Studies on Vitamin B6 Metabolism in Microorganisms

A large number of bacteria were searched for the activity of the synthesis of pyridoxine 5′-phosphate by the transphosphorylation between pyridoxine and p-nitrophenyl phosphate. Several properties of the transphosphorylation by the partially purified enzyme prepared from one of the isolated bacteria, Escherichia freundii K–1, were investigated accompanying with phosphatase activity. The behavior of the phosphotransferase and phosphatase activities in various reaction conditions were almost parallel. It was pointed out that the transphosphorylation might be catalyzed by the function of acid phosphatase. The phosphoryl donor specificity for the enzyme system was found to be broad.

The enzyme which catalyzed the transphosphorylation of pyridoxine accompanying with the hydrolyzation of phosphoryl donor substrates was purified and crystallized from the cell free extract of Escherichia freundii K–1. The purification procedures involved heat treatment, ammonium sulfate fractionation and DEAE-cellulose, hydroxylapatite, and CM-sephadex column chromatographies. The crystalline enzyme showed the sedimentation coefficient of 7.5 S and the diffusion coefficient of 6.15 × 10^?7 cm²/sec. The molecular weight was calculated to be 120,000. Several properties of the purified enzyme were also investigated. It was recognized that the transphosphorylation of pyridoxine might be catalyzed by the action of acid phosphatase.     

Steady state kinetics and effect of SH inhibitors on acid phosphatase from bovine brain.

1. Product inhibition studies and transphosphorylation to methanol using two different substrates indicate that acid phosphatase from bovine brain forms a phosphoryl enzyme and that the phosphorylation step can not be rate limiting. 2. Acid phosphatase from bovine brain is inhibited by 5,5'-dithiobis-(2-nitrobenzoic acid); this inhibition can be counteracted by inorganic phosphate. Incubation of the enzyme with p-nitrophenyl phosphate in the presence of p-chloromercuribenzoate leads, initially, to a higher degree of inhibition than that found with the same concentration of inhibitor in the absence of substrate. Both the titration by 5,5'-dithiobis-(2-nitrobenzoic acid) and inhibition by p-chloromercuribenzoate are consistant with the presence of 2 SH groups per mol of enzyme.     

Isolation and characterization of a human colon carcinoma-secreted enzyme with pancreatic ribonuclease-like activity

A ribonuclease was isolated from serum-free supernatants of the human colon adenocarcinoma cell line HT-29. It was purified by cation-exchange and C18 reversed-phase high-performance liquid chromatography. The protein is basic, has a molecular weight of approximately 16,000, and has an amino acid composition that is significantly different from that of human pancreatic ribonuclease. The amino terminus is blocked, and the carboxyl-terminal residue is glycine. The catalytic properties of this ribonuclease resemble those of the pancreatic ribonucleases in numerous respects. Thus, it exhibits a pH optimum of approximately 6 for dinucleotide cleavage and employs a two-step mechanism in which transphosphorylation to a cyclic 2',3'-phosphate is followed by slower hydrolysis to produce a 3'-phosphate. It does not cleave NpN' substrates in which adenosine or guanosine is at the N position and prefers purines at the N' position. Like bovine ribonuclease A, the HT-29-derived ribonuclease is inactivated by reductive methylation or by treatment with iodoacetate at pH 5.5 and is strongly inhibited by the human placental ribonuclease inhibitor. However, in contrast, the tumor enzyme does not cleave CpN bonds at an appreciable rate and prefers poly(uridylic acid) as substrate 1000-fold over poly(cytidylic acid). It also hydrolyzes cytidine cyclic 2',3'-phosphate at least 100 times more slowly than uridine cyclic 2',3'-phosphate and is inhibited much less strongly by cytidine 2'-monophosphate than by uridine 2'-monophosphate. Other ribonucleases known to prefer poly(uridylic acid) were isolated both from human serum and from liver and were compared with the tumor enzyme. The physical, functional, and chromatographic properties of the serum ribonuclease are essentially identical with those of the tumor enzyme. The liver enzymes, however, differ markedly from the HT-29 ribonuclease. The potential utility of the tumor ribonuclease in the diagnosis of cancer is considered.     

