A single mutation converts the nucleotide specificity of phenol sulfotransferase from PAP to AMP

Sulfotransferases (STs) catalyze all the known biological sulfonations, in which a sulfuryl group from a common sulfonate donor such as 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is transferred to a nucleophilic acceptor. In addition to PAPS, phenol sulfotransferase (PST), a member of the ST family, utilizes other nucleotides as substrates with much less catalytic efficiency [Lin, E. S., and Yang, Y. S. (2000) Biochem. Biophys. Res. Commun. 271, 818-822]. Six amino acid residues of PST have been chosen for mutagenesis studies on the basis of a model of PST and its sequence alignment with those of available cytosolic and membrane-anchored STs. Systematic analyses of the mutants reveal that Ser134 is important for the regulation of nucleotide specificity between 3'-phosphoadenosine 5'-phosphate (PAP) and adenosine 5'-monophosphate (AMP). Kinetic studies also indicate that Ser134 plays a key role in nucleotide binding (K(m)) but not in catalysis (kcat). Consequently, the catalytic efficiency (kcat/K(m)) of PST can be altered by 5 orders of magnitude with a mutation of Ser134. Moreover, the change in nucleotide specificity from PAP to AMP can be achieved by mutation of Ser134 to any of the following residues: Glu, Gln, Arg, and His. Roles of Lys44, Arg126, and Arg253, which interact directly with the 5'- and 3'-phosphate of PAP, were also investigated by mutagenesis and kinetic experiments. On the basis of these findings, we suggest that Ser134 is the key residue that enables PST to discriminate PAP from AMP.     

Two Phenol Sulfotransferase Species from One cDNA: Nature of the Differences

A phenol sulfotransferase from rat liver (EC 2.8.2.9), expressed inEscherichia colifrom a single cDNA, was purified as two separable but catalytically active proteins. The proteins appeared to be identical to each other and to the natural liver sulfotransferase by comparison of their amino acid constitution, amino-terminal end group, and interaction with a polyclonal antibody raised against the liver enzyme. Each of the recombinant forms, α and β, catalyzed the sulfuryl group transfer from 4-nitrophenylsulfate to an acceptor phenol, a reaction in which 3′-phospho-adenosine 5′-phosphate (PAP) is a necessary intermediate. Only form β, however, catalyzes the physiological transfer of a sulfuryl group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the free phenol. Evidence is presented that sulfotransferase α, but not β, has 1 mol of PAP tightly bound per enzyme dimer. The ability to utilize PAPS as a sulfate donor could be altered: form α could be treated and purified as form β to acquire the ability to use PAPS, whereas form β was treated by extended incubation with PAP, lost its ability to use PAPS, and was purified as form α.     

Inhibition and binding studies of coenzyme A and bovine phenol sulfotransferase.

Phenol sulfotransferases (PSTs, EC 2.8.2.1) catalyze sulfonyl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the hydroxyl oxygen of aromatic acceptor substrates. The structural overlap between PAPS and coenzyme A (CoA) suggested a possible role of this common acyl carrier in modulating PST activity. To test this hypothesis, purified recombinant bovine PST was examined by kinetic and affinity chromatographic approaches. After demonstrating PST enzyme inhibition by CoA, systematic variation of CoA and PAPS concentrations indicated simple competitive inhibition with K(i) = 1. 3 microM. PST bound to CoA-agarose, attached via the pantetheinyl thiol group, was eluted with PAP but not by 2-naphthol. This observation was consistent with the pattern of inhibition. Additional members of the sulfotransferase superfamily, as well as acylated CoAs, should be further investigated.     

Kinetic analysis of NodST sulfotransferase using an electrospray ionization mass spectrometry assay

A novel and efficient enzyme kinetics assay using electrospray ionization mass spectrometry was developed and applied to the bacterial carbohydrate sulfotransferase (NodST). NodST catalyzes the sulfuryl group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to chitobiose, generating 3'-phosphoadenosine 5'-phosphate (PAP) and chitobiose-6-OSO(3)(-) as products. Traditional spectrophotometric assays are not applicable to the NodST system since no shift in absorption accompanies sulfuryl group transfer. Alternative assays have employed thin-layer chromatography, but this procedure is time-consuming and requires radioactive materials. The ESI-MS assay presented herein requires no chromophoric substrate or product, and the analysis time is very short. The ESI-MS assay is used to determine NodST kinetic parameters, including K(M), V(max), and K(i) (for PAP). In addition, the mode of inhibition for PAP was rapidly determined. The results were in excellent agreement with those obtained from previous assays, verifying the accuracy and reliability of the ESI-MS assay. This unique technique is currently being used to investigate the enzymatic mechanism of NodST and to identify sulfotransferase inhibitors.     

