Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase

Alkaline phosphatase prepared from mammalian cell cultures was found to have alkaline inorganic pyrophosphatase activity. Both of these activities appear to be associated with a single protein, as demonstrated by: (1) concomitant purification of alkaline phosphatase and alkaline inorganic pyrophosphatase; (2) proportional precipitation of alkaline phosphatase and inorganic pyrophosphatase activities by titrating constant amounts of an enzyme preparation with increasing concentration of antibody; (3) immune electrophoresis, which showed that precipitin bands that have alkaline phosphatase activity also have pyrophosphatase activity; (4) inhibition of pyrophosphatase activity by cysteine, an inhibitor of alkaline phosphatase activity; (5) similar subcellular localization of the two enzyme activities as demonstrated by histochemical methods; (6) hormonal and substrate induction of alkaline phosphatase activity in mammalian cell cultures, which produced a nearly parallel rise in inorganic pyrophosphatase activity.     

Regulation of fructose-1,6-bisphosphatase in yeast by phosphorylation/dephosphorylation

Fructose-1,6-bisphosphatase was precipitated with purified rabbit antiserum from extracts of ³²P-orthophosphate labelled yeast cells, submitted to SDS polyacrylamide gel electrophoresis, extracted from the gels and counted for radioactivity due to ³²P incorporation. Fructose-1,6-bisphosphatase from glucose starved yeast cells contained a very low ³²P label. During 3 min treatment of the glucose starved cells with glucose the ³²P-label increased drastically. Subsequent incubation of the cells in an acetate containing, glucose-free medium led to a label which was again low. Analysis for phosphorylated amino acids in the immunpprecipitated fructose-1,6-bisphosphatase protein from the 3 min glucose-inactivated cells exhibited phospho-serine as the only labelled phosphoamino acid. These data demonstrate a phosphorylation of a serine residue of fructose-1,6-bisphosphatase during this 3 min glucose treatment of glucose starved cells. A concomitant about 60 % inactivation of the enzyme had been shown to occur. The data in addition show a release of the esterified phosphate from the enzyme upon incubation of cells in a glucose-free medium, a treatment which leads to peactivation of enzyme activity. A protein kinase and a protein phosphatase catalysing this metabolic interconversion of fructose-1,6-bisphosphatase are postulated. It is assumed that metabolites accumulating after the addition of glucose exert a positive effect on the kinase activity and/or have a negative effect on the phosphatase activity. A role of the enzymic phosphorylation of fructose-1,6-bisphosphatase in the initiation of complete proteolysis of the enzyme during “catabolite inactivation” is discussed.     

Dephosphorylation of glycogen synthase in rat heart extracts by E. coli alkaline phosphatase

Regulation of the dephosphorylation of glycogen synthase in extracts from rat heart has been studied by adding exogenous phosphatase to the extract. These experiments were possible only because the endogenous protein phosphatase activity of the extract could be inhibited by KF under conditions where alkaline phosphatase activity was not. The concentration of substrate (glycogen synthase from the heart extract) and catalyst (purified E. coli alkaline phosphatase) could be varied independently, by adding known amounts of alkaline phosphatase to the KF-containing heart extracts. Alkaline phosphatase could completely dephosphorylate glycogen synthase while phosphorylase was unchanged. The rate of dephosphorylation was proportional to both the concentration of alkaline phosphatase added to the tissue extract and the amount of glycogen synthase in the extract. The Km for glycogen synthase was close to the concentration found in heart tissue. The Km and the maximum rate of dephosphorylation were both dependent on the phosphorylation state of the glycogen synthase. Less phosphorylated enzyme forms were dephosphorylated faster. These results indicate the necessity for precise control of many variables in studying the rate of glycogen synthase dephosphorylation. Alkaline phosphatase-catalyzed dephosphorylation could be inhibited by physiological concentrations of glycogen. Glycogen synthase dephosphorylation in extracts from fasted-refed rats was less sensitive to glycogen inhibition than in extracts from normal animals. The phosphorylation state of the glycogen synthase in these animals was assessed by kinetic studies to show that differences in phosphorylation state probably could not account for the observations. Fasting led to a decreased rate of dephosphorylation of glycogen synthase due to both an apparent change in kinetic properties of glycogen synthase as a substrate for alkaline phosphatase, and an increased inhibitory effect of glycogen. Stable modifications of glycogen synthase caused by altered nutritional states in the animals are thought to produce these effects.     

