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PubMed1979

Complement fixation for study of placental-type alkaline phosphatase.

Preparations of human placental alkaline phosphatase differing in specific enzyme activities were compared by microcomplement fixation assays using monospecific antisera. While both specific enzyme activity and complement fixation units increased 15,000-fold upon purification, the ratio between these units remained constant. Separation of an alkaline phosphatase preparation into 'A' and 'B' forms by ampholine isoelectric focusing indicated that these forms also possessed the same ratio of immunoreactive enzyme protein to enzyme activity. The correspondence of complement fixation units with specific enzyme activity indicates that complement fixation with monospecific antisera can be used to analyze structural differences among alkaline phosphatase isoenzymes.

Alkaline Phosphatase↗

PubMed1984

[Metabolism of phosphate-limited Streptomyces cultures. I. Purification and characterization of alkaline phosphatase produced by Streptomyces hygroscopicus].

Alkaline phosphatase from Streptomyces hygroscopicus strain JA 5999-R 27-158 was purified and characterized. The enzyme was found in the culture filtrate and in the mycelium. The phosphatase was extracted from the mycelium and purified by adsorption to DEAE-cellulose. To separate impurities, the crude enzyme solution was heated and the phosphatase purified by chromatography through CM-Sepharose and Sephadex G 100. The specific activity of the resulting enzyme was 1000 microMol/min/mg at 25 degrees C. The molecular weight determined by SDS gel electrophoresis was found to be 56 000. The Michaelis-Menten constant determined with p-nitrophenylphosphate as substrate was Km = 1.25 X 10(-3) M. Phosphatase activity was dependent on the presence of Ca++ and the maximum activity of enzyme with p-nitrophenylphosphate as substrate was found at pH 9.2. The pI as detected by isoelectric focusing was at pH 5.6. Temperatures from 30 degrees to 75 degrees C did not affect the stability of the enzyme. The alkaline phosphatase exhibited high substrate specificity; of various phosphomonoesters tested, only p-nitrophenylphosphate, methylumbelliferyl-phosphate, phosphoenolpyruvate, ADP, ATP and tyrosine-O-phosphate was hydrolysed. The activity was inhibited by NAF, Na2P2O7 and EDTA. The involvement of the alkaline phosphatase in the regulation of secondary metabolism was discussed.

Alkaline Phosphatase↗

PubMed1993

Induction of germ-cell alkaline phosphatase by butyrate and cyclic AMP in BeWo choriocarcinoma cells.

BeWo choriocarcinoma cells synthesize two alkaline phosphatase isoenzymes: germ-cell alkaline phosphatase and tissue-unspecific alkaline phosphatase. We have made use of the differential heat-stabilities of these two isoenzymes to study the induction of germ-cell alkaline phosphatase by sodium butyrate and cyclic AMP (cAMP). Sodium butyrate causes a large induction of germ-cell alkaline phosphatase activity (approx. 35-fold after 96 h) after an initial lag period of 12-24 h. We showed that butyrate increases germ-cell alkaline phosphatase mRNA. Dibutyryl cAMP also induces germ cell alkaline phosphatase (approx. 2.5-fold after 96 h). When optimal concentrations of butyrate and dibutyryl cAMP were added simultaneously to cells, they caused a synergistic induction of activity. This suggested that these compounds use separate mechanisms to induce germ-cell alkaline phosphatase activity and that it is the cAMP moiety of dibutyryl cAMP that induces enzyme activity. This was confirmed by the use of two additional cAMP analogues, 8-(4-chlorophenylthio) cAMP and 8-bromo cAMP, and of two compounds, 3-methyl-1-isobutylxanthine and cholera toxin, which raise the endogenous concentration of cAMP. All four compounds caused a 2-fold increase in enzyme activity. Treatment of cells with 8-(4-chlorophenylthio) cAMP, 8-bromo cAMP and cholera toxin increased germ-cell alkaline phosphatase mRNA between 2- and 7-fold. These data suggest that this alkaline phosphatase isoenzyme is regulated at the level of its mRNA by cAMP, in a manner distinct from that of butyrate.

1-Methyl-3-isobutylxanthine↗

PubMed1980

Physiological aspects of alkaline phosphatase in selected cyanobacteria.