Low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase in the developing chick brain: partial characterization and levels during development.

Low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase is largely expressed in chick brain tissue during development. The enzyme was purified from brain extract prepared from 19-day-old chick embryos and from adult chickens using ammonium sulfate fractionation, gel filtration on Sephadex G-75 and two DEAE-Cellulose ion-exchange chromatography steps. The purified enzymes from embryo and adult chick brains show identical molecular weight values (about 18-20 kDa) and biochemical and structural properties such as substrate specificity, sensitivity to inhibitors, and number of free reactive sulphydryl groups. These data suggest that they are the same enzyme protein. Although the total acid phosphatase activity does not change appreciably during development, the activity associated with the low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase markedly increases after birth and reaches the adult values within the first week of life. Taken together, our results suggest an involvement of the low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase in postnatal development and maturation of chick brain tissue. The variations in tyrosine phosphorylation profile of chick brain polypeptides analyzed by Western blotting at the same developmental stages are also reported.     

An 18 kDa Acid Phosphatase from Chicken Heart Possesses Phosphotransferase Activity

A low molecular weight acid phosphatase was purified to homogeneity from chicken heart with a specific activity of 42 U/mg and a recovery of about 1%. Nearly 800 fold purification was achieved. The molecular weight was estimated to be 18 kDa by SDS-polyacrylamide gel electrophoresis. Para-nitrophenyl phosphate, phenyl phosphate and flavin mononucleotide were efficiently hydrolysed by the enzyme and found to be good substrates. Fluoride and tartrate had no inhibitory effect while phosphate, vanadate and molybdate strongly inhibited the enzyme. The acid phosphatase was stimulated in the presence of glycerol, ethylene glycol, methanol, ethanol and acetone, which reflected the phosphotransferase activity. When phosphate acceptors such as ethylene glycol concentrations were increased, the ratio of phosphate transfer to hydrolysis was also increased, demonstrating the presence of a transphosphorylation reaction where an acceptor can compete with water in the rate limiting step involving hydrolysis of a covalent phospho enzyme intermediate. Partition experiments carried out with two substrates, para-nitrophenyl phosphate and phenyl phosphate, revealed a constant product ratio of 1.7 for phosphotransfer to ethylene glycol versus hydrolysis, strongly supporting the existence of common covalent phospho enzyme intermediate. A constant ratio of K _cat/K _m, 4.3×10⁴, found at different ethylene glycol concentrations, also supported the idea that the rate limiting step was the hydrolysis of the phospho enzyme intermediate.     

Subcellular localization of high- and low-molecular weight acid phosphatases from chicken liver

The subcellular localization of high and low molecular weight acid phosphatases in chicken liver was studied. The high molecular weight acid phosphatase is mainly associated with the particulate fraction, particularly with the mitochondrial-lysosomal fraction, whereas the low molecular weight form seems to be a soluble cytoplasmic enzyme. Biochemical properties including optimal pH, molecular weight determination and the effect of some modifier substances indicate that mitochondria-lysosomes and microsomes contain the same high molecular weight acid phosphatase form.     

A single amino acid substitution in the activation loop defines the decoy characteristic of VEGFR-1/FLT-1

VEGFR-1 is a kinase-defective receptor tyrosine kinase (RTK) and negatively modulates angiogenesis by acting as a decoy receptor. The decoy characteristic of VEGFR-1 is required for normal development and angiogenesis. To date, there is no molecular explanation for this unusual characteristic of VEGFR-1. Here we show that the molecular mechanisms underlying the decoy characteristic of VEGFR-1 is linked to the replacement of a highly conserved amino acid residue in the activation loop. This amino acid is highly conserved among all the type III RTKs and corresponds to aspartic acid, but in VEGFR-1 it is substituted to asparagine. Mutation of asparagine (Asn(1050)) within the activation loop to aspartic acid promoted enhanced ligand-dependent tyrosine autophosphorylation and kinase activation in vivo and in vitro. The mutant VEGFR-1 (Asp(1050)) promoted endothelial cell proliferation but not tubulogenesis. It also displayed an oncogenic phenotype as its expression in fibroblast cells elicited transformation and colony growth. Furthermore, mutation of the invariable aspartic acid to asparagine in VEGFR-2 lowered the autophosphorylation of activation loop tyrosines 1052 and 1057. We propose that the conserved aspartic acid in the activation loop favors the transphosphorylation of the activation loop tyrosines, and its absence renders RTK to a less potent enzyme by disfavoring transphosphorylation of activation loop tyrosines.     