Phosphorylable proteins and alkaline phosphatase of brush border membranes from different parts of the rat small intestine

1. The distribution along the small intestine of phosphorylable proteins from the brush border has been studied by gel electrophoresis. 2. Four proteins, with apparent Mr of 190, 160, 140 and 120 kDa were distributed unequally along the gut, which incorporated 32P from gamma 32P (ATP) to different degrees. 3. Alkaline phosphatase activity has been shown to follow the same distribution. 4. Under denaturing conditions 90, 85 and 65 kDa proteins were observed, whilst the proteins of 190, 160, 140 and 120 kDa had disappeared. 5. All these proteins, with the exception of the 190 kDa protein, had also been labelled with 32Pi. Furthermore, a difference in the phosphorylation of the 65 kDa and the 90-85 kDa proteins was observed. 6. The 65 kDa protein like commercial calf alkaline phosphatase had a ratio of phosphorylation from ATP to phosphorylation from Pi less than the 90 and 85 kDa proteins. 7. Mg2+ (2.5-10 mM) decreased phosphorylation of only the 65 kDa protein whilst beta-glycerophosphate inhibited phosphorylation of all forms of alkaline phosphatase. 8. Incorporation of gamma 32P (ATP) into the proteins was enhanced in the presence of 5 mM theophylline or EDTA. 9. The nature of the phosphorylation of these different proteins is discussed.     

An infrared study of the thermal and pH stabilities of the GPI-alkaline phosphatase from bovine intestine

Alkaline phosphatase (EC 3.1.3.1) from bovine intestine mucosa (BIAP) is a homodimeric metalloenzyme, which hydrolyses nonspecifically phosphate monoesters at alkaline pH with release of inorganic phosphate and alcohol. BIAP is either soluble (sBIAP) or membrane-anchored by a glycosylphosphatidylinositol moiety (GPI-BIAP). This anchor might have some contribution in the stabilization of the GPI-linked protein structure. Our purpose was to study the role of the anchor by using two parameters, the enzymatic activity and the protein conformation, which was analyzed by using FTIR spectroscopy. We determined that the two forms of BIAP show some similarities with the previously described structure of alkaline phosphatase isolated from Escherichia coli and human placenta. Meanwhile GPI-BIAP and sBIAP exhibit similar specific activities, the presence of the anchor increases the thermal and pH stabilities of the enzyme activity and conformation.     

Influence of phosphate ions and alkaline phosphatase activity of cells on survival ofEscherichia coli in seawater

When grown in a minimal medium and suspended for 2 hours in distilled water, seawater, phosphate buffer or a polyphosphate solution,E. coli MC4100 cells with high alkaline phosphatase activity survived in seawater for longer periods than cells with low or no activity. However, mutant cells totally deprived of alkaline phosphatase activity held in phosphate-containing media before transfer to seawater showed survival almost as high as the wild type strain, indicating that alkaline phosphatase activity is not the only factor influencing survival. Alkaline phosphatase activity also increased the protection of cells provided by glycine betaine. Survival was enhanced when cells were preincubated in the presence of phosphate or polyphosphate. Thus, the transfer of cells in wastewater could influence their subsequent survival in seawater.     

The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis

When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases ficin and bromelain, the enzyme released was dimeric. Butanol extraction of the plasma membranes at pH 7.6 yielded a water-soluble, aggregated form that PI phospholipase C could also convert to dimers. When the membrane-bound enzyme was solubilized with a non-ionic detergent (Nonidet P-40), it had the Mr of a tetramer; this, too, was convertible to dimers with PI phospholipase C or a protease. Butanol extraction of whole liver tissue at pH 6.6 and subsequent purification yielded a dimeric enzyme on electrophoresis under nondenaturing conditions, whereas butanol extraction at pH values of 7.6 or above and subsequent purification by immunoaffinity chromatography yielded an enzyme with a native Mr twice that of the dimeric form. This high molecular weight form showed a single Coomassie-stained band (Mr = 83,000) on electrophoresis under denaturing conditions in sodium dodecyl sulfate, as did its PI phospholipase C cleaved product; this Mr was the same as that obtained with the enzyme purified from whole liver using butanol extraction at pH 6.6. These results are highly suggestive of the presence of a butanol-activated endogenous enzyme activity (possibly a phospholipase) that is optimally active at an acidic pH. Inhibition of this activity by maintaining an alkaline pH during extraction and purification results in a tetrameric enzyme. Alkaline phosphatase, whether released by phosphatidylinositol (PI) phospholipase C or protease treatment of intact plasma membranes, or purified in a dimeric form, would not adsorb to a hydrophobic medium. PI phospholipase C treatment of alkaline phosphatase solubilized from plasma membranes by either detergent or butanol at pH 7.6 yielded a dimeric enzyme that did not absorb to the hydrophobic medium, whereas the untreated preparations did. This adsorbed activity was readily released by detergent. Likewise, alkaline phosphatase solubilized from plasma membranes by butanol extraction at pH 7.6 would incorporate into phosphatidylcholine liposomes, whereas the enzyme released from the membranes by PI phospholipase C would not incorporate. The dimeric enzyme purified from a butanol extract of whole liver tissue carried out at pH 6.6 did not incorporate. We conclude that PI phospholipase C converts a hydrophobic tetramer of alkaline phosphatase into hydrophilic dimers through removal of the 1,2-diacylglycerol moiety of phosphatidylinositol. Based on these and others' findings, we devised a model of alkaline phosphatase's conversion into its various forms.     

