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PubMed1976

Cortisol modification of HeLa 65 alkaline phosphatase. Decreased phosphate content of the induced enzyme.

Alkaline phosphatase activity of HeLa cells is increased 5-20-fold during growth in medium with cortisol. The increase in enzyme activity is due to an enhanced catalytic efficiency rather than an increase in alkaline phosphatase protein in induced cells. In the present study the chemical composition of control and induced forms of alkaline phosphatase were investigated to determine the enzyme modification that may be responsible for the increased catalytic activity. HeLa alkaline phosphatase is a phosphoprotein and the induced form of the enzyme has approximately one-half of the phosphate residues associated with control enzyme. The decrease in phosphate residues of the enzyme apparently alters its catalytic activity. Other chemical components of purified alkaline phosphatase from control and induced cells are similar; these include sialic acid, hexosamine and sulfhydryl residues.

Alkaline Phosphatase↗

PubMed1979

Electrophoretic variant of fetal intestinal alkaline phosphatase in a patient with cirrhotic liver.

Alkaline phosphatases, which had a unique electrophoretic mobility on polyacrylamide gel electrophoresis, were found in hepatic tissue of a patient with liver cirrhosis. Enzymic and immunological properties of the enzymes examined on electropherogram were similar to those of a fetal intestinal-type alkaline phosphatase in hepatoma with respect to sensitivity to amino acids, heat stability, sensitivity to sodium dodecyl sulfate, and reactivity to anti-intestinal alkaline phosphatase antiserum. The enzymes seem to be a variant of a fetal intestinal alkaline phosphatase. The significance of occurrence of the enzymes in cirrhotic liver is discussed.

Alkaline Phosphatase↗

PubMed1977

The source and clinical significance of alkaline phosphatases in liver diseases.

The authors studied total serum alkaline phosphatase (E.C. 3.1.3.1) activity and its isoenzyme spectrum 1) in relation to the activity of liver diseases, 2) in relation to cholestatic and non-cholestatic liver diseases. The isodistribution of the alkaline phosphatase isoenzymes (the bone, liver and intestinal fraction) in the serum was studied by the heat inactivation and inhibition method and by electrophoretic separation on agar gel. The results showed that alkaline phosphatase had a close diagnostic bearing not only on cholestatic forms of hepatobiliary diseases, but also on the activity of liver disease. The main source of elevated serum alkaline phosphatase activity in the serum of patients with hepatobiliary diseases is the liver isoenzyme. In the serum alkaline phosphatase isoenzymogram, an abnormally large proportion of this isoenzyme is found more frequently than elevation of total alkaline phosphatase activity.

Alkaline Phosphatase↗

PubMed1980

Some properties of acid and alkaline phosphatase in seminal fluid and isolated sperm.

Activities of acid and alkaline phosphatases were examined in spermatozoa isolated from 177 semen samples differing in sperm counts. Alkaline phosphatase was also determined in seminal fluid. The enzymes were assayed using disodium p-nitrophenyl phosphate as substrate and were studied with respect to susceptibility to various concentrations of tartrate (acid) and to heat (alkaline). Electrophoretic separation of alkaline phosphatase from seminal fluid was performed using an Helena apparatus. The results showed that acid phosphatase activity in spermatozoa decreased with increase in sperm densities and that elevation of tartrate from 0.028 to 0.17 M usually correlated an inhibition of the enzyme from 72% to 78% (mean values). Alkaline phosphatase was very low in sperm and generally below the sensitivity of the method used. Activity of alkaline phosphatase in seminal fluid showed a tendency to increase with the increase in sperm counts, but the significance of differences between groups was not statistically valid. Exposure of seminal fluid to 55 degrees C for 16 min resulted in enzyme inactivation of about 90% and in this respect the alkaline phosphatase resembles the enzyme of bone origin. The electrophoretic pattern, however, did not confirm this view and the type of alkaline phosphatase in seminal fluid is not clear.

Acid Phosphatase↗

PubMed1982

Plasma alkaline phosphatase activity in rickets of prematurity.

