Plasma membrane localization of alkaline phosphatase in HeLa cells.

The localization of alkaline phosphatase in HeLa cells was examined by electron microscopic histochemistry and subcellular fractionation techniques. Two monophenotypic sublines of HeLa cells which respectively produced Regan and non-Regan isoenzymes of alkaline phosphatase were used for this study. The electron microscopic histochemical results showed that in both sublines the major location of alkaline phosphatase is in the plasma membrane. The enzyme reaction was occasionally observed in some of the dense body lysosomes. This result was supported by data obtained from a subcellular fractionation study which showed that the microsomal fraction rich in plasma membrane fragments had the highest activity of alkaline phosphatase. The distribution of this enzyme among the subcellular fractions closely paralleled that of the 5'-nucleotidase, a plasma membrane marker enzyme. Characterization of the alkaline phosphatase present in each subcellular fraction showed identical enzyme properties, which suggests that a single isoenzyme exists among fractions obtained from each cell line. The results, therefore, confirm the reports suggesting that plasma membrane is the major site of alkaline phosphatase localization in HeLa cells. The absence of any enzyme reaction in the perimitochondrial space in these cultured tumor cells also indicates that the mitochondrial localization of the Regan isoenzyme reported in ovarian cancer may not be a common phenomenon in Regan-producing cancer cells.     

Dephosphorylation of phosphoproteins of human liver plasma membranes by endogenous and purified liver alkaline phosphatases

Purified alkaline phosphatase and plasma membranes from human liver were shown to dephosphorylate phosphohistones and plasma membrane phosphoproteins. The protein phosphatase activity of the liver plasma membranes was inhibited by levamisole, a specific inhibitor of alkaline phosphatase, and by phenyl phosphonate and orthovanadate, but was relatively insensitive to fluoride (50 mM). Endogenous membrane protein phosphatase activity was optimal at pH 8.0, compared to pH 7.8 for purified liver alkaline phosphatase. Plasma membranes also exhibited protein kinase activity using exogenous histone or endogenous membrane proteins (autophosphorylation) as substrates; this activity was cAMP-dependent. Autophosphorylation of plasma membrane proteins was apparently enhanced by phenyl phosphonate, levamisole, or orthovanadate. The dephosphorylation of phosphohistones by protein phosphatase 1 was not inhibited by levamisole but was inhibited by fluoride. Inhibition of endogenous protein phosphatase activity by orthovanadate during autophosphorylation of plasma membranes could be reversed by complexation of the inhibitor with (R)-(-)-epinephrine, and the dephosphorylation that followed was levamisole-sensitive. Neither plasma membranes nor purified liver alkaline phosphatase dephosphorylated glycogen phosphorylase a. These results suggest that the increased [32P]phosphate incorporation by endogenous protein kinases into the membrane proteins is due to inhibition of alkaline phosphatase and that the major protein phosphatase of these plasma membranes is alkaline phosphatase.     

Alkaline phosphatase in the lactating bovine mammary gland and the milk fat globule membrane. Release by phosphatidylinositol-specific phospholipase C.

1. Alkaline phosphatase is covalently bound to bovine mammary microsomal membranes and milk fat globule membranes through linkage to phosphatidylinositol as demonstrated by the release of alkaline phosphatase following treatment with phosphatidylinositol-specific phospholipase C. 2. The release of alkaline phosphatase from the pellet to the supernatant was demonstrated by enzyme assays and electrophoresis. 3. Electrophoresis of the solubilized enzymes showed that the alkaline phosphatase of the microsomal membranes contained several isozymes, while only one band with alkaline phosphatase activity was seen in the fat globule membrane. 4. Levamisole and homoarginine were potent inhibitors of the alkaline phosphatase activities in both membrane preparations and in bovine liver alkaline phosphatase, but not in calf intestinal alkaline phosphatase.     

A protein phosphotyrosine phosphatase distinct from alkaline phosphatase with activity against the insulin receptor

Rat liver plasma membranes were found to have a relatively high ratio of acid to alkaline phosphatase activity when compared to rabbit liver and human placental membranes, respectively. The rat liver plasma membranes contained PPTl phosphatase activity against the soluble autophosphorylated insulin receptor beta-subunit. The PPT phosphatase activity of the membranes, using 32P-histone 2b as a substrate, was inhibited by 100 microM Zn+2, insensitive to 10 mM EDTA, and displayed maximal activity at neutral pH. Dephosphorylation of the insulin receptor beta-subunit by rat liver membranes was inhibited by Zn+2, and stimulated by EDTA. These results prove that the plasma membrane of a physiologically relevant insulin target tissue contains a PPT phosphatase, distinct from alkaline phosphatase, which catalyzes the dephosphorylation of the insulin receptor beta-subunit.     