The alkaline phosphatase of Plectonema boryanum shows a considerable increase in activity following placement of the cells in a phosphate free medium. Five days of phosphate starvation result in a 14-fold increase of alkaline phosphatase activity. Growth in the presence of inhibitors of transcription and translation indicate that the synthesis of the enzyme is de novo. Orthophosphate causes an immediate inhibition of enzyme activity. Enzyme was extracted from P. boryanum with lysozyme or polymyxin B treatment in order to make comparative studies of cell bound and cell free enzyme. Of several enzyme specific inhibitors tested, mercuric chloride was the most effective. Temperature studies showed that the cell bound enzyme was most active at 40 degrees C while the cell free enzyme was most active at 70 degrees C. The pH optimum was 9 for the cell free enzyme, and 8.8 for the cell bound. The enzyme was tested to determine if it could hydrolyse a number of different organic compounds. It hydrolysed p-nitrophenol phosphate 100%, fructose-6-phosphate 45%, beta-glycerol phosphate 25% and other compounds to a lesser degree. Of seventeen other Cyanobacteria tested for alkaline phosphatase, all were positive, and of these eleven were inducible for the enzyme. Ten of the isolates released some of the enzyme into the culture medium. Michaelis constants for the enzyme were also determined.

Alkaline Phosphatase↗

PubMed1980

Polymyositis-dermatomyositis: diagnostic and prognostic significance of muscle alkaline phosphatase.

The distribution and intensity of alkaline phosphatase deposition in 54 patients with dermatomyositis-polymyositis (PM-DM) was analyzed by the enzyme histochemical method. Increased enzyme reactivity of endomysial capillaries was found in 28% of patients, equally distributed between adult onset PM (Group I) and PM-DM with overlap in other connective tissue diseases (Group V). Patients with high endomysial capillary reactivity (R1 larger than or equal to 60) responded poorly to steroids, had an increased incidence of rheumatoid factor, and had less fiber degeneration/necrosis in their biopsies. Twenty-two percent of patients demonstrated prominent perimysial phosphatase reactivity localized in newly formed collagen and fibroblasts. Thirty patients (55%) demonstrated significant numbers of alkaline-phosphatase-positive fibers positively correlated with increased fiber degeneration/necrosis, endomysial fibrosis, increased numbers of triglyceride-containing muscle fibers, and NADH tetrazolium reductase hyperreactivity. Minimal overlap between the three enzyme distribution patterns was found. Endomysial capillary activity probably represents endothelial alkaline phosphatase induction analogous to the pattern seen normally in lower mammals (rat, rabbit, guinea pig). Alkaline phosphatase fiber reactivity probably represents a particular phase in fiber regeneration/maturation especially after denervation and is positively correlated with an increased incidence of spontaneous fibrillation potentials in PM-DM.

Adult↗

PubMed1978

Histochemical characterization of alkaline phosphatase in developing rat teeth and bone.

Alkaline phosphatase (EC 3.1.3.1.) in developing teeth and in bone has been studied. Prior to hard tissue function a rather high enzyme activity was noted in differentiating odontoblasts, stratum intermedium, and outer enamel epithelium. A lower activity was observed in the cells of the dental papilla and stellate reticulum. After the onset of hard tissue formation the alkaline phosphatase activity was generally increased. Enzyme activity was also found in the proximal part of tall, secretory ameloblasts. In the short postsecretory ameloblasts a high enzyme activity was noted. At the onset of dentin mineralization there was an increase in enzyme activity in the cells of the subodontoblastic layer. In bone the highest alkaline phosphatase activity was found in osteoblasts. A difference was noted between the alkaline phosphatase of hard and soft tissues by means of the addition of inhibitors to the incubation media. Within the hard tissues it was possible to distinguish between two alkaline phosphatases after pretreatment with heat (56 degrees C) or the addition of specific inhibitors (sodium metavandate, ortho-and pyrophosphate). An isoenzyme which was sensitive to these procedures was demonstrated in odontoblasts and in the pulpal connective tissue. Another alkaline phosphatase isoenzyme, which was resistant to pretreatment with heat or the addition of vanadate or phosphate, was demonstrated in the subodontoblastic cell layer, stratum intermedium and the outer cells of the reduced enamel epithelium.

Alkaline Phosphatase↗

PubMedDate not supplied

Expression of alkaline phosphatase in differentiated rat labyrinthine trophoblast tissue.

