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)

Circulating liver plasma membrane fragments (LPMF) were purified from human serum by means of a monoclonal antileucine aminopeptidase antibody, AD-1. This was done by immunoaffinity chromatography or by incubating the sera with AD-1-coated nitrocellulose disks. Alkaline phosphatase (ALP, EC 3.1.3.1) is bound to these LPMF through a glycosylphosphatidylinositol (GPI) anchor and is referred to as membrane-bound liver ALP (Mem-LiALP). Low concentrations of Triton X-100 or high bile salt concentrations released GPI anchor-bearing LiALP (Anch-LiALP) from purified LPMF; once released, Anch-LiALP was slowly and progressively converted to hydrophilic dimeric LiALP [soluble LiALP (Sol-LiALP)], free from its GPI anchor. Low levels of GPI-specific phospholipase D (GPI-PLD) activity were measured in the pure LPMF. Apparently, this membrane-associated GPI-PLD was released by the action of detergents and contributed to the spontaneous conversion of Anch-LiALP to Sol-LiALP. In the absence of detergents, GPI-PLD had little effect on Mem-LiALP, both in purified form as well as in serum. In vitro, isolated Anch-LiALP was converted to Sol-LiALP by both GPI-specific phospholipase C and GPI-PLD. Sol-LiALP in serum, however, appeared to be the product of GPI-PLD activity only. Five- to tenfold higher concentrations of Triton X-100 were needed to release Anch-LiALP from LPMF in serum, compared with those required in a solution of purified LPMF. In serum, as well as in purified conditions, only a small range of detergent of bile salt concentrations permitted the conversion of Mem-LiALP to Sol-LiALP. A model is proposed for the release in the circulation of Mem-LiALP, Anch-LiALP, and Sol-LiALP, involving both LPMF-associated GPI-PLD and liver sinusoid bile salts.
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PMID:Hydrolysis of membrane-bound liver alkaline phosphatase by GPI-PLD requires bile salts. 889 85

The bone morphogenetic proteins (BMPs), a subgroup of the TGF-beta gene super-family, are dimeric molecules involved in the growth, differentiation and repair of a wide variety of tissues. Based on the observation that several of the BMPs co-purify when isolated from bovine bone and that a pattern of co-localization exists during mouse embryogenesis, we co-expressed various combinations of BMPs in Chinese hamster ovary cells to test for possible heterodimer formation and activity. Transient co-expression of BMP-2 with either BMP-5, BMP-6 or BMP-7, or BMP-4 transiently co-expressed with BMP-7, resulted in more BMP activity than expression of any single BMP. Stable cell lines were then made in order to purify and characterize co-expressed BMPs in more detail. Co-expression of BMP-2 with BMP-7 yielded heterodimeric BMP-2/7 with a specific activity about 20-fold higher than BMP homodimers in an in vitro alkaline phosphatase induction assay. These heterodimers were also 5- to 10-fold more potent than BMP-2 in inducing cartilage and bone in an in vivo assay. Similar results were obtained with BMP-2/6 heterodimer. These experiments demonstrate the increased potency of several BMP heterodimers relative to BMP homodimers and support the hypothesis that such heterodimeric forms are likely to have natural biological functions.
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PMID:Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo. 891 35

