Standardized method for the determination of human erythrocyte membrane adenosine triphosphatases

A standardized assay is described for the simultaneous determination of Mg²⁺-ATPase, Na⁺, K⁺-ATPase, and Ca²⁺-ATPase in human erythrocyte (RBC) membrane preparations. Membranes were prepared by lysis of RBCs in hypotonic buffer, and ATPase activity assays were based on the measurement of ³²P-labeled inorganic phosphate release from [γ-³²P]ATP. The results obtained by this method were compared with those of colorimetric determination of inorganic phosphate and of ATP hydrolysis with high-performance liquid chromatography. The activity of the three enzymes was measured in RBC membranes obtained from 30 normal subjects. Repeated sampling of individuals over a 4-month period showed that interindividual differences were substantial, but that in each individual enzymatic activity was maintained in a narrow range by presumed homeostatic mechanisms. Statistical analysis of the data showed no interdependence of the three enzymes; a correlation of activity with age, sex, or phase of the menstrual cycle was not apparent. The values obtained for the Ca²⁺-ATPase did not follow a normal distribution, and it is suggested that this enzyme has two phenotypic variants. The described method is sufficiently precise and economical to be recommended for adoption as standard procedure in clinical research.     

Bioreaction Engineering Leading to Efficient Synthesis of L‐Glyceraldehyd‐3‐Phosphate

Enantiopure L‐glyceraldehyde‐3‐phosphate (L‐GAP) is a useful building block in natural biological and synthetic processes. A biocatalytic process using glycerol kinase from Cellulomonas sp. (EC 2.7.1.30) catalyzed phosphorylation of L‐glyceraldehyde (L‐GA) by ATP is used for the synthesis of L‐GAP. L‐GAP has a half‐life of 6.86 h under reaction conditions. The activity of this enzyme depends on the Mg²⁺ to ATP molar ratio showing maximum activity at the optimum molar ratio of 0.7. A kinetic model is developed and validated showing a 2D correlation of 99.9% between experimental and numerical data matrices. The enzyme exhibits inhibition by ADP, AMP, methylglyoxal and Ca²⁺, but not by L‐GAP and inorganic orthophosphate. Moreover, equal amount of Ca²⁺ exerts a different degree of inhibition relative to the activity without the addition of Ca²⁺ depending on the Mg²⁺ to ATP molar ratio. If the Mg²⁺ to ATP molar ratio is set to be at the optimum value or less, inorganic hexametaphosphate (PPi6) suppresses the enzyme activity; otherwise PPi6 enhances the enzyme activity. Based on reaction engineering parameters such as conversion, selectivity and specific productivity, evaluation of different reactor types reveals that batchwise operation via stirred‐tank reactor is the most efficient process for the synthesis of L‐GAP.     

Regulation of ANP-stimulated guanylate cyclase in the presence of Mn2+ in rat lung membranes

The catalytic activity of guanylate cyclase (GCase) coupled to atrial natriuretic peptide (ANP) receptor depends on the metal co-factor, Mn²⁺ or Mg²⁺. ATP synergistically stimulates the ANP-stimulated GCase in the presence of Mg²⁺. We have now shown the ATP regulation of the ANP-stimulated GCase in the presence of Mn²⁺ in rat lung membranes. ANP stimulated the GCase 2.1-fold compared to the control. ATP enhanced both the basal (basal-GCase) and the ANP-stimulated GCase maximally 1.7- and 2.3- fold compared to the control, respectively, at a concentration of 0.1 mM. The stimulation by ATP was smaller in the presence of Mn²⁺ than in the presence of Mg²⁺. The addition of inorganic phosphate to the reaction mixture altered the GCase activities in the presence of Mn²⁺ with or without ANP and/or ATP. In the presence of 10 mM phosphate, ATP dose-dependently stimulated the basal GCase 5-fold compared to the control at a concentration of 1 mM and augmented the ANP-stimulated GCase, which was 4.2-fold compared to the basal-GCase, 5.5-fold compared to the control at a concentration of 0.5 mM. Protein phosphatase inhibitors, okadaic acid (100 nM), H8 (1 M) and staurosporin (1 M), did not alter the activity. Orthovanadate (1 mM), an inorganic phosphate analogue, significantly stimulated both the basal-GCase and the ANP-stimulated GCase, which were inhibited by ATP. It was assumed that phosphate and orthovanadate might interact with the GCase to regulate the activity in the opposite manner. This was the first report that inorganic phosphate and orthovanadate affected the ATP-regulation of the ANP-stimulated GCase in the presence of Mn²⁺.     

