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PubMed1991

Rat intestinal alkaline phosphatase secretion into lumen and serum is coordinately regulated.

We have reported the presence of intestinal alkaline phosphatase on particles with surfactant-like properties within enterocytes, on the luminal surface (light mucosal scrapings) and in the lumen of adult fat-fed rat intestines ((1989) J. Clin. Invest. 84, 1355). To test the physiological role of these particles, we compared the effect on particle secretion of a known inducer of luminal and serum alkaline phosphatase secretion (fat), with the effect of pharmacological stimulators (cholecystokinin and bethanecol). Fat induced a 2-3-fold increase in membrane-free phosphatase activity in serum, and in particle-bound alkaline phosphatase activity in proximal luminal washings and light mucosal scrapings, reaching a peak in both compartments 7 h after a corn oil feed. Bethanecol given subcutaneously induced a quantitatively similar increase in serum alkaline phosphatase activity and in particle-bound phosphatase activity in proximal light mucosal scrapings, reaching a peak 7.5 min after injection. Cholecystokinin also had a 2-3-fold stimulatory effect, 30 min after injection, on particle-bound phosphatase activity in proximal intestinal light mucosal scrapings and distal intestinal luminal washings. The increase in alkaline phosphatase activity in serum samples reached a peak 60 min after cholecystokinin injection. Thus, three independent stimuli increase both luminal and serum appearance of intestinal alkaline phosphatase. These data support the earlier findings that intestinal alkaline phosphatase secretion into the lumen is mediated by a secreted particle, further show that secretion into serum and lumen is coordinately regulated, and are consistent with the hypothesis that the rise in serum alkaline phosphatase activity could be related to extracellular release of the enzyme from the particles.

Alkaline Phosphatase↗
PubMed1994

Characterization of alkaline phosphatase-reactive neurons in the guinea-pig small intestine.

Endogenous alkaline phosphatase activity has been localized histochemically on the surface of enteric neurons of the guinea-pig small intestine by both light and electron microscopy. The enzyme activity was associated with some myenteric neurons that had Dogiel type I morphology, and the histochemical reaction products typically formed a honeycomb-like structure on labelled cell bodies. No Dogiel type II neurons in the myenteric plexus or submucous neurons showed alkaline phosphatase reactivity. Nerve fibres reactive for alkaline phosphatase were present in the myenteric plexus and ran in bundles in the circular muscle and deep muscular plexus. In addition, reactive varicose axons supplied the submucous plexus and non-ganglionated plexus of the mucosa. The results of interruption of the enteric neuronal pathways demonstrated that alkaline phosphatase-reactive myenteric neurons project anally to other myenteric ganglia, to the circular muscle and to the submucous plexus. Sequential enzyme histochemistry showed that virtually all alkaline phosphatase-reactive neurons also contained nitric oxide synthase, revealed by NADPH-diaphorase reactivity. It was estimated that 14-18% of all myenteric neurons showed alkaline phosphatase reactivity. About one-third of nitric oxide synthase-containing myenteric neurons, however, did not contain alkaline phosphatase activity. At the ultrastructural level, alkaline phosphatase activity was associated specifically with the plasma membranes of nerve cell bodies, axons and dendrites of some myenteric neurons. Reactive nerve fibres made close appositions with non-reactive submucous neurons and, within myenteric ganglia, predominantly with other alkaline phosphatase-reactive neurons. In addition to its presence in neurons, alkaline phosphatase reactivity was also present in some endothelial cells in blood vessels in the submucosa and in capillary pericytes. It is concluded, on the basis of the projections and neurochemistry, that in the guinea-pig small intestine alkaline phosphatase activity is associated with nitric oxide synthase-containing neurons which include inhibitory motor neurons to the circular muscle, and anally-directed interneurons to other myenteric and submucous neurons.

Alkaline Phosphatase↗
PubMed1987

Adverse implications of acid phosphatase levels in the upper range of normal.

