3beta-Hydroxysteroid dehydrogenase activity in human fetal membranes.

A 3beta-hydroxysteroid dehydrogenase (3betaHSD) was demonstrated in term human fetal membranes (chorion and amnion) with both dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) and pregnenolone (3beta-hydroxy-5-pregnen-20-one as substrates, and the subcellular distribution substrate and nucleotide specificity of the enzyme was studied. In both membranes the microsomal fraction (particles which sedimented at 105,000 g after 90 min) had the highest specific activity. The chorion was more active than the amnion but the enzyme in both tissues had similar substrate and nucleotide specificity. NAD was the preferred cofactor, and pregnenolone was a better substrate than dehydroepiandrosterone in the presence of NAD. However, with NADP as cofactor both steroids were equally good substrates. When the 3beta-hydroxysteroid dehydrogenase activity of chorion microsomes was compared with that of placental microsomes, the specific activities were found to be of the same order of magnitude, and the substrate, nucleotide specificity and steroid binding properties were almost identical.     

Purification and properties of acyl-CoA:1-acyl-sn-glycero-3-phosphocholine-O-acyltransferase from bovine brain microsomes

Acyl-CoA:1-acyl-sn-glycero-3-phosphocholine-O-acyltransferase has been purified approximately 3000-fold from bovine brain microsomes by detergent solubilization followed by ion-exchange and affinity chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single protein of molecular weight 43,000. The specificity of the purified enzyme was studied by measuring the catalytic activity with various lysophospholipids and acyl-CoA derivatives. Of the lysophospholipids tested, only lysophosphatidylcholine was a substrate. Less specificity was exhibited toward the acyl-CoA derivatives, although the enzyme showed a clear preference for arachidonoyl-CoA and little or no activity with palmitoyl-CoA or stearoyl-CoA. High concentrations of arachidonoyl-CoA inhibited the enzyme. The velocity was a sigmoidal function of the concentration of lysophosphatidylcholine (LPC) with little activity obtained below 20 microM LPC. The specificity and kinetic properties of the enzyme were altered, however, by incorporation of the enzyme into liposomes composed of a mixture of phospholipids. Decanoyl-CoA and myristoyl-CoA, which were effective substrates for the soluble enzyme, did not serve as acyl donors for the liposome-bound acyltransferase. Furthermore, the liposome-bound enzyme, in contrast to the soluble form of the enzyme, was active at concentrations of LPC below the critical micelle concentration. The liposome-bound enzyme was also substantially less susceptible to thermal denaturation and proteolytic digestion. This modulation of the acyltransferase activity by interaction with phospholipids may relate to the kinetic properties and the regulation of the enzyme in vivo.     

The identification and characterization of protein kinase C activity in fetal membranes

Protein kinase C activity was demonstrated in cytosolic fractions prepared from human amnion and decidua vera tissues. The enzyme has been partially purified and was found to be glycerophospholipid-dependent. Phosphatidylserine was most active in the stimulation of protein kinase C. Ca2+ was also required for the expression of the enzyme activity. In the presence of unsaturated diacylglycerols, maximum activation of protein kinase C was observed at suboptimal concentrations of Ca2+. A possible role of phospholipid-dependent protein kinase C in the regulation of arachidonic acid release in this tissue is discussed.     

Identification of human plasma lysophospholipase D,a lysophosphatidic acid-producing enzyme,as autotaxin,a multifunctional phosphodiesterase

We purified human plasma lysophospholipase D that produces physiologically active lysophosphatidic acid and showed that it is a soluble form of autotaxin, an ecto-nucleotide pyrophosphatase/phosphodiesterase, originally found as a tumor cell motility-stimulating factor. Its lower K(m) value for a lysophosphatidylcholine than that for a synthetic substrate of nucleotide suggests that lysophosphatidylcholine is a more likely physiological substrate for autotaxin and that its predicted physiological and pathophysiological functions could be mediated by its activity to produce lysophosphate acid, an intercellular mediator. Recombinant autotaxin was found to have lysophospholipase D activity; its substrate specificity and metal ion requirement were the same as those of the purified plasma enzyme. The activity of lysophospholipase D for exogenous lysophosphatidylcholine in human serum was found to increase in normal pregnant women at the third trimester of pregnancy and to a higher extent in patients in threatened preterm delivery, suggesting its roles in induction of parturition.     

Lipid metabolism by The gall-bladder. II. The in vitro conversion of lysophosphatidylcholine to phosphatidylcholine.