31P Nuclear Magnetic Resonance Studies on the Phosphorus-Containing Metabolites of the Developing Embryos of a Freshwater Catfish, Clarias batrachus (L.)

Changes in the phosphorus-containing metabolites were monitored by ³¹P nuclear magnetic resonance in the developing embryos of Clarias batrachus. Phosphomonoester, yolk phosphoprotein, phosphocreatine, ATP, and inorganic phosphate (Pi) were consistently observed in all the developmental stages of C. batrachus. None of these phosphometabolites exhibited any significant change in their concentration up to the blastula stage, whereas distinct decrease in all except inorganic phosphate was observed in the fry stage. Concomitantly an increase in the concentration of inorganic phosphate was observed. Further, from the resonance positions of α, β, and γ phosphate groups of ATP, it was evident that the ATP molecules in vivo were liganded either to Ca²⁺ or Mg²⁺. This study also revealed that the intracellular pH of the developing embryos was approximately 7.05 up to the gastrula stage, after which it decreased in the fry stage to 6.98 units. Received August 10, 1998; accepted November 3, 1998.     

Interaction of calcium with mitochondria isolated from Ehrlich ascites tumour cells

Calcium ions are accumulated by intact mitochondria isolated from Ehrlich ascites tumour cells in a buffered system supplemented with ATP or succinate. In the ATP-supplemented system, the tumour mitochondria, in contrast to rat liver mitochondria, retain the accumulated Ca²⁺, do not exhibit a marked “irreversible” ATPase and do not swell. In the succinate-supplemented system, added Ca²⁺ stimulates respiration in either the absence or presence of added inorganic phosphate. Whereas respiration by rat liver mitochondria, measured in the presence of added phosphate, remains continuously activated after the addition of only a small amount of Ca²⁺, that by the tumour mitochondria can be stimulated by several successive additions of 100 μM Ca²⁺ and at all times exhibit appreciable activation ratios.     

3-O-Methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca-ATPase

3-O-Methylfluorescein phosphate hydrolysis, catalyzed by purified erythrocyte Ca²⁺-ATPase in the absence of Ca²⁺, was slow in the basal state, activated by phosphatidylserine and controlled proteolysis, but not by calmodulin. p-Nitrophenyl phosphate competitively inhibits hydrolysis in the absence of Ca²⁺, while ATP inhibits it with a complex kinetics showing a high and a low affinity site for ATP. Labeling with fluorescein isothiocyanate impairs the high affinity binding of ATP, but does not appreciably modify the binding of any of the pseudosubstrates. In the presence of calmodulin, an increase in the Ca²⁺ concentration produces a bell-shaped curve with a maximum at 50 μM Ca²⁺. At optimal Ca²⁺ concentration, hydrolysis of 3-O-methylfluorescein phosphate proceeds in the presence of fluorescein isothiocyanate, is competitively inhibited by p-nitrophenyl phosphate and, in contrast to the result observed in the absence of Ca²⁺, it is activated by calmodulin. In marked contrast with other pseudosubstrates, hydrolysis of 3-O-methylfluorescein phosphate supports Ca²⁺ transport. This highly specific activity can be used as a continuous fluorescent marker or as a tool to evaluate partial steps from the reaction cycle of plasma membrane Ca²⁺-ATPases.     

Standard free energy maps for the hydrolysis of ATP as a function of pH and metal ion concentration: comparison of metal ions

The observed equilibrium constant K_obs for the hydrolysis of ATP to ADP and inorganic phosphate has been calculated as a function of pH and metal ion concentration pM (- log [M]) at 25 °C and μ = 0.2 with the use of literature values of the acid dissociation and complex dissociation constants for the phosphates.The resulting standard free energy changes ΔG °′ are presented by means of contour diagrams for the range pH 4–10 and pM 1–7. These maps summarize the results of some 1900 calculations per diagram, and clearly simulate a differential effect of the metal ions of interest, including Mg²⁺, Ca²⁺, Sr²⁺, Mn²⁺, Li⁺, Na⁺ and K⁺, on the equilibrium hydrolysis of ATP.     