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.     

Comparative effects of fluoride on three enzymes, hydrolyzing pyrophosphate - acid and alkaline phosphatases and inorganic pyrophosphatase

The effects of fluoride on the activities of acid phosphatase (EC 3.1.3.2) from potato and alkaline phosphatase (EC 3.1.3.1) from E. coli during pyrophosphate and p-nitrophenylphosphate hydrolysis and on the activities of inorganic pyrophosphatase (EC 3.6.1.1) from baker's yeast during pyrophosphate hydrolysis were compared. For both phosphatases the type of interaction was found to be independent on the nature of substrate. For acid phosphatase and inorganic pyrophosphatase the inhibition was of non-competitive and uncompetitive types, respectively. In the case of alkaline phosphatase fluoride increased the rate of p-nitrophenol release during p-nitrophenylphosphate hydrolysis at pH greater than or equal to 7.9 without affecting the rate of phosphate release, which is indicative of fluorophosphate formation in the course of the transphosphorylation reaction. The data obtained suggest the existence of essential differences in the mechanisms of fluoride effects on the three enzymes under study.     

The presence of a low molecular weight acid phosphatase in liver tissue that cannot be demonstrated with the histochemical substrate naphthol AS-BI phosphate

Summary Three distinct isoenzymes of acid phosphatase have been separated from extracts of liver tissue of rats by gel filtration. These isoenzymes have molecular weights of 180,000±35,000; 74,000±11,000 and 13,000±2,500. High molecular weight isoenzymes and a low molecular weight isoenzyme of acid phosphatase (molecular weight 13,000±2,100) were also present in extracts of normal human and mouse liver tissue, and of pathologically altered liver tissue of mice in which the activity of acid phosphatase was strongly increased as a result of intraperitoneal injections of dextran solutions. Activity of acid phosphatase was determined with three substrates. The isoenzymes showed different conversion rates for the three substrates. The high molecular weight isoenzymes split the substrates 4-methylumbelliferyl phosphate, p-nitrophenyl phosphate and naphthol AS-BI phosphate. The activity was sensitive to the inhibitors fluoride and L(+)tartrate. In the pathologically altered liver tissue, which had stored dextran, the activity of these isoenzymes was strongly increased. The low molecular weight isoenzyme split 4-methylumbelliferyl phosphate and p-nitrophenyl phosphate but not naphthol AS-BI phosphate. Therefore this isoenzyme cannot be demonstrated with histochemical techniques using the substrate naphthol AS-BI phosphate. In contrast to the activity of the high molecular isoenzymes the activity of the low molecular isoenzyme was not changed in the pathologically altered liver tissue of mice and was not sensitive to the inhibitors fluoride and L(+)tartrate.This study was supported by a grant from the Prinses Beatrix Fonds, s'Gravenhage     

Seasonal Changes in Molecular Forms of Acid Phosphatase and Ribonuclease of Potato Tubers and the Effects of Infection by Phytophthora erythroseptica

Sequential changes in total activity and molecular forms ofacid phosphatase and ribonuclease from potato tubers were studiedby seasonal assays and Sephadex gel filtration. Ribonucleaseand p-nitrophenyl phosphatase activity fluctuated during storageof tubers, while ß-glycerophosphatase declined toa low level coincident with initiation of sprout growth. Inrecently-lifted tubers acid phosphatase activity occurred ina single high molecular weight peak. Two new forms of lowermolecular weight appeared during ageing of stored tubers. Theinfluence of infection by a tuber-rotting fungus, Phytophthoraerythroseptica,on these seasonal changes was variable. No consistent effectson total hydrolase activities were observed, while post-infectionalchanges in molecular forms included a pronounced shift in themajor acid phosphatase peak. The possible significance of thismolecular weight change in infected samples is discussed inthe light of recent evidence concerning the sub-unit structureof acid phosphatase from potato tubers.     

