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PubMed1991

Protein phosphatases in cell division: how vital are they?

In contrast to the wealth of information on cellular function of protein kinases, many of which are known to be the products of proto-oncogenes, little is known about how protein dephosphorylation is involved in growth control of normal and malignant cells. In the present study, roles of protein phosphatases in cell division cycle control were examined by molecular genetic approaches using a lower eukaryote, the fission yeast Schizosaccharomyces pombe. Nine protein phosphatase genes have been so far identified and characterized in this organism. Each of two (dis2+, sds21+, and ppa1+, ppa2+) gene products is highly similar to mammalian type 1 and 2A ser/thr phosphatases, respectively. The ppx1+ product is an intermediate of type 1 and 2A, while the ppb1+ product is similar to Ca(2+)-dependent type 2B. At least two protein tyrosine phosphatase genes (pyp1+ and pyp2+) exist. The cdc25 protein is now established to be a tyrosine phosphatase that activates cdc2 kinase. Some of these phosphatase genes are interrelated but have distinct, essential functions in cell cycle control. Missense mutations, deletions or high dosage expression of these phosphatase genes affect entry into and exit from mitosis, mitotic chromosome disjunction, cell size and cell shape. They seem to interact with the main regulators of mitosis, cdc2, cdc13/cyclin, cdc25 and weel, or with mitotic structural components, such as condensed chromosomes or the spindle apparatus. We show that the product of an essential gene, sds22+, is an important, positive factor in controlling the expression and modulating the activity of dis2 phosphatase.

Amino Acid Sequence↗
PubMed1976

Hypophosphatasia: a cytochemical study of phosphatase activities.

Skeletal abnormalities with defective formation of mature calcified bone are the most prominent clinical features of hypophosphatasia. Low concentrations of serum and tissue alkaline phosphatase and elevated plasma and urinary levels of phosphorylethanolamine (PEA) are also present. Although PEA is hydrolyzed by serum alkaline phosphatase, the relationship between PEA and the deficiency is unclear. PEA has not previously been tested as a cytochemical substrate for the in situ demonstration of human alkaline phosphatase activity. We have studied alkaline phosphatase activity in hypophosphatasia in tissue sections, utilizing PEA and adenosinetriphosphate (ATP) as well as the usual beta-glycerophosphate and naphthol phosphate substrates. Neutral and acid phosphatase activities were also examined. Our results demonstrate that PEA is a substrate for the localization of alkaline phosphatase in normal human tissue, but is not hydrolyzed in hypophosphatasia in the liver, brain or costochondral junction under alkaline conditions. In the kidney in hypophosphatasia only the straight segments of proximal tubules that rim the medullary rays are reactive with PEA. Similar results in hypophosphatasia were obtained at an alkaline pH with ATP, beta-glycerophosphate, and naphthol phosphate. However, the defect in hypophosphatasia is not a generalized deficiency of membrane-associated phosphatases because membranes that were deficient in alkaline phosphatase activity demonstrated normal reactivity with ATP at neutral pH. In addition, thiamine pyrophosphate was also split by Golgi membranes within the cytoplasm. Acid hydrolysis of beta-glycerophosphate by lysosomes was normal.

Alkaline Phosphatase↗
PubMed1976

Phosphohistone and glycogen synthase D phosphatase activities in a liver glycogen pellet preparation.

A liver glycogen pellet preparation previously found to contain synthase D phosphatase activity was shown to contain also phosphohistone phosphatase activity. Pellet phosphohistone phosphatase and synthase D phosphatase competed for the same substrates and appeared to be the same enzyme. ATP, a potent inhibitor, and G-6-P, a potent activator of the synthase phosphatase reaction, had little effect on the phosphohistone phosphatase reaction. These observations suggest that the ATP and G-6-P effects are relatively specific and are probably caused by binding to the synthase D substrate. The observed effects of NaCl and KCl were more complex. They stimulated phosphohistone phosphatase activity but strikingly inhibited synthase phosphatase activity. Sodium fluoride inhibited both reactions.

