Solubilization and partial characterization of a phosphoprotein phosphatase from human myelin.

The phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) solubilized from human central nervous system myelin has been shown to possess a comparatively high degree of specificity towards myelin basic protein, a constituent of the membrane and most likely its natural substrate, rather than the mixed histones. The enzyme has a pH optimum of 7.5. Hydrolysis of both the substrates is stimulated by dithiothreitol and is almost completely dependent upon the presence of divalent metal ions. The maximum rate of dephosphorylation of basic protein is attained in the presence of 125 micrometer Mn2+ whereas a much higher concentration of Mg2+ (50--100 mM) is required for the optimal dephosphorylation of histones. The dephosphorylation of basic protein was also stimulated by Triton X-100 (0.15%, v/v) and was shown to result from a 3-fold increase in the V of the reaction catalyzed by the phosphatase. The apparent Km values for basic protein and histones were unaffected by the presence of Triton X-100 and were found to be approx. 1 and approx. 160 micrometer, respectively. Under optimal conditions of assay, the phosphatase cleaved approx. 32 and approx. 0.7 nmol of orthophosphate.min-1.mg-1 of protein from basic protein and histones, respectively.     

Identification and characterization of a cDNA encoding a dolichyl pyrophosphate phosphatase located in the endoplasmic reticulum of mammalian cells

The CWH8 gene in Saccharomyces cerevisiae has been shown recently (Fernandez, F., Rush, J. S., Toke, D. A., Han, G., Quinn, J. E., Carman, G. M., Choi, J.-Y., Voelker, D. R., Aebi, M., and Waechter, C. J. (2001) J. Biol. Chem. 276, 41455-41464) to encode a dolichyl pyrophosphate (Dol-P-P) phosphatase associated with crude microsomal fractions. Mutations in CWH8 result in the accumulation of Dol-P-P, deficiency in lipid intermediate synthesis, defective protein N-glycosylation, and a reduced growth rate. A cDNA (DOLPP1, GenBank accession number AB030189) from mouse brain encoding a homologue of the yeast CWH8 gene is now shown to complement the defects in growth and protein N-glycosylation, and to correct the accumulation of Dol-P-P in the cwh8Delta yeast mutant. Northern blot analyses demonstrate a wide distribution of the DOLPP1 mRNA in mouse tissues. Overexpression of Dolpp1p in yeast, COS, and Sf9 cells produces substantial increases in Dol-P-P phosphatase activity but not in dolichyl monophosphate or phosphatidic acid phosphatase activities in microsomal fractions. Subcellular fractionation and immunofluorescence studies localize the enzyme encoded by DOLPP1 to the endoplasmic reticulum of COS cells. The results of protease sensitivity studies with microsomal vesicles from the lpp1Delta/dpp1Delta yeast mutant expressing DOLPP1 are consistent with Dolpp1p having a luminally oriented active site. The sequence of the DOLPP1 cDNA predicts a polypeptide with 238 amino acids, and a new polypeptide corresponding to 27 kDa is observed when DOLPP1 is expressed in yeast, COS, and Sf9 cells. This study is the first identification and characterization of a cDNA clone encoding an essential component of a mammalian lipid pyrophosphate phosphatase that is highly specific for Dol-P-P. The specificity, subcellular location, and topological orientation of the active site described in the current study strongly support a role for Dolpp1p in the recycling of Dol-P-P discharged during protein N-glycosylation reactions on the luminal leaflet of the endoplasmic reticulum in mammalian cells.     

A dolichyl phosphate-cleaving acid phosphatase from Tetrahymena pyriformis.

Tetrahymena pyriformis contains an enzyme which hydrolyzed dolichyl phosphate. This activity was solubilized from lyophilized samples of this organism and was relatively stable when stored frozen. The soluble enzyme preparation had an acid pH optimum and hydrolyzed both dolichyl and phytanyl phosphates at equivalent rates. The polyprenylphosphate phosphatase activity was compared with the acid phosphatases which hydrolyzed p-nitrophenyl phosphate and marked differences were found. Dolichyl phosphate hydrolysis required Mg2+ for maximum activity while the bulk of the phosphatase activity was not effected by the absence of this ion. Other differences were that the polyprenylphosphate phosphatase was relatively insensitive to inhibitors such as tartrate and vanadium oxide sulfate which had a pronounced effect on the rate of p-nitrophenyl phosphate hydrolysis. The two activities also appeared to have different subcellular distributions. The polyprenylphosphate phosphatase was markedly inhibited by ethoxy formic anhydride, a reagent which is active against enzymes containing a histidine residue at their active site, while p-nitrophenyl phosphate hydrolysis was unaffected. The polyprenylphosphate phosphatase may be important in regulating the level of dolichyl phosphate in T. pyriformis and thus the rate of glycoprotein synthesis. It is also a useful tool which is capable of liberating dolichol from dolichyl phosphate under mild conditions which will permit the further characterization of the polyprenols.     