Multiple acid phosphatases in avian pectoral muscle--the postmicrosomal supernatant acid phosphatase is elevated in avian dystrophic muscle

There are at least three forms of acid phosphatase in avian pectoralis muscle differing in molecular weight, subcellular location, and response to various substrates and inhibitors. These enzymes are separated by differential sedimentation into postmicrosomal supernatant, lysosomal, and microsomal activities with apparent molecular weights in Triton X-100 of 68,000, 198,000, and 365,000, respectively. All of the enzymes show acid pH optima (pH approximately 5), but the postmicrosomal supernatant form is distinctly different from the other two forms in its resistance to most common phosphatase inhibitors and in its reduced activity against several organic phosphates. Quantitation of these three forms of acid phosphatase in normal and dystrophic avian pectoralis muscle shows that the postmicrosomal supernatant form is significantly elevated in dystrophic muscle; at 33 days ex ovo, 84% of the increased acid phosphatase activity in dystrophic muscle can be attributed to the postmicrosomal supernatant form. The microsomal form is only slightly elevated; the level of the lysosomal form is not altered.     

Glycoprotein nature of yeast alkaline phosphatase. Formation of active enzyme in the presence of tunicamycin.

The nonspecific alkaline phosphatase of yeast (Saccharomyces strain 1710) has been purified by ion exchange, hydrophobic, and affinity chromatography. This vacuolar enzyme has a molecular weight of 130,000 and is composed of subunits (probably of 66,000 molecular weight). It also has a small quantity of covalently associated carbohydrate; hydrolysis yielded mannose and glucosamine. The endo-beta-N-acetylglucosaminidase of Streptomyces plicatus released carbohydrate indicating that the latter was attached to protein through an N-acetylglucosaminylasparginyl bond. Synthesis of active alkaline phosphatase by yeast protoplasts is not depressed by tunicamycin, an inhibitor of dolichol-mediated protein glycosylation. Unlike the enzyme normally produced, the alkaline phosphatase which is formed in the presence of the antibiotic does not interact with concanavalin A and, therefore is deficient in or lacking carbohydrate. We infer that there is no regulatory link in yeast between the glycosylation of a protein and its synthesis. The fact that other Asn-GlcNAc-type glycoprotein enzymes of yeast such as acid phosphatase are not produced in their active forms by tunicamycin-treated protoplasts may mean that, as unglycosylated proteins, they cannot be correctly folded or processed. Protoplasts derepressed for phosphatase production contained substantial amounts of a second alkaline phosphatase which differed from the purified enzyme in substrate specificity, sensitivity to calcium, and reactivity with concanavalin A.     

Studies on Vitamin B6 Metabolism in Microorganisms

Escherichia freundii alkaline phosphatase was found in a membrane fraction and was purified by procedures involving spheroplast formation with lysozyme and EDTA, and DEAE-cellulose and Sephadex G-150 column chromatographies. Then this enzyme along with other phosphatases was investigated on the ability to transfer the phosphoryl group from p-nitrophenyl phosphate to pyridoxine. It was found that the ability of the transphosphorylation varied with these phosphatases. The transphosphorylation to hydroxy compounds such as alcohols, sugars and nucleosides was also compared. Escherichia freundii acid phosphatase showed the highest activity of transphosphorylation among phosphatases tested. The mechanism of transphosphorylation was discussed.

An enzyme, pyridoxamine 5′-phosphate transaminase, was purified from the cell-free extract of Clostridium kainantoi. The purification procedures involved ammonium sulfate fractionation, protamine sulfate treatment and, DEAE-cellulose, hydroxylapatite, DEAE-Sephadex and Sephadex G-200 column chromatographies. The purified enzyme, which had approximately 2700-fold higher specific activity over the original extract, showed a single schlieren pattern in the ultracentrifuge. From the spectral analysis, it seemed that pyridoxamine 5′-phosphate transaminase did not contain pyridoxal 5′-phosphate as a prosthetic group. It was recognized that the transamination was accelerated by the addition of amino acid and was inhibited by diisopropyl phosphofluoride. Glutamic acid formed in the reaction was identified to be a D-isomer. A study on the substrate specificity showed that the enzyme might be possible to be specific for pyridoxamine 5′-phosphate.