The Lpp'OmpA(46-159) hybrid protein can serve as an efficient targeting vehicle for localizing a variety of procaryotic and eucaryotic soluble proteins onto the E. coli surface, thus providing a system for several possible biotechnology applications. Here we show that fusion between Lpp'OmpA(46-159) and bacterial alkaline phosphatase (PhoA), a normally periplasmic dimeric enzyme, are also targeted to the outer membrane. However, protease accessibility experiments and immunoelectron microscopy revealed that, unlike other periplasmic proteins, the PhoA domain of these fusions is not exposed on the cell surface in cells having an intact outer membrane. Conditions that affect the formation of disulfide bonds and the folding of the PhoA domain in the periplasm not only did not facilitate targeting to the cell surface but led to lethality when the fusion was expressed from a high-copy-number plasmid. Furthermore, E. coli expressing the Lpp'OmpA(46-159)-PhoA fusion exhibited strain- and temperature-dependent alterations in outer-membrane permeability. Our results are consistent with previous studies with other vehicles indicating that PhoA is not displayed on the surface when fused to cell-surface expression vectors. Presumably, the enzyme rapidly assumes a tightly folded dimeric conformation that cannot be transported across the outer membrane. The large size and quaternary structure of PhoA may define a limitation of the Lpp'OmpA(46-159) fusion system for the display of periplasmic proteins on the cell surface. Alkaline phosphatase is a unique protein among a group of five periplasmic proteins (beta-lactamase, alkaline phosphatase, Cex cellulase Cex cellulose-binding domain, and a single-chain Fv antibody fragment), which have been tested as passengers for the Lpp'OmpA(46-159) expression system to date, since it was the only protein not displayed on the surface.
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PMID:Characterization of Escherichia coli expressing an Lpp'OmpA(46-159)-PhoA fusion protein localized in the outer membrane. 892 Jan 86

In studies of pressure-induced subunit dissociation of protein aggregates, now widely used to evaluate the association free energy, entropy and enthalpy of very stable complexes, it is assumed that high pressure does not influence their structure/thermodynamic parameters and that some peculiarities of these equilibria, such as the decrease in subunit affinity at larger degrees of dissociation (alpha) and hysteresis in alpha/pressure diagrams are imputable to the slow conformational drift of isolated subunits. To test this premise, the conformation of dimeric alcohol dehydrogenase from horse liver and alkaline phosphatase from Escherichia coli was monitored as a function of pressure (up to 3 kbar) and temperature (0 to 50 degrees C) by means of the intrinsic Trp fluorescence and phosphorescence emission and binding of the 1-anilinonaphatalene-8-sulphonic acid (ANS) fluorophore. The results show a distinct influence of high pressure on the native dimers whose changes in conformation may, depending on whether or not these alterations are promptly reversed, be distinguished in elastic and inelastic changes. Elastic changes are ubiquitous and refer to pronounced modulations of the phosphorescence lifetime which is a monitor of the internal flexibility of the macromolecules. They attest to a tightening of the globular structure in the lower pressure range (below 1.5 kbar) as opposed to an increased fluidity in the higher range. The trend is similar between the two proteins and the tightening/loosening effect is fully consistent with the role that internal cavities and hydration of polypeptide is expected to play in determining the compressibility of these biopolymers. Inelastic perturbations reveal a more profound loosening of the globular fold and were observed only with alcohol dehydrogenase under conditions (low temperature (t < 10 degrees C) and high pressure (p > 2.5 kbar)) that favour protein hydration. They involve slow consecutive reactions that produce drastic reductions in phosphorescence lifetime, spectral red shifts, quenching of fluorescence and phosphorescence emission and modulation of ANS binding. Judging from the full protection afforded by glycerol as cosolvent, or the remarkable enhancement caused by modest concentrations of urea, the driving force of these perturbations appears to be pressure-induced hydration of the protein. Inelastic conformational changes are accompanied by a slow and often incomplete recovery of enzymatic activity. The characteristic times of these processes, their pressure dependence and the slow, thermally activated, reversibility are discussed in the light of hysteresis phenomena and changes of subunit affinity in dissociation equilibria.
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PMID:Pressure effects on the structure of oligomeric proteins prior to subunit dissociation. 894 76

Using a cell-bound immunogen, we have generated a monoclonal antibody, 3D5, that recognizes carboxy-terminal oligo-histidine tags (His tags) on a wide variety of proteins. From this monoclonal antibody, we have generated a single-chain fragment of the variable domains (scFv), a dimeric scFv-alkaline phosphatase fusion and an oligovalent scFv-display phage. The antibody in its various formats is an effective tool used in fluorescence-activated cell sorting analysis, the BIAcore method, Western blots and enzyme-linked immunosorbent assay (ELISA). Western blots and ELISAs can be developed directly by using crude extracts of E.coli cells that produce the scFv-alkaline phosphatase fusion, thus providing an inexhaustable and convenient supply of detection reagent. Alternatively, oligovalent scFv-displaying phage can be used directly from culture supernatants for this purpose. The dissociation constants, KD of the peptide KGGHHHHH (KD = 4 x 10(-7) M) and of imidazole (KD = 4 x 10(-4) M) were determined. Molecular modeling of the Fv fragment suggests the occurrence of two salt bridges between the protonated histidine side chains of the peptide and the acidic groups in the antibody, explaining why the antibody or the substrate may be eluted under mildly basic conditions.
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PMID:Specific detection of his-tagged proteins with recombinant anti-His tag scFv-phosphatase or scFv-phage fusions. 899 61

