Characterization of dolichol kinase from bovine thyroid microsomes

Bovine thyroid microsomes are able to phosphorylate exogenous [1-3H]dolichol as well as endogenous dolichol. The properties and specificity of the dolichol kinase activity have been studied by following the phosphorylation of [1-3H]dolichol to [1-3H]DMP as well as the formation of [32P]DMP from endogenous dolichol and [gamma-32P]CTP. The dolichol kinase activity was not linear with respect to time and exhibited a neutral pH-optimum. Product formation was directly proportional to microsomal protein concentration up to 2.5 mg protein/incubation. The enzyme was found to depend on divalent cations for activity: Mg2+-ions being much more effective than Ca2+- and Mn2+-ions. In accordance, EDTA was strongly inhibitory. The enzyme exhibited specificity for CTP as phosphoryl donor and was found to be inhibited by the reaction product CDP. The apparent Km-value for exogenous dolichol amounted to 4 microM. Those for CTP were estimated to be 3.88 and 10.75 mM with exogenous [1-3H]dolichol depending on the source of CTP. With endogenous dolichol Km-values for CTP of 27.8 and 6.1 microM were calculated in respectively the absence and presence of 5 mM VO4(3-). Triton X-100 (0.15%) was necessary in the [1-3H]dolichol kinase assay (only 3% of enzymatic activity in the absence of detergent), while with [gamma-32P]CTP dolichol kinase detergent was only of minor influence (30% stimulation at 0.02% Triton X-100). The levels of the enzymatic activity could be doubled by the inclusion of 18-21 mM NaF [( 1-3H]dolichol kinase) as phosphatase inhibitor: VO4(3-) had practically no effect. In contrast with [gamma-32P]CTP dolichol kinase, the enzymatic activity could be enhanced 4-fold by addition of 5 mM VO4(3-) while F- resulted into no appreciable effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix

An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.     

Identification and characterization of endothelin converting activity in cultured bovine endothelial cells

Using a specific and sensitive radioimmunoassay (RIA) for the carboxyl terminal tail of endothelin (ET) (His16-Trp21), we have confirmed the presence of the converting activity from synthetic human big ET-1 to ET-1 in the homogenate of cultured bovine aortic endothelial cells. The optimal pHs for the converting activities were found at pH 3.0 and pH 7.0. The activity at pH 3.0 was completely inhibited by pepstatin A, whereas the activity at pH 7.0 was not affected by known various protease inhibitors except EDTA and EGTA. When the products from big ET-1 were analyzed on an ODS and a CN columns, only ET-1 was detected at pH 7.0, but various ET-like immunoreactivities other than ET-1 were detected at pH 3.0. These findings strongly suggest that mature ET-1 is formed from big ET-1 in the endothelial cells by a metal-dependent neutral protease.     

Identification and functional characterization of a presqualene diphosphate phosphatase

Presqualene diphosphate (PSDP) is a bioactive lipid that rapidly remodels to presqualene monophosphate (PSMP) upon cell activation (Levy, B. D., Petasis, N. A., and Serhan, C. N. (1997) Nature 389, 985-990). Here, we have identified and characterized a phosphatase that converts PSDP to PSMP. Unlike the related polyisoprenyl phosphate farnesyl diphosphate (FDP), PSDP was not a substrate for type 2 lipid phosphate phosphohydrolases. PSDP phosphatase activity was identified in activated human neutrophil (PMN) extracts and partially purified in the presence of Nonidet P-40 with gel filtration and anion exchange chromatography. Peptide sequencing of a candidate phosphatase was consistent with phosphatidic acid phosphatase domain containing 2 (PPAPDC2), an uncharacterized protein that contains a lipid phosphate phosphohydrolase consensus motif. Recombinant PPAPDC2 displayed diphosphate phosphatase activity with a substrate preference for PSDP > FDP > phosphatidic acid. PPAPDC2 activity was independent of Mg(2+) and optimal at pH 7.0 to 8.0. Incubation of [(14)C]FDP with recombinant human squalene synthase led to [(14)C]PSDP and [(14)C]squalene formation, and in the presence of PPAPDC2, [(14)C]PSMP was generated from [(14)C]PSDP. PPAPDC2 mRNA was detected in human PMN, and is widely expressed in human tissues. Together, these findings indicate that PPAPDC2 in human PMN is the first lipid phosphate phosphohydrolase identified for PSDP. Regulation of this activity of the enzyme may have important roles for PMN activation in innate immunity.     