Isopentenyl pyrophosphate isomerase from pig liver

Isopentenyl pyrophosphate isomerase (EC 5.3.3.2) from pig liver has been purified 197-fold. The preparation was estimated to contain less than 10% of contaminating protein. The molecular weight determined by gel filtration was 82,500 ± 3,000 and the isoelectric point from isoelectric focusing was in the range 6.0–6.2. N-terminal analysis showed the presence of both leucine and proline. The pH optimum of the enzyme preparation was 6.3. After dialysis against EDTA, activity was restored by either Mn²⁺ or Mg²⁺, the former being more effective. At the optimum pH and concentration of Mn²⁺, K_m and V were 2.7 μm and 6.7 μmol min^?1 mg^?1, respectively. The enzyme was partially inhibited by a variety of terpene mono- and pyrophosphate esters, by inorganic phosphate ions, and by acetate ions; essentially complete inhibition by sulfhydryl-blocking reagents was observed. ATP partially inhibited, the degree of inhibition showing a sigmoid dependence on ATP concentration. Monothiols and dithiothreitol activated the enzyme, as did mevalonic acid.     

Properties of Neurospora crassa plasma membrane ATPase.

A variety of the biochemical properties of the electrogenic plasma membrane ATPase of Neurospora crassa are described. The enzyme catalyzes the hydrolysis of ATP, resulting in the formation of ADP and inorganic phosphate. Optimal activity is observed between pH 6 and 6.5. ATP hydrolysis approaches a maximum rate at an Mg-ATP concentration of 10–20 mm with a half-maximal velocity around 2 mm Mg-ATP. The enzyme requires a divalent cation for activity in the following order of preference at 10 mm: Mg²⁺, Co²⁺ > Mn²⁺ > Zn²⁺ > Fe²⁺, Ca²⁺, Cu²⁺. The enzyme is quite specific for ATP compared to the other nucleotides tested. Treatment of the plasma membranes with sodium deoxycholate inactivates the ATPase and the inactivation can be prevented by the addition of certain acidic phospholipids with the deoxycholate. Other classes of lipids cannot prevent the deoxycholate inhibition. The organic mercurials parachloromercuribenzoate and parachloromercuriphenylsulfonate are potent inhibitors of the ATPase, but N-ethylmaleimide at a similar concentration is not inhibitory. The organic mercurial inhibition is not reversed by mercaptoethanol. Under appropriate conditions, the inhibitory effect of p-chloromercuribenzoate is suppressed in the presence of ATP. Treatment of the plasma membranes with trypsin leads to a marked inhibition of the ATPase activity and this inhibition can be prevented by Mg-ATP. Neither the organic mercurial reactive site(s) nor the trypsin-sensitive site(s) are accessible from the outer surface of the plasma membranes. Some of the implications of the above findings are discussed.     

Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily^1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya⁶. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila^7-14. An acetate kinase which can only utilize PP_i but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus^15,16.In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann^17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD⁺ by the enzymes pyruvate kinase and lactate dehydrogenase^21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine²³. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP⁺ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase²⁴.Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PP_i.     

GTP,as well as ATP,can act as a substrate for the intrinsic protein kinase activity of synaptic plasma membranes

