Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis.

Novel fimbriae were isolated and purified from the human enteropathogen Salmonella enteritidis 27655. These fimbriae were thin (measuring 3 to 4 nm in diameter), were extremely aggregative, and remained cell associated despite attempts to separate them from blended cells by centrifugation. The thin fimbriae were not solubilized in 5 M NaOH or in boiling 0.5% deoxycholate, 8 M urea, or 1 to 2% sodium dodecyl sulfate (SDS) with or without 5% beta-mercaptoethanol. Therefore, an unconventional purification procedure based on the removal of contaminating cell macromolecules in sonicated cell extracts by enzymatic digestion and preparative SDS-polyacrylamide gel electrophoresis (PAGE) was used. The insoluble fimbriae recovered from the well of the gel required depolymerization in formic acid prior to analysis by SDS-PAGE. Acid depolymerization revealed that the fimbriae were composed of fimbrin subunits, each with an apparent molecular mass of 17 kDa. Although their biochemical characteristics and amino acid composition were typical of fimbriae in general, these thin fimbriae were clearly distinct from other previously characterized fimbriae. Moreover, their fimbrin subunits had a unique N-terminal amino acid sequence. Native fimbriae on whole cells were specifically labeled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17-kDa fimbrin protein in Western blots. The polyclonal immune serum did not cross-react with the other two native fimbrial types produced by this strain or with their respective fimbrins on Western blots (immunoblots). Therefore, these fimbriae represent the third fimbrial type produced by the enteropathogen S. enteritidis.     

Purification and characterization of a thermostable α-galactosidase from Thielavia terrestris NRRL 8126 in solid state fermentation

Several seeds and husks of some plants belonging to leguminosae, Graminae, Compositae and Palmae were evaluated as carbon substrates to produce α-galactosidase (α-Gal) by the thermophilic fungus, Thielavia terrestris NRRL 8126 in solid substrate fermentation. The results showed that Cicer arietinum (chick pea seed) was the best substrate for α-Gal production. The crude enzyme was precipitated by ammonium sulphate (60%) and purified by gel filtration on sephadex G-100 followed by ion exchange chromatography on DEAE-Cellulose. The final purification fold of the enzyme was 30.42. The temperature and pH optima of purified α-Gal from Thielavia terrestris were 70 °C and 6.5, respectively. The enzyme showed high thermal stability at 70 °C and 75 °C and the half-life of the α-Gal at 90 °C was 45 min. Km of the purified enzyme was 1.31 mM. The purified enzyme was inhibited by Ag2+, Hg2+, Zn2+, Ba2+, Mg2+, Mn2+ and Fe2+ at 5 mM and 10 mM. Also, EDTA, sodium arsenate, L-cysteine and iodoacetate inhibited the enzyme activity. On the other hand, Ca2+, Cu2+, K+ and Na+ slightly enhanced the enzyme activity at 5 mM while at 10 mM they caused inhibition. The molecular weight of the α-Gal was estimated to be 82 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for biotechnological and medicinal applications.     

Production and purification of Escherichia coli fimbrial antigen F165

Abstract The production of fimbrial antigen F165 by Escherichia coli strains was found to be dependent on the composition of the culture medium and was repressed in the presence of alanine or high levels of glucose, in anaerobic conditions or at growth temperatures of lower than 37°C. Optimal F165 production was found on a minimal medium containing 1% (w/v) casamino acids (MD-1). F165 antigen was isolated from bacteria by mechanical shearing, precipitated with ammonium sulfate, and purified by deoxycholate treatment and gel filtration on Superose 12. The purified fimbriae retained their native morphology as observed by electron microscopy and consisted of two separate protein subunits with apparent molecular weights of 17 500 and 19 000 on sodium dodecyl sulfate-polyacrylamide gels.     

Mechanism of adhesion of Alysiella bovis to glass surfaces

Alysiella bovis adheres to surfaces by means of short, ruthenium red-staining, rod-like fimbriae. The fimbriae remain associated with the cell envelope of A. bovis, even when sonicated or exposed sequentially to toluene, Triton X-100, lysozyme, ribonuclease, and deoxyribonuclease. Adhesion of outer membrane-derived cell wall ghosts of A. bovis to glass was inhibited by IO4-, sodium dodecyl sulfate, urea, pronase, and trypsin. Protease treatment digested the fimbriae from the distal end, and exposure to sodium dodecyl sulfate depolymerized the fimbriae. Exposure of ghosts to 1% sodium dodecyl sulfate preferentially solubilized a 16,500-dalton protein which was subsequently purified by gel filtration and demonstrated to be a glycoprotein (ca. 17% carbohydrate). Antibodies raised against the 16,500-dalton glycoprotein agglutinated whole cells and inhibited adhesion of ghosts to glass.     