A retrospective review of 102 consecutive patients with surgically staged, clinically localized prostatic carcinoma was performed to determine the relationship between pre-treatment enzymatic acid phosphatase values and histopathological extent of the tumor. Of 96 patients with normal pretreatment acid phosphatase titers (thymolphthalein monophosphate substrate) 77 (80 per cent) had values in the lower and 19 (20 per cent) had values in the upper half of the normal range. Of the latter 19 patients 16 (84 per cent) had histological evidence of extraprostatic tumor extension. Similarly, 5 of 6 patients (83 per cent) with elevated pre-treatment acid phosphatase titers had extraprostatic extension and 1 had a persistent postoperative acid phosphatase elevation that normalized with megestrol acetate therapy. Thus, 22 of 25 patients (88 per cent) with acid phosphatase values in or above the upper half of the normal range had either histological or clinical evidence of extracapsular tumor extension. By contrast, 41 of the 77 patients (53 per cent) with acid phosphatase titers in the lower half of the normal range had extracapsular extension. The predictive value for extraprostatic tumor extension of an acid phosphatase level in the upper half of the normal range was 84 per cent. Furthermore, in the 96 patients with normal acid phosphatase titers the incidence of extraprostatic tumor extension was significantly greater (p less than 0.01, chi-square) in those with values in the upper rather than the lower half of the normal range. Acid phosphatase titers in the upper half of the normal range were proportionately more common among patients with high grade and high clinical stage tumors. However, among patients with low grade and low stage tumors an acid phosphatase value in the upper half of the normal range was an independent variable that correlated with the presence of extracapsular tumor extension. These results confirm previously reported adverse prognostic implications of enzymatic acid phosphatase titers in or above the upper half of the normal range.

Acid Phosphatase↗
PubMed2004

Molecular basis for TPR domain-mediated regulation of protein phosphatase 5.

Protein phosphatase 5 (Ppp5) is a serine/threonine protein phosphatase comprising a regulatory tetratricopeptide repeat (TPR) domain N-terminal to its phosphatase domain. Ppp5 functions in signalling pathways that control cellular responses to stress, glucocorticoids and DNA damage. Its phosphatase activity is suppressed by an autoinhibited conformation maintained by the TPR domain and a C-terminal subdomain. By interacting with the TPR domain, heat shock protein 90 (Hsp90) and fatty acids including arachidonic acid stimulate phosphatase activity. Here, we describe the structure of the autoinhibited state of Ppp5, revealing mechanisms of TPR-mediated phosphatase inhibition and Hsp90- and arachidonic acid-induced stimulation of phosphatase activity. The TPR domain engages with the catalytic channel of the phosphatase domain, restricting access to the catalytic site. This autoinhibited conformation of Ppp5 is stabilised by the C-terminal alphaJ helix that contacts a region of the Hsp90-binding groove on the TPR domain. Hsp90 activates Ppp5 by disrupting TPR-phosphatase domain interactions, permitting substrate access to the constitutively active phosphatase domain, whereas arachidonic acid prompts an alternate conformation of the TPR domain, destabilising the TPR-phosphatase domain interface.

Acyl Coenzyme A↗
PubMed1998

Native rat kidney mineralocorticoid receptor is a phosphoprotein whose transformation to a DNA-binding form is induced by phosphatases.

Addition of alkaline phosphatase to rat kidney cytosol diminishes the ability of the mineralocorticoid receptor (MR) to bind aldosterone in a time-, temperature- and concentration-dependent form. A variety of phosphatase inhibitors, including levamisole, are effective in preventing this inactivation. On the other hand, when the steroid-receptor complex is incubated in the presence of alkaline phosphatase, an increment in the rate of receptor transformation is evidenced by a change in the sedimentation coefficient from 8.8 S to 5.1 S, as well as increased DNA-binding capacity. The effects of alkaline phosphatase on activation and transformation can also be observed when the MR is incubated at 20 degreesC in the cytosolic medium, indicating that the catalytic action of an endogenous phosphatase may be involved in the transformation process. The ability of phosphatase inhibitors such as levamisole for suppressing both alkaline phosphatase- and endogenous phosphatase-directed transformation does not correspond well between them. Evidence is presented to affirm that the endogenous phosphatase activity is not due to an alkaline phosphatase-type, but it may be due to a protein serine/threonine phosphatase, as evidenced by the inhibitory effects of okadaic acid. The experimental results also show direct evidence that the MR undergoes phosphorylation in a physiological milieu.

Alkaline Phosphatase↗
PubMed1995

The protein deficient in Lowe syndrome is a phosphatidylinositol-4,5-bisphosphate 5-phosphatase.

