active form, there is considerable difference of opinion as to which buffer is more suitable for particular applications. This review examines the chemical effect of various elution buffers on protein-protein interactions in the context of affinity chromatography and examines strategies that may be used for selection of an appropriate buffer.     

Alkaline phosphatase on activated B cells characterization of the expression of alkaline phosphatase on activated B cells. Kinetics and membrane anchor.

Recently we reported that the expression of the enzyme alkaline phosphatase (APase) is a marker for B cell activation. Enzymatic activity was found only in activated B cells and not T cells. Using flow cytometry we showed that some of the APase was found on the cell membranes (mAPase) and by functional assays, some was spontaneously released into the tissue culture medium. In the present report the expression of mAPase on activated B lymphocytes is more fully characterized. Two mAb specific for rat APase were used to measure the kinetics of the membrane expression of mAPase. Within 48 h of activation, mAPase is detected by flow cytometry and increases coordinately with both the transferrin receptor and IL-2R. Maximal membrane expression of mAPase in terms of number of positive cells and mean fluorescent intensity, is detected by day 4 to 5 of culture. Using hydroxyurea and demecolcine to block cells at G1/S and G2/M, respectively, it appeared that the initial expression of mAPase occurred as cells progressed into S phase of the cell cycle. This was confirmed using two-color flow cytometric analysis with the Hoechst DNA stain 33342 and the FITC-labeled APase-specific mAb. Finally, using phosphatidylinositol-specific phospholipase C we were able to show that 60 to 80% of the mAPase is linked to the membrane via a glycosyl-phosphatidylinositol linkage. From this we have concluded that mAPase can be added to a growing list of glycoproteins that are anchored to the membrane by the glycosyl-phosphatidylinositol linkage and are expressed on differentiating B cells. This list now includes Thy-1, BLAST-1, Jlld, and mAPase.     

Separation and quantification of serum alkaline phosphatase isozymes in the rat by affinity electrophoresis

The purpose of this study was to examine the possibility of separation and quantification of serum alkaline phosphatase (ALP) isozymes in rats by wheatgerm lectin affinity electrophoresis. Cellulose acetate electrophoresis of the liver and bone ALPs without lectin results in overlapping bands, but in the presence of lectin, the mobility of the band of bone enzyme was retarded and well separated from the liver enzyme band. With this affinity electrophoretic method, we determined the serum ALP isozymes in fed and fasting rats grouped by age. As a result, the absolute activity of bone isozyme showed a downward trend with age in the fed and fasting rats. The serum ALP activity was steadily higher in fed rats than in fasting rats, and the increase was due to intestinal ALP isozyme. There was low activity bordering complete absence in liver isozyme under both nutritional conditions. The affinity electrophoretic method provided a rapid, reproducible, and relatively simple technique for further clinical characterization of ALP isozyme in the rat serum.     

Inhibition of human alkaline phosphatase isoenzymes by the affinity reagent reactive yellow 13

The dye Reactive Yellow 13, an affinity reagent for intestinal alkaline phosphatase, inhibits intestinal and other human alkaline phosphatases in solution. The inhibition depends markedly on the presence of a phosphate acceptor such as diethanolamine. The dye is an uncompetitive inhibitor with respect to both substrate and phosphate acceptor in the case of non-intestinal phosphatases. However, in the case of intestinal alkaline phosphatase, the inhibition is noncompetitive with respect to the substrate and competitive with respect to the phosphate acceptor. These observations account for the specific binding of intestinal phosphatase when the dye is used as a ligand in affinity chromatography.     

Structural and functional analysis of human germ cell alkaline phosphatase by site-specific mutagenesis.

Human germ cell alkaline phosphatase (GCAP), which shares 98% amino acid sequence identity with the placental AP (PLAP), is expressed by malignant trophoblasts. Protein sequence analysis suggests that the Ser residue at position 92 is the putative active site of GCAP which contains two recognition sequences (Asn122-Thr-Thr124 and Asn249-Arg-Thr251) for asparagine-linked glycosylation. To examine the roles of the Ser residue and glycan moieties on GCAP activity and processing, we altered the GCAP cDNA by site-directed mutagenesis and expressed the GCAP mutants in COS-1 cells. Substitution of Ser-92 with either a Thr (S92T) or an Ala (S92A) residue yielded a GCAP devoid of catalytic activity, suggesting that the Ser codon 92 is the active site of GCAP. Six GCAP mutants that lack one or both glycosylation sites were constructed by substituting either Asn-122 or Asn-249 with an Asp residue or either Thr-124 or Thr-251 with an Ala residue. The mature GCAP migrated as a 65-kDa product, but GCAP mutants lacking one or both glycosylation sites migrated as 62- or 58-kDa polypeptides, respectively, indicating that both sites were glycosylated. All six glycosylated mutants were active enzymatically and, in addition, were equally sensitive to heat, L-leucine, and EDTA inhibition as the parental enzyme. GCAP as well as its two active-site and six glycosylation mutants could be released from the plasma membrane of transfected COS-1 cells by the proteinase bromelain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmid expression vectors with elements of the E. coli alkaline phosphatase gene

A family of plasmid expression vectors, containing fragments of the E. coli alkaline phosphatase gene (phoA), has been constructed for the lambda PR promoter-directed thermoinducible superproduction of fusions of heterologous polypeptides to the N- or C-terminus of E. coli alkaline phosphatase and its leader peptide. Effective expression and export to periplasm of resulting fusion proteins, which may retain enzymatic activity of the phosphatase, has been shown.     