Lysophosphatidylcholine acyltransferase, which catalyzes the acylation of lysophosphatidylcholine with fatty acid coenzyme A to form phosphatidylcholine, was assayed in gall-bladder mucosa. In guinea pig gall-bladder the activity parallels that of the microsomal enzyme, glucose-6-phosphatase with 3--4-fold enrichment of the activity in the microsomes. Studies with saturated and unsaturated substrates demonstrated highest activity when oleoyl coenzyme A and palmitoyl lysophosphatidylcholine were used and the lowest activity when palmitoyl coenzyme A and palmitoyl lysophosphatidylcholine were used. This activity was demonstrated in the dog, rabbit, cat, calf and human gall-bladder mucosa; however, a wide variation in the amount was observed. Lysophospholipase, which catalyzes the hydrolysis of lysophosphatidylcholine to glycerophosphorylcholine and fatty acid, was also demonstrated in gall-bladder mucosa.     

Acyltransferase activities in adult rat type II pneumocyte-derived subcellular fractions

Acyl-CoA: lysophosphatidylcholine, acyl-CoA: lysophosphatidylethanolamine, and lysophosphatidylcholine:lysophosphatidylcholine acyltransferases were investigated using subcellular fractions derived from adult rat type II pneumocytes in primary culture. Acyl-CoA:lysophospholipid acyltransferase activities were determined to be microsomal, while lysophosphatidylcholine:lysophosphatidylcholine acyltransferase activity was found to be cytosolic. Total palmitoyl CoA:lysophosphatidylcholine acyltransferase activity was 30-fold greater than lysophosphatidylcholine:lysophosphatidylcholine acyltransferase activity, indicating that the former enzyme is more important in the synthesis of dipalmitoyl phosphatidylcholine. Palmitoyl-CoA and oleoyl-CoA lysophosphatidylcholine acyltransferase activities were approximately equal under optimal substrate conditions. Specific activities of the enzyme using arachidoyl-CoA and arachidonoyl-CoA were 46% and 18%, respectively, of those with palmitoyl-CoA. Acyl-CoA:lysophosphatidylethanolamine acyltransferase showed a preference for palmitoyl-CoA as opposed to oleoyl-CoA under optimal conditions. However, when equimolar concentrations of either palmitoyl-CoA and oleoyl-CoA or palmitoyl-CoA and arachidoyl-CoA were assayed together, the relative utilization of the two substrates was found to be dependent on total acyl-CoA concentration. At higher concentrations, the incorporation of palmitoyl-CoA into phosphatidylcholine was less than other acyl-CoAs. However, at lower concentrations palmitoyl-CoA was utilized quite selectively. Whole lung microsomes did not show as marked a preference for palmitoyl-CoA as did type II pneumocyte microsomes under these same conditions. In similar experiments, low total acyl-CoA concentrations produced greater incorporation of oleoyl-CoA into phosphatidylethanolamine. For both enzymes total activity at the lowest concentrations used was at least 45% that at optimal conditions. This demonstrates that the type II pneumocyte acyltransferase system(s) can selectively utilize palmitoyl-CoA. No evidence for direct exchange of palmitoyl-CoA with 1-saturated-2-unsaturated phosphatidylcholine in subcellular fractions from type II pneumocytes was found.     

Characterization of the interactions between lysophosphatides, Triton X-100, and sarcoplasmic reticulum.

The interaction of lysophosphatidylcholines and lysophosphatidylethanolamines with lobster abdominal muscle sarcoplasmic reticulum was studied. Only lysophosphatidylcholines with 16 and 18 carbon acyl chains were effective solubilizing agents. The rate of membrane solubilization was most rapid with the palmitoyl and oleoyl derivatives. All lysophosphatides partially inhibited calcium-dependent ATPase activity between 0.0 and 2.0 μmol of lysophosphatide mg^?1 of membrane protein. Lysophosphatides that were active in solubilizing membranes exhibited a reactivating effect on ATPase activity between 2.0 and 6.0 μmol of lysophosphatide mg^?1 of membrane protein. Arrhenius plots of temperature-dependent ATPase activity showed high activation energies and loss of discontinuities in Arrhenius plots when inhibiting concentrations of the lysophosphatides were present. These results suggest that the inhibiting effect of lysophosphatides on membrane enzyme activity is due to intrusion of the lysophosphatide into the membrane, which results in a less fluid lipid environment around the enzyme. Subsequent membrane solubilization at higher lysophosphatide concentrations may release the enzyme from the inhibiting effects of the lysophosphatide by increasing lipid fluidity neighboring the enzyme. The effects of lysophosphatides on a membrane enzyme system were also examined in the presence of 10 mm Triton X-100. Under these conditions, little effect on membrane enzyme activity was generated by increasing concentrations of the lysophosphatidylcholines (lauryl, palmitoyl, and steroyl), while the unsaturated lysophosphatidylcholine and the lysophosphatidylethanolamines caused a two- to threefold increase in enzyme activity. Temperature-dependent enzyme activity studies showed that discontinuities in the Arrhenius plots of enzyme activity occurred at varying temperatures, depending on the lysophosphatide used. Lowest transition temperatures occurred for lysophosphatidylcholine (oleoyl) and the lysophosphatidylethanolamines. These results suggest that, in the presence of 10 mm Triton X-100, lipid exchange occurs around the sarcoplasmic reticulum ATPase enzyme and the fluidity of this lipid-protein complex is increased by lysophosphatides with unsaturated acyl chains or ethanolamine head groups.     