Identification, purification, and characterization of a novel serine/threonine protein phosphatase from bovine brain.

A novel serine/threonine protein phosphatase is identified, and the catalytic subunit, obtained from a detergent extraction of the pellet generated by a 100,000 x g centrifugation of a whole bovine brain homogenate, is purified and characterized. The protein phosphatase, designated as PP3, has a Mr of 36,000, does not require divalent cations for activity, is stimulated rather than inhibited by inhibitor 2, is inhibited by both okadaic acid and microcystin-LR with an intermediate IC50 compared to type 1 and type 2A protein phosphatases, and preferentially dephosphorylates the beta subunit of phosphorylase kinase. Substrate specificity, immunoblotting with type-specific antisera, and the amino acid sequences of peptides derived from PP3 indicate that PP3 is not an isoform of any known serine/threonine protein phosphatase.     

Identification and characterization of a plastidial phosphatidylglycerophosphate phosphatase in Arabidopsis thaliana

Phosphatidylglycerol (PG) is the only phospholipid in the thylakoid membranes of chloroplasts of plants, and it is also found in extraplastidial membranes including mitochondria and the endoplasmic reticulum. Previous studies showed that lack of PG in the pgp1‐2 mutant of Arabidopsis deficient in phosphatidylglycerophosphate (PGP) synthase strongly affects thylakoid biogenesis and photosynthetic activity. In the present study, the gene encoding the enzyme for the second step of PG synthesis, PGP phosphatase, was isolated based on sequence similarity to the yeast GEP4 and Chlamydomonas PGPP1 genes. The Arabidopsis AtPGPP1 protein localizes to chloroplasts and harbors PGP phosphatase activity with alkaline pH optimum and divalent cation requirement. Arabidopsis pgpp1‐1 mutant plants contain reduced amounts of chlorophyll, but photosynthetic quantum yield remains unchanged. The absolute content of plastidial PG (34:4; total number of acyl carbons:number of double bonds) is reduced by about 1/3, demonstrating that AtPGPP1 is involved in the synthesis of plastidial PG. PGP 34:3, PGP 34:2 and PGP 34:1 lacking 16:1 accumulate in pgpp1‐1, indicating that the desaturation of 16:0 to 16:1 by the FAD4 desaturase in the chloroplasts only occurs after PGP dephosphorylation.     

Phosphoprotein phosphatase activity in the thyroid.

Phosphoprotein phosphatase activity in the calf thyroid was found in various subcellular fractions. The relative amount in each fraction varied according to the substrate used: The 500g fraction had the highest specific activity when protamine was used, while the 5000g fraction was highest when histone was used. Triton X-100 tended to increase activity in all the particulate fractions, the greatest change being found in the 105,000g pellet. DEAE chromatography of the 105,000 g supernatant resolved at least three peaks of phosphoprotein phosphatase activity.     

Circadian rhythm of acid phosphatase activity in rat liver microsomes and dependence of NADH 5 alpha-reductase on phosphatase activity

The specific activity of acid phosphatase in male and female rats follows a circadian rhythm. Preincubation of liver microsomes with testosterone led to an increase of phosphatase activity and a loss of circadian rhythm. NADH 5 alpha-reductase was inactivated by several animal and bacterial acid and alkaline phosphatases while the acid phosphatase from potatoes was ineffective. The extent of inhibition depends on the course of circadian rhythm of NADH 5 alpha-reductase activity. Preincubation of microsomes in the presence of testosterone inhibited the NADH 5 alpha-reduction of testosterone. No such inhibition was observed after preincubation of microsomes with progesterone.     

Identification and purification of a cytosolic phosphotyrosyl protein phosphatase from bovine spleen