Adenosine Triphosphate↗
PubMed1990

Characterization of microcystin-LR, a potent inhibitor of type 1 and type 2A protein phosphatases.

The level of protein phosphorylation is dependent on the relative activities of both protein kinases and protein phosphatases. By comparison with protein kinases, however, there have been considerably fewer studies on the functions of serine/threonine protein phosphatases. This is partly due to a lack of specific protein phosphatase inhibitors that can be used as probes. In the present study we characterize the inhibitory effects of microcystin-LR, a hepatotoxic cyclic peptide associated with most strains of the blue-green algae Microcystis aeruginosa found in the Northern hemisphere, that proves to be a potent inhibitor of type 1 (IC50 = 1.7 nM) and type 2A (IC50 = 0.04 nM) protein phosphatases. Microcystin-LR inhibited the activity of both type 1 and type 2A phosphatases greater than 10-fold more potently than okadaic acid under the same conditions. Type 2A protein phosphatases in dilute mammalian cell extracts were found to be completely inhibited by 0.5 nM microcystin-LR while type 1 protein phosphatases were only slightly affected at this concentration. Thus, microcystin-LR may prove to be a useful probe for the study and identification cellular processes which are mediated by protein phosphatases.

Animals↗
PubMed1985

Characterization of a calmodulin-dependent protein phosphatase from human platelets.

A calmodulin-dependent protein phosphatase has been identified in human platelets by its cross-reactivity with an antibody developed against a bovine brain calmodulin-dependent protein phosphatase and by its calmodulin-stimulated dephosphorylation of 32P-labeled substrates. The platelet enzyme was partially purified to separate it from calmodulin and calmodulin-independent phosphatases. The partially purified enzyme was stimulated by calmodulin, requiring 15 nM calmodulin for half-maximal activation. Calmodulin increased the Vmax of the phosphatase, with no significant effect on its Km. The enzyme was stimulated irreversibly and made calmodulin-independent by limited proteolysis. The optimal pH for the phosphatase was 7.5. After partial purification, phosphatase activity was significantly increased in the presence of Mn2+ and Ca2+ over that observed in the presence of Ca2+ alone. The enzyme effectively dephosphorylated casein, histone, protamine, and platelet actin. The holophosphatase was estimated to have a molecular weight of 76,900 as determined by sedimentation on sucrose gradients. Immunoblotting techniques using an antibody against the brain phosphatase suggests that the enzyme consists of 2 subunits of 60,000 and 16,500 daltons; the 60,000-dalton subunit co-migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 60,000-dalton calmodulin-binding protein in the platelet suggesting that it is the calmodulin-binding subunit of the enzyme. The identification of a calmodulin-dependent protein phosphatase in human platelets suggests a role for Ca2+-dependent dephosphorylation in platelet activation.

Animals↗
PubMed1988

The acid phosphatases of Thermoascus crustaceus, a thermophilic fungus.

Thermoascus crustaceus, a filamentous, thermophilic ascomycete with pathogenic potential was cultured on Sabouraud's liquid medium at temperatures from 27 to 47 degrees C for periods up to 7 days. Growth rate and yield were optimal at 37 degrees C. Morphological changes were confined to the cell walls, the thickness being greatest at 47 degrees C, which were also more resistant to mechanical disruption. Significant amounts of acid phosphatase (EC 3.1.3.2) activity occurred in the spent media of all cultures but were greatest at 37 degrees C. The proportions of acid phosphatase activity which were operationally defined as soluble or bound were also documented; the optimum pH for acid phosphatase activity in all fractions was 5.0. Extracts were subjected to polyacrylamide gel electrophoresis under non-denaturing conditions and the gels were stained for acid phosphatase activity. This revealed four electrophoretically distinct acid phosphatases which had different susceptibilities to inhibition by fluoride, phosphate, or tartrate. Effects of growth temperature, or phosphate supplement in the culture medium, on the acid phosphatase isoenzyme pattern were judged to be minor. Cytochemistry at the electron microscope level indicated acid phosphatase activity on the surface, in the periplasmic space, and in the cytoplasm, but no trends with regard to growth conditions. A substantial temperature range can be tolerated by this species but it is concluded that neither the general shape of the cells nor the acid phosphatase isoenzyme pattern changes substantially; this contrasts with previously documented differences for this class of enzyme in dimorphic Sporotrix schenckii.