The extracellular formation of vitamin B₆ was searched in marine and terrestrial microorganisms. Two bacterial strains were selected and were identified as Vibrio and Flavobacterium, respectively. Marine microorganisms showed the considerable formation of vitamin B₆ and the presence of vitamin B₆ in sea water was also recognized. The cultural and reaction conditions for vitamin B₆ formation by these strains were investigated. Glycerol was commonly the most effective compound on vitamin B₆ formation among the compounds tested. It was suggested that both bacteria did not have the control system on vitamin B₆ biosynthesis by the amount of possible end products.     

Regulation of plant pyruvate dehydrogenase complex by phosphorylation

The ATP-dependent inactivation of the pyruvate dehydrogenase complex (PDC) was examined using ruptured mitochondria and partially purified pyruvate dehydrogenase complex isolated from broccoli and cauliflower (Brassica oleracea) bud mitochondria. The ATP-dependent inactivation was temperature- and pH-dependent. [(32)P]ATP experiments show a specific transphosphorylation of the gamma-PO(4) of ATP to the complex. The phosphate attached to the PDC was labile under mild alkaline but not under mild acidic conditions. The inactivated-phosphorylated PDC was not reactivated by 20 mm MgCl(2), dialysis, Sephadex G-25 treatment, apyrase action, or potato acid phosphatase action. However, partially purified bovine heart PDC phosphatase catalyzed the reactivation and dephosphorylation of the isolated plant PDC. The ATP-dependent inactivation-phosphorylation of the PDC was inhibited by pyruvate. It is concluded that the ATP-dependent inactivation-phosphorylation of broccoli and cauliflower mitochondrial PDC is catalyzed by a PDC kinase. It is further concluded that the PDC from broccoli and cauliflower mitochondria is capable of interconversion between an active (dephosphorylated) and an inactive (phosphorylated) form.     

Rate-determining step of Escherichia coli alkaline phosphatase altered by the removal of a positive charge at the active center

Escherichia coli alkaline phosphatase catalyzes both the nonspecific hydrolysis of phosphomonoesters and a transphosphorylation reaction in which phosphate is transferred to an alcohol via a phosphoseryl intermediate. The rate-determining step for the wild-type enzyme is pH dependent. At alkaline pH, release of the product phosphate from the noncovalent enzyme-phosphate complex determines the reaction rate, whereas at acidic pH hydrolysis of the covalent enzyme-phosphate complex controls the reaction rate. When the lysine at position 328 was substituted with a cysteine (K328C), the rate-determining step at pH 8.0 of the mutant enzyme was altered so that hydrolysis of the covalent intermediate became limiting rather than phosphate release. The transphosphorylation activity of the K328C enzyme was selectively enhanced, while the hydrolysis activity was reduced compared to that of the wild-type enzyme. The ratio of the transphosphorylation to the hydrolysis activities increased 28-fold for the K328C enzyme in comparison with the wild-type enzyme. Several other mutant enzymes for which a positive charge at the active center is removed by site-specific mutagenesis share this characteristic of the K328C enzyme. These results suggest that the positive charge at position 328 is at least partially responsible for maintaining the balance between the hydrolysis and transphosphorylation activities and plays an important role in determining the rate-limiting step of E. coli alkaline phosphatase.     

Characterization of alkaline phosphatase PhoK from Sphingomonas sp. BSAR-1 for phosphate monoester synthesis and hydrolysis

The biocatalytic activity of a so far underexploited alkaline phosphatase, PhoK from Sphingomonas sp. BSAR-1, was extensively studied in transphosphorylation and hydrolysis reactions. The use of high-energy phosphate donors and oligophosphates as suitable phosphate donors was evaluated, as well as the hydrolytic activity on a variety of phosphate monoesters. While substrates bearing free hydroxy group displayed only moderate reactivity as acceptors for transphosphorylation by PhoK, strong hydrolytic activity on a broad variety of phosphate monoesters under alkaline conditions was observed. Site-directed mutagenesis of selected amino acid residues in the active site provided valuable insights on their involvement in enzyme catalysis. The key residue Thr89 so far postulated to engage in enzyme phosphorylation was confirmed to be crucial for catalysis and could be replaced by serine, albeit with much lower catalytic efficiency.     