Characterization of KB cell alkaline phosphatase. Evidence of similarity to placental alkaline phosphatase.

The alkaline phosphatase from KB cells was purified, characterized, and compared to placental alkaline phosphatase, which it resembles immunologically. Two nonidentical nonomeric subunits of the KB phosphatase were found. The two subunits, which have apparent molecular weights of 64,000 and 72,000, can be separated on polyacrylamide gels containing sodium dodecyl sulfate. The Mr = 64,000 KB subunit appears to be identical in protein structure to the monomer of placental alkaline phosphatase. The Mr = 72,000 KB subunit, while differing in the NH2-terminal amino acid, appears also to be very similar to the placental alkaline phosphatase monomer. Both KB phosphatase subunits bind (32P)phosphate, and bind to Sepharose-bound anti-placental alkaline phosphatase. Native KB phosphatase is identical to the placental isozyme in isoelectric point, pH optimum, and inhibition by amino acids, and has a very similar peptide map. The data presented support the hypothesis that the Mr = 64,000 KB phosphatase subunit may the the same gene product as the monomer of placental alkaline phosphatase. This paper strengthens the evidence that the gene for this fetal protein, normally repressed in all cells but placenta, is derepressed in the KB cell line. In addition, this paper presents the first structural evidence that there are two different subunit proteins comprising the placental-like alkaline phosphatase from a human tumor cell line.     

Placental alkaline phosphatase isoenzyme expression by the non-HeLa DoT cervical-carcinoma cell line.

Strain E of chloridazon-degrading bacteria, when grown on L-phenylalanine accumulates cis-2,3-dihydro-2,3-dihydroxyphenylalanine. In experiments with resting cells and during growth the bacterium converts the aromatic carboxylic acids phenylacetate, phenylpropionate, phenylbutyrate and phenyl-lactate into the corresponding cis-2,3-dihydrodiol compounds. The amino acids L-phenylalanine, N-acetyl-L-phenylalanine and t-butyloxycarbonyl-L-phenylalanine were also transformed into dihydrodiols. All seven dihydrodiols, thus obtained, were characterized both by conventional analytical techniques and by the ability to serve as substrates for a cis-dihydrodiol dehydrogenase.     

Functional interrelationships in the alkaline phosphatase superfamily: phosphodiesterase activity of Escherichia coli alkaline phosphatase

Escherichia coli alkaline phosphatase (AP) is a proficient phosphomonoesterase with two Zn(2+) ions in its active site. Sequence homology suggests a distant evolutionary relationship between AP and alkaline phosphodiesterase/nucleotide pyrophosphatase, with conservation of the catalytic metal ions. Furthermore, many other phosphodiesterases, although not evolutionarily related, have a similar active site configuration of divalent metal ions in their active sites. These observations led us to test whether AP could also catalyze the hydrolysis of phosphate diesters. The results described herein demonstrate that AP does have phosphodiesterase activity: the phosphatase and phosphodiesterase activities copurify over several steps; inorganic phosphate, a strong competitive inhibitor of AP, inhibits the phosphodiesterase and phosphatase activities with the same inhibition constant; a point mutation that weakens phosphate binding to AP correspondingly weakens phosphate inhibition of the phosphodiesterase activity; and mutation of active site residues substantially reduces both the mono- and diesterase activities. AP accelerates the rate of phosphate diester hydrolysis by 10(11)-fold relative to the rate of the uncatalyzed reaction [(k(cat)/K(m))/k(w)]. Although this rate enhancement is substantial, it is at least 10(6)-fold less than the rate enhancement for AP-catalyzed phosphate monoester hydrolysis. Mutational analysis suggests that common active site features contribute to hydrolysis of both phosphate monoesters and phosphate diesters. However, mutation of the active site arginine to serine, R166S, decreases the monoesterase activity but not the diesterase activity, suggesting that the interaction of this arginine with the nonbridging oxygen(s) of the phosphate monoester substrate provides a substantial amount of the preferential hydrolysis of phosphate monoesters. The observation of phosphodiesterase activity extends the previous observation that AP has a low level of sulfatase activity, further establishing the functional interrelationships among the sulfatases, phosphatases, and phosphodiesterases within the evolutionarily related AP superfamily. The catalytic promiscuity of AP could have facilitated divergent evolution via gene duplication by providing a selective advantage upon which natural selection could have acted.     