Purification and characterization of bile salt sulfotransferase from human liver

Bile salt sulfotransferase, the enzyme responsible for the formation of bile salt sulfate esters, was purified extensively from normal human liver. The purification procedure included DEAE-Sephadex chromatography, taurocholate-agarose affinity chromatography, and preparative isoelectrofocusing. The final preparation had a specific activity of 18 nmol min-1 mg protein-1, representing a 760-fold purification from the cytosol fraction with a overall yield of 15%. The human enzyme has a Mr of 67,000 and a pI of 5.2. DEAE-Sephadex chromatography of the cytosol fraction revealed only a single species of activity. The limiting Km for the sulfuryl donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), is 0.7 microM. The limiting Km for the sulfuryl acceptor, glycolithocholate (GLC), is 2 microM. Reciprocal plots were intersecting. Product inhibition studies established that adenosine 3',5'-diphosphate (PAP) was competitive with PAPS (Ki = 0.2 microM) and noncompetitive with respect to GLC. GLC sulfate was competitive with GLC (Ki = 2.2 microM) and noncompetitive with respect to PAPS. Also, 3-ketolithocholate, a dead-end inhibitor, was competitive with GLC (Ki = 0.6 microM) and noncompetitive with respect to PAPS. Iso-PAP (the 2' isomer of PAP) was competitive with PAPS (Ki = 0.3 microM) and noncompetitive with GLC. The cumulative results of the steady-state kinetics experiments point to a random mechanism for the binding of substrates and release of products. The purified enzyme displays no activity toward estrone, testosterone, or phenol. Among the reactive substrates tested, the Vmax/Km values are in the order GLC greater than 3-beta OH-5-cholenic acid greater than glycochenodeoxycholate greater than glycocholate. p-Chloromercuribenzoate inactivated the enzyme. Either PAPS or GLC protected against inactivation, suggesting the presence of a sulfhydryl group at the active site.     

Spectrofluorimetric analysis of 7-hydroxycoumarin binding to bovine phenol sulfotransferase

Phenol sulfotransferases (SULT1s, EC 2.8.2.1) catalyze sulfuryl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the hydroxyl oxygen of aromatic acceptor substrates. Previous work with the bovine SULT1A1 has utilized the highly fluorescent substrate 7-hydroxycoumarin (7-HC, umbelliferone) as an acceptor substrate [Biochem. Biophys. Res. Commun. 261 (1999) 815]. Here we report that adenosine-3',5'-bisphosphate (PAP)-dependent binding of 7-HC to bSULT1A1 can be observed due to the appearance of a 400-420-nm shoulder in the emission spectrum, using an excitation wavelength of 280 nm. This emission was observed by placing 7-HC in ethanol, which is consistent with bSULT1A1 phenol binding site hydrophobicity. Titrations with 7-HC indicate a K(d) for 7-HC of 0.58 microM and substoichiometric binding to the homodimeric enzyme. The bSULT1A1:PAP:7-HC complex could be disrupted with pentachlorophenol (PCP), titrations with which indicated 0.5 equivalents per enzyme subunit. Titrations of enzyme plus 7-HC with PAP also indicated 0.5 equivalents per enzyme subunit. These results suggest a model of homodimeric bSULT1A1 in which subunit interactions favor half-site reactivity in the formation of a dead end complex.     

