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PubMed1989

Efficacy of wheat germ lectin-precipitated alkaline phosphatase in serum as an estimator of bone mineralization rate: comparison to serum total alkaline phosphatase and serum bone Gla-protein.

Serum levels of total alkaline phosphatase activity (S-T-AP), wheat germ lectin-precipitated alkaline phosphatase activity (S-L-AP), and bone Gla-protein immunoreactivity (S-BGP) were measured in 26 patients (23 females and 3 males) aged 35-73 years (mean 59 years) with primary hyperparathyroidism (n = 7), hyperthyroidism (n = 9), and hypothyroidism (n = 10) in whom the bone mineralization rate (m) was determined by 47Ca-kinetics (continuously expanding calcium pool model). A weak positive correlation (r = 0.42, P less than 0.05) was found between S-T-AP and m, which in the range from 0-18 mmol Ca/day could be estimated with a standard error of 4.6 mmol/day. A closer correlation (r = 0.65, P less than 0.001) was found between S-L-AP and m which was estimated with an error of 3.9 mmol Ca/day. The AP activity in the supernatant showed no significant correlation to m (r = 0.11, P greater than 0.50). The highest correlation coefficient (r = 0.81, P less than 0.001) was found between S-BGP and m which could be predicted with an error of 3.4 mmol Ca/day. S-BGP showed a closer correlation to S-L-AP (r = 0.71, P less than 0.001) than to S-T-AP (r = 0.58, P less than 0.01). We concluded that S-L-AP predicts bone mineralization at organ level better than S-T-AP in selected metabolic bone disorders and that the supernatant activity shows no relation to bone turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1975

Immunological relationship between human placental and intestinal alkaline phosphatase.

Crystalline human alkaline phosphatase from placenta and intestine was isolated by butanol extraction, acetone precipitation, a heat step (for the placental enzyme), ammonium sulfate precipitation, anion exchange chromatography, gel filtration and crystallisation with ammonium sulfate. Rabbit antibodies showed a partial cross-reaction between both enzymes in double diffusion, quantitative precipitation experiments and in serial precipitin curves. There was no reaction with human alkaline phosphatases from liver, kidney and bone. Three phenotypes of placental alkaline phosphatase with different electrophoretical mobility exhibited identical immunological reactions with their respective antisera. Monospecific antisera were obtained by absorption with crystalline intestinal or placental alkaline phosphatase. These monospecific antisera against the placental or the intestinal alkaline phosphatase can be used for an immunological determination of these two alkaline phosphatases without contamination by other alkaline phosphatases in human serum.

Alkaline Phosphatase↗
PubMedDate not supplied

[Study of the biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli. I. Introduction of directed mutations into the alkaline phosphatase gene].

Various mutations in E. coli alkaline phosphatase gene were obtained by oligonucleotide-directed mutagenesis. They result in amino acid substitutions in the signal peptide cleavage site [Val for Ala(-1)] and in the N terminus of mature polypeptide chain: Ala for Arg(+1) and Gln for Glu(+4); Gln for Glu(+4). Enzyme activity was observed in all E. coli strains transformed by plasmids with cloned mutant genes. In addition, an amber mutation was introduced into the Arg(+1) position, and the synthesis of mutant alkaline phosphatase was shown in E. coli strains containing suppressor tRNAs specific for Ser, Gln, Tyr, Leu, Ala, Glu, Phe, Gly, His, Pro, and Cys.

Alkaline Phosphatase↗
PubMed1985

Specificity of protein phosphotyrosine phosphatases. Comparison with mammalian alkaline phosphatase using polypeptide substrates.

The specificity of cytosolic protein phosphotyrosine (PPT) phosphatases was investigated using different peptides and proteins that were phosphorylated on tyrosine residues by the EGF receptor kinase. The acidic phosphoproteins, serum albumin, casein, and myosin light chains, were dephosphorylated by the PPT phosphatases with apparent Km values of 1.2 to 12.5 microM and apparent velocities of 0.2 to 18 mumol/min/mg. In contrast, [Tyr(32P)]histone and the phosphotyrosine peptides [Val5]angiotensin and RR-src, a peptide with sequence Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, were unreactive with the PPT phosphatases. However, each of these unreactive phosphopolypeptides was dephosphorylated under the same conditions by calf-intestine alkaline phosphatase. The data reveal how PPT phosphatase activity has been ascribed to different cellular enzymes. When acidic phosphotyrosine proteins were used as substrates in assays for PPT phosphatase activity the cytosolic enzymes were isolated, whereas when phosphotyrosine histones were used as substrates only the membrane-bound alkaline phosphatase was detected. Apparently the protein tyrosine kinase and the protein tyrosine phosphatases do not have the same specificity, so substrates such as histone, angiotensin, or RR-src are phosphorylated but not hydrolyzed. Therefore, these polypeptides would be ideal for the characterization of protein tyrosine kinases in cellular extracts.