Summary GTP as well as ATP can act as phosphate donor for the intrinsic protein kinase activity of synaptic plasma membranes. There are many similarities between the activities observed with ATP or GTP. Both need a divalent cation, Mg²⁺ being preferred, both are slightly inhibited by Na⁺, and more strongly by K⁺, both are inhibited by theophylline and adenosine. The K_m for GTP (0.13 mM) is similar to that ATP (0.12 mM). There are, however, some differences in properties. When GTP instead of ATP is the phosphate donor the pH optimum is 6.5 instead of 7.4. In addition NH ₄ ⁺ inhibits the transfer of phosphate from GTP but not from ATP. More importantly, cyclic AMP only stimulates the transfer of phosphate from ATP not from GTP. SDS gel electrophoresis reveals that similar membrane proteins are phosphorylated by GTP and ATP in the presence or absence of cyclic AMP. This suggests that there may be two different types of protein kinase in the synaptic plasma membrane which act on similar membrane proteins. One is stimulated by cyclic AMP and is specific to ATP while the other is unaffected by cyclic nucleotides and can use either ATP or GTP as phosphate donor.Deceased     

Tri-Calciphor (16,16-dimethyl-15-dehydroprostagalndin B1 trimer)-mediated mitochondrial Ca2+ movements: modulation by phosphate

The trimeric derivative of 16,16-dimethyl-15-dehydroprostaglandin B₁ (termed tri-Calciphor), which protects tissues against ischemic damage, induced Ca²⁺ efflux and swelling in mitochondria in the absence of phosphate, Mg²⁺ and ATP. When glutamate/malate rather than succinate was the substrate, higher tri-Calciphor concentrations were required for the ionophoretic activity. Ca²⁺ efflux and mitochondrial swelling induced by tri-Calciphor were completely inhibited by ATP, phopsphate and Mg²⁺ added together, and partially inhibited with phosphate plus either ATP or Mg²⁺. Between 0 and 7 μM added Ca²⁺ and in the presence of phosphate, ATP and Mg²⁺, tri-Calciphor stimulated the uptake of Ca²⁺ by mitochondria and increased the efficiency of buffering of extramitochondrial Ca²⁺. Thus depending on the assay conditions, two different effects involving Ca²⁺ movements and mitochondria are observed with tri-Calciphor.     

Nature of the transport ATPase-digitalis complex. VI. Purification of and ouabain binding to a highly active cardiac Na+, K+-ATPase

A Na⁺,K⁺-ATPase has been isolated from canine heart with a specific activity as high as 200 μmoles of inorganic phosphate/mg protein/hour. Activity is not due to simple detergent activation since specific ouabain binding (i.e., [Mg⁺⁺,Na⁺,ATP] or [Mg⁺⁺,P_i]-ligand dependent) ranged from 200–450 pmoles/mg protein. Specific ouabain binding activities are up to ten times greater than heretofore reported.     

On the red blood cell Ca2+-pump: An estimate of stoichiometry

Summary Efflux of Ca²⁺ from reversibly hemolyzed human red blood cell ghosts was determined by a Ca²⁺ selective electrode, by atomic absorption spectroscopy, and by the use of⁴⁵Ca. Hydrolysis of ATP was determined by measurement of inorganic phosphate (P_i). At 25°C, ghosts loaded with CaCl₂, MgCl₂, Na₂ATP, and Tris buffer (pH 7.4) extruded Ca²⁺, with mean rates ranging from 58.8±3.5 (sd) to 74.7±8.2 (sd) moles·liter ghosts^–1·min^– depending on the method of Ca²⁺ determination. The ratio of Ca²⁺ transported to P_i released in the presence of ouabain without correction for background ATP splitting was 0.83, 0.83, and 0.80, respectively, for the three methods of Ca²⁺ determination. Correction for the ATPase activity not associated with Ca²⁺ transport resulted in a ratio of 0.91:1. In other experiments, the use of La³⁺ to inhibit the Ca²⁺-pump allowed an estimate of the ATPase activity associated with Ca²⁺ extrusion. In the presence of various concentrations of La³⁺, the ratio of Ca²⁺ pumped to P_i liberated was 0.86 or 1.02, depending on the method of Ca²⁺ determination. It is concluded that the stoichiometry of the Ca²⁺-pump of the RBC plasma membrane is one Ca²⁺ pumped per ATP hydrolyzed.     