Alkaline phosphatase of Escherichia coli (a homodimeric protein found in the periplasmic space) contains two intramolecular disulfide bonds (Cys-168-Cys-178 and Cys-286-Cys-336) that are formed after export to the periplasmic space. The location-specific folding character of this enzyme allowed its wide usage as a reporter of protein localization in prokaryotic cells. To study the roles of disulfide bonds in alkaline phosphatase, we eliminated each of them by Cys to Ser mutations. Intracellular stability of alkaline phosphatase decreased in the absence of either one or both of the disulfide bonds. The mutant proteins were stabilized in a DegP protease-deficient strain, allowing accumulation at significant levels and subsequent characterization. A mutant protein that lacked the N-terminally located disulfide bond (Cys-168-Cys-178) was found to have Cys-286 and Cys-336 residues disulfide-bonded, to have a dimeric structure, and to have almost full enzymatic activity. Nevertheless, the mutant protein lost the trypsin-resistant conformation that is characteristically observed for the wild-type enzyme. In contrast, mutants lacking Cys-286 and Cys-336 were monomeric and inactive. These results indicate that the Cys-286-Cys-336 disulfide bond is required and is sufficient for correctly positioning the active site region of this enzyme, but such an active conformation is still insufficient for the conformational stability of the enzyme. Thus, a fully active state of this enzyme can be formed without full protein stability, and the two disulfide bonds differentially contribute to these properties.
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PMID:Roles of disulfide bonds in bacterial alkaline phosphatase. 904 30

The properties of a fusion protein comprising a streptavidin recognition sequence (Strep tag) fused to the C terminus of Escherichia coli alkaline phosphatase are described. The catalytic properties were determined with p-nitrophenyl phosphate and compared to those of the native E. coli alkaline phosphatase. It was found that the Km values were similar in both cases (8 microM for transferase and 2 microM for hydrolase activities) whilst the Vmax values were lower for the fusion protein, possibly due to the presence of misfolded forms. An optical biosensor based on the resonant mirror was used to determine the binding kinetics between the fusion protein and the immobilised streptavidin. The association and dissociation rate constants were determined to be 2.1(+/-0.3) x 10(-2) microM(-1) s(-1) and 11(+/-0.2) x 10(-3) s(-1), respectively, which results in an equilibrium dissociation constant of 0.5 microM. This is larger than previously reported affinities based on titration calorimetry and may be a consequence of the presence of two streptavidin binding sequences on the dimeric alkaline phosphatase simultaneously binding to two subunits of streptavidin.
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PMID:Alkaline phosphatase-Strep tag fusion protein binding to streptavidin: resonant mirror studies. 906 15