Alkaline Phosphatase↗
PubMed1991

Alkaline phosphatase on activated B cells characterization of the expression of alkaline phosphatase on activated B cells. Kinetics and membrane anchor.

Recently we reported that the expression of the enzyme alkaline phosphatase (APase) is a marker for B cell activation. Enzymatic activity was found only in activated B cells and not T cells. Using flow cytometry we showed that some of the APase was found on the cell membranes (mAPase) and by functional assays, some was spontaneously released into the tissue culture medium. In the present report the expression of mAPase on activated B lymphocytes is more fully characterized. Two mAb specific for rat APase were used to measure the kinetics of the membrane expression of mAPase. Within 48 h of activation, mAPase is detected by flow cytometry and increases coordinately with both the transferrin receptor and IL-2R. Maximal membrane expression of mAPase in terms of number of positive cells and mean fluorescent intensity, is detected by day 4 to 5 of culture. Using hydroxyurea and demecolcine to block cells at G1/S and G2/M, respectively, it appeared that the initial expression of mAPase occurred as cells progressed into S phase of the cell cycle. This was confirmed using two-color flow cytometric analysis with the Hoechst DNA stain 33342 and the FITC-labeled APase-specific mAb. Finally, using phosphatidylinositol-specific phospholipase C we were able to show that 60 to 80% of the mAPase is linked to the membrane via a glycosyl-phosphatidylinositol linkage. From this we have concluded that mAPase can be added to a growing list of glycoproteins that are anchored to the membrane by the glycosyl-phosphatidylinositol linkage and are expressed on differentiating B cells. This list now includes Thy-1, BLAST-1, Jlld, and mAPase.

Alkaline Phosphatase↗
PubMedDate not supplied

[Biogenesis and secretion of alkaline phosphatase and its mutants in Escherichia coli. III. Substitution of N-terminal amino acids of alkaline phosphatase affect its biogenesis].

The effect of the N-terminal amino acid substitution on E. coli alkaline phosphatase biogenesis has been studied. The substitutions of Ser, Gln, Tyr, Leu, Gly, Ala, Glu, Phe, His, Cys, Lys and Pro for Arg(+1) were obtained by creating amber mutation at the corresponding position within phoA gene and expressing this mutated gene in E. coli strains that produce the amber-suppressor tRNAs. All mutant proteins were shown to translocate across the cytoplasmic membrane and possess enzyme activity. The introduction of Pro in +1 position disturbs the cleavage of signal peptide whereas the insertion of the other amino acids does not change the rates of processing in comparison with wild-type protein. All amino acid substitutions affect alkaline phosphatase isoenzyme composition. Some experimental evidence were also obtained on the specificity of protease, which split off N-terminal Arg during alkaline phosphatase maturation.

Alkaline Phosphatase↗
PubMed2005

Serum alkaline phosphatase activity is regulated by a chromosomal region containing the alkaline phosphatase 2 gene (Akp2) in C57BL/6J and DBA/2J mice.

Quantitative trait locus (QTL) analyses were conducted to identify chromosomal regions that contribute to variability in serum alkaline phosphatase (AP) enzyme activity in mice derived from the C57BL/6J (B6) and DBA/2J (D2) inbred strains. Serum AP was measured in 400 B6D2 F2 mice at 5 mo and 400 B6D2 F2 mice at 15 mo of age that were genotyped at 96 microsatellite markers, and in 19 BXD recombinant inbred (RI) strains at 5 mo of age. A QTL on the distal end of chromosome 4 was present in all sex- and age-specific analyses with a peak logarithm of odds (LOD) score of 20.36 at 58.51 cM. The Akp2 gene, which encodes the major serum AP isozyme, falls within this QTL region at 70.2 cM where the LOD score reached 13.2 (LOD significance level set at 4.3). Serum AP activity was directly related to the number of D2 alleles of a single nucleotide polymorphism in the 5'-flanking region of the Akp2 gene, although no strain-related differences in hepatic expression of Akp2 RNA were found. A variety of sequence polymorphisms in this chromosomal region could be responsible for the differences in serum AP activity; the Akp2 gene, however, with several known amino acid substitutions between protein sequences of the B6 and D2 strains, is a leading candidate.