Phenol sulfotransferase in humans: properties, regulation, and function

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic and catechol drugs and neurotransmitters. All human tissues that have been studied in detail contain at least two forms of PST. One form is thermolabile (TL), catalyzes the sulfate conjugation of micromolar concentrations of dopamine and other phenolic monoamines, and is relatively resistant to inhibition by 2,6-dichloro-4-nitrophenol (DCNP). The other form is thermostable (TS), catalyzes the sulfate conjugation of micromolar concentrations of simple phenols such as p-nitrophenol, and is relatively sensitive to DCNP inhibition. These two forms of PST have been physically separated and partially purified from several human tissues, including an easily accessible tissue, the blood platelet. The biochemical properties of platelet PST are very similar to those of PST in human brain, liver, and small intestine. Individual differences in the basal activity of TS PST in the platelet are correlated with individual variations in the activity of this form of the enzyme in human cerebral cortex (r = .94, n = 15, P less than 0.001). In addition, both platelet TS and TL PST activities are correlated significantly with the extent of sulfate conjugation of orally administered drugs such as acetaminophen and methyldopa. These latter observations are compatible with the conclusions that platelet PST activity may reflect the activity of the enzyme at sites of drug metabolism, and that variation in PST activity is one factor responsible for individual differences in the sulfate conjugation of orally administered drugs.     

Fluorometric assay for alcohol sulfotransferase

A sensitive fluorometric assay was developed for alcohol sulfotransferase (AST). This was the first continuous fluorometric assay reported for AST. It used 3'-phosphoadenosine 5'-phosphosulfate regenerated from 3-phosphoadenosine 5'-phosphate by a recombinant phenol sulfotransferase (PST) using 4-methylumbelliferyl sulfate as the sulfuryl group donor. The recombinant PST did not use the alcohol substrate under the designed condition, and the sensitivity for AST activity was found to be comparable to that of radioactive assay as reported in the literature. The change of fluorescence intensity of 4-methylumbelliferone corresponded directly to the amount of active AST and was sensitive enough to measure nanogram or picomole amounts of the enzyme activity. This fluorometric assay was used to determine the activities of AST as purified form and in crude extracts of pig liver, rat liver, and Escherichia coli. Some properties of human dehydroepiandrosterone sulfotransferase were determined by this method and were found to be comparable to published data. Under similar assay conditions, the contaminated activities of arylsulfatase in crude extracts were also determined. This method not only is useful for the routine and detailed kinetic study of this important class of enzymes but also has the potential for the development of a high-throughput procedure using microplate reader.     

Human Phenol Sulfotransferase: Correlation of Brain and Platelet Activities

Phenol sulfotransferase (PST; EC 2.8.2.1) catalyzes the sulfate conjugation of phenolic and catechol neurotransmitters and drugs. The human blood platelet has been the most thoroughly studied source of PST because of the possibility that the regulation of the enzyme in this easily accessible tissue might reflect the regulation of PST in the CNS. The human brain and platelet contain at least two forms of PST, forms designated as thermostable (TS) and thermolabile (TL) PST. TS PST catalyzes the sulfate conjugation of micromolar concentrations of phenol and p-nitrophenol and TL PST catalyzes the sulfate conjugation of dopamine and other monoamines. This study was performed to determine whether individual variations in the activities of human platelet TS and TL PST reflect individual variations in cerebral cortical PST activities. PST activities were measured in platelets and in cerebral cortical tissue obtained from 15 patients with epilepsy during clinically indicated neurosurgery. There was a highly significant correlation between the activities of the TS form of PST in cerebral cortex and platelets of these patients (r = 0.940, p less than 0.001), but there was not a significant correlation between activities of the TL form of PST in the two tissues (r = 0.396, p greater than 0.14). In addition to variations in the level of enzyme activity, there are also wide individual variations in the thermal stability of platelet TS PST.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human platelet phenol sulfotransferase: Familial variation in thermal stability of the TS form

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of catechol and phenolic drugs and xenobiotic compounds. Platelets and other tissues contain at least two forms of PST, forms that have been designated the "TL" and the "TS" forms. We measured the thermal stability of platelet TS PST in blood samples from 218 randomly selected unrelated subjects by heating platelet homogenates at 44 degrees C for 15 min. Thermal stability was expressed as the ratio of the enzyme activity remaining after preincubation to that in an unheated sample, a heated/control (H/C) ratio. The frequency distribution of H/C ratios for this population sample was bimodal, with a nadir at an H/C ratio of 0.33. Of the 218 subjects studied, 29 (13.3%) had thermolabile TS PST (H/C less than 0.33). Platelet samples were then obtained from subjects with thermolabile and thermostable TS platelet PST. PST activity in these platelet samples had similar apparent Km constants for substrates. IC50 values for inhibition of TS PST by 2,6-dichloro-4-nitrophenol in these samples were also nearly identical. The results of experiments in which platelet homogenates from subjects with thermolabile and thermostable TS PST were mixed and the results of experiments in which platelet homogenates were subjected to gel filtration chromatography were compatible with the conclusion that individual differences in TS PST thermal stability were properties of PST itself. Finally, there was a significant familial aggregation of the trait of thermolabile TS PST when H/C ratios were measured in platelet homogenates from 231 members of 49 randomly selected families.     