Characterization of fructose 1,6-bisphosphatase from bakers' yeast

Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.     

Isolation and characterization of protease producing bacteria from upper respiratory tract of wild chicken

Bacterial samples isolated from the upper respiratory tract of a healthy broiler chicken and a wild chicken suffering from influenza which were collected locally revealed proteolytic activity as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis. Among five protease producing strains screened, one was selected as promising protease producer. The activity of the protease produced by this organism is stable up to 620C. Optimum yield was achieved after 19 hours of culture, at pH 9.0 and 450C. The desired protein was precipitated from the crude extract by using ammonium sulfate (60%) followed by dialysis and purified by Ion-exchange chromatography. Further investigations are needed to know about the structure elucidation of the purified protein for industrial exploitation.     

Purification and properties of beta-N-acetylglucosaminidase from Escherichia coli.

beta-N-acetylglucosaminidase (EC 3.2.1.30) has been purified from Escherichia coli K-12 to near homogeneity based on polyacrylamide gel electrophoresis in both 0.5% sodium dodecyl sulfate and in 6 M urea at pH 8.5. The purified enzyme shows a pH optimum of 7.7 and the Km for p-nitrophenyl-beta-D-2-acetamido-2-deoxyglucopyranoside is 0.43 mM. The molecular weight of this enzyme, determined by both Sephadex gel filtration and by sodium dodecyl sulfate gel electrophoresis, is equivalent to 36,000. It is shown to be a soluble cytoplasmic enzyme. Studies on the substrate specificites of the purified enzyme indicate that this enzyme is an exo-beta-N-acetylglucosaminidase.     

Purification and biochemical characterization of tellurite-reducing activities from Thermus thermophilus HB8.

Cell-free extracts of Thermus thermophilus HB8 catalyze the in vitro, NADH-dependent reduction of potassium tellurite (K2TeO3). Three different protein fractions with tellurite-reducing activities were identified. Two exhibited high molecular weight and were composed of at least two different polypeptides. The protein in the third fraction was purified to homogeneity and had a single polypeptide chain of 53 to 54 kilodaltons, with an isoelectric point of 8.1. Each enzyme was thermostable, the temperature optimum was 75 degrees C, and 30 mM NaCl, 1.5 M urea, or 0.004% sodium dodecyl sulfate caused 50% inhibition of the enzymes. However, 2% Triton X-100 did not have an inhibitory effect. The enzymes were also able to catalyze the reduction of sodium selenite and sodium sulfite in vitro. NADH was replaceable by NADPH. Divalent cations, such as Ca2+ and Ba2+, had no effect on the activity, while similar concentrations of Zn2+, Ni2+, and Cu2+ abolished the activity. This reductase activity could enable these bacteria both to reduce K2TeO3 and to increase their tolerance toward this salt.     

Purification and some properties of skeletal muscle sarcolemma Ca2+-ATPase]

Ca2+-ATPase of skeletal muscle sarcolemma has been isolated and purified. It is prepared from salt extract of sarcolemma by ammonium sulfate fractionation and further purified by gel chromatography on Sepharose 4B. The purity of preparations was evaluated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. It has been shown that Ca2+-ATPase possesses the same mobility as skeletal muscle myosin under gel chromatography on Sepharose 4B and the same mobility as myosin heavy chains in sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Membrane protein binds to rabbit skeletal muscle actin, and this complex dissociates by ATP. Interaction with actin does not change Ca2+- or Mg2+-stimulated ATPase activity. Enzyme has only one pH optimum at 7,0-7,6. Membrane protein is highly specified to calcium--ATPase activity in the presence of Mn2+ is 10% and in the presence of Sr2+, Mg2+ or Co2+ are 3-5% of the activity in the presence of Ca2+. Other nucleoside triphosphate (UTP and ITP) are hydrolyzed at lower rates than is ATP.     

Purification of phospholipase D from citrus callus tissue

Phospholipase D in extracts of soluble proteins from callus cultures derived from cotyledons of Citrus sinensis (L.) Osbeck is activated by Ca2+ and anionic detergents and has a pH optimum of 6.5. The enzyme was purified 703-fold over the crude protein extract with a yield of 15% by ammonium sulfate precipitation, ion exchange chromatography, gel filtration, hydrophobic interaction chromatography, and preparative acrylamide gel electrophoresis. Preparative electrophoresis was carried out using conventional slab gel equipment and electroelution of the sliced gel. Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified phospholipase revealed two bands of the same staining intensity running at 94.2K and 90.5K.     