Plasma alkaline phosphatase activity was measured in 349 infants aged between 5 and 10 days to establish a normal range for different gestational ages. Significant differences were observed between term and preterm infants, the highest values being associated with the shortest lengths of gestation. Plasma calcium, phosphate, and alkaline phosphatase activity were measured sequentially in 51 preterm infants less than 1500 g at birth. A significant correlation was found between raised plasma alkaline phosphatase activity and radiological changes of osteoporosis, metaphyseal change, and periosteal reaction. Plasma alkaline phosphatase appears to be of value in screening for and monitoring rickets of prematurity.

Alkaline Phosphatase↗

PubMed1977

A fetal intestinal-type alkaline phosphatase in hepatocellular carcinoma tissue.

We examined 19 hepatoma tissues for alkaline phosphatase isoenzyme and found that six have both the Kasahara isoenzyme and an alkaline phosphatase with a unique electrophoretic mobility, in addition to the liver-type enzyme. From two of six carcinoma tissues, the abnormal enzyme was partly purified and subjected to a detailed analysis, which clarified that the abnormal enzyme resembled a fetal intestinal alkaline phosphatase in most of its enzymic and immunologic properties and also in properties that reflect enzyme structure. This fetal intestinal-type alkaline phosphatase was not found in 24 specimens of normal liver from adults. The relevance of fetal intestinal-type alkaline phosphatase to Kasahara isoenzyme and adult intestinal alkaline phosphatase is discussed. The fetal and adult intestinal alkaline phosphatases differ in electrophoretic mobility, heat stability, and reactivity with concanavalin A. The adult-type enzyme has two components; only the electrophoretically slower, neuraminidase-resistant one is described here.

Adult↗

PubMed1981

Cytochemical localization of alkaline phosphatase in intestinal metaplasia of the human stomach.

Alkaline phosphatase in the brush border of areas of intestinal metaplasia of human stomach was studied cytochemically. All absorptive cells in the upper part of the villi of the duodenum had strong alkaline phosphatase activity but, in areas of intestinal metaplasia, the metaplastic glands consisted of alkaline phosphatase-positive and negative absorptive cells. Alkaline phosphatase activity was found in tall dense microvilli of absorptive cells in areas of intestinal metaplasia and in the duodenum. However, in some areas of metaplastic epithelium the activity was very weak in some tall dense microvilli of absorptive cells but strong in those of neighbouring absorptive cells. No alkaline phosphatase activity was found in short sparse microvilli of absorptive cells in areas of intestinal metaplasia. The difference in alkaline phosphatase activity in microvilli of different cells in areas of intestinal metaplasia, which is not seen in the duodenum, indicates abnormal morphological and enzymatic differentiation intestinal metaplasia.

Alkaline Phosphatase↗

PubMed1999

Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme.

The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54¿ omitted¿760 Da. On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E. coli alkaline phosphatase and 22% Bacillus subtilis (Bsu) alkaline phosphatases. High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E. coli alkaline phosphatase. Comparison of Tca alkaline phosphatase and E. coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites. Heat-stable Tca alkaline phosphatase activity was detected in E. coli YK537, harboring pJRAP.

Alkaline Phosphatase↗

PubMed2004

Site-specific localization of two distinct phosphatases along the osteoblast plasma membrane: tissue non-specific alkaline phosphatase and plasma membrane calcium ATPase.

In osteoblasts, alkaline phosphatase has been reported to be restricted to the basolateral domains. In recent studies, we have demonstrated phosphatase activities different from those of tissue non-specific alkaline phosphatase (TNSALP) along the osteoidal aspect of osteoblast membrane at alkaline and neutral pH on undecalcified freshly frozen sections of rat bones. In the present study, we sought to further characterize and define the nature of membrane-associated phosphatases along the osteoidal aspect of osteoblasts. Histochemical properties of the enzymes and their localization in vivo were examined in long bones of normal Wistar rats and TNSALP null mutant mice and their wild type littermates. Molecular profiles of the enzymes in the osteoblast extracts were also examined. The enzymatic activity of the phosphatase along the osteoidal surface of osteoblasts proved to be activated by both Mg2+ and Ca2+. Unlike TNSALP, the activity was inhibited by vanadate but resistant to levamisole, implicating a similarity between this enzyme and plasma membrane Ca2+ transport ATPase (PMCA). Immunohistochemistry showed that PMCA immunoreactions were restricted to the osteoidal domain of the plasma membrane. Native-PAGE analysis of osteoblast extracts suggested the presence of two phosphatases corresponding, respectively, to TNSALP and PMCA. Western blot analysis after SDS-PAGE of osteoblast extracts confirmed the existence of PMCA (140 kDa) and TNSALP (80 kDa). Gel-chemical analysis of the osteoblast extract from TNSALP null mutant mice depicted phosphatase activity, which was resistant to levamisole. These data suggest the presence of a phosphatase different from TNSALP, most plausibly PMCA, on the osteoidal surface of osteoblasts.

