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.     

Purification and partial characterization of xanthine oxidase from human milk.

Xanthine oxidase was purified from human milk in yields comparable with those obtained from bovine milk. The freshly purified enzyme appeared homogeneous in gel permeation FPLC and SDS-PAGE, consistent with its being a homodimer with total M(r) 290,000 +/- 6000. The ultraviolet/visible absorption spectrum differed only slightly from that of bovine milk enzyme and showed an A280/A450 ratio of 5.13 +/- 0.29, indicating a high degree of purity. Xanthine oxidase activities of purified enzyme varied with batches of milk, ranging between 3 and 46 mU/mg protein; values that are some two to three orders of magnitude smaller than those shown by the most highly purified samples of bovine milk enzyme. Direct comparison with commercially-available bovine milk enzyme showed that activities involving xanthine as reducing substrate were 1-6% that of the bovine enzyme, whereas those involving NADH, in contrast, were of the same order for the two enzymes. Anaerobic bleaching experiments indicated that less than 2% of the human enzyme was present as a form active with xanthine. These findings, together with the activity data, are consistent with a very high content, possibly greater than 98%, of demolybdo- and/or desulpho-forms of human enzyme, both of which occur, to a lesser extent, in bovine xanthine oxidase. Molybdenum assay indicated that demolybdo-enzyme could only account for some 26% of this inactive component, suggesting that desulpho-enzyme may account for the remainder.     

A prostatic-like acid phosphatase is present in human lactating milk

Summary The presence of a prostatic-like acid phosphatase is reported in human lactating milk. Its activity is associated with skim milk and it could be separated from the other acid phosphatases only after Triton X-100 treatment. By all the criteria applied, it appears to be very similar to prostatic acid phosphatase. An approximate molecular weight of 96 000 was measured for the native enzyme, which is inhibited by L-(+)tartrate and has similar electrophoretic migration. Besides, it hydrolyzes choline-o-phosphate very well and cross-reacts with an antibody anti-prostatic acid phosphatase. This prostatic-like acid phosphatase has also been detected in a human mammary carcinoma from a lactating patient.     

Human liver acid phosphatases.

Human liver contains three chromatographically distinct forms of non-specific acid phosphatase (EC 3.1.3.2). Acid phosphatases I, II and III have molecular weights of greater than 200 000, of 107 000, and of 13 400, respectively. Following partial purification, isoenzyme II was obtained as a single activity band, as assessed by activity staining with p-nitrophenyl phosphate and alpha-naphthyl phosphate on polyacrylamide gels run at several pH values. With 50mM p-nitrophenyl phosphate as a substrate, enzymes II and III exhibit plateaus of activity over the pH range 3 - 5 and 3.5 - 6, respectively.Acid phosphatase II is not significantly inhibited by 0.5% formaldehyde. The activity of human liver acid phosphatase II and of human prostatic acid phosphatase towards several substrates is compared. The liver enzyme, is marked contrast to the prostatic enzyme, does not hydrolyze O-phosphoryl choline.     

Production of monoclonal antibodies against human 6-pyruvoyl tetrahydropterin synthase and immunocytochemical localization of the enzyme.

Monoclonal antibodies were produced against human pituitary gland 6-pyruvoyl tetrahydropterin synthase, one of the key enzymes in the biosynthesis of tetrahydrobiopterin, by in vitro immunization with the antigen directly blotted from SDS-PAGE to polyvinylidene difluoride membranes. The antibodies produced show crossreactivity in the enzyme linked immunosorbent assay, not only with the human 6-pyruvoyl tetrahydropterin synthase but some also with the same enzyme isolated from salmon liver. 6-Pyruvoyl tetrahydropterin synthase was localized immuno-enzymatically in peripheral blood smears and in skin fibroblasts by the use of these monoclonal antibodies and the alkaline phosphatase monoclonal anti-alkaline phosphatase labeling technique.     

Mammalian protein serine/threonine phosphatase 2C: cDNA cloning and comparative analysis of amino acid sequences.

Complementary DNA encoding rat protein phosphatase 2C alpha was obtained from a liver library and used to isolate the homologous cDNAs from rabbit liver and human teratocarcinoma libraries. The amino acid sequences of the three enzymes deduced from the cDNA (382 amino acids) were extremely similar (greater than 99% identity), the maximum number of differences (between rat and human) being four. Amino acid sequences of peptides corresponding to 238 residues (61%) of the protein phosphatase 2C beta isoform from rabbit skeletal muscle were determined and showed 12 differences from the recently published sequence of the rat liver enzyme deduced from the cDNA (95% identity).     