Acid Phosphatase↗
PubMed1979

Release of alkaline phosphatase activity by aqueous-ether treatment of whole cells of Serratia marcescens.

The effect of aqueous-ether treatment according to the method of Ribi et al. (1961) on the release of alkaline phosphatase from cells of two strains of Serratia marcescens was studied. By this method, lipopolysaccharide-protein (endotoxin) complexes associated with alkaline phosphatase activities were released from both strain 08 and strain Bizio. SDS-polyacrylamide gel electrophoresis followed by enzymatic assay showed the presence of two active components in each strain. Fractions released from strain 08 contained alkaline phosphatase A (140,000 dalton) and alkaline phosphatase B (110,000) daltons) while those from strain Bizio contained alkaline phosphatase A' (190,000 daltons) and alkaline phosphatase B (110,000 daltons). Although it is known that saline plays a role in the release of alkaline phosphatase activities from cell envelope of Gram-negative bacteria the presence of saline in the extracting medium affects only slightly the chemical composition and not at all on the enzymatic nature of the released components. By comparing the enzymatic profiles of the materials released by other techniques, such as polymyxin B treatment and osmotic shock, it appears that alkaline phosphatase activities released by aqueous-ether treatment of whole cells of S. marcescens originate from the periplasmic space.

Alkaline Phosphatase↗
PubMed1984

Cytosolic protein phosphotyrosine phosphatases from rabbit kidney. Purification of two distinct enzymes that bind to Zn2+-iminodiacetate agarose.

Cytosolic protein phosphotyrosine (PPT) phosphatase was measured using a new substrate, Tyr(32P)-labeled bovine serum albumin. Kidney was found as a particularly rich tissue source of PPT-phosphatase activity, containing twice as much as liver and over 10-fold more than brain, heart, lung, or skeletal muscle. An affinity column of Zn2+-iminodiacetate agarose adsorbed up to 60% of the PPT-phosphatase present in kidney extracts. Subsequent chromatography on DEAE-Sepharose separated the phosphatase into two peaks, labeled I and II, that had Mr = 34,000 and 37,000, respectively, upon gel filtration with Sephadex G-75 Superfine. Overall purification of 850- and 1100-fold was achieved with a net 4% yield. Both phosphatases hydrolyzed p-nitrophenylphosphate as well as the protein substrate in the presence of EDTA. Peak I phosphatase activity displayed a neutral pH optimum, had an absolute requirement for sulfhydryl compounds, and was sensitive to trypsin, whereas Peak II activity had an acidic pH optimum and was active without mercaptans. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with S. aureus V8 protease. The results show that multiple forms of PPT phosphatase specifically interact with Zn2+ and provide a basis for further structural and functional comparisons among different members of the phosphoprotein phosphatase family.

4-Nitrophenylphosphatase↗
PubMed1982

Preoperative bilirubin, alkaline phosphatase and amylase levels as predictors of common duct stones.