Alkaline Phosphatase↗

PubMed1994

The alkaline phosphatase in human plexus chorioideus.

The content of alkaline phosphatase isozymes in various brain regions was determined by monoclonal immunocatalytic assays. The levels of the isozymes in human brain tissues were low compared with those in other human tissues, liver, kidney, bone, intestine and placenta. Plexus chorioideus in the brain, however, was found to express significant amounts of alkaline phosphatase activity. The purified isozyme from human plexus chorioideus demonstrated a single 70 kDa protein band on SDS-polyacrylamide gel which coincides with that of tissue-unspecific alkaline phosphatase from human liver. The isozyme expressed in the plexus was confirmed to be the tissue-unspecific alkaline phosphatase isozyme with regard to its reactivity with monoclonal antibodies specific for liver alkaline phosphatase, heat stability, and the inhibition by amino acids. This finding adds new dimensions to the functional role this isozyme may play.

Alkaline Phosphatase↗

PubMed1983

Alkaline phosphatase histochemistry in human germ cell neoplasms.

Alkaline phosphatase is a useful and reliable marker of germ cell neoplasia that has been almost completely overlooked in the recent medical literature and in the practice of surgical pathology. Its presence in immature germ cells and in germ cell cancers was noted as early as 1953, but a systematic study of its use in the diagnosis and classification of germ cell tumors has not appeared in the literature. Using a recently developed plastic embedding technique combined with enzyme histochemistry, a large series of germ cell tumors and gonadal specimens were examined for the presence of alkaline phosphatase. The neoplastic germ cells in all cases of seminoma and embryonal carcinoma showed strong plasma membrane positivity for alkaline phosphatase. Choriocarcinomas (gestational and nongestational) and mature teratomas were negative. These findings suggest that the alkaline phosphatase reaction is a useful adjunct in the diagnosis of germ cell cancers.

Adult↗

PubMed1999

Heterogeneity in alkaline phosphatase isozyme expression in human testicular germ cell tumours: An enzyme-/immunohistochemical and molecular analysis.

In humans, alkaline phosphatases are encoded by one tissue-non-specific alkaline phosphatase (TNAP) gene and three tissue-specific alkaline phosphatase genes, intestinal, placental (PLAP), and germ cell-specific alkaline phosphatase (GCAP). Although the presence of alkaline phosphatases in testicular germ cell tumours (TGCTs) of adolescents and adults has been utilized for both detection and patient monitoring, it is not known in detail which isozymes are expressed. Since alkaline phosphatase is detected in carcinoma in situ (CIS), the common precursor of all TGCTs, it might provide a marker for the early diagnosis of TGCTs. Testicular cancers of germ cell and non-germ cell origin along with testicular parenchyma with and without CIS have been analysed for the expression of the different alkaline phosphatase isozymes. Antibodies to TNAP and PLAP/GCAP showed positivity in CIS, seminoma, and embryonal carcinoma. The heterogeneous staining pattern detected in frozen tissue sections was similar to the pattern found in formalin-fixed, paraffin-embedded material, indicating a biological phenomenon and not a handling artefact. Since PLAP and GCAP cannot be distinguished using immunohistochemistry, the expression of these isozymes was studied at the molecular level using a reverse transcriptase-polymerase chain reaction (RT-PCR) approach, in combination with a primer extension assay. The results show that CIS and seminoma predominantly express GCAP, while in embryonal carcinoma the expression of GCAP versus PLAP varies. Due to the presence of alkaline phosphatase transcripts in normal testicular parenchyma, an RT-PCR-based analysis of alkaline phosphatase is not informative for the early detection of TGCTs in biopsy samples.

Alkaline Phosphatase↗

PubMed1977

Inhibition of alkaline phosphatase by sialic acid.