Properties of p-nitrophenyl phosphatase activity from porcine neutrophils

p-nitrophenyl phosphatase activity is high in porcine neutrophils and was found in plasma membrane and granule fractions isolated from sucrose density gradients after nitrogen cavitation to disrupt the cells. Very little activity was found in the cytosol. The enzyme has optimum activity at alkaline pHs with a pH optimum of 10.3. The pH profile was fairly broad with activity still remaining at physiological pH. Orthovanadate was shown to be a potent competitive inhibitor of the enzyme with a Ki of 14 microM. Phosphate also inhibited but at millimolar concentrations and the two inhibitors bind in a mutually exclusive fashion. Evidence from experiments using divalent ion chelators and zinc ions suggested that the phosphatase is a zinc metalloenzyme. Beryllium was found to be a very potent, non-competitive inhibitor of the neutrophil enzyme (Ki = 1.1 microM). Levamisole and theophylline were both shown to be uncompetitive inhibitors of the porcine phosphatase (Ki = 0.2 mM and 1.2 mM respectively). The neutrophil phosphatase was inhibited by L-homoarginine but unaffected by L-phenylalanine and L-glutamate.     

pH-dependent conversion of liver-membranous alkaline phosphatase to a serum-soluble form by n-butanol extraction

Alkaline phosphatase released from rat liver plasma membrane under usual conditions was electrophoretically not identical with a soluble form in serum which was derived from the liver. The liver-membranous alkaline phosphatase, however, was converted to the serum-soluble form when the liver plasma membrane was treated with n-butanol under the acidic conditions lower than pH 6.5. Such pH-dependent conversion of the enzyme was not observed in plasma membrane of rat ascites hepatoma AH-130 cells. The converting activity for alkaline phosphatase was detected not only in plasma membrane but also in lysosomal membrane of rat liver.     

Alkaline phosphatase activity does not mediate phosphate transport in the renal-cortical brush-border membrane.

We studied (1) the effect of primary modulators of phosphate transport, namely the hypophosphataemic mouse mutant (Hyp) and low-phosphorus diet, on alkaline phosphatase activity in mouse renal-cortex brush-border membrane vesicles and (2) the effect of several primary inhibitors of alkaline phosphatase on phosphate transport. Brush-border membrane vesicles from Hyp-mouse kidney had 50% loss of Na+-dependent phosphate transport, but only 18% decrease in alkaline phosphatase activity. The low-phosphorus diet effectively stimulated Na+/phosphate co-transport in brush-border membrane vesicles (+ 118%), but increased alkaline phosphatase activity only slightly (+13%). Levamisole (0.1 mM) and EDTA (1.0 mM) inhibited brush-border membrane-vesicle alkaline phosphatase activity of 82% and 93% respectively, but had no significant effect on Na+/phosphate co-transport. We conclude that alkaline phosphatase does not play a direct role in phosphate transport across the brush-border membrane of mouse kidney.     

Intracellular alkaline phosphatase activity in cultured human cancer cells

Summary The effect of saponin treatment in demonstrating intracellular portion of alkaline phosphatase activity in human cancer cell lines was evaluated. Previous reports using standard lead-salt techniques visualized enzyme almost exclusively on the plasma membrane and sometimes in the lysosomes. However, by treating cells with saponin before or during the cytochemical incubation, intracellular alkaline phosphatase became demonstrable at the endoplasmic reticulum, Golgi apparatus, Golgi-derived vesicles and mitochondria as well as lysosomes and plasma membrane. These intracellular catalytic activities were significantly inhibited by the specific amino acid inhibitors characteristic for each cell line, and this suggested that intracellular alkaline phosphatase is the same isoenzyme as that present in the plasma membrane. The results of our current and previous studies therefore indicate that saponin reveals latent intracellular alkaline phosphatase activity by changing the membrane's physical state; thereby increasing the availability of both catalytic and antigenic sites of the enzyme to substrate and to antibody respectively.This work was supported by National Institutes of Health Grant No. CA 21967     

The effects of cell population density on the plasma membrane of Acanthamoeba castellanii

