A phosphatase acting on dolichyl phosphate in membranes from neuronal perikarya

Idoyaga-Vargas, Víctor; Belocopitow, Enrique; Mentaberry, Alejandro; Carminatti, Héctor

doi:10.1016/0014-5793(80)80128-1

Cited by 38 publications

(12 citation statements)

References 20 publications

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“…Under the same in vitro conditions, expression of CWH8 also produced an increase in Dol-P phosphatase, although considerably lower than the increase seen for Dol-P-P phosphatase (Table IV). Very significantly, the lipid phosphatase encoded by CWH8 does not hydrolyze PA in contrast to virtually all other Mg 2ϩ -independent lipid phosphatases reported previously (5)(6)(7)(27)(28)(29)(30)(31)(32)(33). Membrane fractions from the insect cells overexpressing CWH8 also did not exhibit diacylglycerol pyrophosphate phosphatase (data not included).…”

Section: Dol-p-p Phosphatase Encoded By Cwh8 Is Labile In Triton X-10mentioning

confidence: 78%

The CWH8 Gene Encodes a Dolichyl Pyrophosphate Phosphatase with a Luminally Oriented Active Site in the Endoplasmic Reticulum of Saccharomyces cerevisiae

Fernández¹,

Rush

Toke

et al. 2001

Journal of Biological Chemistry

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Mutations in the CWH8 gene, which encodes an ER transmembrane protein with a phosphate binding pocket in Saccharomyces cerevisiae, result in a deficiency in dolichyl pyrophosphate (Dol-P-P)-linked oligosaccharide intermediate synthesis and protein N-glycosylation (van Berkel, M. A., Rieger, M., te Heesen, S., Ram, A. F., van den Ende, H., Aebi, M., and Klis, F. M. (1999) Glycobiology 9, 243-253). Genetic, enzymological, and topological approaches were taken to investigate the potential role of Cwh8p in Dol-P-P/Dol-P metabolism. Overexpression of Cwh8p in the yeast double mutant strain, lacking LPP1/ DPP1, resulted in an impressive increase in Dol-P-P phosphatase activity, a relatively small increase in Dol-P phosphatase activity, but no change in phosphatidate (PA) phosphatase activity in microsomal fractions. The Dol-P-P phosphatase encoded by CWH8 is optimally active in the presence of 0.5% octyl glucoside and relatively unstable in Triton X-100, distinguishing this activity from the lipid phosphatases encoded by LPP1 and DPP1. Stoichiometric amounts of P i and Dol-P are formed during the enzymatic reaction indicating that Cwh8p cleaves the anhydride linkage in Dol-P-P. Membrane fractions from Sf-9 cells expressing Cwh8p contained a 30-fold higher level of Dol-P-P phosphatase activity, a slight increase in Dol-P phosphatase activity, but no increase in PA phosphatase relative to controls. This is the first report of a lipid phosphatase that hydrolyzes Dol-P-P/Dol-P but not PA. In accord with this enzymatic function, Dol-P-P accumulated in cells lacking the Dol-P-P phosphatase. Topological studies using different approaches indicate that Cwh8p is a transmembrane protein with a luminally oriented active site. The specificity, subcellular location, and topological orientation of this novel enzyme are consistent with a role in the re-utilization of the glycosyl carrier lipid for additional rounds of lipid intermediate biosynthesis after its release during protein N-glycosylation reactions.Although the biosynthesis of dolichyl-linked saccharide intermediates is initiated on the cytoplasmic side of the endoplasmic reticulum (ER), 1 dolichyl pyrophosphate (Dol-P-P) and several dolichyl monophosphate (Dol-P) molecules are released on the luminal surface during protein N-glycosylation reactions, glycosylphosphatidylinositol anchor synthesis, C-and O-mannosylation of proteins (see reviews in Refs. 1-3). For the dolichyl moiety of Dol-P-P to be re-utilized for additional rounds of lipid intermediate biosynthesis, it must be converted to Dol-P before or after returning to the cytoplasmic leaflet of the ER.There have been many reports of Dol-P-P phosphatase activities in microsomal fractions from mammalian tissues (see reports cited in Ref.3). However, the subcellular location and topological arrangement of the active sites of these enzymes have not been definitively established. A Dol-P-P phosphatase activity reported in brain (4) is curiously enriched in Golgi fractions and is an unlikely candidate to be involved in the ...

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Section: Dol-p-p Phosphatase Encoded By Cwh8 Is Labile In Triton X-10mentioning

confidence: 78%

The CWH8 Gene Encodes a Dolichyl Pyrophosphate Phosphatase with a Luminally Oriented Active Site in the Endoplasmic Reticulum of Saccharomyces cerevisiae

Fernández¹,

Rush

Toke

et al. 2001

Journal of Biological Chemistry

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“…[3H]Dol-P-P was incubated with UDP-Glc or GDP-Man under the conditions indicated in [27], where [3H]Dol-P was used; [3H]-Dol-P-Glc or [3H]Dol-P-Man was produced, respectively, and RF values similar to those of [3H]Dol-P-Gal were obtained as in [13].…”