Lowe syndrome, also known as oculocerebrorenal syndrome, is caused by mutations in the X chromosome-encoded OCRL gene. The OCRL protein is 51% identical to inositol polyphosphate 5-phosphatase II (5-phosphatase II) from human platelets over a span of 744 aa, suggesting that OCRL may be a similar enzyme. We engineered a construct of the OCRL cDNA that encodes amino acids homologous to the platelet 5-phosphatase for expression in baculovirus-infected Sf9 insect cells. This cDNA encodes aa 264-968 of the OCRL protein. The recombinant protein was found to catalyze the reactions also carried out by platelet 5-phosphatase II. Thus OCRL converts inositol 1,4,5-trisphosphate to inositol 1,4-bisphosphate, and it converts inositol 1,3,4,5-tetrakisphosphate to inositol 1,3,4-trisphosphate. Most important, the enzyme converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The relative ability of OCRL to catalyze the three reactions is different from that of 5-phosphatase II and from that of another 5-phosphatase isoenzyme from platelets, 5-phosphatase I. The recombinant OCRL protein hydrolyzes the phospholipid substrate 10- to 30-fold better than 5-phosphatase II, and 5-phosphatase I does not cleave the lipid at all. We also show that OCRL functions as a phosphatidylinositol 4,5-bisphosphate 5-phosphatase in OCRL-expressing Sf9 cells. These results suggest that OCRL is mainly a lipid phosphatase that may control cellular levels of a critical metabolite, phosphatidylinositol 4,5-bisphosphate. Deficiency of this enzyme apparently causes the protean manifestations of Lowe syndrome.

Amino Acid Sequence↗
PubMed1995

The activity of COOH-terminal domain phosphatase is regulated by a docking site on RNA polymerase II and by the general transcription factors IIF and IIB.

Each cycle of transcription appears to be associated with the reversible phosphorylation of the repetitive COOH-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit. The dephosphorylation of RNAP II by CTD phosphatase, therefore, plays an important role in the transcription cycle. The following studies characterize the activity of HeLa cell CTD phosphatase with a special emphasis on the regulation of CTD phosphatase activity. Results presented here suggest that RNAP II contains a docking site for CTD phosphatase that is essential in the dephosphorylation reaction and is distinct from the CTD. This is supported by the observations that (a) phosphorylated recombinant CTD is not a substrate for CTD phosphatase, (b) RNAP IIB, which lacks the CTD, and RNAP IIA are competitive inhibitors of CTD phosphatase and (c) CTD phosphatase can form a stable complex with RNAP II. To test the possibility that the general transcription factors may be involved in the regulation of CTD phosphatase, CTD phosphatase activity was examined in the presence of recombinant or highly purified general transcription factors. TFIIF stimulates CTD phosphatase activity 5-fold. The RAP74 subunit of TFIIF alone contained the stimulatory activity and the minimal region sufficient for stimulation corresponds to COOH-terminal residues 358-517. TFIIB inhibits the stimulatory activity of TFIIF but has no effect on CTD phosphatase activity in the absence of TFIIF. The potential importance of the docking site on RNAP II and the effect of TFIIF and TFIIB in regulating the dephosphorylation of RNAP II at specific times in the transcription cycle are discussed.

Binding Sites↗
PubMed1990

Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells.

Dynamic reorganization of the actin microfilament networks is dependent on the reversible phosphorylation of myosin light chain. To assess the potential role of protein phosphatases in this process in living nonmuscle cells, we have microinjected the purified type-1 and type-2A phosphatases into the cytoplasm of mammalian fibroblasts. Our studies reveal that elevating type-1 phosphatase levels led to the rapid (within 30 min) and fully reversible disassembly of the actin microfilament network as determined by immunofluorescence analysis. In contrast, microinjection of equivalent amounts of the purified type-2A phosphatase had no effect on actin microfilament organization. Metabolic labeling of cells after injection of purified phosphatases was used to analyze changes in protein phosphorylation. Concomitant with the disassembly of the actin microfilaments induced by type-1 phosphatase, there was an extensive dephosphorylation of myosin light chain. No such change was observed when cells were injected with type-2A phosphatase. In addition, after extraction of fibroblasts with Triton X-100, the type-1 phosphatase could be specifically localized by immunofluorescence to a fibrillar network of microfilaments. Furthermore, neutralizing type-1 phosphatase activity in vivo by microinjection of an affinity-purified antibody, prevented the reorganization of actin microfilaments that we had previously described following injection of cAMP-dependent protein kinase. These data support the notion that type 1 and type-2 phosphatases have distinct substrate specificity in living cells, and that type-1 phosphatase plays a predominant role in the dephosphorylation of myosin light chain and thus in the modulation of actin microfilament organization in vivo in intact nonmuscle cells.