Human Brain Phenol Sulfotransferase: Biochemical Properties and Regional Localization

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of catecholamines and phenol and catechol drugs. The human blood platelet contains a thermolabile (TL) form of PST that catalyzes the sulfate conjugation of dopamine and other monoamines and a thermostable (TS) form that catalyzes the sulfate conjugation of micromolar concentrations of phenol and p-nitrophenol. Experiments were performed to determine whether the brain contains forms of PST analogous to the TL and TS forms found in the human platelet, and to determine whether there are regional variations in human brain PST activity. We found that the human brain contains at least two forms of PST, forms that are similar to the platelet TS and TL forms of the enzyme with respect to substrate specificity, apparent Km constants, thermal stability, and sensitivity to inhibitors. Optimal conditions were determined for the measurement of these two activities in brain homogenates. The stability of PST activities in the human brain after death was determined in five samples of cerebral cortex that were obtained during clinically indicated neurosurgical procedures. An average of 76 +/- 8% and 80 +/- 9% (mean +/- SEM) of the basal TL and TS PST activities, respectively, remained in these five samples of cerebral cortex after 8 h of storage under simulated post-mortem conditions. Six human brains were then obtained less that 8 h after death from patients who had no neurological disease prior to death. The mean activities of the TL and TS forms of PST were measured in 17 different regions of the six brains. If the pituitary was excluded from consideration, TL and TS PST activities both varied approximately fivefold among these regions, and both activities were highest in cerebral cortex. However, the average TS activity in the anterior pituitary, a tissue of non-neural origin embryologically, was 6.5-fold greater than the highest average TS PST activity found in cerebral cortex.     

Degradation of human pancreastatin-52 by human kidney extract.

We examined the in vitro degradation of human pancreastatin-52 (hPST-52) and a larger molecular form (approximate 15 kDa) of human PST by an enzyme extract from human kidney. The PST-degrading activity was determined from the amount of immunoreactive PST remaining after incubation of hPST-52 or the larger molecular form with the enzyme extract. Human PST-52 was degraded to smaller molecular forms within 30 min, but the larger molecule was not degraded within 90 min. Phosphoramidon, an inhibitor of endopeptidase, metal ion chelators (EDTA and 1, 10-phenanthroline) and Cu2+ prevented the degradation of hPST-52. These results indicated that the enzyme in the kidney extract degraded hPST-52 and smaller forms of the peptide, but had no effect on the 15 kDa form.     

A nucleotide-gated molecular pore selects sulfotransferase substrates

Human SULT2A1 is one of two predominant sulfotransferases in liver and catalyzes transfer of the sulfuryl moiety (-SO(3)) from activated sulfate (PAPS, 3'-phosphoadenosine 5-phosphosulfate) to hundreds of acceptors (metabolites and xenobiotics). Sulfation recodes the biologic activity of acceptors by altering their receptor interactions. The molecular basis on which these enzymes select and sulfonate specific acceptors from complex mixtures of competitors in vivo is a long-standing issue in the SULT field. Raloxifene, a synthetic steroid used in the prevention of osteoporosis, and dehydroepiandrosterone (DHEA), a ubiquitous steroid precusor, are reported to be sulfated efficiently by SULT2A1 in vitro, yet unlike DHEA, raloxifene is not sulfated in vivo. This selectivity was explored in initial rate and equilibrium binding studies that demonstrate pronounced binding antisynergy (21-fold) between PAPS and raloxifene, but not DHEA. Analysis of crystal structures suggests that PAP binding restricts access to the acceptor-binding pocket by restructuring a nine-residue segment of the pocket edge that constricts the active site opening, or "pore", that sieves substrates on the basis of their geometries. In silico docking predicts that raloxifene, which is considerably larger than DHEA, can bind only to the unliganded (open) enzyme, whereas DHEA binds both the open and closed forms. The predictions of these structures with regard to substrate binding are tested using equilibrium and pre-steady-state ligand binding studies, and the results confirm that a nucleotide-driven isomerization controls access to the acceptor-binding pocket and plays an important role in substrate selection by SULT2A1 and possibly other sulfotransferases.     

