Purification and partial amino acid sequencing of bovine kidney alkaline phosphatase

Bovine kidney alkaline phosphatase (ALPase) was purified by the sequential application of monoclonal anti-bovine cartilage ALPase affinity, DEAE-cellulose, and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide-gel electrophoresis showed the presence of a single band corresponding to a molecular weight of 80,000. The N-terminal amino acid sequence of bovine kidney alkaline phosphatase was determined as follows: Leu-Val-Pro-Glu-Lys-Asp-Pro-?-Tyr-Trp-Arg-Asp-Gln-Ala-Gln.     

Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme

The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54? omitted?760 Da. On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E. coli alkaline phosphatase and 22% Bacillus subtilis (Bsu) alkaline phosphatases. High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E. coli alkaline phosphatase. Comparison of Tca alkaline phosphatase and E. coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites. Heat-stable Tca alkaline phosphatase activity was detected in E. coli YK537, harboring pJRAP.     

Nucleotide sequence of the Vibrio alginolyticus calcium-dependent, detergent-resistant alkaline serine exoprotease A

The nucleotide sequence of the Vibrio alginolyticus alkaline serine exoprotease A (ProA) gene cloned in Escherichia coli was determined. The exoprotease A gene (proA) consisted of 1602 bp which encoded a protein of 534 amino acids (aa) with an Mr of 55,900. The region upstream from the gene was characterized by a putative promoter consensus region (-10 -35), a ribosome-binding site and ATG start codon. The proA gene encodes a typical 21-aa N-terminal signal sequence which, when fused to alkaline phosphatase by means of transposon TnphoA, was able to mediate transport of the alkaline phosphatase to the periplasm in E. coli. Deletions of up to 106 aa from the C terminus of ProA did not result in the loss of extracellular protease activity. Additional V. alginolyticus genes were not involved in the secretion into the medium of the cloned ProA in E. coli. The amino acid sequence of ProA showed low overall homology to a Serratia marcescens serine exoprotease but significant homology was detected with other subtilisin family exoproteases. The fungal proteinase K, another sodium dodecyl sulfate-resistant protease, had 44% aa homology with ProA.     

Constitutive phosphorylation of the vesicular inhibitory amino acid transporter in rat central nervous system

gamma-Aminobutyric acid (GABA) and glycine are stored into synaptic vesicles by a recently identified vesicular inhibitory amino acid transporter [VIAAT, also called vesicular GABA transporter (VGAT)]. Immunoblotting analysis revealed that rat brain VIAAT migrated as a doublet during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a predominant slower band in all areas examined except olfactory bulb and retina. The slower band corresponded to a phosphorylated form of VIAAT as it was converted to the faster one by treating brain homogenates with alkaline phosphatase or with an endogenous phosphatase identified as type 2A protein-serine/threonine phosphatase using okadaic acid. In contrast, the recombinant protein expressed in COS-7 or PC12 cells co-migrated with the faster band of the brain doublet and was insensitive to alkaline phosphatase. To investigate the influence of VIAAT phosphorylation on vesicular neurotransmitter loading, purified synaptic vesicles were treated with alkaline phosphatase and assayed for amino acid uptake. However, neither GABA nor glycine uptake was affected by VIAAT phosphorylation. These results indicate that VIAAT is constitutively phosphorylated on cytosolic serine or threonine residues in most, but not all, regions of the rat brain. This phosphorylation does not regulate the vesicular loading of GABA or glycine, suggesting that it is involved at other stages of the synaptic vesicle life cycle.     

Rat alkaline phosphatase. II. Structural similarities between the osteosarcoma, bone, kidney, and placenta isoenzymes

A mouse monoclonal antibody raised against rat osteosarcoma alkaline phosphatase (AP) was covalently coupled to protein A-Sepharose and used to purify this enzyme from preparations of rat osteosarcoma, calvaria, kidney, and placenta in a single-step procedure. The tissue-specific isoenzymes purified in this manner showed identity in the immunodiffusion reaction with a polyclonal anti-AP antibody, but differed in apparent molecular weight and degree of polydispersity on sodium dodecyl sulfate-polyacrylamide gels. Treatment with N-glycanase abolished these differences, yielding proteins with an apparent molecular weight of 52,000 Da and identical V8 protease digestion patterns. Alkaline phosphatase from these tissues showed no significant difference in amino acid composition and identity in the first 20 N-terminal amino acids. These findings provide structural evidence which supports the hypothesis that the tissue-specific alkaline phosphatase isoenzymes share a common protein sequence subject to different glycosylation pattern.     