Actin Cytoskeleton↗
PubMed1996

Regulated binding of PTP1B-like phosphatase to N-cadherin: control of cadherin-mediated adhesion by dephosphorylation of beta-catenin.

Cadherins are a family of cell-cell adhesion molecules which play a central role in controlling morphogenetic movements during development. Cadherin function is regulated by its association with the actin containing cytoskeleton, an association mediated by a complex of cytoplasmic proteins, the catenins: alpha, beta, and gamma. Phosphorylated tyrosine residues on beta-catenin are correlated with loss of cadherin function. Consistent with this, we find that only nontyrosine phosphorylated beta-catenin is associated with N-cadherin in E10 chick retina tissue. Moreover, we demonstrate that a PTP1B-like tyrosine phosphatase associates with N-cadherin and may function as a regulatory switch controlling cadherin function by dephosphorylating beta-catenin, thereby maintaining cells in an adhesion-competent state. The PTP1B-like phosphatase is itself tyrosine phosphorylated. Moreover, both direct binding experiments performed with phosphorylated and dephosphorylated molecules, and treatment of cells with tyrosine kinase inhibitors indicate that the interaction of the PTP1B-like phosphatase with N-cadherin depends on its tyrosine phosphorylation. Concomitant with the tyrosine kinase inhibitor-induced loss of the PTP1B-like phosphatase from its association with N-cadherin, phosphorylated tyrosine residues are retained on beta-catenin, the association of N-cadherin with the actin containing cytoskeleton is lost and N-cadherin-mediated cell adhesion is prevented. Tyrosine phosphatase inhibitors also result in the accumulation of phosphorylated tyrosine residues on beta-catenin, loss of the association of N-cadherin with the actin-containing cytoskeleton, and prevent N-cadherin mediated adhesion, presumably by directly blocking the function of the PTP1B-like phosphatase. We previously showed that the binding of two ligands to the cell surface N-acetylgalactosaminylphosphotransferase (GalNAcPTase), the monoclonal antibody 1B11 and a proteoglycan with a 250-kD core protein, results in the accumulation of phosphorylated tyrosine residues on beta-catenin, uncoupling of N-cadherin from its association with the actin containing cytoskeleton, and loss of N-cadherin function. We now report that binding of these ligands to the GalNAcPTase results in the absence of the PTP1B-like phosphatase from its association with N-cadherin as well as the loss of the tyrosine kinase and tyrosine phosphatase activities that otherwise co-precipitate with N-cadherin. Control antibodies and proteoglycans have no such effect. This effect is similar to that observed with tyrosine kinase inhibitors, suggesting that the GalNAcPTase/proteoglycan interaction inhibits a tyrosine kinase, thereby preventing the phosphorylation of the PTP1B-like phosphatase, and its association with N-cadherin. Taken together these data indicate that a PTP1B-like tyrosine phosphatase can regulate N-cadherin function through its ability to dephosphorylate beta-catenin and that the association of the phosphatase with N-cadherin is regulated via the interaction of the GalNAcPTase with its proteoglycan ligand. In this manner the GalNAcPTase-proteoglycan interaction may play a major role in morphogenetic cell and tissue interactions during development.

Actins↗
PubMed1982

In vitro stimulation of alkaline phosphatase activity in immature embryonic chick pelvic cartilage by adenosine 3'5'-monophosphate.