Differences in phosphatidate hydrolytic activity of human alkaline phosphatase isozymes

Hydrolytic activities of human alkaline phosphatase isozymes were investigated using phosphatidases with various fatty acyl chains (egg phosphatidate and dioleoyl, distearoyl, dipalmitoyl, dimyristoyl and dilauroyl phosphatidates). In the presence of sodium deoxycholate, purified human placental and intestinal alkaline phosphatases hydrolyzed all the phosphatidates examined. The hydrolytic activity was maximal in the presence of 10 g/l sodium deoxycholate. Of the phosphatidates, dilauroyl phosphatidate was the best substrate. Using the same unit of the enzyme, the phosphatidate hydrolytic activity of placental alkaline phosphatase was 2- to 3-times higher than that of the intestinal enzyme. In contrast, liver alkaline phosphatase did not hydrolyze phosphatidates with long fatty acyl chains (C16-18) even in the presence of sodium deoxycholate. The liver enzyme hydrolyzed dimyristoyl and dilauroyl phosphatidates very slowly. These results show that the phosphatidates with long fatty acyl chains were useful to differentiate placental and intestinal alkaline phosphatases from the liver enzyme, and suggest that the former enzymes play a different physiological role from the liver enzyme.     

Phosphoester specificity of purified human liver alkaline phosphatase.

Kinetic parameters for the hydrolysis of a number of physiologically important phosphoesters by purified human liver alkaline phosphatase have been determined. The enzyme was studied at pH values of 7.0 to 10.0. The affinity of the enzyme for the compounds was determined by competition experiments and by their direct employment as substrates. Phosphodiesters and phosphonates were not hydrolysed but the latter were inhibitors. Calcium and magnesium ions inhibited the hydrolysis of ATP and PP1 and evidence is presented to show that the metal complexes of these substrates are not hydrolysed by alkaline phosphatase. A calcium-stimulated ATPase activity could not be demonstrated for the purified enzyme or the enzyme in the presence of a calcium-dependent regulator protein. Nevertheless, the influence of magnesium and calcium ions on the ATPase activity of alkaline phosphatase means that precautions must be taken when assaying for Ca2+-ATPase in the presence of alkaline phosphatase. The low substrate Km values and the hydrolysis which occurs at pH 7.4 mean that the enzyme could have a significant phosphohydrolytic role. However, liver cell phosphate concentrations, if accessible to the enzyme, are sufficient to strongly inhibit this activity.     

Characterization of human foetal intestinal alkaline phosphatase. Comparison with the isoenzymes from the adult intestine and human tumour cell lines.

The molecular structure of human foetal intestinal alkaline phosphatase was defined by high-resolution two-dimensional polyacrylamide-gel electrophoresis and amino acid inhibition studies. Comparison was made with the adult form of intestinal alkaline phosphatase, as well as with alkaline phosphatases isolated from cultured foetal amnion cells (FL) and a human tumour cell line (KB). Two non-identical subunits were isolated from the foetal intestinal isoenzyme, one having same molecular weight and isoelectric point as placental alkaline phosphatase, and the other corresponding to a glycosylated subunit of the adult intestinal enzyme. The FL-cell and KB-cell alkaline phosphatases were also found to contain two subunits similar to those of the foetal intestinal isoenzyme. Characterization of neuraminidase digests of the non-placental subunit showed it to be indistinguishable from the subunits of the adult intestinal isoenzyme. This implies that no new phosphatase structural gene is involved in the transition from the expression of foetal to adult intestinal alkaline phosphatase, but that the molecular changes involve suppression of the placental subunit and loss of neuraminic acid from the non-placental subunit. Enzyme-inhibition studies demonstrated an intermediate response to the inhibitors tested for the foetal intestinal, FL-cell and KB-cell isoenzymes when compared with the placental, adult intestinal and liver forms. This result is consistent with the mixed-subunit structure observed for the former set of isoenzymes. In summary, this study has defined the molecular subunit structure of the foetal intestinal form of alkaline phosphatase and has demonstrated its expression in a human tumour cell line.     