Recycling of dolichyl monophosphate to the cytoplasmic leaflet of the endoplasmic reticulum after the cleavage of dolichyl pyrophosphate on the lumenal monolayer

During protein N-glycosylation, dolichyl pyrophosphate (Dol-P-P) is discharged in the lumenal monolayer of the endoplasmic reticulum (ER). Dol-P-P is then cleaved to Dol-P by Dol-P-P phosphatase (DPPase). Studies with the yeast mutant cwh8Delta, lacking DPPase activity, indicate that recycling of Dol-P produced by DPPase contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis on the cytoplasmic leaflet. Whether Dol-P formed in the lumen diffuses directly back to the cytoplasmic leaflet or is first dephosphorylated to dolichol has not been determined. Incubation of sealed ER vesicles from calf brain with acetyl-Asn-Tyr-Thr-NH(2), an N-glycosylatable peptide, to generate Dol-P-P in the lumenal monolayer produced corresponding increases in the rates of Man-P-Dol, Glc-P-Dol, and GlcNAc-P-P-Dol synthesis in the absence of CTP. No changes in dolichol kinase activity were observed. When streptolysin-O permeabilized CHO cells were incubated with an acceptor peptide, N-glycopeptide synthesis, requiring multiple cycles of the dolichol pathway, occurred in the absence of CTP. The results obtained with sealed microsomes and CHO cells indicate that Dol-P, formed from Dol-P-P, returns to the cytoplasmic leaflet where it can be reutilized for lipid intermediate biosynthesis, and dolichol kinase is not required for recycling. It is possible that the flip-flopping of the carrier lipid is mediated by a flippase, which would provide a mechanism for the recycling of Dol-P derived from Man-P-Dol-mediated reactions in N-, O-, and C-mannosylation of proteins, GPI anchor assembly, and the three Glc-P-Dol-mediated reactions in Glc(3)Man(9)GlcNAc(2)-P-P-Dol (DLO) biosynthesis.     

Characterization of dolichyl diphosphate phosphatase from rat liver

Dolichyl diphosphate phosphatase (DolPPase) has been characterized in rat liver. Subcellular distribution studies indicate that the enzyme is localized in the endoplasmic reticulum. The in vitro enzymatic activity is stimulated by EDTA, due to release of inhibition by trivalent cations found in the assay tubes. All di- and trivalent cations tested were inhibitory, with the trivalent ions Al3+ and Fe3+ showing greater than 70% inhibition at a concentration of 10 microM. The assay requires the presence of a detergent for optimal activity, with Triton X-100 giving maximum activity at 0.1%. The substrate specificity of DolPPase toward polyprenyl diphosphates has been determined and indicates that there is little preference of the enzyme for substrates of different chain length, and either stereochemical orientation or degree of saturation of the alpha-isoprene unit. Km values of 11-14 microM were obtained for all substrates tested. Preliminary studies on the transmembrane topology of the DolPPase using latency assays, indicate that the active site of the enzyme may reside on the cytoplasmic face of the endoplasmic reticulum.     

Enzymatic activities in cultured human lymphocytes that dephosphorylate dolichyl pyrophosphate and dolichyl phosphate

Enzymatic activities which dephosphorylate dolichyl phosphate (Dol-P) and dolichyl pyrophosphate (Dol-P-P) have been observed in membranes from cultured human lymphocytes. Neither activity requires divalent metals. Dol-P phosphatase is inhibited by inorganic phosphate but not by other phosphate-containing compounds. Dol-P-P phosphatase is inhibited by bacitracin but not by phosphate-containing compounds including the methylene analogue of pyrophosphate. These reactions are similar to those previously found in the cycle of bacterial wall peptidoglycan biosynthesis. A chemical synthesis of [32P]Dol-P and [32P]Dol-P-P is reported.     

Solubilization and characterization of phosphoprotein phosphatase(s) from bovine corpus-luteum plasma membranes.

