Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An inactivated gene for Bacillus amyloliquefaciens extracellular ribonuclease (barnase) has previously been cloned and sequenced following transposon mutagenesis. The intact gene could not be assembled in Escherichia coli and is presumed to be lethal. Therefore, we introduced specific mutations into the barnase gene to prevent its lethal effect. A Gln-73 mutant gene was stable in E. coli but only produced low amounts of barnase antigen. Mutants containing Asp, Gln or Arg, instead of His-102, at the active site were identified by immunological screening for barnase antigen. None of the mutant proteins with alterations at aa residue 102 possessed RNase activity. The level of barnase (Asp-102) was higher in E. coli than in B. subtilis but the protein was not processed to the correct size in E. coli. To obtain correct processing, the barnase (Asp-102) structural gene was fused to the E. coli alkaline phosphatase promoter and signal sequence (phoA). Cells containing this construct secreted correctly processed barnase (Asp-102) into the periplasmic space and culture supernatant at a level of 20 mg/l. Barnase (Asp-102) was purified and found to have an identical N-terminus and a thermal unfolding curve that was nearly identical to that of active barnase (His-102). The cloning and expression of barnase in E. coli will allow detailed analysis of barnase protein folding by molecular genetic approaches.
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PMID:Expression of Bacillus amyloliquefaciens extracellular ribonuclease (barnase) in Escherichia coli following an inactivating mutation. 329 26

Membrane and soluble forms of alkaline phosphatase (ALP) were selectively prepared from human placental microsomes by treatment with 1-butanol at pH 8.5 and 5.5, respectively. The purified membrane (mALP) and soluble (sALP) forms were analyzed for chemical compositions. mALP was found to contain 1 mol each of palmitate, stearate, and glycerol/subunit of ALP, which were absent in sALP. Both the forms contained 1 mol of inositol and 2 mol of ethanolamine/subunit. However, none of these compounds was detectable in another soluble form prepared by treatment with papain, which is known to cleave the carboxyl-terminal region. The results suggest that mALP contains diacylglycerol, the removal of which results in its conversion to sALP. We then prepared [3H]ethanolamine-labeled ALP by incubating choriocarcinoma cells (JEG-3) with the isotope. 3H-Labeled sALP was mixed with unlabeled sALP and treated with papain. A 3H-labeled single component was purified from the digests by sequential chromatography through anti-ALP-IgG-Sepharose, concanavalin A-Sepharose, Bio-Gel P-6, and TSK G-2000 columns. Chemical analyses revealed that the purified sample contains the tripeptide Thr-Thr-Asp, ethanolamine, glucosamine, mannose, inositol, and phosphate. Molar ratios of the latter five compounds were calculated to be 2, 1, 3, 1, and 2, respectively, by taking Asp as 1 mol. The tripeptide sequence was identified at positions 482-484 in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 513, containing a hydrophobic amino acid sequence. Taken together, these results suggest that the mature ALP molecule lacks the predicted carboxyl-terminal peptide extension and is attached at Asp484 with a glycosylphospholipid, the components of which are characterized above. The glycosylphospholipid thus attached is considered to function as the membrane anchor of ALP.
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PMID:Chemical characterization of the membrane-anchoring domain of human placental alkaline phosphatase. 339 21

The nucleotide sequence of the alkaline phosphatase (APase) gene (phoA) of Escherichia coli strain 294 has been determined. Pre-APase has a total of 471 amino acids (aa) including a signal sequence of 21 aa. The derived aa sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at positions 16 and 36, and glutamic acid instead of glutamine at position 197. Two open reading frames (ORF1 and ORF2) located downstream from phoA or upstream from proC have been found. ORF1 encodes a putative presecretory protein of 106 aa with a signal sequence of 21 or 22 aa. If this protein is actually produced, it may be one of the smallest periplasmic proteins in E. coli.
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PMID:Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli. 353 24

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.
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PMID:Purification and partial amino acid sequencing of bovine kidney alkaline phosphatase. 359 90

Human liver alkaline phosphatase (AP) has been purified to homogeneity. The enzyme has a molecular weight of 150,000 in its native state and consists of two identical subunits of Mr 75,000. After treatment with endoglycosidase F the molecular weight is reduced to 50,000 indicating a high degree of glycosylation. The amino-terminal sequence up to 22 residues was found to be Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-Lys-Tyr-(Ala)-Arg-Asp-Gln-Ala-Gln-?- Thr-Leu-Lys-Tyr. The amino-terminal portions of human and bovine liver AP are identical. The amino termini of the human liver and human placental AP isozymes have appreciable homology. Conformationally the amino termini are very similar.
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PMID:Human liver alkaline phosphatase, purification and partial sequencing: homology with the placental isozyme. 395 57

Bovine liver alkaline phosphatase has been purified to homogeneity by procedures that include reverse-phase HPLC. The pure enzyme has an apparent Mr of 160,000 and is composed of what appears to be two identical monomers of Mr 82,000. About 80% of the material yielded the amino-terminal sequence Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-?-Tyr-?-Arg-Asp-Gln-Ala-Gln. The minor component was extended at the amino terminus by two residues that have not yet been identified, i.e., ?-?-Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-?-Tyr-?-Arg-Asp-Gln-Ala-Gln.
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PMID:Purification and partial sequencing of bovine liver alkaline phosphatase. 403 95