In this report, we describe the generation of specific antibodies to rat alkaline phosphatase and the temporal and regional characteristics of alkaline phosphatase expression during maturation of the rat chorioallantoic placenta. An antipeptide antiserum was generated to the amino terminal 15 amino acids of rat alkaline phosphatase. The antiserum specifically recognized alkaline phosphatase. Alkaline phosphatase expression was monitored in the junctional and labyrinth zones of the chorioallantoic placenta by Western and Northern blot analyses. Alkaline phosphatase protein and mRNA were present in both the junctional and labyrinth zones on day 13 of gestation. As gestation advanced, alkaline phosphatase mRNA and protein expression decreased below the limits of detection in the junctional zone, while alkaline phosphatase expression increased in the labyrinth zone. Labyrinthine alkaline phosphatase migrated predominantly as a 95-kDa species, whereas rat kidney expressed exclusively the 75-kDa species. Enzymatic deglycosylation of the 75- and 95-kDa alkaline phosphatase species resulted in the generation of a 55-kDa species. In summary, alkaline phosphatase expression is a useful indicator of trophoblast differentiation.

Alkaline Phosphatase↗

PubMed1981

Multiple forms of bovine kidney alkaline phosphatase.

Partial purification of bovine kidney alkaline phosphatase produced two fractions (F1, F2). Double immunodiffusion experiments using antiserum against the pooled kidney fractions showed a complete fusion of the precipitin line of bovine kidney F2 with those of bovine kidney F1, liver, bone, and placental alkaline phosphatases. No reaction was observed between the antiserum and alkaline phosphatase from either bovine intestine or human kidney. Crossed immunoelectrophoresis of fraction F2 produced two peaks that crossed each other and stained for enzyme activity. This finding is discussed in light of recent reports of multiple antigenic forms of alkaline phosphatase form human and rabbit kidney.

Alkaline Phosphatase↗

PubMed1986

Substrate specificity of alkaline phosphatase from human polymorphonuclear leukocytes.

The ability of alkaline phosphatase in purified preparations from human neutrophils and liver to utilize ATP or inorganic pyrophosphate as substrate depended upon the Mg2+ concentration. With pyrophosphate present (1.0 mmol/l), activity peaked at Mg2+ concentrations of 0.25 to 0.50 mmol/l and fell sharply above this. By contrast, p-nitrophenylphosphatase activity was activated with Mg2+ concentration up to 0.75 mmol/l but above this was constant to 5.0 mmol/l. Hydrolysis was abolished by L-levamisole, a specific inhibitor of alkaline phosphatase. Testing butanol extracts of neutrophils from 50 healthy subjects showed good correlation of enzyme activity with p-nitrophenylphosphate and ADP (r = 0.90), and between p-nitrophenylphosphate and pyridoxal phosphate (r = 0.96) as substrate, consistent with hydrolysis of all three phosphoesters by one enzyme. Inhibition studies yielded no evidence of a specific pyridoxal phosphatase. Alkaline phosphatase from human neutrophils has the same broad substrate specificity as other molecular forms of the human enzyme and, like other forms, has little or no activity towards phosphoesters complexed with Mg2+.

Adenosine Diphosphate↗

PubMed1988

Purification and characterization of intestinal alkaline phosphatase from harp seal.

1. Alkaline phosphatase (EC 3.1.3.1.) from harp seal (Phagophilus groenlandicus) has been purified by concanavalin A-Sepharose chromatography to homogeneity with a specific activity of 1200-1500 units/mg of protein. 2. The mol. wt of the enzyme and its subunits were estimated as 260,000 and 70,000, respectively. By chromatofocusing the isoelectric point of this enzyme is 5.5. 3. With p-nitrophenylphosphate, pH-optimum and KM for the enzyme are 9.8 and 0.9 mM, respectively. 4. The enzyme was strongly inhibited by Sn4+, Fe3+ and Zn2+, whereas Mg2+ and Mn2+ were effective activators of the enzyme. Seal alkaline phosphatase was slightly inhibited by high concentrations of Ca2+ and Cr3+. 5. The enzyme activity reached a maximum at 55-60 degrees C. It was shown that the heat stability of seal and calf intestinal alkaline phosphatases were equal at 37 and 56 degrees C.

Alkaline Phosphatase↗

PubMed1985

Effects of orally administered cadmium on alkaline phosphatase isoenzymes in rat tissues.