The predictive value of the preoperative level of bilirubin, alkaline phosphatase and amylase as indicators of choledocholithiasis was determined by prospectively evaluating 304 consecutive patients undergoing cholecystectomy. Elevated levels of bilirubin and alkaline phosphatase are associated with an increased incidence of common duct stones, and the percentage incidence of stones increases with rising bilirubin and alkaline phosphatase levels. Alkaline phosphatase levels as great as 200 are associated with common duct stones in a low percentage of instances, being equivalent to that for unsuspected stones. Levels of 200 or greater are associated with a marked increase in the incidence of common duct stones. An elevated serum or urine amylase level, or both, is of little, if any, value as a predictor of common duct stones. Alkaline phosphatase appears to be a better indicator of common duct stones than does bilirubin, but neither bilirubin nor alkaline phosphatase in themselves are statistically significant indicators. Bilirubin and alkaline phosphatase in combination is a statistically significant predictor of common duct stones at all levels. The combination of a bilirubin level of greater than 3.0 and an alkaline phosphatase level of greater than 250 has a 76.2 per cent probability of an associated common duct stone. The quite important role of operative cholangiography in demonstrating unsuspected stones and in preventing unnecessary common duct explorations is reinforced.

Adult↗
PubMed1981

Periplasmic phosphatases in Salmonella typhimurium LT2. A biochemical, physiological, and partial genetic analysis of three nucleoside monophosphate dephosphorylating enzymes.

Three periplasmic nucleoside monophosphate-splitting phosphatases from Salmonella typhimurium, 2':3'-cyclic nucleotide 2'-phosphodiesterase, nonspecific acid phosphatase I, and nonspecific acid phosphatase II, were separated by column chromatography. They are characterized with respect to their substrate specificities, Km values, pH optima, molecular weights, and sensitivity to inhibition by Pi and EDTA. Nonspecific acid phosphatase II has not been reported previously. The physiological roles of the various phosphatases are assessed by studies on mutant strains. Selection procedures were developed for the isolation of mutants defective in the synthesis of one or more of the phosphatase activities. Analysis of these mutant strains revealed that 2':3'-cyclic nucleotide 2'-phosphodiesterase is the major 3'-nucleotide dephosphorylating activity, and nonspecific acid phosphatase II is most active against 5'-nucleotides at concentrations below 1 mM. Triple mutants, lacking all periplasmic nucleotide-splitting activities, are viable. Regulatory mutants with elevated levels of nonspecific acid phosphatase II activity were isolated. The gene for 2':3'-cyclic nucleotide 2'-phosphodiesterase (pde) was located between purA and argI at 135 min on the S. typhimurium linkage map.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1983

Resolution and reassociation of three distinct components from pig heart phosphoprotein phosphatase.

A partially purified pig heart phosphoprotein phosphatase was dissociated into three distinct components, namely alpha, beta, and gamma, by gel filtration on Sephacryl S-200 followed by chromatography on DEAE-Sephadex in the presence of 6 M urea. Although alpha itself had phosphatase activities toward P-H2B histone, P-H1 histone, phosphorylase a, and glycogen synthase b, beta and gamma had no activity toward these substrates even in the presence of 1 mM Mn2+. The beta component (Mr = 80,000) combined with alpha (Mr = 31,000) in the absence of urea to produce Form 2 (Mr = 123,000) with concomitant increase in P-H1 histone phosphatase activity and Mg2+ requirement for P-H2B histone phosphatase activity (Imazu, M., Imaoka, T., Usui, H., Kinohara, N., and Takeda, M. (1981) J. Biochem. 90, 851-862). The gamma component (Mr = 62,000) reassociated with Form 2 to produce Form 1 (Mr = 199,000) which was similar to the original phosphoprotein phosphatase in substrate specificity and Mg2+ requirement. Binding of gamma to Form 2 strongly suppressed the phosphatase activities toward phosphorylase a and glycogen synthase b with marginal effects on the other phosphatase activities and Mg2+ requirement. However, gamma alone could not associate with alpha. The gamma component was sensitive to treatment with heat (60 degrees C for 2 min) or trypsin and was resistant to treatment with DNase or RNase. The pig heart phosphoprotein phosphatase was further purified to near homogeneity, as judged by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis revealed that the purified enzyme (Mr = 171,000) was composed of three polypeptide components, namely alpha', beta', and gamma' with molecular weights of 34,000, 69,000, and 56,000, respectively. The component stoichiometry was determined to be alpha' 1 beta' 1 gamma' 1 by densitometric tracing of the Coomassie blue-stained bands on the acrylamide gel. After dissociation of alpha ' and other components by gel filtration of the purified enzyme on Sephacryl S-200 in the presence of 6 M urea, one alpha ' combined with one beta' to produce Form 2' of Mr = 106,000. Since Form 1 and the purified enzyme as well as Form 2 and Form 2' had similar catalytic properties and s20,w values, respectively, component compositions are suggested to be alpha 1 beta 1 gamma 1 for Form 1 and alpha 1 beta 1 for Form Form 2.

