Molecular evolution of the tissue-nonspecific alkaline phosphatase allows prediction and validation of missense mutations responsible for hypophosphatasia.

ALPL encodes the tissue nonspecific alkaline phosphatase (TNSALP), which removes phosphate groups from various substrates. Its function is essential for bone and tooth mineralization. In humans, ALPL mutations lead to hypophosphatasia, a genetic disorder characterized by defective bone and/or tooth mineralization. To date, 275 ALPL mutations have been reported to cause hypophosphatasia, of which 204 were simple missense mutations. Molecular evolutionary analysis has proved to be an efficient method to highlight residues important for the protein function and to predict or validate sensitive positions for genetic disease. Here we analyzed 58 mammalian TNSALP to identify amino acids unchanged, or only substituted by residues sharing similar properties, through 220 millions years of mammalian evolution. We found 469 sensitive positions of the 524 residues of human TNSALP, which indicates a highly constrained protein. Any substitution occurring at one of these positions is predicted to lead to hypophosphatasia. We tested the 204 missense mutations resulting in hypophosphatasia against our predictive chart, and validated 99% of them. Most sensitive positions were located in functionally important regions of TNSALP (active site, homodimeric interface, crown domain, calcium site, …). However, some important positions are located in regions, the structure and/or biological function of which are still unknown. Our chart of sensitive positions in human TNSALP (i) enables to validate or invalidate at low cost any ALPL mutation, which would be suspected to be responsible for hypophosphatasia, by contrast with time consuming and expensive functional tests, and (ii) displays higher predictive power than in silico models of prediction.

Serum alkaline phosphatase isoenzymes in SD rats detected by polyacrylamide-gel disk electrophoresis.

Serum alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. In the present study, we assessed the usefulness of a commercially available polyacrylamide-gel disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co. Ltd., Tokyo, Japan) for identifying serum ALP isoenzymes in rats of the Sprague-Dawley strain (SD rats), which are commonly used in toxicity studies. We also examined age-related changes in serum ALP isoenzymes in SD rats. In order to identify the origin of each ALP isoenzyme, tissue ALP extracts from the liver, bone and small intestine (SI) and serum samples were treated with neuraminidase, antiintestinal ALP antibody, ALP inhibitor levamisole, and/or wheat germ agglutinin. It became clear that pretreatment of serum with neuraminidase is necessary for rat serum ALP isoenzyme analysis. The kit revealed that the main serum ALP isoenzymes in fasted 8-week-old intact rats were bone- and SI-derived and they tended to decrease with age. Serum liver-derived isoenzyme was slightly detected in both sexes of all ages examined, but it greatly increased in cholestasis model rats with bile-duct ligation, and rats of this model also had large molecular ALP detected in the stacking gel, suggesting hepatic damage. High-molecular intestinal ALP isoenzyme was slightly observed at the most cathodal side of the resolving gel. These results suggest that the present method is a useful tool for detecting serum ALP isoenzymes in SD rats and that concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity.

Analytical subcellular fractionation of rat pituitary homogenates with special reference to the subcellular localization and properties of alkaline phosphatases.

Alkaline phosphatase activities of the virgin rat anterior pituitary were studied with a highly sensitive fluorometric assay. Tissue whole homogenates were fractionated on sucrose density gradients in a Beaufay automatic zonal rotor and the gradient fractions assayed for alkaline phosphatase, prolactin and various organelle marker enzymes. Alkaline phosphatase was distributed between two peaks on the gradient. The low-density (1.10-1.15 g . cm-3) alkaline phosphatase component co-sedimented with the plasma membrane marker, 5'-nucleotidase, had an apparent Km for 4-methylumbelliferyl phosphate of approx. 59 microM, and was inhibited by levamisole. The high-density (1.20-1.25 g . cm-3) peak was resistant to levamisole-inhibition, had an apparent Km of approx. 30 microM and its distribution was distinct from plasma membrane, Golgi, lysosome, endoplasmic reticulum, mitochondria and prolactin granule markers on the isopycnic gradients.

Characterization and purification of alkaline phosphatase from Elephas trogontherii (Steppe elephant) bone.