Dephosphorylation of glycogen synthase in rat heart extracts by E. coli alkaline phosphatase

Summary Regulation of the dephosphorylation of glycogen synthase in extracts from rat heart has been studied by adding exogenous phosphatase to the extract. These experiments were possible only because the endogenous protein phosphatase activity of the extract could be inhibited by KF under conditions where alkaline phosphatase activity was not. The concentration of substrate (glycogen synthase from the heart extract) and catalyst (purified E. coli alkaline phosphatase) could be varied independently, by adding known amounts of alkaline phosphatase to the KF-containing heart extracts. Alkaline phosphatase could completely dephosphorylate glycogen synthase while phosphorylase was unchanged. The rate of dephosphorylation was proportional to both the concentration of alkaline phosphatase added to the tissue extract and the amount of glycogen synthase in the extract. The K_m for glycogen synthase was close to the concentration found in heart tissue. The K_m and the maximum rate of dephosphorylation were both dependent on the phosphorylation state of the glycogen synthase. Less phosphorylated enzyme forms were dephosphorylated faster. These results indicate the necessity for precise control of many variables in studying the rate of glycogen synthase dephosphorylation.Alkaline phosphatase-catalyzed dephosphorylation could be inhibited by physiological concentrations of glycogen. Glycogen synthase dephosphorylation in extracts from fasted-refed rats was less sensitive to glycogen inhibition than in extracts from normal animals. The phosphorylation state of the glycogen synthase in these animals was assessed by kinetic studies to show that differences in phosphorylation state probably could not account for the observations. Fasting led to a decreased rate of dephosphorylation of glycogen synthase due to both an apparent change in kinetic properties of glycogen synthase as a substrate for alkaline phosphatase, and an increased inhibitory effect of glycogen. Stable modifications of glycogen synthase caused by altered nutritional states in the animals are thought to produce these effects.%GSI represents the percentage of glycogen synthase activity that is active without glucose 6-P.     

Evidence of two forms of poly(A) polymerase in germinated wheat embryos and their regulation by a novel protein kinase

Two forms of poly(A) polymerase (PAPI and PAPII) from germinated wheat embryos have been resolved on DEAE-cellulose ion-exchange chromatography by a linear gradient of 0-500 mM (NH(4))(2)SO(4). Further purification shows that both forms are monomeric in nature with an identical molecular weight, approximately 65 kDa. The phosphoprotein nature of PAPI and PAPII has been established by in vivo labelling with (32)P-orthophosphate. Acid hydrolysis of both (32)P-labelled purified PAPI and PAPII has revealed that phosphorylations generally take place in serine and threonine residues. PAPI and PAPII have also been characterised with respect to V(max) and K(m) for poly(A). The V(max) and K(m) values of PAPI are 28.57 and 11.37 microg, respectively, whereas 34.48 and 7.04 microg of PAPII. In vitro dephosphorylation of the purified enzyme by alkaline phosphatase leads to a significant loss of the enzyme activity, which is regained upon phosphorylation by a 65 kDa protein kinase (PK) purified from wheat embryos. The extent of phosphorylation by protein kinase shows that PK has similar affinity towards both PAPI and PAPII, whereas the phosphate incorporation in PAPII is twofold higher than PAPI suggesting their distinct chemical nature.     