Polyphosphatase PPN1 of Saccharomyces cerevisiae: Switching of Exopolyphosphatase and Endopolyphosphatase Activities

The polyphosphatase PPN1 of Saccharomyces cerevisiae shows an exopolyphosphatase activity splitting phosphate from chain end and an endopolyphosphatase activity fragmenting high molecular inorganic polyphosphates into shorter polymers. We revealed the compounds switching these activities of PPN1. Phosphate release and fragmentation of high molecular polyphosphate prevailed in the presence of Co²⁺ and Mg²⁺, respectively. Phosphate release and polyphosphate chain shortening in the presence of Co²⁺ were inhibited by ADP but not affected by ATP and argininе. The polyphosphate chain shortening in the presence of Mg²⁺ was activated by ADP and arginine but inhibited by ATP.     

The relation of the 515 nanometers absorbance change to adenosine triphosphate formation in chloroplasts and digitonin subchloroplast particles

The flash-induced absorbance changes at 515 nanometers has been studied in chloroplasts and in digitonin subchloroplast particles of lettuce. The effect of various conditions and uncouplers was tested on the decay kinetics of this absorbance change and on ATP formation in the presence of phenazine methosulphate, either by continuous or flash illumination. It has been found that in chloroplasts, carbonyl cyanide m-chloromethoxyphenylhydrazone and nigericin in the presence of K⁺ accelerate the decay of the 515 change and inhibit ATP formation. However, under a variety of conditions the rate of decay of the 515 absorbance change was found to be unrelated to ATP formation. Preillumination, addition of valinomycin in the presence of K⁺, addition of Na⁺, or divalent cations accelerate the decay of the 515 absorbance change markedly but have no effect on ATP formation. Addition of phosphorylation reagents has no effect on the decay rate beyond that obtained by Mg²⁺ and inorganic phosphate. NH₄Cl, and to some extent atebrin, while inhibiting ATP formation, do not affect the decay of the 515 absorbance change.     

Mitochondrial ATP-Mg/phosphate carriers transport divalent inorganic cations in complex with ATP

The ATP-Mg/phosphate carriers (APCs) modulate the intramitochondrial adenine nucleotide pool size. In this study the concentration-dependent effects of Mg²⁺ and other divalent cations (Me²⁺) on the transport of [³H]ATP in liposomes reconstituted with purified human and Arabidopsis APCs (hAPCs and AtAPCs, respectively, including some lacking their N-terminal domains) have been investigated. The transport of Me²⁺ mediated by these proteins was also measured. In the presence of a low external concentration of [³H]ATP (12 μM) and increasing concentrations of Me²⁺, Mg²⁺ stimulated the activity (measured as initial transport rate of [³H]ATP) of hAPCs and decreased that of AtAPCs; Fe²⁺ and Zn²⁺ stimulated markedly hAPCs and moderately AtAPCs; Ca²⁺ and Mn²⁺ markedly AtAPCs and moderately hAPCs; and Cu²⁺ decreased the activity of both hAPCs and AtAPCs. All the Me²⁺-dependent effects correlated well with the amount of ATP-Me complex present. The transport of [¹⁴C]AMP, which has a much lower ability of complexation than ATP, was not affected by the presence of the Me²⁺ tested, except Cu²⁺. Furthermore, the transport of [³H]ATP catalyzed by the ATP/ADP carrier, which is known to transport only free ATP and ADP, was inhibited by all the Me²⁺ tested in an inverse relationship with the formation of the ATP-Me complex. Finally, direct measurements of Mg²⁺, Mn²⁺, Fe²⁺, Zn²⁺ and Cu²⁺ showed that they are cotransported with ATP by both hAPCs and AtAPCs. It is likely that in vivo APCs transport free ATP and ATP-Mg complex to different degrees, and probably trace amounts of other Me²⁺ in complex with ATP.     

