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PubMed1984

DNA and protein content as cellular biochemical parameters. A discussion with two examples: acid phosphatase and cathepsin D in rat liver and hepatoma and acid phosphatase in human breast normal tissue and adenocarcinoma.

The advantage of using DNA content as a biochemical parameter because the results it gives are directly related to cellularity is discussed. As examples, comparisons of acid phosphatase and cathepsin D activities in rat liver and hepatoma and of acid phosphatase in human normal breast tissue and adenocarcinoma are considered. Contradictory results are obtained, depending whether they are related to DNA content, fresh tissue weight, or protein content.

Acid Phosphatase↗
PubMed1979

Pulmonary phosphatidic acid phosphatase. Properties of membrane-bound phosphatidate-dependent phosphatidic acid phosphatase in rat lung.

1. The membrane-bound phosphatidate-dependent phosphatidic acid phosphatase activity of rat lung has been investigated in cytosol and microsomal fractions using as a substrate [32P]phosphatidate bound to heat inactivated rat liver microsomes. Both activities demonstrated broad pH optima with a maximum of 7.4--8 for the cytosol and a maximum of 6.5--7.5 with microsomal preparations. 2. At low concentrations (0--5 mM) Mg2+ produced a slight stimulation of the cytosol activity but at higher concentrations an inhibition was observed. Low concentrations (1.0--2.0 mM) of EDTA abolished the cytosol activity and reduced the microsomal activity to half. In both cases, the addition of Mg2+ in the presence of EDTA resulted in an activity which was more than 2-fold greater than that observed in the absence of chelator or divalent cation. 3. The cytosol activity was relatively resistant to the addition of ionic and nonionic detergents. In general, the addition of a number of phosphate esters increased rather than decreased the release of 32Pi, indicating a relative specificity for phosphate groups associated with a hydrophobic environment. The addition of aqueous dispersions of phosphatidate, lysophosphatidic acid or phosphatidylglycerophosphate markedly reduced the hydrolysis of membrane-bound [32P]phosphatidate. The cytosol activity was slightly inhibited by the addition of phosphatidylcholine. 4. In an attempt to estimate the relative contributions of the cytosol and microsomal activities in vivo, these activities were assayed using [32P]phosphatidate endogenously generated on rat lung microsomes. With the 32P-labelled microsomes, the hydrolysis remained linear over the 45 min of the experiment. Addition of high speed supernatant produced a rapid release of 32Pi during the first 10 min followed by a more gradual release similar to that oberved with the microsomes alone. The cytosol activity remained greater than the microsomal activity at all times studied. 5. When [14C]phosphatidate-labelled microsomes were incubated in the presence of nonradioactive CDPcholine, the addition of cytosol markedly stimulated the incorporation of radioactivity into phosphatidylcholine. This observation suggests that the phosphatidic acid phosphatase activity associated with the cytosol has a role in phosphatidylcholine (and presumably surfactant) biosynthesis in rat lung.

Animals↗
PubMed1977

The quality control of alkaline phosphatase determinations with placental phosphatase.

Human placenta has been recommended as the source of the alkaline phosphatase used for the preparation of control or reference materials. The effect of certain inhibitors, which may be present in the reagents, on control materials containing the placental isoenzyme, is shown to differ significantly from their effect on the isoenzymes in patient's sera. This finding indicates that if control materials are used which contain only human placental alkaline phosphatase, changes in accuracy resulting from differences in reagent quality may be missed, or alternatively a change in accuracy may be indicated by the quality control results which does not apply to the patient's sera.

Alkaline Phosphatase↗
PubMed1993

Sucrose-phosphate synthase phosphatase, a type 2A protein phosphatase, changes its sensitivity towards inhibition by inorganic phosphate in spinach leaves.

The activity of a type 2A protein phosphatase from spinach leaves was monitored using phosphorylated sucrose-phosphate synthase (SPS) as a substrate. After partial purification the overall activities of sucrose-phosphate synthase phosphatase (SPS-P) recovered from leaves harvested in the dark and in the light did not vary. However, SPS-P preparations from darkened leaves were more strongly inhibited by inorganic phosphate and certain phosphorylated compounds than preparations from illuminated or mannose fed leaves. We conclude, that activation of SPS involves an interconversion of multiple forms of SPS-P activity.