Dimeric nature and amino acid compositions of homogeneous canine prostatic human liver and rat liver acid phosphatase isoenzymes. Specificity and pH-dependence of the canine enzyme.

Two isoenzymes of rat liver acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) have been purified to homogeneity, at least one of these for the first time. Both of the rat liver isoenzymes have identical specific activities towards p-nitrophenyl phosphate. Molecular weights of the native enzymes are 92 000 for rat liver isoenzyme I and 93 000 for isoenzyme II, while the subunit molecular weights are 51 000 and 52 000 respectively. Data on substrate specificity and pH dependence are presented for the homogeneous canine prostatic enzyme, which is also isolated as a dimeric enzyme of (native) molecular weight 89 000. Carbohydrate analysis data are presented for canine prostatic acid phosphatase and it is further noted that both isoenzymes of rat liver acid phosphatase are also glycoproteins. The amino acid compositions of the two rat liver isoenzymes are presented together with those of the similar dimeric acid phosphatase of human liver and of canine prostate. Comparison of these results with published data for the amino acid composition of human prostatic acid phosphatase shows substantial similarities. However, significant differences are seen in the amino acid composition of rat liver acid phosphatase isoenzyme I as compared to a previous literature report. Most notably, 17 histidine residues are found per mol of isoenzyme I and 18 for isoenzyme II.     

Alkaline phosphatase from Echinococcus multilocularis: purification and characterization.

1. Kinetic and physical parameters of purified alkaline phosphatase from Echinococcus multilocularis metacestodes, livers of infected gerbils and control animals were determined. 2. Km value for p-nitrophenyl phosphate was about 0.05 +/- 0.02 mM for the three enzymes. 3. Vmax values were 357 +/- 67 nmol/min/mg proteins for metacestode enzyme, and 6.7 +/- 1.1 and 6.7 +/- 0.8 nmol/min/mg proteins for liver enzyme of infected and control animals, respectively. 4. Mr and pI were different for the parasite and hepatic enzyme. 5. The parasite enzyme was less sensitive to the elevation of temperature than hepatic enzyme. 6. The isatin inhibition was a competitive inhibition type for parasite and uncompetitive type for host liver enzyme.     

The autorelease of alkaline phosphatase from the plasma membrane during the incubation of cultured liver cell homogenates

When a rat hepatoma cell (R-Y121B) homogenate was incubated at 37 degrees C, 30-70% of the total alkaline phosphatase was released into the supernatant fluid from the precipitate fractions. The release reached a plateau level after 10 h of incubation at 37 degrees C. The optimum pH value for the release was 7.4. Alkaline phosphatase activity increased during the incubation of the cell homogenates, but this increase was independent of the enzyme release. Serum increased not only alkaline phosphatase activity in the cultured cells but also enzyme release in their homogenates. In addition, we examined a rat liver homogenate and the following 11 cell lines: 3 hepatoma cell lines, including the R-Y121B cell line, 4 liver cell lines, 2 human urinary bladder carcinoma cell lines, a kidney cell line, and a mouse adrenal tumor cell line. Only in the cultured liver cell line and hepatoma cell lines, 30-60% of the total enzyme was released into the soluble fraction from the precipitate fractions; the release was not observed in the other cell lines, nor in the rat liver homogenate. The release of alkaline phosphatase took place in both heat-stable and heat-labile alkaline phosphatases. Alkaline phosphatase, extracted from cell homogenates, showed two bands during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mobilities of the two bands changed inversely with or without sodium dodecyl sulfate. In general, the alkaline phosphatase which showed slow mobility with sodium dodecyl sulfate was more readily released from the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Native and latent forms of liver phosphorylase phosphatase. The non-identity of native phosphorylase phosphatase and synthase phosphatase.