A protein tyrosine kinase with an apparent Mr of 60,000 was highly purified from bovine spleen and used to phosphorylate poly(Glu, Tyr) (4:1) on tyrosine residues for the study of phosphotyrosyl protein phosphatases from this tissue. About 70% of the phosphotyrosyl protein phosphatase activity in extracts of bovine spleen was adsorbed on DEAE-Sepharose. Chromatography of the eluted phosphotyrosyl protein phosphatases on phosphocellulose indicated the presence of at least two species, one that did not bind to the phosphocellulose and a second species that did bind and was eluted at about 0.5 M NaCl. The phosphatase that did not bind to phosphocellulose was further purified by successive chromatography on poly(L-lysine)-Sepharose, L-tyrosine-agarose, poly(Glu,Tyr)-Sepharose, and Sephacryl S-200. The enzyme had an apparent Mr of 50,000 as estimated by gel filtration and 52,000 as estimated by NaDodSO4- polyacrylamide gel electrophoresis. The phosphatase exhibited a pH optimum of 6.5-7.0, was inhibited by Zn2+ and vanadate ions, and was stimulated by EDTA. Sodium fluoride and sodium pyrophosphate, inhibitors of phosphoseryl protein phosphatases, had no effect on the enzyme. Protein inhibitors of type 1 phosphoseryl/threonyl phosphatase were also ineffective.     

Purification and characterization of a phosphoprotein phosphatase from bovine adrenal cortex.

A phosphoprotein phosphatase which is active against chemically phosphorylated protamine has been purified about 500-fold from bovine adrenal cortex. The enzyme has a pH optimum between 7.5 and 8.0, and has an apparent Km for phosphoprotamine of about 50 muM. The hydrolysis of phosphoprotamine is stimulated by salt, and by Mn2+. Hydrolysis of phosphoprotamine is inhibited by ATP, ADP, AMP, and Pi, but is not affected by AMP or cyclic GMP. The purified phosphoprotein phosphatase preparation also dephosphorylates p-nitrophenyl phosphate and phosphohistone, and catalyzes the inactivation of liver phosphorylase, the inactivation of muscle phosphorylase a (and its conversion to phosphorylase b), and the inactivation of muscle phosphorylase b kinase. Phosphatase activities against phosphoprotamine and muscle phosphorylase a copurify over the last three stages of purification. Phosphoprotamine inhibits phosphorylase phosphatase activity, and muscle phosphorylase a inhibits the dephosphorylation of phosphoprotamine. These results suggest that one enzyme possesses both phosphoprotamine phosphatase and phosphorylase phosphatase activities. The stimulation of phosphorylase phosphatase activity, but not of phosphoprotamine phosphatase activity, by caffeine and by glucose, suggests that the different activities of this phosphoprotein phosphatase may be regulated separately.     

Identification of the Pseudornonas aeruginosa acid phosphatase as a phosphorylcholine phosphatase activity

Summary Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg²⁺, the K_m values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The K _inf1 ^sups values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.     

Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase

A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions.     

Membrane lipid dynamics and enzymic activity in bovine adrenal cortex microsomes

The lipid dynamics of the adrenocortical microsomal membranes was studied by monitoring the fluorescence anisotropy and excited state lifetime of a set of anthroyloxy fatty acid probes (2-, 7-, 9- and 12-(9-anthroyloxy)-stearic acid (AP) and 16-(9-anthroyloxy)palmitic acid (AS). It was found that a decreasing polarity gradient from the aqueous membrane interface to the membrane interior, was present. This gradient was not modified by the proteins, as evidenced by comparison of complete membranes and derived liposomes, suggesting that the anthroyloxy probes were not in close contact with the proteins. An important change of the value of the mean rotational relaxation time as a function of the position of the anthroyl ring along the acyl chain was evidenced. In the complete membranes, a relatively more fluid medium was evidenced in the C₁₆ as compared to the C₂ region, while the rotational motion appeared to be the most hindered at the C₇–C₉ level. In the derived liposomes, a similar trend was observed but the mobility was higher at all levels. The decrease of the mean rotational relaxation time was more important for 12-AS and 16-AP. Temperature dependence of the mean rotational relaxation time of 2-AS, 12-AS and 16-AP in the complete membranes revealed the existence of a lipid reorganization occurring around 27°C and concerning mainly the C₁₆ region. The extent to which the acyl chain reacted to this perturbation at the C₁₂ level depended on pH. The presence of proteins increased the apparent magnitude of this reorganization and also modified the critical temperature from approx. 23°C in the derived liposomes to approx. 27°C in the complete membranes. Thermal dependence of the maximum velocity of the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$, the second enzyme in the enzymatic sequence, responsible for the biosynthesis of the $\text{3-}\text{oxo}\text{-Δ}{}^4\text{-}\text{steroids}$ in the adrenal cortex microsomes, was studied. The activation energy of the catalyzed reaction was found to be low and constant ($\text{2–5}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) in the temperature range 16–40°C at pH 7.5, 8.5 and 9, corresponding to the minimum, intermediate and maximum rate, respectively. A drastic increase of the activation energy ($\text{20}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) was observed at temperature below 16°C at pH 7.5. A correlated change of the $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}$ as function of temperature was detected; at 36°C $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}\text{= 8.3}$ while at 13°C the value shifted to 8.7. The pH range of the group ionization was narrower at 13°C. In contrast with the behaviour of the $\text{3β-}\text{hydroxy}\text{-Δ}{}^5\text{-}\text{steroid dehydrogenase}$, the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$ was apparently unaffected by the lipid reorganization at 27°C. It is suggested that this enzyme possesses a different and more fluid lipid environment than the bulk lipids.     

Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase.

Protein histidine phosphorylation in eukaryotes has been sparsely studied compared to protein serine/threonine and tyrosine phosphorylation. In an attempt to rectify this by probing porcine liver cytosol with the phosphohistidine-containing peptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide (phosphopeptide I), we observed a phosphatase activity that was insensitive towards okadaic acid and EDTA. This suggested the existence of a phosphohistidine phosphatase different from protein phosphatase 1, 2A and 2C. A 1000-fold purification to apparent homogeneity gave a 14-kDa phosphatase with a specific activity of 3 micro mol.min-1.mg-1 at pH 7.5 with 7 micro m phosphopeptide I as substrate. Partial amino-acid sequence determination of the purified porcine enzyme by MS revealed similarity with a human sequence representing a human chromosome 9 gene of hitherto unknown function. Molecular cloning from a human embryonic kidney cell cDNA-library followed by expression and purification, yielded a protein with a molecular mass of 13 700 Da, and an EDTA-insensitive phosphohistidine phosphatase activity of 9 micro mol.min-1.mg-1 towards phosphopeptide I. No detectable activity was obtained towards a set of phosphoserine-, phosphothreonine-, and phosphotyrosine peptides. Northern blot analysis indicated that the human phosphohistidine phosphatase mRNA was present preferentially in heart and skeletal muscle. These results provide a new tool for studying eukaryotic histidine phosphorylation/dephosphorylation.     

Identification and characterization of a protein-tyrosine phosphatase in Leishmania: Involvement in virulence

Leishmania parasites are eukaryotic protozoans responsible for a variety of human diseases known as leishmaniasis, which ranges from skin lesions to fatal visceral infections. Leishmania is transmitted by the bite of an infected sandfly where it exists as promastigotes and, upon entry into a mammalian host, differentiates into amastigotes, which replicate exclusively in macro-phages. The biochemical pathways enabling Leishmania to differentiate and survive in the mammalian host are poorly defined. We have therefore examined the role of protein-tyrosine phosphorylation, which is essential in regulating cell function in higher eukaryotes. Using the recently completed Leishmania genome, we have identified and cloned a Leishmania protein-tyrosine phosphatase (PTP) gene (LPTP1) by virtue of its homology with the human protein-tyrosine phosphatase 1B gene (hPTP1B). The enzyme activity of recombinant LPTP1 was confirmed using a combination of PTP-specific substrates and inhibitors. We further demonstrate, by creating LPTP1 null mutants through gene targeting, that LPTP1 is necessary for survival as amastigotes in mice, but it is dispensable for survival as promastigotes in culture. Human PTPs, including the PTP1B enzyme, are actively pursued drug targets for a variety of diseases. The observations with the LPTP1 mutants in mice suggest that it may also represent a drug target against the mammalian amastigote stage. However, in silico structure analysis of LPTP1 revealed a striking similarity with hPTP1B in the active site suggesting that, although this is an attractive drug target, it may be difficult to develop an inhibitor specific for the Leishmania LPTP1.     

Identification and partial characterization of an extracellular acid phosphatase activity of Leishmania donovani promastigotes.

An extracellular acid phosphatase was detected in the growth media of Leishmania donovani promastigotes. The enzyme was released at all stages of the growth cycle and in amounts which accounted for 90% of the total amount of this enzyme in the culture. The exoenzyme exhibited a pH optimum of 4.5 to 5.0 and was active with a variety of organic phosphates. The enzymatic activity was excluded from Sephacryl S-300 and was retained by ultrafilters with nominal molecular weight cutoffs of up to 300,000. The results of comparative studies indicated that the extracellular enzyme was distinct from a surface membrane-bound acid phosphatase of L. donovani promastigotes which has been previously described.