Cyclic AMP content in embryonic chick pelvic cartilage increases significantly as the embryo ages from 8 to 10 d. This in ovo elevation in cyclic AMP content precedes maximal cartilage alkaline phosphatase activity by some 24 h. We studied whether this temporal relationship may be causally related, using an in vitro organ culture. Incubation of pelvic cartilage from 9- and 10-d embryos in medium containing monobutyryl cyclic AMP (BtcAMP) resulted in significant increases in alkaline phosphatase activity (220 and 66 percent, respectively) as compared to that of cartilages incubated in medium alone. This stimulation was both concentration- and time-dependent with maximal response at 0.5 mM BtcAMP and 4-h incubation, respectively. Similar incubations of cartilage in medium containing 1-methyl-3-isobutyl xanthine (MIX), 0.25 mM, also resulted in increased alkaline phosphatase activity (114 percent). However, pelvic cartilage from 11-d embryos incubated in medium containing BtcAMP or MIX showed no increase in alkaline phosphatase activity. We postulated that developmental age was the factor responsible for this difference in response and that immature cartilage (that with little or no alkaline phosphatase activity) would respond to BtcAMP whereas mature cartilage (that with significant alkaline phosphatase activity) would not. This was tested by incubating end sections of 11-d cartilage, which have little alkaline phosphatase activity, and center sections, which have significantly alkaline phosphatase activity, with both BtcAMP and MIX. Alkaline phosphatase activity in end sections (immature cartilage) was stimulated by BtcAMP and MIX, whereas it was not stimulated in the center sections. Actinomycin D and cycloheximide inhibited BtcAMP and MIX stimulation of alkaline phosphatase activity. Thus, the in vitro data suggest that cyclic AMP is a mediator for the stimulation of alkaline phosphatase activity in embryonic cartilage.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Characterization and sequence of PhoC, the principal phosphate-irrepressible acid phosphatase of Morganella morganii.

Phosphatase activities were investigated in Morganella morganii, which is one of the few enterobacterial species producing high-level phosphate-irrepressible acid phosphatase activity (HPAP phenotype), and the gene encoding the major phosphate-irrepressible acid phosphatase was cloned, sequenced, and its product characterized. Using p-nitrophenyl phosphate as substrate, Morganella produced a major phosphate-irrepressible acid phosphatase (named PhoC) which is associated with the HPAP phenotype, a minor phosphate-irrepressible acid phosphatase, and a phosphate-repressible alkaline phosphatase. The presence of the PhoC activity prevented induction of alkaline phosphatase when a PhoC-hydrolysable organic phosphate ester, such as glycerol 2-phosphate, was the sole phosphate source. PhoC is a secreted nonspecific acid phosphatase apparently composed of four 25 kDa polypeptide subunits. The enzyme is resistant to EDTA, P(i), fluoride and tartrate. The M. morganii PhoC showed 84.6% amino acid sequence identity to the PhoN nonspecific acid phosphatase of Providencia stuartii, 45.3% to the PhoN nonspecific acid phosphatase of Salmonella typhimurium, and 37.8% to the principal acid phosphatase (PhoC) of Zymomonas mobilis. Comparison of sequence data and of regulation of these enzymes suggested a different phylogeny of members of this gene family within the Enterobacteriaceae.

Acid Phosphatase↗
PubMed1995

Phosphatase production and activity in Citrobacter freundii and a naturally occurring, heavy-metal-accumulating Citrobacter sp.

The ability of a naturally occurring Citrobacter sp. to accumulate cadmium has been attributed to cellular precipitation of CdHPO4, utilizing HPO4(2-) liberated via the activity of an overproduced, Cd-resistant acid-type phosphatase. Phosphatase production and heavy metal accumulation by batch cultures of this strain (N14) and a phosphatase-deficient mutant were compared with two reference strains of Citrobacter freundii. Only strain N14 expressed a high level of acid phosphatase and accumulated lanthanum and uranyl ion enzymically. Acid phosphatase is regulated via carbon-starvation; although the C. freundii strains overexpressed phosphatase activity in carbon-limiting continuous culture, this was approximately 20-fold less than the activity of strain N14 grown similarly. Citrobacter strain N14 was originally isolated from a metal-contaminated soil environment; phosphatase overproduction and metal accumulation were postulated as a detoxification mechanism. However, application of Cd-stress, and enrichment for Cd-resistant C. freundii ('training'), reduced the phosphatase activity of this organism by about 50% as compared to Cd-unstressed cultures. The acid phosphatase of C. freundii and Citrobacter N14 had a similar pattern of resistance to some diagnostic reagents. The enzyme of the latter is similar to the PhoN acid phosphatase of Salmonella typhimurium described by other workers; the results are discussed with respect to the known phosphatases of the enterobacteria.

Acid Phosphatase↗
PubMed1987

Identification of two isoenzymes of protein phosphatase 2C in both rabbit skeletal muscle and liver.