The Bacillus subtilis 168 alkaline phosphatase III gene: impact of a phoAIII mutation on total alkaline phosphatase synthesis.

The first alkaline phosphatase (APase) structural gene mutant of Bacillus subtilis 168 was constructed by using a clone identified by hybridization to a synthetic degenerative oligonucleotide. The design of the probe was based on the first 29 amino acids of the sequenced mature APase III protein, which had been isolated from the secreted fraction of vegetative, phosphate-starved cells. DNA sequencing of the clone revealed the first 80 amino acids of the APase III protein, including a typical procaryotic signal sequence of 32 amino acids preceding the start of the mature protein. The 29 amino acids encoded by the predicted open reading frame immediately following the signal sequence are identical to the first 29 amino acids of the sequenced mature protein. This region shows 80% identity to strand A of the beta sheet that is very well conserved in Escherichia coli and mammalian APases. A phoAIII structural mutant was constructed by insertional mutagenesis with a fragment internal to the coding region. The effects of this mutation on APase production in B. subtilis 168 were analyzed under both phosphate starvation and sporulation conditions. The mutation in APase III reduced the total vegetative APase specific activity by approximately 40% and sporulation APase specific activity by approximately 45%. An APase protein was isolated from sporulating cells at stage III and was identified as APase III by protein sequencing of the amino terminus and by its absence in the phoAIII mutant. The APase III gene has been mapped to approximately 50 degrees on the B. subtilis chromosome.     

Introduction of foreign peptides in surface loops of alkaline phosphatase

The introduction of foreign peptides into alkaline phosphatase that have a minimal influence on the enzymatic activity of a protein is described in the work. Calculated based on the methods of molecular dynamics the longest surface loops of alkaline phosphatase, which are not adjacent to both active center of a dimeric enzyme and contact surface between its monomers, were used as sites of the introduction of foreign peptides. The length of loops was estimated by several methods. Two loops were used to genetically engineer the introduction of peptides. Experiments demonstrated that the introduction of several peptides after the Ala218 residue of alkaline phosphatase insignificantly influences on the enzyme activity. According to experimental data, the selection of the loop for introducing the foreign peptide can be determined using the methods of molecular dynamics based on calculating the mobility of dihedral angles. In order to create macromolecules with new features by introducing foreign polypeptides in enzymatically active proteins, it is possible in practice to use an estimation of the loop length by means of the methods of molecular dynamics.     

Ani s 1, the major allergen of Anisakis simplex: purification by affinity chromatography and functional expression in Escherichia coli

Third stage larvae of the nematode Anisakis simplex often infect marine fish and invertebrates. When the larvae are ingested orally via seafood, they can cause IgE-mediated allergic reactions as well as anisakiasis. Of the known A. simplex allergens, Ani s 1 (Kunitz/bovine pancreatic trypsin inhibitor family protein) has been demonstrated to be a major allergen, being expected to be a useful tool for diagnosis of A. simplex allergy. For a diagnostic purpose, sufficient amounts of either natural Ani s 1 (nAni s 1) or recombinant Ani s 1 (rAni s 1) with an IgE-binding capacity should be stably supplied whenever needed. In this study, therefore, we first developed a simple and rapid purification method for Ani s 1 that is based on affinity chromatography using anti-Ani s 1 antibodies as ligands. The method was shown to produce nAni s 1 with a higher yield than the previously reported methods. Then, an attempt was made to express rAni s 1 in Escherichia coli as a His-tagged protein. rAni s 1 obtained as an inclusion body was solubilized in a solvent containing denaturing and reducing reagents and purified by nickel-chelate chromatography. Refolding of rAni s 1 was accomplished by dialysis in the presence of arginine, followed by that in the absence of arginine. Fluorescence ELISA and inhibition ELISA data revealed that rAni s 1 is IgE reactive enough to be used as a diagnostic tool.     

Giardia lamblia: expression of alkaline phosphatase activity in infected rat intestine

Alkaline phosphatase (IAP) is a marker of intestinal microvillus membrane. Changes in IAP activity have been studied as a function of Giardia lamblia (G. lamblia) infection using rat as the experimental model. At day 11 and 15 post-infection, enzyme activity was reduced (p<0.01) compared to controls. The enzyme levels were essentially similar to control values by day 30 post-infection. Analysis of the enzyme activity in cell fractions across crypt-villus axis revealed a marked decrease in enzyme activity in the villus tip and mid villus regions but a considerable increase (p<0.01) in enzyme activity in the crypt base of 11 day post-infected animals compared to that in controls. The observed changes in IAP activity were confirmed by assaying the enzyme activity in acrylamide gels using bromo-chloro-indolyl phosphate staining under non-denaturing conditions. These findings indicate differential changes across the crypt-villus axis, but impaired alkaline phosphatase levels in G. lamblia infected rat intestine.