Isolation and characterization of three lysophospholipases from the murine macrophage cell line WEHI 265.1.

Anion exchange chromatography of WEHI 265.1 cell homogenates resolved the lysophospholipase activity into three peaks, when assayed using lysophosphatidylcholine as a substrate. Peaks 1 and 2 were purified by sequential hydrophobic interaction and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peaks 1 and 2 indicated homogeneous proteins with apparent masses of 28 and 27 kDa, respectively. Peak 3 lysophospholipases was partially purified by hydrophobic, hydroxyapatite and gel filtration chromatography. Peak 3 lysophospholipase also had calcium-dependent phospholipase A2 activity, which further co-purified with the lysophospholipase activity. The three lysophospholipases were characterized with respect to substrate specificity, additional enzymatic activities and the effects of lipids, metal ions and other compounds on enzymatic activity. Peaks 1, 2 and 3 hydrolyzed lysophosphatidylcholine most readily, but lysophosphatidylethanolamine also served as substrate for each enzyme. Furthermore, all three enzymes hydrolyzed platelet activating factor and acetylated lysophosphatidylcholine. Each lysophospholipase was inhibited by free fatty acids and by palmitoyl carnitine, although the relative sensitivities to these agents differed among the enzymes. The lysophospholipase activities of peaks 1 and 2, but not peak 3, were inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate and N-ethylmaleimide. Although they had similar masses, the amino acid compositions of peaks 1 and 2 differed, indicating that these are distinct proteins rather than posttranslational modifications of the same gene product.     

Thioesterase activity and subcellular localization of acylprotein thioesterase 1/lysophospholipase 1

Acylprotein thioesterase 1 (APT1), also known as lysophospholipase 1, is an important enzyme responsible for depalmitoylation of palmitoyl proteins. To clarify the substrate selectivity and the intracellular function of APT1, we performed kinetic analyses and competition assays using a recombinant human APT1 (hAPT1) and investigated the subcellular localization. For this purpose, an assay for thioesterase activity against a synthetic palmitoyl peptide using liquid chromatography/mass spectrometry was established. The thioesterase activity of hAPT1 was most active at neutral pH, and did not require Ca²⁺ for its maximum activity. The K_M values for thioesterase and lysophospholipase (against lysophosphatidylcholine) activities were 3.49 and 27.3 μM, and the V_max values were 27.3 and 1.62 μmol/min/mg, respectively. Thus, hAPT1 revealed much higher thioesterase activity than lysophospholipase activity. One activity was competitively inhibited by another substrate in the presence of both substrates. Immunocytochemical and Western blot analyses revealed that endogenous and overexpressed hAPT1 were mainly localized in the cytosol, while some signals were detected in the plasma membrane, the nuclear membrane and ER in HEK293 cells. These results suggest that eliminating palmitoylated proteins and lysophospholipids from cytosol is one of the functions of hAPT1.     

Purification and characterization of thiol aminopeptidase from the cytosolic fraction of human placenta

A thiol-dependent aminopeptidase was purified from the cytosolic fraction of human placenta. The purified enzyme consisted of a single polypeptide chain with a mol wt of 95,000. The enzyme was most active in the neutral region with Ala-pNA as substrate, and the activity was increased about 20-fold in the presence of some -SH compounds. The results of substrate specificity studies indicated that the enzyme hydrolyzes bonds involving the amino groups of neutral and basic amino acid residues. However, higher thiol-dependent activity was only detected with neutral ones. The enzyme was strongly inhibited by microbial aminopeptidase inhibitors, puromycin, o-phenanthroline, and sulfhydryl reactive-reagents. As to several naturally occurring peptides tested, the enzyme showed N-terminal Tyr-releasing activity toward enkephalins and kinin-converting activity.     