Changes of alkaline phosphatase in small intestine, liver, kidney, bone and serum of rats administered 100 ppm (890 nM) of Cd2+ in the drinking water were observed during a 12 months period. After 2 weeks of cadmium administration, decreases in bone and small intestine alkaline phosphatases in serum of Cd-exposed rats were observed by a polyacrylamide gradient gel electrophoresis and the alterations continued through the 9th month of administration. At one month, the activities of the alkaline phosphatase fractions, p-1 and p-2, obtained from Sephadex G-200 column gel filtration of bone extracts from Cd-exposed rats were 32% and 43% of those in the controls, respectively, whereas Cd accumulation in the bone was very low (9 nmol/g wet weight). After 3 months, osteoporotic changes of bone and erosion of submucosa layer of the small intestine were observed by light microscopy. Alkaline phosphatase in small intestine of the Cd-exposed rats was 60% of that in the controls after 3 months. At 12 months, the decreased activity of bone alkaline phosphatase in Cd-exposed rats recovered to the same level as activity in the non-exposed rats. Moreover, the activity in kidney of the Cd-exposed rats was 80% of that in the controls. However, histological conversion from osteoporotic to osteomalacic changes in bone and kidney lesions by Cd administration were not observed by light microscopy. Liver alkaline phosphatase activity of the Cd-exposed rats did not change even at 12 months, whereas 1.2 mumol of Cd per g wet weight accumulated in this organ.

Alkaline Phosphatase↗

PubMed1995

Echinococcus granulosus, E. multilocularis and mammalian liver-type alkaline phosphatases: a comparative study.

The alkaline phosphatases (EC 3.1.3.1) from Echinococcus granulosus and E. multilocularis (Cestoda) were compared to each other and to a liver-type enzyme. The purified proteins (210 and 220 kDa, respectively) had a tetrameric structure composed of 4, 56/53 kDa subunits. Enzymatic removal of their N-linked sugar moieties abolished the differences in their apparent molecular weight under reducing conditions. After phase separation in Triton X-114, the E. multilocularis enzyme was the most amphiphilic, and treatment with PI-P1C reduced the amount of the parasite alkaline phosphatases that were in a hydrophobic form by about 50%. Both parasite enzymes were highly resistant to heat denaturation and insensitive to the inhibitors L-phenylalanine and L-leucine. In addition, L-homoarginine, levamisole and ZnCl2 can be used to differentiate the parasite and mammalian liver-type enzymes from each other. The Echinococcus alkaline phosphatases have original biochemical properties when compared to the mammalian liver-type enzyme.

Alkaline Phosphatase↗

PubMed1999

Role of metal ions on the secondary and quaternary structure of alkaline phosphatase from bovine intestinal mucosa.

Alkaline phosphatase (EC 3.1.3.1) from bovine intestinal mucosa (BIAP) is an homodimeric metalloenzyme, containing one Mg2+ and two Zn2+ ions in each active site. ApoBIAP, prepared using ion-chelating agents, exhibited a dramatic decrease of its hydrolase activity, concomittant to conformational changes in its quaternary structure. By rate-zonal centrifugation and electrophoresis, we demonstrated, for the first time, that the loss of divalent ions leads to some monomerization process for a metal-depleted alkaline phosphatase. Divalent ions are also involved in the secondary and tertiary structures. Metal-depletion induced more exposure of some Trp residues and hydrophobic regions to the solvent (as proved by intrinsic and ANS fluorescences). These changes might correspond to the disappearance of alpha-helices and/or turns with a concomittant appearance of unordered structures and beta-sheets (as probed by FTIR spectroscopy). For BIAP, three steps of temperature-induced changes were exhibited, while for apoBIAP, only one step was exhibited at 55 degrees C. Our work on BIAP showed two main differences with alkaline phosphatase from Escherichia coli. The loss of the divalent ions induces protein monomerization and the total recovery of enzyme activity by divalent ion addition to apoBIAP was not obtained.

Alkaline Phosphatase↗

PubMed1997

Purification and characterization of alkaline phosphatase from Thermotoga neapolitana.

A hyperthermophilic alkaline phosphatase was purified from Thermotoga neapolitana by heat treatment at 100 degrees C in the presence of Co2+ followed by ion-exchange and affinity chromatographies. The enzyme was purified 2,880-fold with 44% yield. The purified enzyme showed a single protein band of M(r) 45,000 on SDS-PAGE and an apparent molecular weight of 87,000 estimated by gel filtration chromatography. This suggested a homogenous dimer structure. The optimal pH and temperature for enzyme activity were 9.9 and 85 degrees C, respectively. Under optimal conditions, T. neapolitana alkaline phosphatase displayed 30% higher activity than calf intestine alkaline phosphatase did with p-nitrophenyl-phosphate as substrate. The hyperthermostable enzyme had a half-life of 238 min at 90 degrees C and K(m) and Vmax values of 183 microM and 1,352 U mg-1, respectively. Co2+ enhanced the enzyme activity, thermostability, and ligand affinity during column chromatography. The alkaline phosphatase was twice as active with Co2+ than with either Zn2+ or Mn2+ as the metal cofactor.