Animals↗
PubMed1984

Effect of vanadate, a potent alkaline phosphatase inhibitor, on 45Ca and 32Pi uptake by matrix vesicle-enriched fractions from chicken epiphyseal cartilage.

Although alkaline phosphatase has been long associated with the mineralization process, its exact function remains to be elucidated. To clarify its possible role in matrix vesicle-mediated mineralization, we tested the effect of vanadate, a phosphate analogue and powerful competitive inhibitor of alkaline phosphatase activity, on calcium and phosphate uptakes by a matrix vesicle-enriched microsomal fraction. Vanadate was also tested in a hydroxyapatite-seeded ion uptake system to determine possible direct effects on mineral formation. The effect of vanadate on vesicle mineral ion uptake was complex; low dosages of vanadate (2-20 microM) were stimulatory to Ca2+ uptake, but were inhibitory to Pi. Higher dosages (greater than 67 microM) were inhibitory to both ions. The effect of vanadate on ion uptake was strongly influenced by the stage of vesicle loading; major effects were seen during the lag and early uptake phases, and minimal effects were seen in the terminal stages. Concentrations of vanadate highly inhibitory to vesicle ion uptake had minimal effects on ion accretion by a hydroxyapatite-seeded system. Inhibition of alkaline phosphatase activity by vanadate broadly paralleled inhibition of Pi and Ca2+ uptake; however, at low vanadate concentrations, inhibition of Pi uptake closely paralleled that of alkaline phosphatase. The data indicate that vanadate binds with high affinity to Pi-loading sites, blocking initial Pi uptake. Complexation between vanadate and Ca2+ may be responsible for the stimulation of Ca2+ uptake at early stages of vesicle ion loading with low levels of vanadate by enhancing binding of Ca2+ to the vesicles. It may also account for the selective inhibition of Ca2+ uptake during the rapid stage of vesicle ion loading with high levels of vanadate by reducing Ca2+ ion activity. The close parallelism between inhibition of early Pi uptake and of alkaline phosphatase activity supports the concept that alkaline phosphatase is involved in Pi transport during the early stages of matrix vesicle ion loading. However, the fact that only about half of the Pi uptake was affected by vanadate, despite the progressive inhibition of alkaline phosphatase activity, indicates that alkaline phosphatase is not solely responsible for Pi uptake by the matrix vesicle-enriched fraction.

Alkaline Phosphatase↗
PubMed1981

Immunochemical evaluation of the organ specificity of prostatic acid phosphatase.

In a survey of normal and cancerous human tissues we determined the distribution of immunoreactive prostatic acid phosphatase, using rabbit antiserum to acid phosphatase purified from prostatic fluid. In all normal tissues and blood cells studied except leukocytes we found less than 0.1% (expressed as micrograms per gram of wet weight of tissue) of the quantity of immunoreactive prostatic acid phosphatase detected in normal prostate tissue by radioimmunoassay. A small quantity of cross-reactive antigen (2.5 microgram/10(8) cells) was found in leukocytes. In all normal and cancerous nonprostate tissues surveyed by an immunohistochemical technique we detected no immunoreactive prostatic acid phosphatase, except in kidney tissue. Faint but reproducible staining was detected in the lumen of distal tubules and collecting ducts and within interstitial capillaries. Immunoreactive prostatic acid phosphatase was detected in the urine of pre- and post-pubertal males and females. We propose that this material is from serum (low concentrations of immunoreactive prostatic acid phosphatase are present in the serum of men and women) and that it is excreted into urine by the kidneys. Full proof of this must await future experimentation. The specificity of our antiserum for prostatic acid phosphatase was demonstrated by the fact that the Mr 100 000 and 20 000 liver acid phosphatase isoenzymes did not cross with our antiserum in either the radioimmunoassay or double-diffusion analysis. Similarly, preparations of isoenzymes 5A and 5B are human serum albumin did not cross react.