Sequencing, cloning, and expression of human red cell-type acid phosphatase, a cytoplasmic phosphotyrosyl protein phosphatase.

Low molecular weight phosphotyrosyl protein phosphatases of human placenta and human red cell were purified and sequenced by a combination of Edman degradation and tandem mass spectrometry. Screening of a human placental lambda gt11 cDNA library yielded overlapping cDNA clones coding for two distinct human cytoplasmic low molecular weight phosphotyrosyl protein phosphatases (HCPTPs). The two longest clones, designated HCPTP1-1 and HCPTP2-1, were found to have identical nucleotide sequences, with the exception of a 108-base pair segment in the middle of the open reading frame. Polymerase chain reaction studies with human genomic DNA suggest that the difference between HCPTP1-1 and HCPTP2-1 does not result from alternative RNA splicing. Studies with a human chromosome 2-specific library confirmed that these sequences are located on chromosome 2, which is known to be the location of red cell acid phosphatase locus ACP1. The coding sequences of HCPTP1-1 and HCPTP2-1 were placed downstream from a bacteriophage T7 promoter and the proteins were expressed in Escherichia coli. The resulting recombinant enzymes (designated HCPTP-A and HCPTP-B, respectively) showed molecular weights of 18,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and both of them exhibited immunoreactivity with antisera raised against authentic human placental and bovine heart enzymes. The expressed proteins were highly active towards the phosphatase substrates p-nitrophenyl phosphate, beta-naphthyl phosphate, and O-phospho-L-tyrosine, but not alpha-naphthyl phosphate, threonine phosphate, or O-phospho-L-serine. HCPTP-A and -B possessed effectively identical amino acid compositions, immunoreactivities, inhibition by formaldehyde, and kinetic properties when compared with two human red cell acid phosphatase isoenzymes. It is concluded that HCPTP-A and -B are the fast and slow forms of red cell acid phosphatase, respectively, and that this enzyme is not unique to the red cell but is instead expressed in all human tissues.     

The effects of hypoxanthine on methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells

When cultured human choriocarcinoma (BeWo) cells are exposed to methotrexate, proliferation ceases and cells undergo a complex differentiative response that resembles development of normal trophoblast. Although thymidylate starvation has been shown to be causative in methotrexate-induced expression of syncytiotrophoblastic markers by BeWo cells, the role of purine deprivation is uncertain since previous studies utilized growth media containing exogenous purines. This work investigated the effects of hypoxanthine on methotrexate-induced cell enlargement, expression of placental alkaline phosphatase, and morphological differentiation to the syncytiotrophoblast-like phenotype. When methotrexate exposures (1 microM, 48 h) were conducted in a purine-free basal medium supplemented with dialyzed fetal bovine serum, RNA synthesis was greatly reduced and cell enlargement did not occur. Specific methods for removing purines (charcoal extraction and xanthine oxidase treatment) decreased the ability of serum to support cell enlargement during methotrexate exposures, whereas addition of hypoxanthine to culture fluids restored its ability to support maximal increases in cell mass, confirming that purines were the factors lost during dialysis. In contrast, morphologically differentiation to the syncytiotrophoblast-like phenotype and increased expression of placental alkaline phosphatase were unaffected by the availability of purines during exposure to methotrexate.     

Tartrate-resistant acid phosphatase of human lung: apparent identity with osteoclastic acid phosphatase

Extracts of human lung tissue contain appreciable activities of a tartrate-resistant acid phosphatase which is apparently identical with the analogous enzyme in bone extracts, with respect to electrophoretic mobility, apparent molecular weight (ca. 37,000), Michaelis constants and relative rates of hydrolysis of various substrates. The acid phosphatase appears to be a constituent of alveolar macrophages. Lung provides a convenient source for the preparation of tartrate-resistant acid phosphatase.