Plasma membranes were isolated from both exponential and stationary phase cells and their properties compared, to determine whether alterations are sustained coincident with the transition to plateau phase growth. Polyacrylamide gel electrophoresis revealed no significant differences in macromolecular composition between the two types of membrane. However, the specific activity of alkaline phosphatase (EC 3.1.3.1), an enzyme which shows enrichments in purified plasma membrane fractions relative to homogenates, was markedly reduced in preparations from stationary as compared with exponentially growing cells. The total activity per cell did not change, but in cell fractionation experiments the stationary phase cells yielded a higher proportion of the enzyme in microsomal fractions than did exponentially growing cells. This indicates that once plateau phase is attained, a greater proportion of the membrane bearing alkaline phosphatase activity is internalized as opposed to being associated with the plasmalemma.Alkaline phosphatase is known to be present on the contractile vacuole membrane. During discharge this vacuole becomes associated with the plasmalemma, an event which presumably accounts for at least part of the alkaline phosphatase in plasma membrane preparations. Thus one interpretation of the decreased levels of alkaline phosphatase in plasma membrane fractions from stationary phase cells is that they reflect a decline in the rate of water expulsion. This in turn suggests that the plasmalemma of stationary phase cells may have undergone changes leading to a decreased rate of water influx.     

Alterations in hepatic 5'-nucleotidase in the tumor-bearing rat

The effect of the growth of the Walker 256 carcinoma on the level of 5'-nucleotidase and alkaline phosphatase in the whole liver and in an isolated hepatocyte membrane preparation of its host was investigated. Alkaline phosphatase activities of whole liver and plasma membrane were increased approximately 5-fold by tumor growth. A 50% decrease in whole liver 5'-nucleotidase activity was observed in tumor-bearing rats while the 5'-nucleotidase activity per milligram membrane protein was unaltered. Tumor growth would therefore appear to affect a pool of 5'-nucleotidase which is not associated with the plasma membrane.     

Hepatic alkaline phosphatase isoenzymes: isolation, characterization and differential alteration.

Although it is generally believed that hepatic alkaline phosphatase is localized to liver plasma membranes, 63% is present in the cytosol fraction after ultracentrifugation of rat liver homogenates. Divalent cation requirements, heat inactivation, pH optima, Km and chemical inhibition characteristics of partially purified alkaline phosphatase enzymes prepared from membrane and cytosol fractions suggested different structural forms. Furthermore, bile duct obstruction and ethinyl estradiol administration preferentially increased membrane-bound alkaline phosphatase activity, while cytosol activity was unaltered. In contrast, phenobarbital treatment decreased membrane-bound alkaline phosphatase and increased cytosol activity. These studies support the presence of two forms of hepatic alkaline phosphatase in rat liver which are regulated by different control mechanisms.     

Induction of plasma membrane alkaline phosphatase in rat liver

We have determined alkaline phosphatase activity in total liver plasma membrane fractions from rats subjected to a partial hepatectomy and sham operated with or without manipulation of the liver. In all these cases, an increase of the enzyme activity was observed. Kinetic studies of alkaline phosphatase activity performed on plasma membrane fractions from rats subjected to a partial hepatectomy suggest that alkaline phosphatase increase is produced by de novo biosynthesis of enzyme molecules. Determination of alkaline phosphatase activity in purified plasma membrane subfractions corresponding to each of the three functional regions of the hepatocyte surface (blood sinusoidal, lateral and bile canalicular), indicates that the increase of the enzyme activity observed after partial hepatectomy is selectively induced in the bile canalicular domain of the hepatocyte plasma membrane.     

Further evidence for the existence of an intrinsic bicarbonate-stimulated Mg2+-ATPase in brush border membranes isolated from rat kidney cortex

Summary The aim of this study was to provide further evidence for the existence of a nonmitochondrial bicarbonate-stimulated Mg²⁺-ATPase in brush border membranes derived from rat kidney cortex. A plasma membrane fraction rich in brush border microvilli and a mitochondrial fraction were isolated by differential centrifugation. Both fractions contain a Mg²⁺-ATPase activity which can be stimulated by bicarbonate. The two Mg²⁺-ATPases are stimulated likewise by chloride, bicarbonate, and sulfite or inhibited by oligomycin and aurovertin, though to different degrees. In contrast to these similarities, only the Mg²⁺-ATPase activity of the mitochondrial fraction is inhibited by atractyloside, a substance which blocks an adenine nucleotide translocator in the inner mitochondrial membrane. On the other hand, filipin, an antibiotic that complexes with cholesterol in the membranes inhibits exclusively the Mg²⁺-ATPase of the cholesterol-rich brush border membranes. Furthermore it could be demonstrated by the use of bromotetramisole, an inhibitor of alkaline phosphatase activity, that the Mg²⁺-ATPase activity in the membrane fraction is not due to the presence of the highly active alkaline phosphatase in these membranes. These results support the assumption that an intrinsic bicarbonate-stimulated Mg²⁺-ATPase is present in rat kidney brush border membranes.     