Analysis of Halobacterium halobium gas vesicles

Gas vesicles, isolated from lysed Halobacterium halobium cells, gave an amino acid analysis which accounted for 78% of the weight, and the balance was mainly salt and water. One percent of tightly bound d-galactose was found, as well as 2% of phosphate that was not released by treatment which promotes beta-elimination, by hydrolytic release of the galactose, by carboxymethylation of lysine, or by alkaline phosphatase digestion. Only a trace of lipid was detected, and it appeared to have a polyisoprenoid structure. The vesicles were not solubilized by extremes of pH, by agents such as urea, guanidine hydrochloride, formic acid, and detergents, or by organic solvents. Succinylation and carboxymethylation gave partial dispersion, but the products were heterogeneous and of high molecular weight. The amino acid composition of vesicles was independent of fragment size. No band was obtained by polyacrylamide gel electrophoresis, with neutral, acidic, and alkaline systems, with or without sodium dodecyl sulfate and urea, before or after chemical modification. No amino terminus was detected. Electrofocusing of a vesicle dispersion showed a major component with a pI of 4.0 and an amino acid composition of the whole vesicles, and a minor band with pI 3.4 which had an amino acid composition different from whole vesicles. Vesicle protein was resistant to digestion by Pronase, trypsin, thermolysin, and papain. The precipitin reaction with rabbit antivesicle serum was not inhibited by galactose or inorganic phosphate. Succinylated and carboxymethylated vesicles cross-reacted with antivesicle serum. Cell lysates contained material which reacted with antiserum, but it was heterogeneous and mainly larger than 5 x 10(6) daltons. Material from nonvacuolated mutants reacted weakly with antiserum, but the amino acid composition of the precipitated antigen was different from that of vesicles and of soluble cross-reacting material from vacuolated cells.     

An amphiphilic structure of the ninth component of human complement. Evidence from analysis of fragments produced by alpha-thrombin

Purified human C9 was treated separately with three proteolytic enzymes: trypsin, plasmin, and alpha-thrombin, and the digestion products were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Trypsin initially cleaved the Mr = 71,000 C9 to produce a Mr = 47,000 fragment plus numerous smaller fragments and prolonged digestion reduced the molecule to small polypeptides. Plasmin produced a Mr = 37,000 fragment which was stable to further digestion, plus fragments smaller than Mr = 10,000. Human alpha-thrombin cleaved C9 (7.8% carbohydrate) at a single internal site to produce a Mr = 37,000 fragment (11.3% carbohydrate) and a Mr = 34,000 fragment (3.9% carbohydrate). Statistical analysis of the amino acid compositions of the fragments and alkaline polyacrylamide gel electrophoresis showed that C9 is highly amphiphilic; the Mr = 34,000 fragment contains a majority of the acidic amino acids and migrates rapidly on alkaline gels; the Mr = 37,000 fragment is hydrophobic with a slow electrophoretic mobility. The two fragments remain noncovalently associated, but were separated by sodium dodecyl sulfate-hydroxylapatite chromatography. The NH2-terminal sequence analysis of native C9, of alpha-thrombin-cleaved C9, and for the isolated fragments showed that the acidic Mr = 34,000 fragment is the NH2-terminal C9a domain and the more hydrophobic Mr = 37,000 fragment is the carboxyl-terminal C9b domain. Hemolytic activity of C9 was unaffected by alpha-thrombin cleavage.     

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.     

Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis: application to peptide mapping of human complement component C3

The combination of high-resolution Tricine-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (H. Sch?gger and G. von Jagow (1987) Anal. Biochem. 166, 368-379) and electroblotting onto polyvinylidene difluoride (PVDF) membranes represents a powerful technique for the isolation of small amounts of peptides and protein fragments (Mr 1000-20,000) in a suitable form for amino acid sequencing, directly on the blotting membrane. Conditions for electrophoresis and electroblotting were optimized with respect to high transfer yield and suitability for both amino acid analysis and sequence determination of stained PVDF-bound peptides. Transfer yields were 50-80%, amino acid compositions including Cys were correct, and picomole quantities were sequenced with initial and repetitive yields as high as those we normally obtain for peptides in solution. The method was used for peptide mapping of polymorphic forms of human complement component C3.     

Chemical modification of Bacillus thuringiensis subsp. thuringiensis (HD-524) trypsin-activated endotoxin: implication of tyrosine residues in lepidopteran cell lysis

A purified protein fraction from a solubilized and trypsin-digested extract of Bacillus thuringiensis subsp. thuringiensis (HD-524) fermentation powder was lytic to cells from several lepidopteran lines. Maximum yield was obtained by alkaline carbonate-thiocyanate solubilization of washed powder followed by trypsin digestion and Sephacryl (S-300) chromatography. The alkaline carbonate-solubilized fraction consisted predominantly of two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with MW of 144 +/- 0.9 kDa and 134 +/- 1.4 kDa. After trypsin treatment and column chromatography, the cytolytic fraction consisted of a major band with a MW of 60.0 +/- 1.8 kDa and a minor band of 69 +/- 0.9 kDa. Cells from Trichoplusia ni (TN368) were most susceptible to lysis with 50% of cells lysed at 3 micrograms/ml, followed by Spodoptera frugiperda cells (SF21AE) exhibiting 50% cell lysis at 5 micrograms/ml and Lymantria dispar cells (Ld652Y) showing 40% lysis at 10 micrograms/ml. Chemical modification of the polypeptides was performed to determine the role of certain amino acid residues in the cytolytic activity. The group-specific reagent tetranitromethane was used to nitrate and oxidize tyrosine and cysteine residues, respectively. Cysteine residues alone were also modified with p-hydroxymercuribenzoic acid. Lysine residues were modified with O-methylisourea. Of the three types of amino acid residues, only the modification of tyrosine resulted in reduced cell lysis.     

Isolation and partial characterization of exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A.

Homogeneous fragments of exosporium were isolated and purified from mature spores of a highly sporogenic mutant derived from Clostridium botulinum type A strain 190L. The exosporium was composed of three lamellae and showed a hexagonal array when negatively stained. The hexagonal array of isolated exosporium was resistant to sodium dodecyl sulfate, urea, dithiothreitol, and proteolytic enzymes such as trypsin, pronase, and nagarse, except for pepsin. The hexagonal array was partially disintegrated with 5 M guanidine-HCl and almost completely disrupted with 8 M urea in combination with 1% mercaptoethanol under alkaline conditions. The purified exosporium fraction was composed mainly of protein (69.1%) and lipids (13.8%). A small amount of amino sugars (2.5%) was present, but neutral sugars could not be detected. The exosporium protein had a predominantly acidic amino acid composition accompanied by low levels of cystine, methionine, and histidine.     

Characterization of the major duck hepatitis B virus core particle protein.

The amino acid composition of the major duck hepatitis B virus (DHBV) core particle proteins was determined. The results of this analysis indicated that cores are composed of a single major protein that initiates translation from the second available AUG in the DHBV core gene. Proteins isolated from core particles purified from the cytoplasm of DHBV-infected duck hepatocytes exhibited heterogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, independent of the stage of viral DNA maturation. Incubation of native cores with alkaline phosphatase removed this heterogeneity, indicating that phosphorylation of external amino acids was responsible. Core protein isolated from mature DHBV purified from serum of infected animals did not display heterogeneity, suggesting a possible role for dephosphorylation in virus maturation.     

Purification and characterization of a repressible alkaline phosphatase from Thermus aquaticus.