Alkaline Phosphatase↗
PubMed1986

Quantitative method for determining serum alkaline phosphatase isoenzyme activity II. Development and clinical application of method for measuring four serum alkaline phosphatase isoenzymes.

A method for quantitating the liver, bone, intestinal and placental alkaline phosphatase activity of serum, using an algorithm for converting selective inactivation by guanidine hydrochloride, L-phenylalanine, and heat into equivalent isoenzyme activity is described. The method can individually quantify mixtures of isoenzymes to within a margin of 3%; it has acceptable reproducibility and has been used to develop both age and sex related reference ranges. Analysis time is about 30 minutes. The clinical reliability of this method has been shown in a study of 101 patients, in 79% of whom isoenzyme results were compatible with the final clinical diagnosis; in 10% a clinical diagnosis resulted from isoenzyme analysis, and in a further 11% the source of the increased alkaline phosphatase activity was identified and supported by electrophoresis, with a definite clinical diagnosis yet to be made.

Adult↗
PubMed1983

Physicochemical studies on leukocyte alkaline phosphatase.

Leukocyte alkaline phosphatase (ALP) was subjected to polyacrylamide gradient gel electrophoresis and its observed characteristic mobility compared with the ALP of placenta, intestine, liver, and bone. Comparison runs showed that leukocyte ALP moved more anodal to the origin than the others. Physicochemical differences between the leukocyte and other ALP were examined, based on studies of inhibition by L-phenylalanine, L-homoarginine, urea and EDTA, and of heat inactivation. Leukocyte ALP was inhibited strongly by L-homoarginine and urea but not by L-phenylalanine, which seemed similar to the effect of inhibition of liver and bone ALP. However, heat inactivation appeared to be helpful in distinguishing leukocyte from liver and bone ALP.

Alkaline Phosphatase↗
PubMed2005

Covalent protein immobilization on glass surfaces: application to alkaline phosphatase.

Lyophilized alkaline phosphatase (ALPase) was immobilized on aminated glass surfaces using the in vacuo cross-linking process [Simons, B.L., King, M.C., Cyr, T., Hefford, M.A., Kaplan, H., 2002. Zero-length cross-linking of lyophilized proteins. Protein Sci. 11, 1558-1564]. In this procedure, amide bonds were formed between carboxyl groups on the protein and amino groups on the glass surface. After the non-covalently attached enzyme was removed the immobilized ALPase not only retained its activity but could also be used, washed and reused at least six times without significant loss of activity. An average of 1.4+/-0.6 mg of reusable ALPase per gram of glass fibre was immobilized based on the activity of the soluble equivalent.

Alkaline Phosphatase↗
PubMed2002

Jenfluor ap--a novel fluorogenic substrate for in situ detection of alkaline phosphatase activity.

Alkaline phosphatase (AP) activity is often targeted in enzyme-related histochemistry as probe enzyme to detect neoplastic cells, as marker for primordial germ cells as well as in preimplantation studies, osteoblast differentiation, phosphate starvation in bacteria, yeast and phytoplankton. Moreover, AP-marker activity is a very useful tool in immunohistochemistry to detect gene sequences, antigens and antibodies. Here we describe a novel high resolution fluorescence method to localize AP-activity in cells and tissue sections based on a naphthol-AS azo coupling procedure (Jenfluor ap). This method provides amorphous photostable fluorescent final reaction products without any diffusion artifacts which are visible in conventional fluorescence microscopes as well as in confocal laser scanning and near infrared multiphoton laser scanning microscopes. The superiority of the Jenfluor ap method in comparison to the known Fast Red TR salt as well as the ELF stains is discussed.

Alkaline Phosphatase↗
PubMed1995

Bovine enamel organ cells express tissue non-specific alkaline phosphatase mRNA.

Alkaline phosphatase (AP) is expressed at high levels in all mineralizing tissues, and the isoform identified in developing enamel has biochemical properties similar to that found in bone. While the bone AP is referred to as the liver/bone/kidney or tissue non-specific (TNS) form, other APs are highly specific for tissue of expression. To determine unequivocally the AP isoform made by enamel organ cells, we constructed a fetal bovine enamel organ cDNA library, which yielded eight AP cDNA clones. In each case, the DNA sequence was homologous to the partial cDNA reported for bovine kidney AP (Garattini et al., 1987). It is concluded that enamel organ cells express the TNS-AP isoform. The extended 3' untranslated region of the cDNA has considerable homology to human TNS-AP, and the conservation of sequence suggests that the 3' end may have a role in post-transcriptional regulation of expression.

Alkaline Phosphatase↗
PubMed1994

Effective method for discriminating between oral bacterial and human alkaline phosphatase activity.