The phosphorescence spectrum and decay of Trp109 in Escherichia coli alkaline phosphatase was measured for the enzyme in 10 mM Tris/HCl, pH 7.4, at 21 degrees C. Changes in the spectrum and decay from the steady-state in response to non-covalent phosphate binding suggested a phosphate-induced alteration in the local environment surrounding Trp109 which lies buried below the active site. The seemingly inflexible structure in the region of Trp109, as judged by its very long phosphorescence lifetime, appeared unaltered when the enzyme was symmetrically bound with phosphate. However, the protein with phosphate bound to only one site displayed a marked increase in flexibility that extended over both subunits. For ratios of phosphate/enzyme (mol/mol) between 1.0 and 2.0, the observation of exponential phosphorescence decays with lifetimes that are a function of dilution provided evidence for the rapid exchange between phosphate half-saturated and fully-saturated enzymes consistent with observed enzyme turnover rates. The lifetimes under these conditions result in the calculation of a Kd for the dissociation of phosphate from the doubly occupied enzyme of 1.1 +/- 0.1 microM. The non-exponential decays at P/Ed (phosphate/dimeric enzyme) ratios less than 1.0 revealed that the exchange of phosphate between phosphate-free and half-saturated enzymes was not occurring on the timescale of the phosphorescence decay times, which implied that the half-saturated molecule cannot be contributing significantly to catalysis under steady-state conditions. The observation that the phosphorescence decay at a P/Ed ratio of 1.0 is exponential with a lifetime characteristic of the half-saturated species indicates that the binding of the first phosphate is significantly greater than the second, or that the binding exhibits negative cooperativity.
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PMID:Room temperature phosphorescence study of phosphate binding in Escherichia coli alkaline phosphatase. 912 21

Using concanavalin A-Sepharose affinity chromatography (Con A) we found that the serum of normal fasted adult rats contains two alkaline phosphatase (APase) glycoforms, one weakly bound (II) and the other strongly (III) bound to the column. Both serum APase glycoforms had an apparent molecular mass of 163 kD on Sepharose CL-6B and 118 kD on SDS-PAGE under nondenaturing conditions. We consider the molecular forms as dimeric, since monomers of 60.3 and 58.5 kD for the Con A weakly and strongly bound glycoform, respectively, were obtained. However, these two dimeric glycoforms were different in their pH optimum, affinity to p-nitrophenyl phosphate as substrate, the degree of L-phenylalanine inhibition and relative thermostability. Judging by the relative thermostability and by L-phenylalanine inhibition, it seems that both serum APase glycoforms in fasted rats are mainly of duodenal mucosal cell origin. The Con A weakly bound (II) glycoform could be derived from the cytosol, and the Con A strongly bound (III) one from both the cytosolic and membranous fractions of duodenal mucosal cells. However, in addition to the heat-stable component, the Con A strongly bound serum APase glycoform also contains a minor heat-labile and L-phenylalanine-resistant component which could be of nonspecific tissue origin since such a fraction was not discovered by us in rat duodenal mucosal cells.
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PMID:Microheterogeneity of rat serum alkaline phosphatase in fasting state: characterization of two duodenal alkaline phosphatase glycoforms. 914 9

Diagnostic enzymology measures the serum or plasma levels of enzymes that were originally located within the cell, or were attached to its plasma membrane with their active sites exposed to the external milieu. The process by which they are released varies under different physiological and pathological conditions. In this way, shedding of hepatocyte plasma membranes is thought to be responsible for the release of liver plasma membrane fragments (LiPMF) into the circulation in metastatic, infiltrative and cholestatic liver diseases. Several membrane-bound enzymes, such as gamma-glutamyltransferase (gamma-GT), alkaline phosphatase (ALP), leucine aminopeptidase (LAP) and 5'-nucleotidase (5'-Nu) are expressed at the surface of the shedded LiPMF. These enzymes are attached to the cell membrane by means of hydrophobic interactions between the anchoring domain of the enzyme and lipid components of the cell membrane, e.g. through a specific glycan phosphatidylinositol (GPI) anchor. There is a striking homology between these LiPMF and the membrane fragments shedded or actively formed by other cells, such as bone matrix vesicles-rich in bone ALP-, membrane fragments of the syncitiotrophoblast-rich in placental ALP-, and membrane fragments present in duodenal fluid-rich in intestinal ALP. With the exception of LiPMF, membrane-bound (Mem-) forms of ALP are only very exceptionally found in human serum. Normally, the soluble (Sol-ALP) dimeric fractions of the enzyme predominate in serum, but liver, bone, placental and intestinal ALP can also be present as GPI-anchor bearing (Anch-) hydrophobic isoforms. Models for the release in the circulation of Mem-, Anch- and Sol-liver and intestinal ALP, involving both plasma membrane-associated GPI-phospholipase-D (GPI-PLD) and liver bile salts are proposed.
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PMID:How do plasma membranes reach the circulation? 943 85


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