Changes in phosphometabolites and intracellular pH in the tail muscle of the prawnPalaemon serratus as shown by in vivo31P-NMR

Summary ³¹P NMR spectra were recorded from tail muscles of the prawnPalaemon serratus, at rest, after exhaustive work and during subsequent recovery. At rest, the spectra indicated concentrations of phosphoarginine and ATP in good agreement with those obtained from resting fast skeletal muscles in mammals, which are characterized by a high phosphocreatine/P_i ratio. Following exhaustive work, phosphoarginine dropped by ca. 60% and ATP by 20%, while inorganic phosphate increased by 160%. The increase in inorganic phosphate immediately after contractions and in the first minutes of recovery corresponded partially to the changes in phosphoarginine and ATP. During recovery, the decrease of inorganic phosphate balanced the resynthesized phosphoarginine which was fully replenished within 30–40 min. The position of the inorganic phosphate resonance peak was used to monitor changes in intracellular pH (pH_i). The average pH_i in resting tail muscles was 7.20. After stimulation it was observed to decrease by 0.22 units. The return to pre-stimulation value was not achieved within 45 min. A NMR index (ATP+Arg-P)/(ATP+Arg-P+P_i) was calculated to characterize the extent of energetic changes caused by exercise.     

Differences between CTP and ATP as substrates for the (Na + K)-ATPase

CTP was a poorer substrate than ATP when substituted in the (Na + K)-ATPase reaction assay, not only in terms of K_m but also of V. CDP was a poorer inhibitor than ADP, so product inhibition cannot account for CTP being a poorer substrate. In the Na-ATPase reaction, which the enzyme also catalyzes, substituting CTP for ATP resulted in greater activity, arguing against CTP being less effective than ATP in forming the enzyme-phosphate intermediate common to both reactions. Ligands that favor the E₂ conformational state of the enzyme, K⁺, Mg²⁺, and Mn₂⁺, inhibited the (Na + K)-CTPase reaction more than the (Na + K)-ATPase. Conversely, Triton X-100, which favors the E₁ conformational state of the enzyme, K⁺, Mg²⁺, and Mn²⁺, inhibited the (Na + K)-CTPase ATPase reaction but stimulated the (Na + K)-CTPase. Although the (Na + K)-ATPase reaction sequence probably involves cyclical interconversion between E₁ and E₂ conformational states (and is thus inhibitable by ligands favoring either state), the K-phosphatase reaction catalyzed by the enzyme apparently functions entirely in the E₂ state. This reaction is better stimulated by CTP plus Na⁺ than by ATP plus Na⁺; moreover, CTP lessens inhibition by Triton X-100, and ATP lessens inhibition by inorganic phosphate (which reacts with the E₂ state). These observations indicate that CTP is a poorer substrate than ATP because it is less effective in promoting conversion of E₂ to E₁, essential for the (Na + K)-dependent reaction mechanism. However, contrary to this rationale, dimethyl sulfoxide stimulated the (Na + K)-CTPase reaction although by other criteria, including inhibition of the (Na + K)-ATPase, the reagent appears to favor the E₂ over the E₁ conformational state.     

Enzymatic dephosphorylation of endogenous and exogenous dolichyl monophosphate by calf brain membranes

Calf brain membranes have been shown to enzymatically dephosphorylate endogenous and partially purified, exogenous dolichyl [³²P]monophosphate. The properties and specificity of the dolichyl monophosphatase activity have been studied by following the release of [³²P]phosphate from exogenous dolichyl [³²P]monophosphate added in a dispersion with Triton X-100. The calf brain phosphatase (1) is inhibited by Mn²⁺, Mg²⁺, Ca²⁺, fluoride, and phosphate; (2) exhibits a neutral pH optimum; and (3) has an apparent K_m of 200 μm for dolichyl monophosphate. Dolichyl monophosphatase activity can be distinguished from phosphatidate phosphatase on the basis of their responses to fluoride and phosphate. Based on differential thermolability and the effects of divalent cations and EDTA, the calf brain dolichyl monophosphatase can also be discriminated from the general phosphatase activity assayed with p-nitrophenyl phosphate. Dolichyl monophosphatase activity can be solubilized by treating microsomes with Triton X-100. The enzymatic dephosphorylation of exogenous dolichyl [³²P]monophosphate catalyzed by particulate and detergent-solubilized preparations is negligibly affected by equimolar concentrations of ATP and an assortment of phosphomonoesters, including phosphatidic acid and hexadecyl phosphate. A reduction of approximately 40% in dolichyl monophosphatase activity is observed in the presence of equimolar amounts of retinyl monophosphate. Overall, these results represent good evidence for the presence of a neutral polyisoprenyl monophosphatase in central nervous tissue.