Purification, Properties, and Specificity of Rat Brain Cytosolic Fatty Acyl Coenzyme A Hydrolase

Abstract: Rat brain cytosolic acyl-CoA hydrolase has been purified 3,500-fold to apparent homogeneity using heat treatment, ammonium sulfate fractionation followed by anion exchange, hydrophobic interaction, and hydroxyapatite chromatography. The purified enzyme remains stable only in the presence of a high concentration (30%, vol/vol) of ethylene glycol. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified enzyme shows a single band of 40.9 kDa. However, on high-performance size-exclusion chromatography the migration rate of the enzyme corresponds with an apparent molecular mass of 148 kDa, indicating that the native enzyme may be a tetramer. The enzyme catalyzes the hydrolysis of fatty acyl-CoAs from six to 18 carbon chains long, having the highest activity for lauroyl (12:0)-CoA. For the purified enzyme the K _m for palmitoyl-CoA is 5.8 µ M and the V _max is 1,300 µmol/min/mg of protein. The enzyme is inhibited by bovine serum albumin, various detergents, lysophosphatidylcholine, and palmitoyl carnitine. Among the sulfhydryl agents, only p -hydroxymercuribenzoate inhibited the enzyme. The enzyme is also inactivated by treatment with a high concentration of diethyl pyrocarbonate, an active center histidine-reacting agent, but not by phenylmethylsulfonyl fluoride (10 m M ), a serine esterase inhibitor. The purified enzyme does not appear to possess any O -ester hydrolase, lysophospholipase, transacylase, or acyltransferase activity.     

Differences in phosphatidate hydrolytic activity of human alkaline phosphatase isozymes

Hydrolytic activities of human alkaline phosphatase isozymes were investigated using phosphatidases with various fatty acyl chains (egg phosphatidate and dioleoyl, distearoyl, dipalmitoyl, dimyristoyl and dilauroyl phosphatidates). In the presence of sodium deoxycholate, purified human placental and intestinal alkaline phosphatases hydrolyzed all the phosphatidates examined. The hydrolytic activity was maximal in the presence of 10 g/l sodium deoxycholate. Of the phosphatidates, dilauroyl phosphatidate was the best substrate. Using the same unit of the enzyme, the phosphatidate hydrolytic activity of placental alkaline phosphatase was 2- to 3-times higher than that of the intestinal enzyme. In contrast, liver alkaline phosphatase did not hydrolyze phosphatidates with long fatty acyl chains (C16-18) even in the presence of sodium deoxycholate. The liver enzyme hydrolyzed dimyristoyl and dilauroyl phosphatidates very slowly. These results show that the phosphatidates with long fatty acyl chains were useful to differentiate placental and intestinal alkaline phosphatases from the liver enzyme, and suggest that the former enzymes play a different physiological role from the liver enzyme.     

Purification and characterization of human placental aminopeptidase A

Human placental aminopeptidase A (AAP) was purified 3,900-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100, then subjected to trypsin digestion, zinc sulfate fractionation, chromatographies with DE-52, Sephacryl S-300, and hydroxylapatite, affinity chromatography with Bestatin-Sepharose 4B, and finally immunoaffinity chromatography with the antibody against microsomal leucine aminopeptidase (LAP). Aminopeptidase A was completely separated from leucine aminopeptidase by the immunoaffinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 280,000 by gel filtration. The purified enzyme was most active at pH 7.1 with L-aspartyl-beta-naphthylamide (L-Asp-NA) as substrate; the Km value for this substrate was 4.0 mmol/l in the presence of Ca2+. Human placental aminopeptidase A was markedly activated by alkaline earth metals (Ca2+, Sr2+, Ba2+), but strongly inhibited by metal chelating agents such as EDTA and o-phenanthroline. The highest activity was observed with L-glutamyl-beta-naphthylamide, while only minimal hydrolysis was found with some neutral and basic amino acid beta-naphthylamides.     

Phospholipase C from human melanoma: purification and characterization of a phosphatidylinositol-selective enzyme.