Alkaline phosphatase (ALPase) activity was quantitatively compared in various kinds of oral bacteria. High ALPase activity was detected in 3 species of periodontal bacteria, Porphyromonas gingivalis, Prevotella intermedia and Capnocytophaga sputigena. The ALPase activity detected in these bacteria was almost completely inhibited in the presence of 1% sodium dodecyl sulfate (SDS). By contrast, the activity of mammalian ALPase isoenzymes was not inhibited at all even in the presence of 1% SDS. These results indicate that the ALPase assay in combination with 1% SDS can identify the origin of ALPase detected in gingival crevicular fluid as being from bacteria or from a host response. Clinical examination with adult periodontitis revealed that ALPase activity in gingival crevicular fluid from the patients consisted of a combination of SDS-sensitive and SDS-resistant activities. These findings indicate that ALPase activity detected in gingival crevicular fluid originates not only from bacteria but also from a host response.

Adult↗
PubMed1989

Enhancement of the in vitro and in vivo antitumor activities of phosphorylated mitomycin C and etoposide derivatives by monoclonal antibody-alkaline phosphatase conjugates.

Alkaline phosphatase (AP) was covalently linked to the two antitumor monoclonal antibodies, L6 (anticarcinoma) and 1F5 (anti-B lymphoma), forming conjugates that could bind to antigen-positive tumor cells. The conjugates were able to convert the prodrugs, mitomycin phosphate (MOP) and etoposide phosphate (EP), into an active mitomycin C derivative, mitomycin alcohol, and etoposide, respectively. MOP and EP were less toxic to cultured cells from the H2981 lung adenocarcinoma than their respective hydrolysis products, mitomycin alcohol and etoposide, by a factor greater than 100, and they were also less toxic in mice. Pretreatment of H2981 cells with L6-AP greatly enhanced the cytotoxic effects of MOP and EP, while 1F5-AP caused no such enhancement. A strong antitumor response was observed in H2981-bearing mice that were treated with L6-AP followed 24 h later by either MOP or a combination of MOP and EP. This response was superior to that of MOP or combinations of MOP and EP given alone.

Adenocarcinoma↗
PubMed1989

Late events in B cell activation. Expression of membrane alkaline phosphatase activity.

Alkaline phosphatase (APase) has been previously described as a membrane marker correlating with B cell proliferation after stimulation by selected B cell mitogens. We have found, however, that the appearance of B cell membrane APase correlates more closely with differentiation than with proliferation. This conclusion has been drawn from the following observations: 1) APase activity appears well after peak B cell thymidine uptake, 2) mitogens which stimulate only B cell proliferation (Salmonella typhimurium mitogen) fail to induce expression of the enzyme, and 3) when proliferation of mitogen-activated B cells is inhibited, APase activity is not suppressed and may even be augmented. In addition to membrane expression, APase is also spontaneously shed into the surrounding milieu, perhaps as a result of endogenous phospholipase activity. By using a group of well-characterized inhibitors, the APase activity was shown to belong to class I (similar to the bone/liver/kidney class). Because APase always appears in differentiating but not proliferating cells, we would propose that the enzyme appearance is a late marker of B cell activation, associated with cell progression to differentiation and consequent IgM synthesis.

Alkaline Phosphatase↗
PubMed2006

8-quinolyl phosphate as a substrate for the fluorimetric determination of alkaline phosphatase.

BACKGROUND: Alkaline phosphatase (ALP) is an important target for clinical analysis. 8-Quinolyl phosphate (QP) was developed as a new substrate for the fluorimetric determination of ALP activity. METHODS: QP is a strong fluorescent substance and the product of the enzyme reaction is 8-hydroxyquinoline (HQ), which has no fluorescence. Under the optimal conditions for the determination of ALP, the decreased fluorescence intensity via the enzyme reaction is proportional to ALP activity. The fluorescence intensity was measured at lambdaex/lambdaem=318/495 nm before and after the enzyme reaction. RESULTS: QP reacted with ALP in the buffer solution of pH=9.5 and incubated for 20 min at 37.0 degrees C were selected as the optimal conditions for the determination of ALP. The linear range and detection limit for the determination of ALP are 1.0-16.0 and 0.229 U/l, respectively. With this method, ALP could be applied to assess ALP in human serum and the results were evaluated by comparison with a standard colorimetric assay using p-nitrophenyl phosphate as ALP substrate. CONCLUSIONS: This method is simple, practical and can be used as an alternative to assess ALP in clinical analysis.

Alkaline Phosphatase↗