Incorporation of urease into liposomes.

1. Plasma membranes were isolated from ascites hepatoma AH-130 and rat livers with or without partial hepatectomy or bile duct ligation. Reciprocal manifestations of two marker enzymes for plasma membranes were observed in these membrane preparations; alkaline phosphatase activity was found much higher in the hepatoma membrane than in any preparations of the liver membranes, whereas 5'-nucleotidase activity was much lower in the former than in the latter. 2. Effects of lectins and anti-plasma membrane antiserum on these two marker enzymes were examined. The results showed that about 50% of apparent activity of 5'-nucleotidase found in the hepatoma membrane was exhibited by alkaline phosphatase. 3. Localizations of alkaline phosphatase and 5'-nucleotidase in polyacrylamide gels after electrophoresis were demonstrated using 5'-AMP and 5-Br, 4-Cl-indoxylphosphate as substrate. There was a difference in electrophoretic mobility between the alkaline phosphatase of the hepatoma and that of the liver.     

Intracellular localization of phosphodiesterase induced by cyclic adenosine 3',5'-monophosphate in Dictyostelium discoideum

The intracellular distribution of phosphodiesterase [EC 3.1.4.17] induced by cyclic adenosine 3',5'-monophosphate (cAMP) in Dictyostelium discoideum was studied. When cAMP-treated cells were homogenized and fractionated according to the method of de Duve et al. ((1955) Biochem, J. 60, 604), the specific activity of phosphodiesterase was highest in the light mitochondrial fraction. Peaks of specific activities of alkaline phosphatase (marker enzyme of membrane) and catalase (marker enzyme of peroxisomes) also appeared in the same fraction as phosphodiesterase. However, after centrifugation of the light mitochondrial fraction in a sucrose density gradient, the activity of phosphodiesterase was clearly separated with that of catalase (density 1.19 g/ml) and showed three peaks at lower density (1.10, 1.13, 1.17 g/ml) with good reproducibility. Some parts (1.13, 1.17 g/ml) of the activity in the gradient overlapped with alkaline phosphatase activity, but in the density fraction of 1.10 g/ml the activity of alkaline phosphatase was hardly detectable. When the light mitochondrial fraction was treated with Emulgen 108, or sonicated, phosphodiesterase was more easily solubilized than alkaline phosphatase and catalase, and was found in supernate after centrifugation at 20,000 X g for 30 min. In order to distinguish the locations of the three enzymes, the supernatant of the light mitochondrial fraction treated with Emulgen 108 was subjected to charge shift electrophoresis. The electrophoretic mobilities of phosphodiesterase and catalase were unaffected by ionic detergent. However, alkaline phosphatase shifted towards the anode in the presence of anionic detergent (sodium deoxycholate), and shifted towards the cathode in cationic detergent (cetyltrimethylammonium bromide), relative to nonionic detergent (Emulgen 108) alone. Thus, some part of the phosphodiesterase induced by cAMP may be associated with the plasma membrane, but the remainder is localized in some kind of intracellular particle of lower density. Moreover, the association with the membrane or particle is more easily dissociated than that of alkaline phosphatase, and the liberated phosphodiesterase is rather hydrophilic.     

Spectrophotometric and cytochemical analyses of phosphatase activity in Beta vulgaris L

Spectrophotometric and cytochemical methods were used to investigate the localization and/or the sensitivity of phosphatase activities in aldehyde-fixed beet leaves and membrane fractions. The nonspecific acid phosphatase substrates, p-nitrophenyl phosphate and beta-glycerol phosphate, each exhibited unique spectrophotometric patterns of hydrolysis as a function of pH. Additionally, beta-glycerol phosphatase activity was primarily present on the tonoplast, whereas p-nitrophenyl phosphatase was present on the plasma membrane. Because of the unique pH response of each enzyme and their different localization, we conclude that they cannot be entirely "nonspecific." The spectrophotometric pattern of ATP hydrolysis differed from that of p-nitrophenol phosphate in that it decreased at pH 5.0-5.5 and was greatly inhibited by 10 mM sodium fluoride; however, both activities were on the plasma membrane. Therefore, we conclude that these activities represent either two enzymes or only one enzyme that differs in its ability to hydrolyze these two substrates. Generally, enzymatically produced lead deposits on the plasma membrane of non-vascular cells were as frequent and large as those on phloem cells; frequently, deposits on sieve element plasma membranes were relatively small. We therefore conclude that there is no evidence for the presence of relatively intense ATPase activity on the plasma membrane of phloem cells in beet leaf, in contrast to other species. Studies with membrane fractions indicated that formaldehyde could completely inhibit the inhibitor-sensitive phosphatase activities in mitochondrial and vacuolar fractions while preserving significant activity in the plasma membrane fraction.     