Acid Phosphatase↗
PubMed1982

Effect of skeletal alkaline phosphatase inhibitors on bone cell proliferation in vitro.

Chicken skeletal alkaline phosphatase is subject to competitive inhibitions by vanadate (Ki = 0.38 mM in carbonate, Ki = 0.08 mM in Tris, both at pH 7.4) and phenylphosphonate (Ki = 15 mM in carbonate, Ki = 1.3 mM in Tris, both at pH 7.4), and uncompetitive inhibition by levamisole (Ki = 0.08 mM in carbonate, Ki = 0.10 mM in Tris, both at pH 7.4). The competitive inhibitors were more effective in Tris buffer because nonreactive ternary complexes were formed between alkaline phosphatase, the inhibitor and Tris. The effects of vanadate, phenylphosphonate and levamisole on the proliferation of embryonic chick calvarial cells in vitro were biphasic. Low doses of each agent stimulated 3H-thymidine incorporation into TCA-insoluble material; higher doses were inhibitory. Neither effect could be attributed to inhibition of alkaline phosphatase activity (e.g. 20 microM vanadate should inhibit alkaline phosphatase by 3% but stimulated cell proliferation by 187%; 50 microM vanadate should inhibit alkaline phosphatase by 7% but inhibited 3H-thymidine incorporation by 90%). None of the alkaline phosphatase inhibitors tested affected the cellular concentration of the enzyme during the 24-hour incubation. These studies indicate that alkaline phosphatase inhibitors can have nonspecific effects on bone cells in culture, and that for cells in the osteoblast cell line, an inhibition of alkaline phosphatase activity is not consistently related to a decrease in cell proliferation.

Alkaline Phosphatase↗
PubMed1982

The effect of 1,25(OH)2D3 on alkaline phosphatase in osteoblastic osteosarcoma cells.

The effects of 1,25-dihydroxyvitamin D3 on growth, cell protein, and alkaline phosphatase were examined in cultures of a clonal osteoblast-like rat osteosarcoma cell line at different stages of phenotypic maturation, defined by the level of alkaline phosphatase activity. In "immature" cultures with low alkaline phosphatase specific activity, 1,25-dihydroxyvitamin D3 at 10(-8)-10(-7) M inhibited cell growth and elevated alkaline phosphatase activity; lower hormone concentrations had no effect. In cultures with higher alkaline phosphatase activity, the hormone had biphasic effects: at concentrations of 10(-8)-10(-7) M, 1,25-dihydroxyvitamin D3 inhibited growth and elevated alkaline phosphatase and total cell protein; however, lower hormone concentrations (10(-10) and 10(-9) M) reduced alkaline phosphatase activity. This effect was more pronounced in more "mature" cultures where half-maximum inhibition of alkaline phosphatase activity occurred near 10(-11) M. At this stage, the hormone caused a small increase in cell number. The data suggest that 1,25-dihydroxyvitamin D3 exerts differential effects on skeletal cells depending on their state of growth or maturation. This finding may offer new insights into the effects of 1,25-dihydroxyvitamin D3 on bone and may help resolve apparently conflicting experimental findings on the effects of the hormone on osteoblast-like cells in culture.

Alkaline Phosphatase↗
PubMed1978

Serum prostate-specific acid phosphatase: development and validation of a specific radioimmunoassay.