Plasma membrane fractions I and II isolated from bovine corpus luteum contain phosphoprotein phosphatases. Enzyme activities associated with both membrane fractions showed pH optima in the neutral range and were most active with phosphoprotamine as the exogenous substrate. The enzyme activity was partially inhibited by Co2+, Zn2+ and Fe2+. Dithioerythritol, glutathione (reduced) and 2-mercaptoethanol stimulated the enzyme activity, whereas N-ethylmaleimide and N-phenylmaleimide were inhibitory. Similarly, various cyclic nucleotides and nuclsoside triphosphates also inhibited phosphoprotein phosphatase activities. The phosphatase activity was also observed with endogenous phosphorylated membrane proteins as substrate. The endogenous phosphorylation of membranes was rapid and attained a maximal level after 15--20 min of incubation. Initially endogenous dephosphorylation was also very rapid, but did not reach completion. In addition to phosphoprotein phosphatase, membrane preparations also possessed very active cyclic-AMP-dependent protein kinase activity. Phosphoprotein phosphatase activity from plasma membranes was solubilized by ionic and nonionic detergents. Optimal solubilization was achieved with 0.1% sodium deoxycholate. Sucrose density gradient centrifugation of deoxycholate-solubilized fraction I and fraction II membranes resolved phosphoprotein phosphatase activity into two species with apparent sedimentation coefficients of 6.7 S (Mr 130000) and 4.8 S (Mr 90000). Cyclic-AMPstimulated protein kinase activity sedimented as a broad peak with a sedimentation coefficient of 5.5 S (Mr 110000).     

Golgi-enriched membrane fractions from rat brain and liver contain long-chain polyisoprenyl pyrophosphate phosphatase activity.

The subcellular distribution of polyisoprenyl pyrophosphate phosphatase activity has been examined in rat brain by assaying the release of 32Pi from [beta-32P]dolichyl pyrophosphate (Dol-P-P) as described previously (Scher,M.G. and Waechter, C.J. (1984) J. Biol. Chem., 259, 14580-14585). The highest specific activities of Dol-P-P phosphatase in rat brain were found in the Golgi-enriched light microsomal, synaptic plasma membrane and heavy microsomal fractions. A comparative analysis of the distribution of galactosyltransferase and dolichol kinase reveals that Dol-P-P phosphatase activity co-fractionates with galactosyltransferase activity, and that the high level found in the Golgi-enriched fraction is not due to cross-contamination with heavy microsomes. When beta-labelled C95 Dol-P-P and the C95 allylic polyisoprenyl pyrophosphate (Poly-P-P) were compared as substrates for the Golgi-enriched light microsomal and heavy microsomal fractions, similar Km values were calculated for the two pyrophosphorylated substrates for each membrane fraction. Based on these kinetic analyses, the enzyme(s) catalysing this reaction do not distinguish between substrates containing saturated or allylic alpha-isoprene units. When Dol-P-P phosphatase activity was assessed in submicrosomal fractions obtained from rat liver by two separate procedures, the highest specific activity was also detected in the Golgi-enriched fraction. While the specific activities for Dol-P-P phosphatase and sialyltransferase were in the relative order of Golgi greater than smooth endoplasmic reticulum (ER) greater than rough ER, the relative order of dolichol kinase was rough ER greater than smooth ER greater than Golgi.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and characterization of pellet-associated human brain alpha-L-fucosidase activity.

The pellet-associated portion of human brain alpha-L-fucosidase (which represents approx. 20% of the homogenate activity) was solubilized with 0.5% (w/v) Triton X-100, characterized with regard to several properties and compared with the corresponding properties of the soluble supernatant-fluid enzyme in an attempt to find a second alpha-L-fucosidase in human brain. The solubilized and soluble alpha-L-fucosidase activities exhibited complete stability after storage at 2-4 degrees C for up to 29 days, comparable thermostability after preincubation at 50 degrees C, comparable apparent Km values (0.07-0.08 mM) for 4-methylumbelliferyl alpha-L-fucopyranoside, comparable hydrophobicity, comparable isoelectric-focusing profiles (six major forms, with pI values between 4.5 and 5.8) and comparable immunoprecipitation curves (with the IgG fraction of antisera prepared against human liver alpha-L-fucosidase). Differences in three properties were found between solubilized and soluble alpha-L-fucosidase activities: the solubilized activity was less stable to storage at -20 degrees C, had a 0.5-pH-unit neutral shift in its pH optimum (6.0) and had smaller Mr forms after gel filtration on Sephadex G-200. The overall results indicate that the pellet-associated and soluble portions of human brain alpha-L-fucosidase are quite similar in most of their properties. Thus there is still no compelling evidence for the existence of a second mammalian alpha-L-fucosidase.     