Four isozymes of alkaline phosphatase (AP) were purified from Elephas trogontherii (Steppe elephant) from different locations in the bone (outer and inner peripheral, cytosolic, and integral) using Sephadex G-200 gel filtration and TEAE-cellulose anion-exchange chromatography. The specimen was obtained from Erzurum Museum and its age was approx. 0.3-0.5 million years old. No fungi or bacteria were present in the bone sample. The enzyme activity was determined by using p-nitrophenylphosphate as a substrate. SDS-PAGE of all the isozymes gave a single band at the same location. The molecular mass of the four isozymes as determined by using gel filtration was about 60 kDa. Optimum pHs for the four isozymes were between 8-8.5. The optimum temperatures of the isozymes were: outer peripheral, 37.5 degrees C, cytosolic, 37.5 degrees C, inner peripheral, 35 degrees C and integral, 40 degrees C. The values of V(max) and K(m), as well different optimum temperatures indicated that isozymes were structurally different.

Bone matrix vesicle-bound alkaline phosphatase for the assessment of peripheral blood admixture to human bone marrow aspirates.

PURPOSE: Peripheral blood (PB) admixture should be minimized during numerical and functional, as well as cytokinetic analysis of bone marrow (BM) aspirates for research purposes. Therefore, purity assessment of the BM aspirate should be performed in advance. We investigated whether bone matrix vesicle (BMV)-bound bone alkaline phosphatase (ALP) could serve as a marker for the purity of BM aspirates. RESULTS: Total ALP activity was significantly higher in BM serum (97 (176-124)U/L, median (range)) compared to PB serum (63 (52-73)U/L, p < 0.001). Agarose gel electrophoresis showed a unique bone ALP fraction in BM, which was absent in PB. Native polyacrylamide gel electrophoresis revealed the high molecular weight of this fraction, corresponding with membrane-bound ALP from bone matrix vesicles (BMV), as evidenced by electron microscopy. A serial PB admixture experiment of bone cylinder supernatant samples, rich in BMV-bound ALP, confirmed the sensitivity of this proposed quality assessment method. Furthermore, a BMV ALP fraction of ≥ 15% is suggested as cut-off value for minimal BM quality. Moreover, the BM purity declines rapidly with larger aspirated BM volumes. CONCLUSION: The exclusive presence of BMV-bound ALP in BM could serve as a novel marker to assess purity of BM aspirates.

A superfamily of metalloenzymes unifies phosphopentomutase and cofactor-independent phosphoglycerate mutase with alkaline phosphatases and sulfatases.

Sequence analysis of the probable archaeal phosphoglycerate mutase resulted in the identification of a superfamily of metalloenzymes with similar metal-binding sites and predicted conserved structural fold. This superfamily unites alkaline phosphatase, N-acetylgalactosamine-4-sulfatase, and cerebroside sulfatase, enzymes with known three-dimensional structures, with phosphopentomutase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, phosphoglycerol transferase, phosphonate monoesterase, streptomycin-6-phosphate phosphatase, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1, and several closely related sulfatases. In addition to the metal-binding motifs, all these enzymes contain a set of conserved amino acid residues that are likely to be required for the enzymatic activity. Mutational changes in the vicinity of these residues in several sulfatases cause mucopolysaccharidosis (Hunter, Maroteaux-Lamy, Morquio, and Sanfilippo syndromes) and metachromatic leucodystrophy.

A divergent archaeal member of the alkaline phosphatase binuclear metalloenzyme superfamily has phosphoglycerate mutase activity.

The hyperthermophilic archaeon Methanococcus jannaschii uses several non-canonical enzymes to catalyze conserved reactions in glycolysis and gluconeogenesis. A highly diverged gene from that organism has been proposed to function as a phosphoglycerate mutase. Like the canonical cofactor-independent phosphoglycerate mutase and other members of the binuclear metalloenzyme superfamily, this M. jannaschii protein has conserved nucleophilic serine and metal-binding residues. Yet the substrate-binding residues are not conserved. We show that the genes at M. jannaschii loci MJ0010 and MJ1612 encode thermostable enzymes with phosphoglycerate mutase activity. Phylogenetic analyses suggest that this gene family arose before the divergence of the archaeal lineage.