The interaction of human organ alkaline phosphatases (orthophosphoric-monoester phosphohydrolases (alkaline optimum), EC 3.1.3.1) with sugars was studied. Hexosamines, N-acetylneuraminic acid (NANA or sialic acid), N-acetylmuramic acid and N-acetylglycolylneuraminic acid inhibited human organ alkaline phosphatase activities. Of these, sialic acid was the most effective inhibitor. The pH profiles for the enzymes in the absence and presence of sialic acid were similar. The sialic acid - enzyme complex was more heat stable than the free enzyme between 20 and 45 degrees C. Lineweaver-Burk plots of 1/v versus 1/S at various concentrations of sialic acid showed intersecting straight lines indicating that the mechanism of inhibition was a mixed type. The Ki value obtained from the plots of 1/v versus the square of sialic acid concentration was 0.07 mM for the hepatic, sialidase-treated hepatic, and intestinal alkaline phosphatases. The respective Hill coefficients varied somewhat with the alkaline phosphatase isoenzyme. Hyperbolic curves were obtained when the percentage of remaining activity was plotted against the substrate concentration at different concentrations of sialic acid. The Hill coefficient was lowered in the presence of sialic acid. The sialidase-treated hepatic enzymes used gave the most effective conversion. Partial denaturation of the enzyme with urea, or pronase digestion had a little if any effect on the sialic acid inhibition with constant time.

Alkaline Phosphatase↗

PubMed1984

Effects of hypophysectomy on tissue alkaline phosphatase in the rat.

To examine the effect of hypophysectomy on tissue alkaline phosphatase (EC 3.1.3.1) activity, studies were performed on mature rats. Hypophysectomy reduced the alkaline phosphatase activity in duodenum, kidney, and femoral bone. The decrease of alkaline phosphatase was greater in the intestine than in other organs studied. Intestinal alkaline phosphatase fell rapidly within 4 days post-hypophysectomy. In contrast, bone and kidney alkaline phosphatase activity had an initial lag period for 4 days after the hypophysectomy. Serum alkaline phosphatase also declined after hypophysectomy. The bone isoenzyme of serum alkaline phosphatase was reduced to a greater extent than the intestinal isoenzyme.

Alkaline Phosphatase↗

PubMed1991

A water-mediated salt link in the catalytic site of Escherichia coli alkaline phosphatase may influence activity.

Escherichia coli alkaline phosphatase catalyzes the hydrolysis of a wide variety of phosphomonoesters at similar rates, and the reaction proceeds through a phosphoenzyme intermediate. The active site region is highly conserved between the E. coli and mammalian alkaline phosphatases. The three-dimensional structure of the E. coli enzyme indicates that Lys-328, which is replaced by histidine in all mammalian alkaline phosphatases, is bridged to the phosphate through a water molecule. This water molecule is also hydrogen bonded to Asp-327, a bidendate ligand of the one of the two zinc atoms. Here we report the use of site-specific mutagenesis to convert Lys-328 to both histidine and alanine. Steady-state kinetic studies above pH 7.0 indicate that both mutant enzymes have altered pH versus activity profiles compared to the profile for the wild-type enzyme. At pH 10.3, in the presence of Tris, the Lys-328----Ala enzyme is approximately 14-fold more active than the wild-type enzyme. At the same pH in the absence of Tris the Lys-328----Ala enzyme is still 6-fold more active than the wild-type enzyme. Both mutant enzymes have lower phosphate affinities than the wild-type enzyme at all pH values investigated. Pre-steady-state kinetics at pH 5.5 reveal that the Lys-328----Ala enzyme behaves very similar to the phosphate-free wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1976

Alkaline phosphatase of human and calf small intestine. Purification and immunochemical characterization.