The directly measurable (native) phosphorylase phosphatase present in a fresh mouse liver extract is bound to particulate glycogen and is not inhibited by heat-stable inhibitors. Treatment of the extract with trypsin or ethanol at room temperature caused a more than 10-fold increase in phosphorylase phosphatase activity. This increased activity stems from the activation of completely inactive (latent) enzyme, the major part of which is present in the high-speed supernatant. The trypsin-revealed activity can be completely blocked by heat-stable inhibitors. Treatment of the animal with glucocorticoids increases, and fasting decreases the activity of the native phosphorylase phosphatase. The level of latent enzyme, however, is unaffected by these treatments. The major portion of synthase phosphatase in the fresh liver extract is bound to glycogen. This enzyme is inhibited by the heat-stable inhibitor-2 and inactivated by trypsin or ethanol as well as by several treatments that have little effect on phosphorylase phosphatase. Upon DEAE-cellulose chromatography at 0 degrees C of a fresh liver extract, phosphorylase phosphatase and synthase phosphatase were resolved as separate, single peaks. If the preparation was not kept at 0 degrees C during the entire procedure, two peaks of each enzyme were observed. Under these conditions the first peak of phosphorylase phosphatase and of synthase phosphatase coincided. From these findings it is concluded that synthase phosphatase and phosphorylase phosphatase, in their native form, are distinct enzymes.     

Selective preparation and characterization of membranous and soluble forms of alkaline phosphatase from rat tissues. A comparison with the serum enzyme

We developed a method for selective preparation of two forms of alkaline phosphatase from rat tissues. The enzyme was extracted by n-butanol treatment at pH 5.5 and pH 8.5 as soluble and aggregated (membranous) forms, respectively. The soluble form prepared from liver was found to be identical with the serum enzyme. Complete solubilization of the membrane-bound enzyme without detergents had a great advantage in its purification. Rat hepatoma AH-130 cells enriched in alkaline phosphatase were first used for purification of the liver-type enzyme. The hepatoma enzyme, purified by chromatographies on concanavalin-A-Sepharose, Sephacryl S-300 and hydroxyapatite was used for production of antibodies specific for the liver-type isozyme. An immunoaffinity column, prepared with anti-(hepatoma-enzyme) IgG was utilized for the enzyme purification from other tissues including the membranous form. Analyses of amino acid composition of the purified enzymes revealed that all the liver-type enzymes from hepatoma, liver, kidney and serum had the same composition, whereas the intestinal type consisted of the composition distinctly different from that in the liver type. In addition, there was no significant difference in amino acid composition between the soluble and membranous forms, suggesting a possible involvement in the membranous form of a hydrophobic component other than its polypeptide domain. The present method for selective preparation of the soluble and membranous forms of alkaline phosphatase will be useful for a further investigation on the interaction of the enzyme with membranes.     

Immunological characterization of human acid phosphatase gene products

The immunological cross-reactivity of heterogeneous acid phosphatase isozymes from different human tissues has been studied using monospecific antisera prepared against four homogeneous acid phosphatases. The enzyme characterized as tartrate-inhibitable, prostatic acid phosphatase is also found to be present in leukocytes, kidney, spleen, and placenta. The tartrate-inhibitable (liver) lysosomal enzyme is also found in kidney, fibroblasts, brain, placenta, and spleen, but it is not detectable in erythrocytes and prostate. In several tissues, 10–20% of the tartrate-inhibitable enzyme is not precipitated by any of the antisera used; an exceptionally high amount (54%) of such an enzyme is present in human brain. Antiserum against a low molecular weight tartrate-resistant liver enzyme (14 kDa) does not cross-react with the erythrocyte enzyme. (10–20 kDa). All other tissues except placenta, prostate, and fibroblast cells show a cross-reactivity with the 14-kDa acid phosphatase antiserum. Thus, the low molecular weight human liver acid phosphatase is distinct from the erythrocyte enzyme, and there are also at least three different tartrate-inhibitable acid phosphatases in human tissues. Chromosomal assignments have been made for only two of the (at least) five acid phosphatases that are present in adult human tissues.This study was supported by DHHS Research Grant GM 27003 from the U.S. National Institute of General Medical Sciences and by Grant SFB-104 from the Deutsche Forschungsgemeinschaft.     

A new fluorescence method for alkaline phosphatase histochemistry.

We have developed a new fluorescence method for the histochemical localization of alkaline phosphatase activity. Calcium phosphate deposited at the sites of alkaline phosphatase activity in a Gomori-type reaction are identified by calcium binding fluorochromes. The calcium binding fluorochromes calcein, calcein blue, and xylenol orange were investigated, with each fluorochrome being included in the alkaline phosphatase incubating medium and used in a single-step procedure. Alkaline phosphatase activity was studied in freeze-substituted, resin-embedded human liver and jejunal biopsies, and each fluorochrome produced intense fluorescence of different colors at sites of alkaline phosphatase activity. Calcein, calcein blue, and xylenol orange produced green, blue, and red fluorescence, respectively. Sites of enzyme activity were accurately localized without evidence of diffusion, and there was an absence of non-enzyme-catalyzed binding of any of the fluorochromes to tissue. This fluorescence method, which is particularly suited to investigating the localization and distribution of the activity of different enzymes in the same section, was used to investigate the distribution and co-localization of alkaline phosphatase and aminopeptidase M in human liver and jejunum.     