Subcellular distribution of N-ethylmaleimide-sensitive and -insensitive phosphatidic acid phosphohydrolase in rat brain

The dephosphorylation of phosphatidic acid by phosphatidic acid phosphohydrolase (PAP) is important in both cell-signalling and in glycerolipid metabolism. However, these roles are apparently performed by two different enzymes, which can be distiuguisged by their sensitivity in vitro to N-ethylmaleimide (NEM) Both of these enzymes are present in rat brain as well as a wide range of other rat tissues. However, the quantity and specific activity of each enzyme varies considerably between different tissues, as does the ratio of the two enzymes in each tissue. Tissues rich in glycerolipids are abundant in NEM-sensitive PAP, whereas there is no obvious pattern to the distribution of the NEM-insensitive enzyme in the different tissues tested. Studies on brain cortex, which is relatively rich in both forms of PAP, indicate that the NEM-insensitive PAP is located in the synaptosomes, and the NEM-sensitive enzyme present in the cytosol and microsomes. The NEM-sensitive PAP can also be translocated from the cytosol to the microsomes by oleate. When assayed against a range of phosphatidic acids, NEM-sensitive PAP showed a preference for phosphatidic acids with short acyl chains and for those containing arachidonate, whereas NEM-insensitive PAP had a preference for short and unsaturated acyl chains. The two isozymes also had different activity profiles against these substrates suggesting that they are in fact different enzymes. The implications for these results on the putative roles of the two forms of PAP are discussed.     

Phenol sulfotransferase inheritance

1. Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of many phenolic and catechol neurotransmitters. Human tissues contain both thermostable (TS) and thermolabile (TL) forms of PST that differ in their substrate specificities, inhibitor sensitivities, physical properties, and regulation. 2. Individual variations in the levels of activity of both TS and TL PST in the human platelet are strongly influenced by inheritance. 3. Individual differences in the level of platelet TS PST activity are correlated with individual variations in the activity of this form of the enzyme in human cerebral cortex, liver, and intestinal mucosa. 4. There are also individual familial differences in the thermal stability of TS PST in the platelet. These differences are correlated with individual variations in the thermal stability of TS PST in cerebral cortex, liver, and intestinal mucosa. 5. Individual variations in the thermal stability of TS PST in hepatic tissue are associated with the presence of one or both of a pair of TS PST isozymes that can be separated by ion-exchange chromatography and that differ in their thermal stabilities. 6. This series of observations suggests that a structural gene polymorphism may be one mechanism by which inheritance controls TS PST in humans. The isozymes of TS PST in liver may represent the products of alternative alleles for this polymorphism, alleles that might control the structure of TS PST in many human tissues.     

Amine N-sulfotransferase

A highly purified amine N-sulfotransferase has been isolated from guinea pig liver that catalyzes sulfuryl group transfer from 3'-phosphoadenosine 5'-phosphosulfate to one of a large number of either primary or secondary amines forming the appropriate sulfamate and adenosine 3',5'-bisphosphate. Amines as different as aniline, 2-naphthylamine, octylamine, 1,2,3,4-tetrahydroisoquinoline and 1,2,3,4-tetrahydroisoquinoline, desmethylimipramine, and cyclohexylamine serve as acceptors; the product of the last of these substrates is the sugar-substitute cyclamate. Amine N-sulfotransferase activity is dependent on the presence of an unprotonated amino group. The purified enzyme preparation also has O-sulfotransferase activities, suggesting that transfer to oxygen could represent an intrinsic function of the N-sulfotransferase.     

Sulfation of hypertensive and hypotensive drugs by monkey brain phenol sulfotransferase

The substrate specificity and affinity of two forms of phenol sulfotransferase (PST) from Rhesus macaque brain cortex were studied. Catecholamines, their methylated metabolites (normetanephrine, metanephrine) and methylated precursor, -methylDOPA, were examined as substrates for both the cationic (PST I) and the anionic (PST II) forms of the enzyme. Sulfation of hypertensive drugs (phenylephrine, octopamine, metaraminol), hypotensive drugs (-methylDOPA, minoxidil), and related agents without a free hydroxy group on the benzene ring were also studied. Results indicated that both PST forms sulfated -methylDOPA and minoxidil, but only PST II transferred the sulfate group to catecholamines and most of the adrenergic agents examined.     