Alkaline phosphatase from human and calf small intestines has been prepared and purified until homogeneous, as judged by polyacrylamide gel electrophoresis, by means of the following techniques: n-butanol extraction, ammonium sulfate precipitation, acetone fractionation, ion exchange chromatography and isoelectric focusing in a sucrose density gradient. Three and two alkaline phosphatase froms from human and calf small intestines, respectively could be isolated by preparative isolectric focusing. The relative amounts of these components are not constant, but they have the same catalytic properties, suggesting that they may embody a common protein core with an identical active centre(s). Precipitating antisera for alkaline phosphatase from human and calf intestine have been prepard in rabbits by intramuscular, dermal, subcutaneous and intravenous administration of the pure major component of each enzyme species. Both antisera precipitate completely their homologous as well as their heterologous antigens (intestinal enzyme) and showed partial identity with placental alkaline phosphatase. There was no reaction with alkaline phosphatase from bone, liver heart, spleen, lung, stomach, pancreas, brain, bile-gall bladder and erythrocytes. Alkaline phosphatase preparation from human kidney contains a minor component of the intestinal type, beside many multiple forms which, on treatment with neuramindase, became identical in their electrophoretic and biochemical properties. At least eight multiple forms of the placental enzyme could be shown by isoelectric focusing in polyacrylamide gel. On treatment with neuraminidase these forms became less charged and only four forms remained. All forms were immunologically identical using either anti placental-enzyme or anti intestinal-AP serum. Monospecific antisera against human or calf intestinal alkaline phosphatase were obtained by absorption with purified placental enzyme. This monospecific anti intestinal-AP serum could be used for an immunological quantitative determination of the intestinal isoenzyme in sera or other liquids in the presence of other alkaline phosphatase isoenzymes.

Alkaline Phosphatase↗

PubMed1990

Purification and comparison of corticosteroid-induced and intestinal isoenzymes of alkaline phosphatase in dogs.

Corticosteroid-induced alkaline phosphatase (CALP) and intestinal alkaline phosphatase (IALP) from dogs were purified to homogeneity, as determined by polyacrylamide gel electrophoresis. Purification involved an un-interrupted system using DEAE-cellulose, concanavalin A-agarose, and monoclonal antibody affinity columns. The monoclonal antibody was prepared by use of IALP as the antigen. The 2 isoenzymes were compared, using molecular weight determinations, amino acid analyses, peptide mapping, N-terminal sequencing of the first 10 amino acids, carbohydrate analyses, and recognition by anti-IALP monoclonal antibody. The data indicated that canine IALP and CALP are identical with regard to recognition by monoclonal antibody and N-terminal amino acid sequence, nearly identical in amino acid content and peptide maps, but different in carbohydrate content. It was concluded that CALP is a product of the same gene as IALP and that differences in glycosyl transferase activities between liver and intestines or the presence of glycosidase activities in or around the intestinal mucosae result in the marked difference in carbohydrate content.

Adrenal Cortex Hormones↗

PubMed2001

Multiple unfolding intermediates of human placental alkaline phosphatase in equilibrium urea denaturation.

Alkaline phosphatase is an enzyme with a typical alpha/beta hydrolase fold. The conformational stability of the human placental alkaline phosphatase was examined with the chemical denaturant urea. The red shifts of fluorescence spectra show a complex unfolding process involving multiple equilibrium intermediates indicating differential stability of the subdomains of the enzyme. None of these unfolding intermediates were observed in the presence of 83 mM NaCl, indicating the importance of ionic interactions in the stabilization of the unfolding intermediates. Guanidinium chloride, on the other hand, could stabilize one of the unfolding intermediates, which is not a salt effect. Some of the unfolding intermediates were also observed in circular dichroism spectroscopy, which clearly indicates steady loss of helical structure during unfolding, but very little change was observed for the beta strand content until the late stage of the unfolding process. The enzyme does not lose its phosphate-binding ability after substantial tertiary structure changes, suggesting that the substrate-binding region is more resistant to chemical denaturant than the other structural domains. Global analysis of the fluorescence spectral change demonstrated the following folding-unfolding process of the enzyme: N <--> I(1) <--> I(2) <--> I(3) <--> I(4) <--> I(5) <--> D. These discrete intermediates are stable at urea concentrations of 2.6, 4.1, 4.7, 5.5, 6.6, and 7.7 M, respectively. These intermediates are further characterized by acrylamide and/or potassium iodide quenching of the intrinsic fluorescence of the enzyme and by the hydrophobic probes, 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The stepwise unfolding process was interpreted by the folding energy landscape in terms of the unique structure of the enzyme. The rigid central beta-strand domain is surrounded by the peripheral alpha-helical and coil structures, which are marginally stable toward a chemical denaturant.

Alkaline Phosphatase↗