Protein phosphatase 2C was isolated from rabbit skeletal muscle by a procedure that involved chromatography on DEAE-cellulose, precipitation with ammonium sulphate, gel-filtration on Sephadex G-100, affinity chromatography on thiophosphorylated myosin-P-light-chain--Sepharose and chromatography on Mono Q. The enzyme was purified about 35,000-fold and 0.3-0.4 mg was isolated from 2500 g skeletal muscle within 5 days. The final step resolved the activity into two peaks, termed protein phosphatases 2C1 and 2C2, that possessed identical substrate specificities and enzymatic properties. About 2.5-fold more protein phosphatase 2C2 was isolated than protein phosphatase 2C1. Protein phosphatases 2C1 and 2C2 migrated as single bands on SDS/polyacrylamide gels yielding apparent molecular masses of 44 kDa and 42 kDa, respectively, and the native proteins were both monomeric at pH 7.5 as judged by their elution from Sephadex G-100 and Sephacryl S200. Peptide maps of protein phosphatases 2C1 and 2C2, obtained after separate digestions with four different proteinases, were different, indicating that they are isoenzymes. Protein phosphatases 2C1 and 2C2 were purified from rabbit liver by the same procedure, and 0.2 mg (2C1 + 2C2) was isolated from 120 g hepatic tissue. Hepatic protein phosphatases 2C1 and 2C2 were also isolated in a molar ratio of about 1:2.5, and their enzymatic properties and apparent molecular masses in the presence and absence of SDS were identical to the skeletal muscle enzymes. Protein phosphatases 2C1 from muscle and liver displayed identical peptide maps, as did protein phosphatases 2C2 from these two tissues. It is concluded that the same two isoenzymes of protein phosphatase 2C are present in skeletal muscle and liver.

Animals↗
PubMed1983

Prostatic acid phosphatase in serum and bone marrow in patients with prostatic carcinoma.

Sixty-two per cent of 61 patients with prostatic carcinoma showed elevated levels of serum acid phosphatase, analysed by radioimmunoassay (RIA). Enzymatically determined serum acid phosphatase was raised in only 38% of the same patients. Bone marrow acid phosphatase determined by RIA was raised in 41%. In untreated metastatic patients with prostatic carcinoma, radioimmunologically determined serum acid phosphatase was elevated in 12 of 13 patients, whereas bone marrow acid phosphatase (RIA) and enzymatically determined serum prostatic acid phosphatase were increased only in about half of the patients. In a control group the upper reference limit of bone marrow acid phosphatase determined by RIA was significantly raised above that obtained by serum analyses. This indicates that nonprostatic acid phosphatases (possibly from bone marrow cells) cross-react with prostatic acid phosphatase in an unpredictable way, even when using a specific radioimmunoassay. In patients with metastatic carcinoma of the prostate, the results of bone marrow acid phosphatase determinations, analysed by RIA, seem to lack diagnostic and/or prognostic information additional to that obtainable by serum acid phosphatase (RIA) analysis.

Acid Phosphatase↗
PubMed1973

Alkaline phosphatase. Possible induction by cyclic AMP after cholera enterotoxin administration.

The present studies were undertaken to determine the role, if any, of cyclic 3',5'-adenosine monophosphate (cyclic AMP) as a chemical inducer of rat liver alkaline phosphatase. Cholera enterotoxin, given intravenously to rats, led to a rapid rise in the activity of hepatic adenyl cyclase that was 7(1/2) times greater than control values in 6 h. Cyclic AMP levels were also significantly increased above control values while the activity of cyclic nucleotide phosphodiesterase was unchanged. Hepatic alkaline phosphatase activity was increased 5(1/2) times above control in 12 h, but its rise followed that of adenyl cyclase and cyclic AMP by several hours. Cycloheximide inhibited the rise of hepatic alkaline phosphatase but not that of adenyl cyclase. The administration of glucagon, a known stimulator of hepatic adenyl cyclase, and of dibutyryl cyclic AMP, led to similar striking increases in hepatic alkaline phosphatase activity. This alkaline phosphatase increase was blocked by the prior administration of cycloheximide. Bile duct ligation, a known stimulator of hepatic alkaline phosphatase activity, failed to produce any significant changes in adenyl cyclase or cyclic AMP. Concomitant treatment of rats with bile duct ligation and cholera enterotoxin or bile duct ligation and glucagon, had no additive effect on the increase in hepatic alkaline phosphatase activity, although the increase occurred earlier. These results suggest that: (a) cyclic AMP may act as an inducer of hepatic alkaline phosphatase: (b) the stimulation of hepatic alkaline phosphatase by cholera enterotoxin is mediated by cyclic AMP; (c) the rise in hepatic alkaline phosphatase following bile duct ligation is not mediated by cyclic AMP; (d) the same alkaline phosphatase in rat liver may be induced by two (or more) mechanisms, only one of which requires cyclic AMP.