Immunocytochemical localization of a tartrate-resistant and vanadate-sensitive acid nucleotide tri- and diphosphatase

Purified rabbit antiserum to a tartrate-resistant and vanadate-sensitive acid phosphatase (nucleotide tri- and diphosphatase) prepared from rat bone was used in immunocytochemical studies. The antigen was localized in sections of fixed, decalcified tissue (head from rat) using the peroxidase-antiperoxidase bridge (PAP) or the avidin-biotin-peroxidase complex (ABC) technique. Both techniques resulted in similar and specific immunostaining in the following cells and tissues: osteoclasts situated in resorption lacunae, epithelium overlying enamel-free areas of tips of cusps of unerupted molars, cilia of respiratory epithelium, and tissue macrophages. This distribution corresponds to the cellular sites of tartrate-resistant acid phosphatase activity, as revealed by enzyme histochemistry. With the ABC method, staining in osteoclasts was obtained with antiserum dilutions of up to 1:10,000. Biochemical studies revealed that vanadate-sensitive acid ATPase activity in liver subcellular fractions was almost exclusively confined to lysosomes. Thus, the immunostaining has revealed the presence of the tartrate-resistant and vanadate-sensitive nucleotide phosphatase in many cells associated with tissue resorption and phagocytosis.     

Multiple forms of poly(A) polymerases purified from HeLa cells function in specific mRNA 3''-end formation.

Poly(A) polymerases (PAPs) from HeLa cell cytoplasmic and nuclear fractions were extensively purified by using a combination of fast protein liquid chromatography and standard chromatographic methods. Several forms of the enzyme were identified, two from the nuclear fraction (NE PAPs I and II) and one from the cytoplasmic fraction (S100 PAP). NE PAP I had chromatographic properties similar to those of S100 PAP, and both enzymes displayed higher activities in the presence of Mn2+ than in the presence of Mg2+, whereas NE PAP II was chromatographically distinct and had approximately equal levels of activity in the presence of Mn2+ and Mg2+. Each of the enzymes, when mixed with other nuclear fractions containing cleavage or specificity factors, was able to reconstitute efficient cleavage and polyadenylation of pre-mRNAs containing an AAUAAA sequence element. The PAPs alone, however, showed no preference for precursors containing an intact AAUAAA sequence over a mutated one, providing further evidence that the PAPs have no intrinsic ability to recognize poly(A) addition sites. Two additional properties of the three enzymes suggest that they are related: sedimentation in glycerol density gradients indicated that the native size of each enzyme is approximately 50 to 60 kilodaltons, and antibodies against a rat hepatoma PAP inhibited the ability of each enzyme to function in AAUAAA-dependent polyadenylation.     

Role of phosphatidic acid phosphatase 2a in uptake of extracellular lipid phosphate mediators

Phosphatidic acid (PA) phosphatase 2a (PAP2a) is an integral membrane glycoprotein that hydrolyzes a number of structurally related lipid phosphate substrates when presented in mixed phospholipid and detergent micelles. The physiological substrate specificity and functions of this enzyme are unclear. Using reconstitution studies we demonstrate that PAP2a hydrolyses both PA and LysoPA substrates in a lipid bilayer. To investigate the activity of PAP2a against cellular substrates we generated HEK293 cell variants stably overexpressing the enzyme. Although one of these lines exhibited a 27-fold increase in PAP2 activity measured in vitro, levels of PA were not significantly reduced in comparison with control cells. Cell surface labeling and activity measurements demonstrate that a portion of the enzyme was localized to the cell surface. Pagano and Longmuir (J. Biol. Chem. 260 (1985) 1909) described the rapid uptake of PA by cultured cells, but the mechanisms and proteins involved were not identified. We found that overexpression of PAP2a was accompanied by a 2.1-fold increase in uptake of a fluorescent PA analog but that uptake of other phospholipids and diacylglycerols was unaltered. The increase in lipid uptake was completely dependent on PAP activity and unaffected by endocytosis inhibitors. Our results indicate that PAP2a is a cell surface enzyme that plays an active role in the hydrolysis and uptake of lipids from the extracellular space.