Different patterns of isozymes were obtained by starch-gel electrophoresis of alkaline phosphatase from Escherichia coli strains differing only by strA or ram mutations, or both, in the 30S ribosomal subunit. The isozyme spread was reduced in strA and increased in ram strains; this strictly parallels the restriction and enhancement of translational ambiguity produced by these mutations. Streptomycin present during growth had an effect similar to ram on both isozymes and ambiguity. The three isozymes analyzed have different N-terminal residues: aspartic acid, valine, and threonine. Different patterns of isozymes were also obtained in a wild-type strain through the specific action of exogenous arginine. A link between the mechanism of the effect of arginine and that of the ribosome is not obvious. The possibility is discussed that in both cases, although by different mechanisms, N-terminals are formed with different sensitivity to limited degradative attack.
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PMID:Ribosomal alterations controlling alkaline phosphatase isozymes in Escherichia coli. 455 93

A diagonal-electrophoresis method for the selective purification of serine phosphate peptides was applied to tryptic, chymotryptic and peptic digests of oxidized ovalbumin. This method is based on the release of the phosphate group bound to serine by treatment with alkaline phosphatase on paper. The identified serine phosphate peptides were purified by paper electrophoresis at pH6.5 and 2.0, dephosphorylation with bacterial alkaline phosphatase, and paper electrophoresis at pH2.0 again, in that order. The presence of two groups of serine phosphate peptides was apparent from the amino acid composition. One group contained no lysine, cysteic acid, proline, leucine or isoleucine (sequence 1) and the other had all those amino acids (sequence 2). Further degradation with subtilisin of those peptides and ;dansyl'-Edman sequence analysis established their partial sequences. The proposed sequences are as follows (with ;SerP' representing serine phosphate): sequence 1, -Ala-Gly-Arg-Glu-Val-Val-Gly-SerP-Ala-Glu-Ala-Gly-Asp-Val-Ala-Ala-Ser-(Val,Glx(2),Ser,Phe)-Arg-; sequence 2, -Asp-Lys-Leu-Pro-Gly-Phe-Gly-Asp-SerP-Ile-Glx-Ala-Glx-CySO(3)H-Gly-(Thr,Ser,Val)-(Asp,His,Val)-. The partial sequence of one of the phosphopeptides, Asp-(Glu,Ile,SerP), reported by Flavin (1954) was used to establish the proposed sequence 2.
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PMID:An application of diagonal electrophoresis to the selective purification of serine phosphate peptides. Serine phosphate peptides from ovalbumin. 488 Nov 41

Analogs of phosphophoryn, a calcium-binding phosphorylated protein found in mineralized dentin, were synthesized by solid phase peptide synthesis. The dentin phosphophoryn appears to contain some sequence blocks of (Asp-PhosphoSer)n. As this sequence is difficult to synthesize, polymers of (alpha-L-Glu-L-Ser) were prepared. The 30-peptide, (alpha-L-Glu-L-Ser)15, was phosphorylated by reaction with orthophosphoric acid in the presence of trichloroacetonitrile in anhydrous dimethylsulfoxide. Calcium ion binding studies were carried out with both the 30-peptide and phosphorylated 30-peptide. The conformation of the original 30-peptide, (Glu-Ser)15, was examined, in the presence and absence of calcium ion, by circular dichroism measurements. Purified bovine phosphophoryn, previously studied by the same techniques, was partially dephosphorylated by alkaline phosphatase, and its calcium ion binding properties were determined. Dephosphorylation to 31% of the initial phosphorus content reduced the number of high affinity sites to approximately 30% of the initial value. However, the stoichiometry of binding indicated that both phosphate and carboxylate groups participate in the high affinity binding and that the binding constant was decreased only slightly. Partial phosphorylation of the 30-mer raised the calcium binding constant, Ka, from 2.1 x 10(2) to 3.3 x 10(3) M-1 and increased the amount of binding from an electrostatic equivalent number of sites to a stoichiometric equivalent number. Concomitant with binding, there was a transition from random coil to beta-like structure. These data suggest that the repetitive (Asp-PhosphoSer)n regions in phosphophoryn and the (Glu-PhosphoSer)n sequence of the synthetic polymer have special conformations which favor the unidentate binding of calcium to the carboxyl groups and phosphate groups. and which enhance the binding affinities of the carboxyl groups in such sequences in a cooperative fashion.
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PMID:Cooperativity in calcium ion binding to repetitive, carboxylate-serylphosphate polypeptides and the relationship of this property to dentin mineralization. 678 Apr 83

1. Acid phosphatase (AcPase) from potato tubers was purified by tannic acid fractionation, DEAE-cellulose chromatography, filtration on Bio-Gel P-150 and affinity chromatography on Con A-Sepharose. The enzyme was purified 260-fold and was electrophoretically homogeneous; its mol. mass is about 69 000. 2. The carbohydrate component accounts for 16.6% of the total enzyme weight and includes mannose (5.6%), rhamnose (3.4%), glucose (2.5%), galactose (1.5%) and glucosamine (3.6%). In the amino acid composition aspartic acid, glutamic acid, serine and glycine account for 37.7% of total amino acid residues. 3. Optimum pH is at 5.0-5.3. The enzyme activity was reduced by half after 30 min incubation at 60 degrees C, and was fully abolished after 2 h incubation at 70 degrees C. The enzyme is a nonspecific phosphomonoesterase; aromatic phosphomonoesters and inorganic pyrophosphate can serve as substrates. Apparent Km values were 1.25 mM and 40 mM for p-nitrophenylphosphate and inorganic pyrophosphate, respectively. The enzyme is inhibited by MoO42-, Zn2+, Hg2+ and urea. Inhibition caused by urea was reversible at urea concentration below 9 M.
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PMID:Acid phosphatase of potato tubers (Solanum tuberosum L). Purification, properties, sugar and amino acid composition. 715 77


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