Catalysis↗
PubMed1992

Multimeric analysis of von Willebrand factor by vertical sodium dodecyl sulphate agarose gel electrophoresis, vacuum blotting technology and sensitive visualization by alkaline phosphatase anti-alkaline phosphatase complex.

To detect von Willebrand factor multimers in plasma samples and factor VIII concentrates, a vertical discontinuous SDS electrophoresis was developed. A vacuum blotting system allowed to improve the transfer to the nitrocellulose membrane. The visualization of the separated multimers was sensitized by applying an alkaline phosphatase anti-alkaline phosphatase staining technique. The reported method clearly shows structural abnormalities of von Willebrand factor and deficiency of high multimers, the vacuum transfer is efficient and the sensitivity of the staining system is very high.

Alkaline Phosphatase↗
PubMed1991

Protein phosphatase 2Bw and protein phosphatase Z are Saccharomyces cerevisiae enzymes.

cDNAs encoding three protein phosphatases, termed PP2Bw (Da Cruz e Silva, E.F. and Cohen, P.T.W. (1989) Biochim. Biophys. Acta 1009, 293-296), PPZ1 and PPZ2 that have been isolated from a Clontech 'rabbit brain' library are shown to be Saccharomyces cerevisiae clones. PPZ1 and PPZ2 are two novel yeast phosphatases showing 93% amino acid sequence identity to one another. PPZ1 shows approx. 60% sequence identity to S. cerevisiae or mammalian PP1 and approx. 40% identity to S. cerevisiae or mammalian PP2A. These and other observations suggest that the two isoforms of PPZ have functions distinct from those of PP1.

Amino Acid Sequence↗
PubMed1999

A histidine thiol 100 kDa, tetrameric acid phosphatase from lentil, Lens esculenta, seeds with the characteristics of protein tyrosine phosphatases.

A non-specific acid phosphatase (APase) hydrolysing L-tyrosine-O-phosphate and 3'-AMP was purified to electrophoretic homogeneity from mature lentil seeds with apparent native molecular mass of 100 kDa and subunit molecular mass of 24 kDa. These activities appear to reside on the same protein which shows a single band in native and SDS-PAGE. The pH optimum is 5.5, while the K(m) (mM) and V(max) (micromoles/min/mg protein) for p-nitrophenyl phosphate (pNPP) are 0.7 and 9.2 and for L-tyrosine-O-phosphate 1.4 and 10.1, respectively, at 30 degrees C and for 3'-AMP, 2 and 4.4 at 37 degrees C. The protein also hydrolyses other phosphomonoesters to a lesser extent. L-Tyrosine-O-phosphate, 3'-AMP and pNPP hydrolysis is potently inhibited by micromolar orthovanadate and also to nearly the same extent by sodium fluoride, potassium tartrate and metal ions. Histidine and cysteine are likely to be involved in the catalysis. Thermal inactivation studies indicate that the active site conformations for pNPP and 3'-AMP hydrolytic activities are different. The enzyme shows the characteristics of the animal protein tyrosine phosphatase.

Adenosine Monophosphate↗
PubMed2003

Membrane-bound acid phosphatase (MAP) from Entamoeba histolytica has phosphotyrosine phosphatase activity and disrupts the actin cytoskeleton of host cells.

Protein tyrosine phosphatases (PTPases) have been described as virulence factors in different pathogenic microorganisms. The pathogenic process by Enatamoeba histolytica is a multifactorial phenomenon that occurs in 3 steps: adhesion, cytolytic and cytotoxic effect, and phagocytosis. Lytic enzymes may participate during the second part of this process. In this work, we determined that purified membrane-bound acid phosphatase (MAP) from E. histolytica trophozoites has PTPase activity. The enzyme specifically dephosphorylated O-phospho-L-tyrosine at optimum pH of 5.0, with little activity towards O-phospho-L-serine, O-phospho-L-threonine, and ATP. It was inhibited by ammonium molybdate and sodium tungstate, and trifluoperazine did not show any effect. A monoclonal antibody against the catalytic domain of the human placental PTPase 1B, cross-reacted with a 55 kDa molecule present in the solubilized fraction. The interaction of the amoebic PTPase with HeLa cells resulted in the alteration of the cell actin cytoskeleton by disruption of the actin stress fibres.