Evidence on the presence of two distinct alkaline phosphatases in Serratia marcescens.

Certain strains of Serratia marcescens synthesized two different types of alkaline phosphatase (APase), constitutive (CAPase) and inducible (IAPase) APases, in low phosphate medium. Synthesis of the IAPase was repressed in the presence of high phosphate. Purification and separation of these electrophoretically distinct APases was achieved by using fractional (NH(4))(2)SO(4) precipitation, adsorption on a DEAE-cellulose column and elution of enzymes by a linear sodium chloride gradient. Starch gel electrophoresis of certain fractions revealed the separation of not only IAPase from CAPase but its separation into four distinct isozymes. CAPase gave maximum enzyme activity around pH 9.5, whereas for IAPase a broad range of enzyme activity was found between pH 8.5 and 10.5. Reversible inactivation at low pH occurred for IAPase but very little with CAPase. CAPase was more thermolabile than IAPase at 95 degrees C. The two APases were found to be distinct in their kinetic as well as immunological properties, suggesting two distinct enzyme species.

The alkaline phosphatase PhoX is more widely distributed in marine bacteria than the classical PhoA.

Phosphorus (P) is a vital nutrient for all living organisms and may control the growth of bacteria in the ocean. Bacteria induce alkaline phosphatases when inorganic phosphate (P(i)) is insufficient to meet their P-requirements, and therefore bulk alkaline phosphatase activity measurements have been used to assess the P-status of microbial assemblages. In this study, the molecular basis of marine bacterial phosphatases and their potential role in the environment were investigated. We found that only a limited number of homologs to the classical Escherichia coli alkaline phosphatase (PhoA) were present in marine isolates in the Bacteroidetes and gamma-proteobacteria lineages. In contrast, PhoX, a recently described phosphatase, was widely distributed among diverse bacterial taxa, including Cyanobacteria, and frequently found in the marine metagenomic Global Ocean Survey database. These taxa included ecologically important groups such as Roseobacter and Trichodesmium. PhoX was induced solely upon P-starvation and accounted for approximately 90% of the phosphatase activity in the model marine bacterium Silicibacter pomeroyi. Analysis of the available transcriptomic datasets and their corresponding metagenomes indicated that PhoX is more abundant than PhoA in oligotrophic marine environments such as the North Pacific Subtropical Gyre. Those analyses also revealed that PhoA may be important when Bacteroidetes are abundant, such as in algal bloom episodes. However, PhoX appears to be much more widespread. Its identification as a gene that mediates organic P acquisition in ecologically important groups, and as a marker of P(i)-stress, constitutes an important step toward a better understanding of the marine P cycle.

A new domain family in the superfamily of alkaline phosphatases.

During the course of our large-scale genome analysis a conserved domain, currently detectable only in the genomes of Drosophila melanogaster, Caenorhabditis elegans and Anopheles gambiae, has been identified. The function of this domain is currently unknown and no function annotation is provided for this domain in the publicly available genomic, protein family and sequence databases. The search for the homologues of this domain in the non-redundant sequence database using PSI-BLAST, resulted in identification of distant relationship between this family and the alkaline phosphatase-like superfamily, which includes families of aryl sulfatase, N-acetylgalactosomine-4-sulfatase, alkaline phosphatase and 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGM). The fold recognition procedures showed that this new domain could adopt a similar 3-D fold as for this superfamily. Most of the phosphatases and sulfatases of this superfamily are characterized by functional residues Ser and Cys respectively in the topologically equivalent positions. This functionally important site aligns with Ser/Thr in the members of the new family. Additionally, set of residues responsible for a metal binding site in phosphatases and sulphtases are conserved in the new family. The in-depth analysis suggests that the new family could possess phosphatase activity.

Detection of minor immunological differences among human "universal-type" alkaline phosphatases.

Two clones of monoclonal antibodies against swine alkaline phosphatase (ALPase; orthophosphoric monoester phosphohydrolase, alkaline optimum, EC 3.1.3.1), which were useful in distinguishing human kidney and bone ALPases from liver ALPase, were successfully raised in mice. On the other hand, polyclonal antibody cross-reacted not only with human kidney ALPase but also with all other human universal type ALPases. The difference in cross-reactivity of monoclonal and polyclonal antibodies may be caused by the specific antigenicity of human enzymes. The monoclonal antibodies were able to recognize minor heterogeneity that could not be distinguished by their enzymatic properties. The present monoclonal antibody preparations will be utilized for clinical as well as basic investigations to detect minor heterogeneity among universal-type ALPases.