Isolation and properties of actin, myosin, and a new actinbinding protein in rabbit alveolar macrophages.

Actin, myosin, and a high molecular weight actin-binding protein were extracted from rabbit alveolar macrophages with low ionic strength sucrose solutions containing ATP, EDTA, and dithiothreitol, pH 7.0. Addition of KCl, 75 to 100 mM, to sucrose extracts of macrophages stirred at 25 degrees caused actin to polymerize and bind to a protein of high molecualr weight. The complex precipitated and sedimented at low centrifugal forces. Macrophage actin was dissociated from the binding protein with 0.6 M KCl, and purified by repetitive depolymerization and polymerization. Purified macrophage actin migrated as a polypeptide of molecular weight 45,000 on polyacrylamide gels with dodecyl sulfate, formed extended filaments in 0.1 M KCl, bound rabbit skeletal muscle myosin in the absence of Mg-2+ATP and activated its Mg-2+ATPase activity. Macrophage myosin was bound to actin remaining in the macrophage extracts after removal of the actin precipitated with the high molecular weight protein by KCl. The myosin-actin complex and other proteins were collected by ultracentrifugation. Macrophage myosin was purified from this complex or from a 20 to 50% saturated ammonium sulfate fraction of macrophage extracts by gel filtration on agarose columns in 0.6 M Kl and 0.6 M Kl solutions. Purified macrophage myosin had high specific K-+- and EDTA- and K-+- and Ca-2+ATPase activities and low specific Mg-2+ATPase activity. It had subunits of 200,000, 20,000, and 15,000 molecular weight, and formed bipolar filaments in 0.1 M KCl, both in the presence and absence of divalent cations. The high molecular weight protein that precipitated with actin in the sucrose extracts of macrophages was purified by gel filtration in 0.6 M Kl-0.6 M KCl solutions. It was designated a macrophage actin-binding protein, because of its association with actin at physiological pH and ionic strength. On polyacrylamide gels in dodecyl sulfate, the purified high molecular weight protein contained one band which co-migrated with the lighter polypeptide (molecular weight 220,000) of the doublet comprising purified rabbit erythrocyte spectrin. The macrophage protein, like rabbit erythrocyte spectrin, was soluble in 2 mM EDTA and 80% ethanol as well as in 0.6 M KCl solutions, and precipitated in 2 mM CaCl2 or 0.075 to 0.1 M KCl solutions. The macrophage actin-binding protein and rabbit erythrocyte spectrin eluted from agarose columns with a KAV of 0.24 and in the excluded volumes. The protein did not form filaments in 0.1 M KCl and had no detectable ATPase activity under the conditions tested.     

Overexpression and characterization of a lipase from Bacillus subtilis

A novel plasmid, pBSR2, was constructed by incorporating a strong lipase promoter and a terminator into the original pBD64. A mature lipase gene from Bacillus subtilis strain IFFI10210, an existing strain for lipase expression, was cloned into the plasmid pBSR2 and transformed into B. subtilis A.S.1.1655. Thus, an overexpression strain, BSL2, was obtained. The yield of lipase is about 8.6 mg protein/g of wet weight of cell mass and 100-fold higher than that in B. subtilis strain IFFI10210. The recombinant lipase was purified in a three-step procedure involving ammonium sulfate fractionation, ion exchange, and gel filtration chromatography. Characterizations of the purified enzyme revealed a molecular mass of 24 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, maximum activity at 43 degrees C and pH 8.5 for hydrolysis of p-nitrophenyl caprylate. The values of Km and Vm were found to be 0.37 mM and 303 micromol mg-1 min-1, respectively. The substrate specificity study showed that p-nitrophenyl caprylate is a preference of the enzyme. The metal ions Ca2+, K+, and Mg2+ can activate the lipase, whereas Fe2+, Cu2+, and Co2+ inhibited it. The activity of the lipase can be increased about 48% by sodium taurocholate at the concentration of 7 mM and inhibited at concentrations over 10 mM.     