The submicrosomal distribution of dolichyl phosphate and dolichyl phosphate phosphatase in rat liver

Rat liver microsomes were isolated and fractionated into Golgi, smooth endoplasmic reticulum (SER), and rough endoplasmic reticulum (RER), and the purity of these preparations was determined. The dolichyl phosphate (Dol-P) content of whole microsomes and of each of the submicrosomal fractions was estimated using high pressure liquid chromatography. Dol-P accounts for 4 and 40% of the sum of the alcohol, the fatty acyl esters of dolichol, and monophosphate forms present in whole liver and in purified microsomes, respectively. Concentrations equal to 58, 77, and 108 ng of Dol-P/mg of protein were found in Golgi, SER, and RER, respectively. These values represent 3, 36, and 54% of the sum of the alcohol, the fatty acyl esters of dolichol, and monophosphate forms present in each of these same fractions, respectively. Increases in the Dol-P content of rat liver were observed as early as 12 h after turpentine-induced inflammation and increased 2-fold over 36 h. In this system, Dol-P accounts for no more than 50% of the sum of all phosphorylated and pyrophosphorylated dolichol intermediates present. The specific activity for dolichyl phosphate phosphatase was highest by more than a factor of 2 in Golgi membrane. Specific activities obtained for SER and RER were 42 and 11% of those present in Golgi. The major requirement for Dol-P is thought to be for the saccharide and oligosaccharide transferase reactions which are presumed to take place in RER. The discovery of significant quantities of Dol-P in Golgi and SER is consistent with a possible role of Dol-P in the transport of sugars required for glycoprotein synthesis and processing from a cytosolic to luminal orientation.     

Guanosine monophosphate synthetase from Ehrlich ascites cells. Multiple inhibition by pyrophosphate and nucleosides.

GMP synthetase (xanthosine-5'-phosphate: ammonia ligase (AMP-forming), EC 6.3.4.1) from Ehrlich ascites cells was found to be subject to multiple inhibition by its reaction product, PPi, and some analogs of adenosine. PPi and the nucleoside (N) inhibitors were also capable of individually inhibiting this enzyme. Under no conditions did the inhibition appear to be irreversible or "pseudoinactivating" in nature. The individual inhibition by PPi was competitive with respect to ATP (KI = 0.42 mM). Conversely, in the absence of PPi, the binding of N was noncompetitive with ATP, but shifted to a competitive pattern when PPi was present. Furthermore, with the inhibitors in concert, there was an apparent lowering of the KI values for both inhibitors. This data was consistent with either PPi functioning to tighten the binding of N at a noncatalytic site (positive cooperativity) or with PPi actually opening a second binding site for N in addition to the non-catalytic site. Although this study did not distinguish which of these events was occurring, it did reveal that the intensity of the effect of PPi appeared to be constant. That is, for various N inhibitors with a range of independently determined KI values from 26 to 1650 muM, the ratio of their KI values determined in the absence of PPi to the values determined in the presence of PPi was always 38 +/- 1.     

Mutagenesis of the phosphatase sequence motif in diacylglycerol pyrophosphate phosphatase from Saccharomyces cerevisiae.

Diacylglycerol pyrophosphate (DGPP) phosphatase, encoded by the DPP1 gene, is a membrane-associated enzyme in the yeast Saccharomyces cerevisiae. The enzyme removes the beta phosphate from DGPP to form phosphatidate. The substrate and product of the DGPP phosphatase reaction play roles in lipid signaling and in cell metabolism. The deduced primary structure of the DGPP phosphatase protein contains a three-domain phosphatase sequence motif. In this work, we examined the hypothesis that the phosphatase sequence motif in the enzyme is involved in the DGPP phosphatase reaction. The amino acid residues Arg(125), His(169), and His(223) in domains 1, 2, and 3, respectively, of the phosphatase sequence motif were changed to alanine residues by site-directed mutagenesis. The mutant DPP1(R125A), DPP1(H169A), and DPP1(H223A) alleles were cloned into a yeast shuttle vector and then expressed in a dpp1Delta lpp1Delta double mutant that lacks DGPP phosphatase activity. Northern blot and immunoblot analyses showed that the mutations in the phosphatase sequence motif did not affect the expression of the enzyme. The DGPP phosphatase activities of the R125A, the H169A, and the H223A mutant enzymes were 0.05, 9, and 0.03%, respectively, of the DGPP phosphatase activity of the wild-type enzyme. Enzymes with mutations in more than one domain of the phosphatase sequence motif had no measurable DGPP phosphatase activity. The R125A and H233A mutant DGPP phosphatase enzymes had reduced V(max) and elevated K(m) values for DGPP when compared with the wild-type enzyme. The H169A mutant enzyme had reduced V(max) and K(m) values when compared with the control. The specificity constants (V(max)/K(m)()) for DGPP of the R125A mutant and H233A mutant enzymes were 4610-fold and 15 367-fold lower, respectively, when compared to the wild-type enzyme. The studies reported here indicated that the phosphatase sequence motif played an important role in the reaction catalyzed by the S. cerevisiae DGPP phosphatase.