Acid Phosphatase↗
PubMed1999

Restoration of potent protein-tyrosine phosphatase activity into the membrane-distal domain of receptor protein-tyrosine phosphatase alpha.

Most transmembrane, receptor-like protein-tyrosine phosphatases (RPTPs) contain two cytoplasmic catalytic protein-tyrosine phosphatase (PTP) domains, of which the membrane-proximal domain, D1, contains the majority of the activity, while the membrane-distal domain, D2, exhibits little or no activity. We have investigated the structural basis for reduced activity in RPTP-D2s, using RPTPalpha as a model system. Sequence alignment of PTP domains indicated that two motifs, the KNRY motif and the WpD motif, are highly conserved in all PTP domains, but not in RPTP-D2s. In RPTPalpha-D2, the Tyr in the KNRY motif is substituted by Val (position 555) and the Asp in the WpD motif by Glu (position 690). Mutation of Val555 and Glu690 had synergistic effects on RPTPalpha-D2 activity, in that the PTP activity of RPTPalpha-D2-V555Y/E690D was greatly enhanced to levels that were similar to or approaching those of RPTPalpha-D1. Therefore, Val555 and Glu690 are responsible in large part for reduced RPTPalpha-D2 activity. In addition, we established that the increased PTP activity is due to restoration of effective transition-state stabilization in RPTPalpha-D2-V555Y/E690D. Since the KNRY motif and the WpD motif are mutated in all RPTP-D2s, it is highly unlikely, due to lack of transition-state stabilization, that the residual RPTP-D2 catalytic activity plays a role in the function of RPTPs.

Amino Acid Sequence↗
PubMed1968

Studies on alkaline phosphatase. Phosphorylation of calf-intestinal alkaline phosphatase by 32P-labelled pyrophosphate.

1. A purified preparation of alkaline phosphatase from calf-intestinal mucosa was phosphorylated by (32)P-labelled PP(i) at a serine residue on the enzyme. Under the conditions employed, up to 0.15mum-labelled sites were obtained from 1mum-[(32)P]PP(i). 2. The phosphorylated enzyme was labile, the rate of dephosphorylation being similar to the overall rate of substrate hydrolysis. 3. A stopped-flow technique was used to determine the number of phosphomonoesterase active sites, which agreed with the number of (32)P-labelled sites. 4. It is concluded that calf-intestinal alkaline phosphatase is both a phosphomonoesterase and a pyrophosphatase.

Alkaline Phosphatase↗
PubMed2006

The role of the C-terminal domain of protein tyrosine phosphatase-1B in phosphatase activity and substrate binding.

Protein tyrosine phosphatase 1B (PTP-1B) has been implicated in the regulation of the insulin receptor. Dephosphorylation of the insulin receptor results in decreased insulin signaling and thus decreased glucose uptake. PTP-1B-/- mice have increased insulin sensitivity and are resistant to weight gain when fed a high fat diet, validating PTP-1B as a potential target for the treatment of type 2 diabetes. Many groups throughout the world have been searching for selective inhibitors for PTP-1B, and most of them target inhibitors to PTP-1B-(1-298), the N-terminal catalytic domain of the enzyme. However, the C-terminal domain is quite large and could influence the activity of the enzyme. Using two constructs of PTP-1B and a phosphopeptide as substrate, steady state assays showed that the presence of the C-terminal domain decreased both the Km and the k(cat) 2-fold. Pre-steady state kinetic experiments showed that the presence of the C-terminal domain improved the affinity of the enzyme for a phosphopeptide 2-fold, primarily because the off-rate was slower. This suggests that the C-terminal domain of PTP-1B may contact the phosphopeptide in some manner, allowing it to remain at the active site longer. This could be useful when screening libraries of compounds for inhibitors of PTP-1B. A compound that is able to make contacts with the C-terminal domain of PTP-1B would not only have a modest improvement in affinity but may also provide for specificity over other phosphatases.