We describe radioimmunoassay for human prostatic acid phosphatase [orthophosphoric-monoester phospho-hydrolase (acid optimum), EC 3.1.3.2] in serum, with use of monospecific antisera raised in rabbits against highly purified acid phosphatase from human prostates. The antiserum did not cross react with partly purified acid phosphatases from human spleen, erythrocytes, or synovial tissues. 125I-labeled acid phosphatase was prepared by a Chloramine T method, and the bound and free antigen was separated in the assay by use of anti-rabbit gamma-globulin raised in sheep. Uniform low nonspecific binding of the [125I]acid phosphatase was achieved by using acid-phosphatase-free serum to prepare standard curves and diluted samples of serum with high acid phosphatase activities. Concentrations of immunoreactive acid phosphatase in the serum of healthy men ranged from less than 1 to 10 microgram/liter and for 12 patients with advanced prostatic carcinoma between 100 and 500 microgram/liter. The concentrations of the enzyme in sera of patients with benign prostatic hyperplasia were very similar to those in sera of the reference group.

Acid Phosphatase↗
PubMed1981

Altered form of placental alkaline phosphatase produced by JAR choriocarcinoma cells in culture.

The alkaline phosphatase activity expressed by JAR choriocarcinoma cells was compared to the placental isoenzyme of human alkaline phosphatase by several criteria. JAR cell alkaline phosphatase was similar to the placental isoenzyme with respect to heat and urea stability and sensitivity to most inhibitors, but it differed significantly from placental alkaline phosphatase in its sensitivity to L-phenylalanylglycylglycine. In contrast to the serum form of placental alkaline phosphatase, the JAR cell enzyme was highly hydrophobic as determined by octyl agarose chromatography and appeared to be larger than the placental isoenzyme based on gel filtration and polyacrylamide gel electrophoresis. The slowly migrating hydrophobic JAR cell activity could be converted into a fast-migrating hydrophilic form similar to the serum placental isoenzyme by treatment with trypsin-like proteases. Conversion to the hydrophilic form was accompanied by a decrease in subunit molecular weight from 68,000 to 66,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 33P-labeled enzyme. Peptide mapping of the 33P-labeled molecules by limited proteolysis in the presence of sodium dodecyl sulfate showed JAR cell alkaline phosphatase to be closely related to the placental isoenzyme with respect to primary structure. However, placental alkaline phosphatase appeared to be richer in sialic acid residues than was the JAR cell enzyme. We conclude that JAR cells produce a form of placental alkaline phosphatase that contains a hydrophobic region not associated with the free serum enzyme and that the tumor cell enzyme is modified or processed differently than is the enzyme found in the term placenta.

Alkaline Phosphatase↗
PubMed1994

Purification and characterization of a phosphatase from HeLa cells which dephosphorylates the C-terminal domain of RNA polymerase II.

The repetitive C-terminal domain (CTD) of RNA polymerase (RNAP) II is extensively phosphorylated concomitant with the initiation of transcription and must be dephosphorylated before RNAP II can begin another round of transcription. A CTD phosphatase was purified more than 7,500-fold from a HeLa cell extract. SDS-polyacrylamide gel electrophoresis shows a predominant protein of 205 kDa and a less abundant protein of 150 kDa co-eluting with the CTD phosphatase activity. Sedimentation and gel filtration analysis suggest that CTD phosphatase has an elongated structure with a M(r) of 200,000. This enzyme is a type 2C phosphatase in that it requires Mg2+ for activity and is resistant to okadaic acid. CTD phosphatase appears to processively dephosphorylate the CTD and is specific in that it does not dephosphorylate phosphorylase a, the alpha or beta subunits of phosphorylase kinase or RNAP II phosphorylated with casein kinase II. CTD phosphatase dephosphorylates RNAP IIO purified from calf thymus or generated in vitro by two previously described CTD kinases. These results suggest that CTD phosphatase has the properties expected for a protein phosphatase that catalyzes the conversion of RNAP IIO to RNAP IIA and may play a key role in the transcription cycle of RNAP II.

Ethers, Cyclic↗