Phosphorylation of glucokinase from rat liver in vitro by protein kinase A with a concomitant decrease of its activity

Glucokinase, purified from rat liver, was phosphorylated to an extent of 1 mol [32P]-phosphate/mol of enzyme when incubated with [32P]ATP and protein kinase A from pig or rabbit muscle. The phosphate was bound to serine residues. K0.5 increased and Vmax decreased upon phosphorylation. The phosphate group was removed during incubation of the phosphorylated glucokinase with alkaline phosphatase. Enzymatically inactive glucokinase was not phosphorylated by the protein kinase.     

Cloning and expression of a highly active recombinant alkaline phosphatase from psychrotrophic Cobetia marina

Alkaline phosphatase catalyzes the hydrolysis of phosphomonoesters and is widely used in molecular biology techniques and clinical diagnostics. We expressed a recombinant alkaline phosphatase of the marine bacterium, Cobetia marina, in Escherichia coli BL21 (DE3). The recombinant protein was purified with a specific activity of 12,700 U/mg protein, which is the highest activity reported of any bacterial alkaline phosphatase studied to date. The molecular mass of the recombinant protein was 55–60 kDa, as determined by SDS–PAGE, and was observed to be a dimer by gel filtration analysis. The enzyme was optimally active at 45°C and the recombinant alkaline phosphatase efficiently hydrolyzed a phosphoric acid ester in luminescent and fluorescent substrates. Therefore, this enzyme can be considered to be extremely useful as a label conjugated to an antibody.     

Identification of hematoxylin-stainable protein in epidermal keratohyalin granules as phosphorylated cystatin alpha by protein kinase C.

The hematoxylin-stainable protein (HSP) in keratohyalin granules of the newborn rat epidermis was found to have the same amino acid composition and the same inhibitory and immunological properties as cystatin alpha. However, only its pI value (4.7) differed from that of cystatin alpha (5.3). Alkaline phosphatase treatment of HSP changed its pI value from 4.7 to 5.3. This pI change was inhibited by EDTA, an inhibitor of alkaline phosphatase. Furthermore, 32P from [gamma-32P]ATP was incorporated into recombinant cystatin alpha by a protein kinase C (PKC) preparation in the presence of phosphatidyl serine and Ca2+ ions as co-factors. The incorporation increased dose-dependently with the added cystatin alpha and was inhibited significantly by H-7, a specific inhibitor of PKC. SDS-PAGE autoradiography of the 32P-labeled proteins showed that 32P was incorporated into the cystatin alpha. This incorporation was not observed by the action of cAMP-dependent protein kinase. Therefore, it is highly possible that the HSP is a phosphorylated cystatin alpha and that the phosphorylation is catalyzed specifically by PKC.     

Further characterization of the phosphate moiety of the adenovirus type 2 DNA-binding protein

The adenovirus type 2 DNA-binding protein is phosphorylated. Alkaline phosphatase treatment removes phosphate groups resulting in a decrease in molecular weight from 72000 to 70000. The dephosphorylated protein binds to single-stranded and double-stranded DNA as well as the phosphorylated protein does. Controlled chymotrypsin treatment cleaves the DNA-binding protein into two subspecies of Mr about 45000 and 25000. The 45000-Mr polypeptide contains most of the methionine residues but no phosphate and binds to DNA. The 25000-Mr polypeptide contains all the phosphate groups and shows no binding to DNA. Isoelectric focusing gels show heterogeneity of the DNA-binding protein and 15 subspecies with different charges can be observed after partial dephosphorylation by alkaline phosphatase. After extensive dephosphorylation two or three basic species with a molecular weight around 70000 are observed. Quantitative immunoprecipitation from cells labeled to equilibrium with inorganic 32PO4 gives a molar ratio of phosphate to protein of 4--7 and direct chemical determination of the phosphate residues yields 4 mol Pi/mol protein. These results suggest that there exist subspecies of the protein moiety of the adenovirus DNA-binding protein. The DNA-binding protein isolated from infected cells after a short 'pulse' of [35S]methionine has a molecular weight which corresponds to that of the dephosphorylated protein. After a 'chase' period the molecular weight increases to 72000, but alkaline phosphatase treatment converts it to a species with the same molecular weight as the newly synthesized DNA-binding protein, indicating that the modification of the protein is due to phosphorylation.     

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degrees C. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degrees C. Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme. An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells.     

Sensitive fluorogenic substrate for alkaline phosphatase

Alkaline phosphatase serves both as a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered on P–O bond cleavage in a process mediated by a “trimethyl lock.” Although this substrate requires a nonenzymatic second step to manifest fluorescence, we demonstrated that the enzymatic first step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications.     