The rabbit differs from other mammalian in the tissue distribution of alkaline phosphatase isoenzymes

There are only two gene loci code for alkaline phosphatase of mammalian other than human and great apes: one for the intestinal form and other for the liver/kidney/bone form. The former form is present only in the intestine and the latter form occurs in other tissues such as liver, kidney and bone. In the present study, the rabbit was found to be different from other mammalian in the tissue distribution of alkaline phosphatase isoenzymes: only in the rabbit, most of the enzyme in the kidney and liver was the third form which differs from the liver/kidney/bone form, and this form was enzymatically and immunologically similar to the intestinal form of ALPase.     

The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum.

1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, before immunoaffinity purification of the liver enzyme, resulted in the same specific activity and subunit Mr. The native Mr of the sulphobetaine 14-solubilized enzyme was consistent with the enzyme being a dimer of two identical subunits and was higher than that of the butanol-extracted enzyme, presumably due to the binding of the detergent micelle. 2. Pure bone and liver alkaline phosphatase were used to raise further antibodies to the two isoenzymes. Altogether, 27 antibody-producing cell lines were cloned from 12 mice. Several of these antibodies showed a greater than 2-fold preference for bone alkaline phosphatase in the binding assay used for screening. No antibodies showing a preference for liver alkaline phosphatase were successfully cloned. None of the antibodies showed significant cross-reaction with placental or intestinal alkaline phosphatase. Epitope analysis of the 27 antibodies using liver alkaline phosphatase as antigen gave rise to six groupings, with four antibodies unclassified. The six major epitope groups were also observed using bone alkaline phosphatase as antigen. 3. Serum from patients with cholestasis contains soluble and particulate forms of alkaline phosphatase. The soluble serum enzyme had the same size and charge as butanol-extracted liver enzyme on native polyacrylamide-gel electrophoresis. Cellulose acetate electrophoresis separated the soluble and particulate serum alkaline phosphatases as slow- and fast-moving forms respectively. In the presence of sulphobetaine 14 all the serum enzyme migrated as the slow-moving form on cellulose acetate electrophoresis. Monoclonal anti-(alkaline phosphatase) immunoadsorbents did not bind the particulate form of alkaline phosphatase in cholestatic serum but bound the soluble form. In the presence of sulphobetaine 14 all the cholestatic serum alkaline phosphatase bound to the immunoadsorbents. 4. The electrophoretic and immunological data are consistent with both particulate and soluble forms of alkaline phosphatase in cholestatic serum being derived from the hepatocyte membrane.     

The modulator protein dissociates the catalytic subunit of hepatic protein phosphatase G from glycogen.

1. The phosphorylase phosphatase and glycogen-synthase phosphatase activities associated with the glycogen particles from rat liver were progressively inhibited by incubation with modulator protein. However, the phosphorylase phosphatase activity of the catalytic subunit was entirely recovered after destruction of the modulator and the regulatory subunit(s) by trypsin. 2. Inhibition of protein phosphatase G by modulator was associated with a translocation of the phosphorylase phosphatase activity (measured after incubation with trypsin) from glycogen to the soluble fraction. The degree of inhibition of phosphatase G corresponded closely to the extent to which the phosphorylase phosphatase activity was released from the glycogen particles. Incubation of glycogen-free protein phosphatase G with modulator did not change the affinity of the enzyme for added glycogen, but decreased the amount of phosphatase that could be bound to glycogen. 3. The phosphorylase phosphatase activity that was released from the glycogen particles by modulator migrated on gel filtration as a complex (Mr 106,000) of the catalytic subunit with modulator. Phosphorylase phosphatase activity could be transferred from glycogen-bound protein phosphatase G to modulator that was covalently bound to Sepharose. After elution from the column, the enzyme was identified as the free catalytic subunit (Mr 37,000).