Adenylyl Cyclases↗
PubMed1995

Determination of alkaline phosphatase isozymes in amniotic fluid.

A simple method for the determination of the three isozymes of alkaline phosphatase (EC 3.1.3.1) contained in amniotic fluid (fetal intestinal, placental, and liver-bone-kidney) is presented. Total alkaline phosphatase activity was assayed in 10,000 g supernatants of amniotic fluid from 30 normal women between the 16th and 20th week of pregnancy. Electrophoretic patterns and inhibition by L-phenylalanine and L-homoarginine studies showed that all the fetal intestinal isozyme was precipitated in the pellet after centrifugation at 100,000 g for 90 min. Thus, the difference between total alkaline phosphatase activity and activity in the 100,000 g supernatant corresponds to fetal intestinal alkaline phosphatase. Placental isozyme can be determined by assaying alkaline phosphatase in the 100,000 g supernatant after heating at 56 degrees C for 90 min. Liver-bone-kidney isozyme activity is obtained by subtracting placental alkaline phosphatase activity from that of the 100,000 g supernatant. Mean percentages of the total alkaline phosphatase for each of the isozymes in amniotic fluid were 81% for fetal intestinal alkaline phosphatase, 7.5% for placental alkaline phosphatase and 12.0% for liver-bone-kidney alkaline phosphatase. Determination of fetal intestinal alkaline phosphatase by this method could be applied to the diagnosis of cystic fibrosis in fetuses having a 1:4 risk of being affected.

Adult↗
PubMed1998

Assessment of serum total and bone alkaline phosphatase measurement in clinical practice.

The aim of the study was to measure serum levels of the bone-specific isoenzyme of alkaline phosphatase in normal subjects and patients with metabolic bone disease by using an immunoadsorption assay. We studied 140 healthy adults, 122 patients affected by metabolic bone disease and 15 patients with cholestatic liver disease. Mean values of the bone-specific isoenzyme of alkaline phosphatase in healthy men were significantly higher than those found in premenopausal women (17.8 +/- 4.2 U/l vs 15.6 +/- 4.6 U/l, p < 0.02); postmenopausal women had significantly higher levels of bone-specific isoenzyme of alkaline phosphatase (22.6 +/- 6.4 U/l) than premenopausal women (p < 0.0001). After the menopause total alkaline phosphatase increased by 46%, while the increase in bone-specific isoenzyme of alkaline phosphatase was 39%. No significant correlations were found between bone-specific isoenzyme of alkaline phosphatase and either age or years since menopause, in postmenopausal subjects. In patients with bone-specific isoenzyme of alkaline phosphatase above the upper limit of normal, the assay had a sensitivity of 100% only in patients with Paget's disease of bone. In patients with cholestatic liver disease we found no correlation between bone-specific isoenzyme of alkaline phosphatase and either total alkaline phosphatase and gamma-glutamyl transpeptidase, while a positive correlation was found between total alkaline phosphatase and gamma-glutamyl transpeptidase. Our results confirm the role of bone-specific isoenzyme of alkaline phosphatase assay in clinical research; however, its usefulness in clinical practice is unclear once liver involvement has been excluded.

Adult↗
PubMed1980

Effect of ethanol treatment on high molecular weight phosphoprotein phosphatases of rat liver.

When the crude phosphoprotein phosphatase fraction of rat liver cytosol was treated with 80% aqueous ethanol at room temperature, the activity with phosphorylase alpha as substrate was increased by 110%, but those with glycogen synthase D and phosphohistone were decreased by 53 and 34%, respectively. Chromatography of the ethanol-treated fraction on DE-52 revealed that while phosphoprotein phosphatase IA (Mr=69,000) remained to exist even though it was reduced, phosphatases IB (Mr=-300,00) and II (Mr=160,000) were totally replaced by a new phosphatase form with an approximate molecular weight of 35,000. This low molecular weight form has been designated phosphatase III. When partially purified phosphatases IB and II were separately treated with ethanol, they were converted to phosphatase III. These results suggest that phosphoprotein phosphatases IB and II, but IA, contain phosphatase III as a subunit. Phosphatases IB and II, however, must differ in structure since "IB to III" is accompanied by an increase in phosphorylase phosphatase activity much greater than that for "II to III"

Animals↗