Amino Acid Sequence↗
PubMed2002

Inositol polyphosphate 5-phosphatases: lipid phosphatases with flair.

Recent studies have identified the inositol polyphosphate 5-phosphatases as a large family of signal modifying enzymes comprising 10 mammalian and 4 yeast family members. A number of investigations including gene-targeted deletion of 5-phosphatases in mice have demonstrated that these enzymes regulate many important cellular events including hematopoietic cell proliferation and activation, insulin signaling, endocytosis, and actin polymerization.

Animals↗
PubMed1986

Monoclonal antibody study of Philadelphia chromosome-positive blastic leukemias using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technic.

The leukemic cells from 15 cases of Philadelphia chromosome-positive blastic leukemia were immunophenotyped by the alkaline phosphatase anti-alkaline phosphatase (APAAP) immunocytochemical technic using nine monoclonal antibodies (MoAb) reactive with various myeloid or lymphoid antigens. On the basis of morphology, cytochemistry, terminal deoxynucleotidyl transferase (TdT) reactivity, and electron microscopy, five of the cases had been classified as lymphoid; eight, myeloid; one, mixed myeloid-lymphoid; and one, undifferentiated. The blasts from all five lymphoid cases were reactive with lymphocyte differentiation antigen MoAb, and four of five reacted with MoAb to anti-common acute lymphoblastic leukemia-associated antigen (CALLA) (BA3). The blasts from all eight myeloid cases were reactive with MY7, a marker of myelomonocytic differentiation. Some of the blasts from three of the eight myeloid cases reacted with HP1-1D and AP3, markers of megakaryocytic differentiation; megakaryocyte differentiation was confirmed by electron microscopy. In the case classified as mixed myeloid-lymphoid, the blasts showed morphologic and immunophenotypic heterogeneity; ultrastructural studies demonstrated lymphoid, basophil, and erythroid differentiation. The blasts from the case classified as undifferentiated were immunophenotypically heterogeneous. In all cases in which the leukemic cells were also immunophenotyped by flow cytometry, the results correlated well with those obtained by the APAAP technic. The APAAP technic is a reliable method for immunophenotyping leukemias. Advantages of this method include its applicability to routinely prepared blood and bone marrow smears and cytocentrifuge preparations, lack of endogenous peroxidase background staining, and a permanent record.

Adult↗
PubMed1994

Why are mammalian alkaline phosphatases much more active than bacterial alkaline phosphatases?

Mammalian alkaline phosphatases are 20-30-fold more active than the corresponding bacterial enzymes even though their amino acid sequences are 25-30% absolutely conserved. In the active-site region there are two noticeable differences between the sequences of the bacterial and mammalian enzymes. In the Escherichia coli enzyme positions 153 and 328 are Asp and Lys, respectively, but in the mammalian enzymes His is observed at both of these positions. Site-specific mutagenesis, genetic and X-ray crystallographic data, which will be summarized here, suggest that the His substitutions at positions 153 and 328 are primarily responsible for the differences in properties between the bacterial and mammalian alkaline phosphatases.

Alkaline Phosphatase↗
PubMed1987

The alkaline phosphatase anti-alkaline phosphatase technique in dermatopathology.

The APAAP technique is an unlabelled antibody bridge technique which can be used on either frozen or paraffinembedded sections. One applies first a monoclonal antibody, then a polyclonal bridge antibody, and finally a soluble complex of alkaline phosphatase and monoclonal mouse anti-alkaline phosphatase. Subsequently, the enzyme label is developed with a naphthol salt and new fuchsin as a dye. This technique was used in our laboratory on frozen and/or paraffin embedded sections by using 15 different monoclonal antibodies, which are commercially available. The reaction product was bright red and could easily be distinguished from the brown color of melanin, which makes the APAAP technique particularly suitable for dermatopathology.

Alkaline Phosphatase↗
PubMed1998

The effects of retinoic acid on alkaline phosphatase activity and tissue-non-specific alkaline phosphatase gene expression in human periodontal ligament cells and gingival fibroblasts.