Section: Preparation Of Substratesmentioning

confidence: 99%

“…c) The enzyme that lowers Dol-P concentration through a transformation of the latter into dolichol, Dol-P phosphatase [12,13,15,16].…”

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confidence: 99%

Dolichyl‐Phosphate Phosphatase and Dolichyl‐Diphosphate Phosphatase in Rat‐Liver Microsomes

Belocopitow

Boscoboinik

1982

European Journal of Biochemistry

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Dolichyl-phosphate phosphatase and dolichyl-diphosphate phosphatase activities of a liver-cell microsomal preparation were solubilized by treatment with Triton X-100. The I00000 x g supernatant was then passed through a column of Sepharose-4B -concanavalin A. Both enzyme activities were found in the percolate. This treatment eliminated inhibition by ATP and glucose 6-phosphate in both phosphatase activities. In each case the activities were inhibited by higher concentrations of enzyme preparation due to the presence of phospholipids. The inhibitory effects of either phosphatidylcholine or phosphatidylethanolamine were due to competition for detergent. On the other hand, the effect produced by phosphatidic acid appeared to be different, since it did not change the optimal concentration of Triton X-100 for the two enzymes. Dolichyl-phosphate phosphatase was strongly inhibited by both Pi and PPi, whereas dolichyl-diphosphate phosphatase was only slightly inhibited by Pi and not at all by PPi. Dolichyl-diphosphate phosphatase was more inhibited by divalent cations than dolichyl-phosphate phosphatase. The apparent K,,, of dolichyl-phosphate phosphatase for dolichyl phosphate was 0.15 mM. Dolichol also inhibited dolichyl-phosphate phosphatase, but it produced a stronger inhibition on dolichyl-diphosphate phosphatase. The inhibitory effect of dolichol was not entirely due to detergent competition.The enzymatic system involved in the biosynthesis of asparagine-linked glycoproteins is located in the endoplasmic reticulum. In this biosynthetic process the formation of the peptide linkage precedes the formation of the covalent bond between oligosaccharide and amino acid. During the last decade experimental evidence has shown that, at least in the case of asparagine-type glycoproteins, the oligosaccharide moiety is built up linked to dolichol diphosphate. The Dol-P-P moiety is released when to oligosaccharide moiety is transferred to the peptide chain [1,2]. Since the biosynthesis of the oligosaccharide lipid is initiated with dolichyl monophosphate as an acceptor of the first glycosyl groups, it seems reasonable to assume the existence of a conversion step from Dol-P-P to Dol-P, which would complete the cycle.Some suggestions [3] and further experimental evidence [4-71 indicate that an increased concentration of dolichyl phosphate would in turn increase the rate of glycoprotein synthesis. The Dol-P concentration would be determined by the activity of the following three enzymes.a) The enzyme that forms Dol-P from dolichol, dolichol phosphokinase [8-111, which catalyzes the reaction Dolichol + CTP -+ Dol-P + CDP .b) The enzyme that forms Dol-P by a cleavage of the pyrophosphate bond of Dol-P-P, Dol-P-P phosphatase [I2 -141. The reaction is Dol-P-P + Dol-P + Pi.c) The enzyme that lowers Dol-P concentration through a transformation of the latter into dolichol, Dol-P phosphatase [12,13,15,16].Abbreviations. Dol-P-P, dolichyl diphosphate; Dol-P, dolichyl phosphate.In the studies reported below we have focused on the characteri...

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“…A dolichyl monophosphate phosphatase has been recently described in membranes obtained from neuronal perikarya (Idoyaga-Vargas et al, 1980). This enzyme may be involved in the regulation of the biosynthesis of asparagine-type glycoproteins.…”

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confidence: 99%

Postnatal Changes in the Activity Ratio of Specific and Nonspecific Cholinesterases from Neuronal Perikarya

Ochoa¹,

Brusco²,

Idoyaga-Vargas

et al. 1982

Journal of Neurochemistry

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A soluble fraction from rat brain neuronal perikarya was shown to contain both the specific and nonspecific forms of the enzyme acetylcholinesterase (EC's 3.1.1.7. and 3.1.1.8., respectively). The ratio of the enzyme activities varied along the course of brain development: the nonspecific form being predominant from 1 to 15 days of age and the specific one showing the pattern of rising activity from day 15 onward. We suggest a possible relationship between this changing in cholinesterase activities and the establishment of synapses within the rat cerebral cortex.

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A phosphatase acting on dolichyl phosphate in membranes from neuronal perikarya

Cited by 38 publications

References 20 publications

The CWH8 Gene Encodes a Dolichyl Pyrophosphate Phosphatase with a Luminally Oriented Active Site in the Endoplasmic Reticulum of Saccharomyces cerevisiae

The CWH8 Gene Encodes a Dolichyl Pyrophosphate Phosphatase with a Luminally Oriented Active Site in the Endoplasmic Reticulum of Saccharomyces cerevisiae

Dolichyl‐Phosphate Phosphatase and Dolichyl‐Diphosphate Phosphatase in Rat‐Liver Microsomes

Postnatal Changes in the Activity Ratio of Specific and Nonspecific Cholinesterases from Neuronal Perikarya

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