The alkaline phosphatases of human liver: an immunochemical study.

Four out of 18 human liver alkaline phosphatase (AP) preparations reacted with anti-liver and anti-intestinal AP. The liver AP that reacted with anti-intestinal AP, designated as intestine-like AP, was precipitated at 50% ammonium sulfate whereas the major liver AP precipitated at 66% saturation. Gel filtration showed that liver AP, intestine-like AP and intestinal AP contained AP with apparent molecular weights of 130,000 daltons; the intestine-like AP contained a second but smaller component of 70,000-80,000 daltons. AP extracted from intestine also contained this smaller component; its electrophoretic mobility was that of an a2-globulin, whereas that of the intestine-like AP had a mobility of beta-globulin. The similarity of the intestine-like AP to the AP variant found in hepatomas is stressed.

pH-dependent modulation of alkaline phosphatase activity in Serratia marcescens.

Serratia marcescens is an opportunistic pathogen responsible for causing nosocomial infections, corneal ulcer, necrotizing fasciitis, cellulites, and brain abscess. Alkaline phosphatase (APase) is believed to play an important role in the survival of several intracellular pathogens and their adaptation. We have studied the effect of low phosphate concentration and acid pH on the APase activities of S. marcescens. In a low phosphate medium, some strains of S. marcescens synthesize two different types of APases, a constitutive (CAPase) and an inducible (IAPase). Both the CAPase and IAPase isoenzymes completely lost their enzyme activities at pH 2.3, within 10 min of incubation at 0 degrees C. Acid-treated IAPase isoenzymes I, II, III, and IV solutions when adjusted to pH 7.8 showed recovery of 70%, 52%, 72%, and 60% of the lost activities, respectively. When the pH of the CAPase reaction mixture was raised to pH 7.8, the enzyme activity regained only 5% of its initial activity. Variations in protein concentration also affected the pH-dependent reversible changes of the IAPase activity. The higher the protein concentration, the faster the inactivation of enzyme activity observed at acidic pH at 0 degrees C. Conversely, the lower the protein concentration, the higher the rate of reactivation of enzyme activity observed for IAPase at alkaline pH. Protein interaction studies revealed a lack of similarity between CAPase and IAPase, suggesting separate genetic origin of these potentially virulent genes of S. marcescens.

Modification of alkaline phosphatases by treatment with glycosidases.

The tissue-specific variants of alkaline phosphatase that are characteristic of human liver and bone are believed to possess identical protein cores; nevertheless, they differ in certain properties such as electrophoretic mobility and stability to heat. Their electrophoretic mobilities are modified by digestion with various glycosidases. Furthermore, the difference in heat stability between them is reduced by treatment with a glycosidase preparation from Trichomonas foetalis. These results are consistent with the view that these enzyme variants differ only in their carbohydrate moieties.

Rat liver alkaline phosphatases. Evidence hepatocyte and portal triad enzymes differ.

Using biochemical and electron microscopic histochemical techniques, we studied membrane-bound alkaline phosphatase activities of rat hepatocytes and portal triads. Activity in portal triads was localized to capillaries surrounding bile ducts (peribiliary plexus) and arterioles. Despite the reputation of alkaline phosphatase as a "biliary enzyme," activity was not observed in bile ducts. Livers were separated into hepatocyte and portal triad fractions with collagenase. Enzyme from hepatocytes migrated faster during electrophoresis and eluted later during anion-exchange chromatography than that from portal triads. Thus, hepatocyte enzyme is more negatively charged (and also possibly smaller) than portal triad enzyme. Twelve hours after bile duct obstruction, new activity appeared on lateral and sinusoidal membranes of hepatocytes; appearance of portal triads did not change with obstruction. Electrophoretic mobilities of the two forms were not altered by obstruction. We conclude that two distinct liver alkaline phosphatases exist, one in hepatocytes, the other in portal triad blood vessels.