Alkaline phosphatase (ALP) in human periodontal ligament (HPDL) cells is classified as a tissue-non-specific alkaline phosphatase (TNSALP) by its enzymatic and immunological properties. Since retinoic acid (RA) has been shown as a potent inducer of TNSALP expression in various osteoblastic and fibroblastic cells, we investigated the effects of RA on the level of ALP activity and expression of TNSALP mRNAs in HPDL cells. Cultured cells were treated with desired RA concentrations (0, 10(-7), 10(-6), 10(-5) M) in medium containing 1% bovine serum albumin without serum. ALP activity was determined by the rate of hydrolysis of p-nitrophenyl phosphate and was also assayed in the presence of specific inhibitors. In order to identify the TNSALP mRNA type expressed by HPDL, a set of oligonucleotide primers corresponding to 2 types of human TNSALP mRNA (i.e. bone-type and liver-type) were designed, and mRNA isolated from HPDL was amplified by means of reverse transcription-polymerase chain reaction (RT-PCR). After treatment with RA (10(-6) M) for 4 d, there was a significant increase in the ALP activity of HPDL cells. The use of inhibitors and thermal inactivation experiments showed that the increased ALP activity had properties of the TNSALP type. RT-PCR analysis revealed that bone-type mRNA was highly stimulated in HPDL cells by RA treatment, but the expression of liver-type mRNA was not detected. These results indicated that the upregulation of ALP activity in HPDL cells by RA was due to the increased transcription of bone-type mRNA of the TNSALP gene.

Alkaline Phosphatase↗
PubMed1997

Comparison of total alkaline phosphatase and three assays for bone-specific alkaline phosphatase in childhood and adolescence.

We compared serum levels of total alkaline phosphatase (TAP) and bone-specific alkaline phosphatase (BAP) as determined by three different assays (lectin affinity electrophoresis, immunoradiometric assay, enzyme-linked immunosorbent assay) in subjects aged 5-20 years suffering from X-linked hypophosphatemic rickets (n = 14), chronic renal failure (n = 10) and chronic cholestatic liver disease (n = 16). Results were compared to controls of the same age and were expressed as standard deviation scores (SDS). TAP correlated significantly with BAP (r > 0.9 for each assay; p < 0.001) in controls. In children with cholestatic diseases, TAP (median SDS + 2.0) was elevated, but BAP, as measured by the electrophoretic assay, was within the reference range for most patients (median SDS: -0.4; p = 0.003 for the difference between the median SDS of TAP and BAP). In contrast, results for BAP as determined by the two immunoassays were not significantly different from TAP in any of the three patient groups (p > 0.05 in each group for both assays). In this study, the two immunoassays did not have a detectable advantage over lectin affinity electrophoresis in the determination of BAP.

Adolescent↗
PubMed1987

Detection of chlamydial inclusions in cell culture or biopsy tissue by alkaline phosphatase-anti-alkaline phosphatase staining.

An immunological technique for detecting Chlamydia trachomatis and Chlamydia psittaci inclusions in infected McCoy cell cultures was developed by using a genus-specific monoclonal antibody to Chlamydia spp., rabbit anti-mouse immunoglobulin G bridging antibody, alkaline phosphatase-anti-alkaline phosphatase (APAAP) monoclonal antibody conjugate, and naphthol AS-phosphate/fast red substrate. Chlamydial inclusions stained red and were easily detected against a background of blue hematoxylin-stained nuclei. After 18 h, inclusions of C. trachomatis serovar L2 LGV434/Bu and C. psittaci strain 6BC were stained by APAAP but not by iodine or Giemsa. At 48 h inclusion counts were significantly higher in the APAAP cultures. Both the APAAP procedure and conventional staining detected 35 of 239 (15%) cultures 48 h after inoculation with urethral or endocervical specimens. However, at 24 h after inoculation 22 of 35 (63%) were positive by APAAP staining while negative by iodine. This immunostain also allowed identification of chlamydial inclusions in endometrial biopsies from patients with tubal factor infertility or pelvic inflammatory disease.

Alkaline Phosphatase↗