Purification and characterization of pregastric esterase from calf

Calf pregastric esterase (PGE) was purified from calf gullet tissues. The tissue was excised and lyophilized, and lipid materials were extracted with acetone and n-butanol at -20 degrees C. Proteins were extracted from the delipidated tissue with a buffer containing a chaotropic salt (NaSCN) to solubilize hydrophobically bound protein aggregates. Calf PGE precipitated from the crude extract at pH 5.0. The precipitated, solubilized proteins were subjected to anion-exchange chromatography on DEAE-Sephacel, and the enzymatic activity was eluted using a linear gradient from 0.10 to 0.50 M NaCl at pH 8.0. Fractions with high specific activities were then chromatographed twice using gel filtration on Sephadex G-100. The resultant enzyme was shown to be pure upon discontinuous electrophoresis in 12% polyacrylamide containing 0.1% sodium dodecyl sulfate (SDS-PAGE). From SDS-PAGE gel patterns, a molecular weight of 49,000 was determined. The amino acid composition of the enzyme allowed calculation of an "average hydrophobicity" (Bigelow index) of 1150 cal/residue. This indicates that calf PGE is relatively hydrophobic, being similar to proteins such as alpha-lactalbumin and bovine serum albumin in average hydrophobicity.     

Galactose-Specific Fimbrial Adhesin of Enteroaggregative <Emphasis Type="Italic">Escherichia coli</Emphasis>: A Possible Aggregative Factor

A galactose-specific adhesin was isolated from the fimbriae of an enteroaggregative Escherichia coli (EAEC) strain. The adhesin was found to be a high molecular weight aggregate of the 18-kDa monomer. The dimeric (36 kDa) and tetrameric (76 kDa) forms appeared in sodium dodecyl sulphate polyacrylamide gel electrophoresis when a higher concentration of the adhesin was used. The IgG_AD (IgG against adhesin) obtained from the immune sera raised in rabbits against purified adhesin could detect all three forms of the adhesin even from the crude fimbrial preparation. The IgG_AD failed to recognize the adhesin in the presence of galactose, thereby suggesting the antibody-binding site and the sugar-binding site on the adhesin might be same or overlapping. Furthermore, the IgG_AD could localize the adhesin exclusively on the fimbriae as observed in immunogold electron microscopy. The aggregative adherence of the bacteria to HEp-2 cells was reduced to 70% in the presence of the IgG_AD. A glycoprotein (34 kDa) present in the membrane fraction of HEp-2 cells interacted with the purified adhesin in a galactose-specific manner. The IgG_AD could recognize the adhesin from the crude fimbrial preparation of 9 out of 10 clinical isolates of EAEC strains but failed to identify any protein from the crude fimbrial preparation of Salmonella typhimurium (fim +ve as well as fim −ve strain), Vibrio cholerae (WO7) or Escherichia coli DH5α.     

Enzyme system involved in the synthesis of thiamin triphosphate. I. Purification and characterization of protein-bound thiamin diphosphate: ATP phosphoryltransferase

An enzyme system catalyzing the synthesis of thiamin triphosphate consists of an enzyme (protein-bound thiamin diphosphate:ATP phosphoryltransferase), thiamin diphosphate bound to a macromolecule as substrate, ATP, Mg2+, and a low molecular weight cofactor. This system was established by combining a purified enzyme and an essentially pure, macromolecule-bound substrate prepared from rat livers. This macromolecule was found to be a protein, and the transphosphorylation of thiamin diphosphate to thiamin triphosphate with ATP and enzyme was shown to occur on this macromolecule which binds thiamin diphosphate. Free thiamin, thiamin monophosphate, thiamin diphosphate, and thiamin triphosphate have no effect on this reaction. Thus, the overall reaction is: thiamin diphosphate-protein + ATP in equilibrium thiamin triphosphate-protein + ADP. So-called thiamin diphosphate:ATP phosphoryltransferase (EC 2.7.4.15) activity was not detected in rat brain or liver. The enzyme was extracted from acetone powder of a crude mitochondrial fraction of bovine brain cortex and purified to homogeneity with a 0.6% yield after DEAE-cellulose chromatography, a first gel filtration, hydroxylapatite chromatography, chromatofocusing, and a second gel filtration. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its molecular weight was estimated to be 103,000. The pH optimum was 7.5, and the Km was determined to be 6 X 10(-4) M for ATP. ATP was found to be the most effective phosphate donor among the nucleoside triphosphates. Amino acid analysis of the purified enzyme revealed an abundance of glutaminyl, glutamyl, and aspartyl residues. Sulfhydryl reagents inhibited the enzyme reaction. Metals such as Fe2+, Zn2+, Pb2+, and Cu2+ strongly inhibited the activity. The enzyme was unstable, and glycerol (20%) and dithiothreitol (1.0 mM) were found to preserve the enzyme activity.     