Alkaline Phosphatase↗

PubMed1994

Osseous plate alkaline phosphatase is anchored by GPI.

Alkaline phosphatase activity was released up to 100% from the membrane by using 0.1 U of phosphatidylinositol-specific phospholipase C from B. thuringiensis. The M(r) of solubilized enzyme was 145,000 by Sephacryl S-300 gel filtration and 66,000 by SDS-PAGE, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyze p-nitrophenyl phosphate (PNPP) (264.3 mumol min-1 mg-1),ATP (42.0 mumol min-1 mg-1) and pyrophosphate (28.4 mumol min-1 mg-1). The hydrolysis of ATP and PNPP by solubilized enzyme exhibited "Michaelian" kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (Kd = 1.5 mM) but zinc ions were powerful non-competitive inhibitors (Kd = 6.2 microM) of solubilized enzyme. Treatment of solubilized alkaline phosphatase with Chellex 100 reduced the original PNPPase activity to 5%. Cobalt (K0.5 = 10.1 microM), magnesium (K0.5 = 29.5 microM) and manganese ions (K0.5 = 5 microM) restored the activity of the apoenzyme with positive cooperativity, suggesting that phosphatidylinositol-specific phospholipase C-solubilized alkaline phosphatase is a metalloenzyme. The stimulation of the apoenzyme by calcium ions (K0.5 = 653 microM) was lower than that observed for the other ions (26%) and exhibited site-site interactions (n = 0.7). Zinc ions had no effect on the apoenzyme of the solubilized enzyme.

Alkaline Phosphatase↗

PubMed1992

Kinetics and characteristics of bovine neutrophil alkaline phosphatase during acute Escherichia coli mastitis.

Alkaline phosphatase activity of isolated bovine blood neutrophils was investigated before and during experimentally induced Escherichia coli mastitis. Activities markedly increased 1 wk after infection in neutrophils of cows suffering from moderate or from severe disease. Elevated neutrophil alkaline phosphatase activity did not correlate with a specific stage of maturating postmitotic neutrophils appearing in circulation during mastitis. Neutrophil alkaline phosphatase from healthy cows before infection and from mastitic cows was characterized by means of thermostability, specific inhibitor patterns, slab gel electrophoresis, and kinetic parameter analysis. The leukocyte enzyme from healthy and mastitic cows displayed very similar characteristics, suggesting that the increase in activity during mastitis is most probably related to the enhanced expression of the normal alkaline phosphatase enzyme.

Alkaline Phosphatase↗

PubMed1968

Human kidney and urinary alkaline phosphatases.

1. Four fractions of kidney alkaline phosphatase were prepared by chromatography on DEAE-Sephadex. An investigation of their properties suggests that the fractions represent modifications of a single kidney enzyme. 2. Urinary alkaline phosphatase resembles kidney enzyme in most of its properties, but differs in K(m) and in the degree by which it is activated by Mg(2+) ions. 3. Estimates of the molecular weights of kidney and urinary alkaline phosphatase gave values of 150000-170000 for kidney phosphatase and 75000 for the urinary enzyme. 4. It is suggested that urinary alkaline phosphatase is a sub-unit of kidney phosphatase, but it has not been possible to simulate the formation of urinary enzyme by treating kidney enzyme with urea or H(+) ions.

Alkaline Phosphatase↗

PubMed1975

Serum alkaline phosphatase isoenzymes in sickle cell anemia.

Alkaline phosphatase isoenzymes in sera from patients with sickle cell anemia were separated by electrophoresis on starch gel. Physical and biochemical criteria identified bone alkaline phosphatase as the principal, although not necessarily the sole, enzyme fraction that increases during symptomatic sickle cell crises. Moreover, there appeared to be concordance between crisis severity, serum levels of alkaline phosphatase, and isoenzyme patterns; electrophoretic and biochemical abnormalities could be detected even when the patients were asymptomatic. The present data suggest that the serum alkaline phosphatase level may be an additional indicator of the degree, frequency, and persistence of tissue injuries that occur in sickle cell anemia.

Alanine Transaminase↗