Construction of an alkaline phosphatase-liposome system: a tool for biomineralization study

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper, we standardize a method for construction an alkaline phosphatase liposome system to mimic matrix vesicles and examine a some kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into liposomes constituted from dimyristoylphosphatidylcholine (DMPC), dilaurilphosphatidylcholine (DLPC) or dipalmitoylphosphatidylcholine (DPPC). This process was time-dependent and >95% of the enzyme was incorporated into the liposome after 4h of incubation at 25 degrees C. Although, incorporation was more rapid when vesicles constituted from DPPC were used, the incorporation was more efficient using vesicles constituted from DMPC. The 395nm diameter of the alkaline phosphatase-liposome system was relatively homogeneous and more stable when stored at 4 degrees C.Alkaline phosphatase was completely released from liposome system only using purified phosphatidylinositol-specific phospholipase C (PIPLC). These experiments confirm that the interaction between alkaline phosphatase and lipid bilayer of liposome is via GPI anchor of the enzyme, alone. An important point shown is that an enzyme bound to liposome does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but a liposome environment affects its kinetic properties, specifically for pyrophosphate.The standardization of such system allows the study of the effect of phospholipids and the enzyme in in vitro and in vivo mineralization, since it reproduces many essential features of the matrix vesicle.     

Regulation of Phosphate Accumulation in the Unicellular Cyanobacterium Synechococcus

The phosphorus contents of acid-soluble pools, lipid, ribonucleic acid, and acid-insoluble polyphosphate were lowered in Synechococcus in proportion to the reduction in growth rate in phosphate-limited but not in nitrate-limited continuous culture. Phosphorus in these cell fractions was lost proportionately during progressive phosphate starvation of batch cultures. Acid-insoluble polyphosphate was always present in all cultural conditions to about 10% of total cell phosphorus and did not turn over during balanced exponential growth. Extensive polyphosphate formation occurred transiently when phosphate was given to cells which had been phosphate limited. This material was broken down after 8 h even in the presence of excess external orthophosphate, and its phosphorus was transferred into other cell fractions, notably ribonucleic acid. Phosphate uptake kinetics indicated an invariant apparent K(m) of about 0.5 muM, but V(max) was 40 to 50 times greater in cells from phosphate-limited cultures than in cells from nitrate-limited or balanced batch cultures. Over 90% of the phosphate taken up within the first 30 s at 15 degrees C was recovered as orthophosphate. The uptake process is highly specific, since neither phosphate entry nor growth was affected by a 100-fold excess of arsenate. The activity of polyphosphate synthetase in cell extracts increased at least 20-fold during phosphate starvation or in phosphate-restricted growth, but polyphosphatase activity was little changed by different growth conditions. The findings suggest that derepression of the phosphate transport and polyphosphate-synthesizing systems as well as alkaline phosphatase occurs in phosphate shortage, but that the breakdown of polyphosphate in this organism is regulated by modulation of existing enzyme activity.     

Phosphatase activity and its rôle in the mineralization of organic phosphorus in coastal sea water

Alkaline phosphatase activity in sea-water samples taken from Tokyo, Sagami, and Suruga Bays in Japan was measured by a sensitive fluorometric method. There is a relationship between the phosphatase activity and bacterial biomasses in these three bays. The phosphatase-producing bacteria accounted for 40, 46, and 41% of heterotrophic bacteria in Tokyo, Sagami and Suruga Bays, respectively. Significant amounts of phosphatase-hydrolysable organic phosphorus were found in the euphotic zones of these bays and this organic phosphorus fraction accounted for 19 and 50% of organic phosphorus in Tokyo and Sagami Bays, respectively. Phosphatase-hydrolysable organic phosphorus was decomposed completely in the euphotic zone suggesting that this organic fraction is re-cycled in the primary production of the bays. Decomposition of natural organic phosphorus by enzymes was followed by measuring the release of inorganic orthophosphate from samples saturated with chloroform. Release of inorganic phosphorus proceeds rapidly for 2 or 3 days followed by a slow release. Enzymes present in the samples contributed to the decomposition of 50% of the organic phosphorus and the phosphatase enzyme was responsible for the decomposition of one-third of the hydrolysable organic phosphorus in the samples. Potential phosphatase activity in the samples was found to be indicative of the extent and rate of decomposition of organic phosphorus in coastal waters.