Membrane-bound 5'-nucleotidase inhibitor in Ehrlich ascites tumour cells and newborn mouse liver

5'-Nucleotidase activity in Ehrlich ascites tumour cells was undetectable. The cell homogenate, when mixed with adult mouse liver homogenate, inhibited the 5'-nucleotidase activity of the latter, without affecting its p-nitrophenyl phosphate-hydrolysing activity. The inhibitor activity was enriched (6.8-fold) in a membrane fraction which was enriched in (Na+ + K+)-ATPase (14-fold) and alkaline phosphatase (8-fold). 5'-Nucleotidase activity in this membrane fraction could be detected only after separating the inhibitor activity from the enzyme on Sephadex G-50. The inhibitor activity was decreased by 27% when heat-treated, 33% when treated with 6 M urea and was almost completely lost when treated with trypsin. It was dialysable from a tubing with a molecular exclusion limit of 10,000, but was retained in a tubing with an exclusion limit of 3000. From these results we conclude that a small molecular weight protein inhibitor(s) of 5'-nucleotidase is present in the plasma membrane of Ehrlich ascites tumour cells. Also, the presence of such an inhibitor in the newborn mouse liver but not in the adult liver suggests that it may have some role in cellular ageing and cancer.     

Purification and properties of H+-translocating, Mg2+-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae

H+-translocating, Mg2+-ATPase was solubilized from vacuolar membranes of Saccharomyces cerevisiae with the zwitterionic detergent N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and purified by glycerol density gradient centrifugation. Partially purified vacuolar membrane H+-ATPase, which had a specific activity of 18 units/mg of protein, was separated almost completely from acid phosphatase and alkaline phosphatase. The purified enzyme required phospholipids for maximal activity and hydrolyzed ATP, GTP, UTP, and CTP, with this order of preference. Its Km value for Mg2+-ATP was determined to be 0.21 mM and its optimal pH was 6.9. ADP inhibited the enzyme activity competitively, with a Ki value of 0.31 mM. The activity of purified ATPase was strongly inhibited by N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, tributyltin, 7-chloro-4-nitrobenzoxazole, diethylstilbestrol, and quercetin, but was not affected by oligomycin, sodium azide, sodium vanadate, or miconazole. It was not inhibited at all by antiserum against mitochondrial F1-ATPase or mitochondrial F1-ATPase inhibitor protein. These results indicated that vacuolar membrane H+-ATPase is different from either yeast plasma membrane H+-ATPase or mitochondrial F1-ATPase. The vacuolar membrane H+-ATPase was found to be composed of two major polypeptides a and b of Mr = 89,000 and 64,000, respectively, and a N,N'-dicyclohexylcarbodiimide binding polypeptide c of Mr = 19,500, whose polypeptide composition was also different from those of either plasma membrane H+-ATPase or mitochondrial F1-ATPase of S. cerevisiae.     

Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes

Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane entity in all membrane preparations and support the view that there is little overlap in the enzymatic equipment of the various types of cytomembranes.     

Characterization of D-myo-inositol 1,4,5-trisphosphate phosphatase in rat liver plasma membranes

D-myo-Inositol 1,4,5-trisphosphate has been previously demonstrated to act as a second messenger for the hormonal mobilization of intracellular calcium in rat liver. In this study, the breakdown of D-myo-inositol 1,4,5-trisphosphate by a phosphatase activity was characterized. Using partially purified subcellular fractions, it was found that D-myo-inositol 1,4,5-trisphosphate phosphatase (I-P3ase) specific activity was highest in the plasma membrane fraction, while D-myo-inositol 1,4-bisphosphate phosphatase specific activity was highest in the cytosolic and microsomal fractions. The plasma membrane I-P3ase was Mg2+-dependent with optimal activity observed at 0.5-1.5 mM free Mg2+. The enzyme had a neutral pH optimum, suggesting that it was neither an acid nor alkaline phosphatase. Neither LiCl nor NaF inhibited the I-P3ase activity. However, both L-cysteine and dithiothreitol stimulated the activity 2-fold. Spermine (2.0 mM) inhibited the I-P3ase activity by 50%, while putrescine and spermidine had little or no effect.