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2

An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.     

地鳖纤溶活性蛋白的纯化及性质研究

通过硫酸铵分段沉淀、DEAE-纤维素柱和SephadexG-75柱层析从雌地鳖(Eupolyphagesinensiswalker)体内分离纯化到一种相对分子质量约为41.3kD的纤溶活性蛋白,纤维蛋白平板测定表明,该蛋白具有纤溶作用,经SDS-PAGE电泳显示为一条带,含糖量为10.5%。其水解纤维蛋白的比活力为547.86u/mg。该成分受蛋白抑制剂和丝氨酸蛋白酶抑制剂PMSF的抑制,但EDTA对其影响不大,提示该成分属于丝氨酸蛋白酶类。该成分在40℃下基本稳定,最适温度40℃,最适pH为8.0,其激活纤维蛋白溶解酶(PLG)的机制与尿激酶(UK)有一定区别。推测其可能是一种新的地鳖纤溶酶组分。     

Striated fibers of the rho form of Mycoplasma: in vitro reassembly, composition, and structure.

The rho-form of Mycoplasma contains a striated, axial fiber and associated terminal structure. The presence of this organelle was correlated with the synthesis of two proteins, A and B, of molecular weights of approximately 85,000 and 26,000, respectively, each accounting for about 10% of the total cell protein. Their amino acid compositions showed them to have distinct polypeptide chains. After osmotic lysis of rho-form cells the organelles disappeared; protein A accompanied the membrane fraction, whereas protein B was partly released in soluble form. After lysis by Nonidet P-40 in a medium composed of 4 M glycerol, 50 mM phosphate, and 10 mM MgSO4 at pH 6 (GPM-6), the organelles were preserved and released with ultrastructure unchanged. Protein A was recovered in the soluble fraction and protein B in the particulate (crude fiber) fraction. Treatment of the crude fiber fraction with 0.5 M NaCl in GPM-6 or with a solution containing 4 M glycerol, 10 mM morpholinoethanesulfonate, and 1 mM ethylenediaminetetraacetate at pH 7.0 caused the fibers to disassemble into subunits. By subsequent changes in the ionic conditions and temperature it was possible to cause the subunits to reassemble into ordered aggregates having the same ultrastructure as the native rho-fibers. The optimum temperature for reassembly in the presence of 4 M glycerol was 37 C, the optimum pH was 6.5 to 7.0, and the presence of Mg-2+, replaceable by Ca-2+, SR-2+, or Ba-2+, was essential. Protein B was the only protein detected in the purified, reconsituted fibers.     

Purification, characterization and immunolocalization of fimbrial protein from Porphyromonas (bacteroides) gingivalis.

Rapid and reproducible method is described here for the purification of the 43 kDa fimbrial protein from P. gingivalis by preferential fractionation in the presence of 1% SDS and 0.2M of a bivalent cation at pH 6.5. Homogeneity of the purified 43 kDa was confirmed by SDS-PAGE and Western blot analysis using monoclonal and polyclonal antibodies raised against this protein. Amino acid composition and the amino acid sequence of the first 30 amino acid residues of the purified fimbriae are consistent with the composition and sequence predicted from the cloned gene of the fimbrial subunit. Circular dichroism spectra shows high levels of beta-sheet structure. The purified 43 kDa polymer shows fimbriae-like morphology under the electron microscope. Ultrastructural localization of the 43 kDa protein by the immunogold technique revealed specific labeling of the fimbriae with a diameter of approximately 3.5 to 5.0 nm. Localization of this protein suggest that the 43 kDa component is a fimbrial subunit.     

Partial purification of alkaline phosphatase from bovine polymorphonuclear neutrophils and some properties

Alkaline phosphatase was purified from bovine polymorphonuclear neutrophils by butanol extraction and a combination of ion exchange, gel filtration and affinity chromatography. The enzyme was partially purified 2300-fold with a 4.7% yield and a sp. act. of 206 units/mg of protein. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a single activity band with the mol. wt of 165,000. The pH optima for the enzyme were 10.0 with p-nitrophenylphosphate and phenylphosphate and were 9.0 when beta-glycerophosphate, AMP and ADP were used. The enzyme was activated by Mg2+, Mn2+, Co2+ and Ni2+ but was inhibited by Zn2+. The enzyme was inhibited by EDTA and the EDTA-inactivated enzyme was reactivated by Mg2+, Mn2+ and Co2+ but not Zn2+.