A repressible alkaline phosphatase has been isolated from the extreme bacterial thermophile, Thermus aquaticus. The enzyme can be derepressed more than 1,000-fold by starving the cells for phosphate. In derepressed cells, nearly 6% of the total protein in a cell-free enzyme preparation is alkaline phosphatase. The enzyme was purified to homogeneity as judged by disc acrylamide electrophoresis and sodium dodecyl sulfate electrophoresis. By sucrose gradient centrifugation it was established that the enzyme has an approximate molecular weight of 143,000 and consists of three subunits, each with a molecular weight of 51,000. Tris buffer stimulates the activity of the enzyme, which has a pH optimum of 9.2. The enzyme has a broad temperature range with an optimum of 75-80 degrees. The enzyme catalyzes the hydrolysis of a wide variety of phosphorylated compounds as do many of the mesophilic alkaline phosphatases. The Michaelis constant(Km) for the enzyme is 8.0 X 10(-4) M. Amino acid analysis of the protein revealed little in the amino acid composition to separate it from other mesophilic enzymes which have been previously studied.     

Purification and partial characterization of feline classical pancreatic lipase

Classical pancreatic lipase has been purified and partially characterized in many species. The objective of this project was to purify feline classical pancreatic lipase (fPL) from pancreatic tissue and partially characterize this protein. Pancreata were collected from cats (Felis catus) euthanized for unrelated research projects. Fat was removed by trimming away grossly visible fat and by extraction in organic solvents. The delipidated pancreatic extract was further purified by extracting the enzymes in a Tris-buffer containing two different protease inhibitors, benzamidine and phenylmethylsulfonyl fluoride, followed by anion-exchange, size-exclusion, and cation-exchange chromatography. Feline pancreatic lipase was successfully purified from feline pancreatic tissue. The purified product showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular mass of approximately 52.5 kDa. Exact molecular mass was determined by mass spectrometry as 52.4 kDa. Approximate specific absorbance at 280 nm of fPL was 1.18 for a 1 mg/ml solution. N-terminal amino acid sequence of the first 25 amino acid residues showed the sequence Lys-Glu-Ile-?-Phe-Pro-Arg-Leu-Gly-?-Phe-Ser-Asp-Asp-Ala-Pro-Trp-Ala-Gly-Ile-Ala-Gln-Arg-Pro-Leu. This sequence showed close homology with the amino acid sequence of classical pancreatic lipase in other species.     

Purification and partial amino acid sequencing of alkaline phosphatase from rachitic rat epiphyseal cartilage

1. Alkaline phosphatase of rachitic epiphyseal cartilage was purified to apparent homogeneity by sequential application of monoclonal affinity, DEAE-cellulose, and Sepharose CL-6B chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the enzyme showed the presence of a dominant band corresponding to a molecular weight of 80,000. 2. The N-terminal amino acid sequence was determined as follows: Phe-Val-Pro-Glu-Lys5-Glu-Lys-Asp-Pro-Ser10-Tyr-Trp-Arg-Gln-+ ++Gln15-Ala-Gln-Glu- Thr-Leu20-Lys-Asn-Ala-Leu-Lys25-Leu-Gln-Lys-?-Asn-Val-Asn-?- Ala-Lys35-?-Ile-?- Met-Phe40-Leu-(Gly?)-Asp-(Ala/Gly?)-Met45-?-Val-?- (Val/Gly?).     

ISOLATION OF AN ACID-SOLUBLE BASIC PROTEIN FROM MONKEY BRAIN

—A basic protein, soluble in 0·1 m -perchloric acid, has been purified from brain of Macaca irus. The protein is homogeneous as indicated by ultracentrifugation, gel filtration, gel isoelectric focusing and gel electrophoresis at pH 2·9, 4·3 and 7·5. The molecular weight is estimated to be 16,000 by electrophoresis in sodium dodecyl sulphate–polyacrylamide gels. This result is in agreement with the value of 16,728 obtained from the amino acid analysis. The protein dimerizes under alkaline conditions. The predominant amino acid is glycine (15%) and the protein also contains 4% cysteine. The ratio of acidic to basic amino acids is 1·6, but a high amide content gives the protein a basic character. An isoelectric point of 9·5 is observed in gel isoelectric focusing.     

Western blotting and immunochemical detection of histones electrophoretically resolved on acid-urea-Triton- and sodium dodecyl sulfate-polyacrylamide gels

We have developed a method for the efficient transfer of histones from acetic acid-urea-Triton X-100 (AUT)-polyacrylamide minislab gels to nitrocellulose. The AUT gel was equilibrated with 50 mM acetic acid and 0.5% sodium dodecyl sulfate and then with 62.5 mM Tris-HCl, pH 6.8, and 2.3% sodium dodecyl sulfate. An alkaline transfer buffer [25 mM 3-(cyclohexylamino)-1-propanesulfonic acid, pH 10, with 20% methanol] was used to electrophoretically transfer the strongly basic proteins from AUT or sodium dodecyl sulfate gels to nitrocellulose. The applicability of this approach in the immunochemical detection of ubiquitinated histone species is demonstrated.     

Cross-linking of wheat gluten proteins during production of hard pretzels

The impact of the hot alkaline dip, prior to pretzel-baking, on the types and levels of cross-links between wheat proteins was studied. Protein extractability of pretzel dough in sodium dodecyl sulfate containing buffer decreased during alkaline dipping [45?s, 1.0% (w/v) NaOH, 90°C], and even more during baking (3?min at 250°C) and drying (10?min at 135°C). Reducing agent increased the extractability partly, indicating that both reducible (disulfide, SS) and non-reducible (non-SS) protein cross-links had been formed. The decrease in cystine levels suggested β-elimination of cystine releasing Cys and dehydroalanine (DHA). Subsequent reaction of DHA with Lys and Cys, induced the unusual and potentially cross-linking amino acids lysinoalanine (LAL) and lanthionine (LAN), respectively, in alkaline dipped dough (7?μmol LAN/g protein) and in the end product (9?μmol LAL and 50?μmol LAN/g protein). The baking/drying step increased sample redness, decreased Lys levels more than expected based on LAL formation (57?μmol/g protein), and induced a loss of reducing sugars (99?μmol/g protein), which suggested the potential contribution of Maillard-derived cross-links to the observed extractability loss. However, levels of Maillard products which possibly cross-link proteins, are small compared to DHA-derived cross-links. Higher dipping temperatures, longer dipping times, and higher NaOH concentrations increased protein extractability losses and redness, as well as LAL and LAN levels in the end product. No indications for Maillard-derived cross-links or LAL in pretzel dough immediately after dipping were found, even when severe dipping conditions were used.     

Purification and characterization of a novel bacteriocin produced by Enterococcus faecalis strain RJ-11

Lactic acid bacteria exhibiting activity against the gram-positive bacterium Bacillus subtilis were isolated from rice bran. One of the isolates, identified as Enterococcus faecalis RJ-11, exhibited a wide spectrum of growth inhibition with various gram-positive bacteria. A bacteriocin purified from culture fluid, designated enterocin RJ-11, was heat stable and was not sensitive to acid and alkaline conditions, but it was sensitive to several proteolytic enzymes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that enterocin RJ-11 had a molecular weight of 5,000 in its monomeric form. The amino acid sequence determined for purified enterocin RJ-11 exhibited high levels of similarity to the sequences of enterocins produced by Enterococcus faecium.     

Nucleotide sequence and characterization of the gene for secreted alkaline phosphatase from Lysobacter enzymogenes.

Lysobacter enzymogenes produces an alkaline phosphatase which is secreted into the medium. The gene for the enzyme (phoA) was isolated from a recombinant lambda library. It was identified within a 4.4-kb EcoRI-BamH1 fragment, and its sequence was determined by the chain termination method. The structural gene consists of an open reading frame which encodes a 539-amino-acid protein with a 29-residue signal sequence, followed by a 119-residue propeptide, the 281-residue mature phosphatase, and a 110-residue carboxy-terminal domain. The roles of the propeptide and the carboxy-terminal peptide remain to be determined. A molecular weight of 30,000 was determined for the mature enzyme from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence was compared with sequences available in the current protein data base, and a region of the sequence was found to show considerable homology with sequences in mammalian type 5 iron-containing purple acid phosphatases.