Phospholipase C was purified from human melanoma grown as solid tumors in nude mice. The specific activity of the pure enzyme was approx. 100 mumol/min per mg; its apparent molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 150 kDa. The enzyme required calcium for activity and was activated by deoxycholate in the presence of the substrate phosphatidylinositol. The melanoma phospholipase C has a distinctly different substrate preference than those identified from normal tissues; it prefers phosphatidylinositol to phosphatidylinositol bisphosphate. The tumor enzyme was approx. 4-5-fold more active using phosphatidylinositol than phosphatidylinositol bisphosphate as the substrate.     

Estrogen sulfotransferase of mouse placenta: behaviour and characteristics during partial purification

Mouse placental estrogen sulfotransferase (ST) was partially purified by fast protein liquid chromatography (FPLC) gel filtration in combination with FPLC anion exchange. Owing to the highly unstable nature of the enzyme, large increases in specific activity were not obtained. Storage of the ST in the presence of thiol groups at -20 degrees C stabilized the enzyme considerably. Forty-three percent of the cytosolic ST was bound to an Affi-Gel blue column and eluted as a broad peak at approximately 0.8 M NaCl. The use of the latter procedure, in combination with FPLC gel filtration, did not increase the specific activity substantially. Larger increases in specific activity were obtained using agarose-hexane-adenosine-3',5'-diphosphate affinity chromatography. The bound ST activity was eluted under a single peak at 1 mM ADP. Increases in specific activity following use of this column averaged 54-fold but could reach 90-fold. Attempts at further purification of this material resulted in low recovery and decreased specific activity. Velocity versus substrate concentration curves show that estrone and particularly estradiol inhibit the partially purified mouse placental sulfotransferase above 0.1-0.25 microM substrate concentrations.     

Purification and properties of human placental aminopeptidase B.

Aminopeptidase B (EC 3.4.11.6; L-arginyl-beta-naphthylamidase) was purified 1,800-fold from human placental cytoplasm and characterized. The enzyme was subjected to ammonium sulfate fractionation and a series of chromatographies on DE-52, hydroxylapatite, Bio-gel A 0.5 m and L-arginine-Sepharose. The native molecular mass of the enzyme was estimated to be 220,000 by gel filtration. The molecular mass was estimated to be about 83,000 by SDS/PAGE in the absence of 2-mercaptoethanol, suggesting that the enzyme exists in a polymeric form. The isoelectric point of the enzyme was 5.4. The purified enzyme was most active at pH 7.2 with L-arginyl-beta-naphthylamide as substrate and the Km value for this enzyme was 0.3 mmol/l. Human placental aminopeptidase B was markedly activity by Cl-. Bestatin and arphamenin, low molecular weight peptides, showed appreciable inhibition of this enzyme. However, amastatin and puromycin did not inhibit the enzyme. Bacitracin markedly activated this enzyme.     

Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization.

Phospholipase C (heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ammonium sulfate precipitation followed by a novel application of DEAE-Sephacel chromatography. Enzymatic activity remained associated with DEAE-Sephacel even in the presence of 1 M NaCl, but was eluted with a linear gradient of 0 to 5% tetradecyltrimethylammonium bromide. Elution from DEAE-Sephacel was also obtained with 2% lysophosphatidylcholine, and to a lesser extent with 2% phosphorylcholine, but not at all with choline. The enzyme was highly active toward phospholipids possessing substituted ammonium groups (e.g., phosphatidycholine, lysophosphatidylcholine, and sphingomyelin); however, it had little if any activity toward phospholipids lacking substituted ammonium groups (e.g., phosphatidylethanolamine, phosphatidylserine, and phosphaditylglycerol). Collectively, these data suggest that phospholipase C from P. aeruginosa exhibits high affinity for substituted ammonium groups, but requires an additional hydrophobic moiety for optimum binding. The specific activity of the purified enzyme preparation increased 1,900-fold compared with that of culture supernatants. The molecular weight of the phospholipase C was estimated to be 78,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 column chromatography and was 76,000 by high-performance size exclusion chromatography. The isoelectric point was 5.5. Amino acid analysis showed that phospholipase C was rich in glycine, serine, threonine, aspartyl, glutamyl, and aromatic amino acids, but was cystine free.     

Cloning and expression of human placental L-Dopa decarboxylase

L-Dopa decarboxylase (DDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyses the decarboxylation of L-Dopa to dopamine. In this study we show the expression of DDC in human placental tissue and present data on the molecular cloning and in vitro expression of the active recombinant enzyme. Our analyses indicated the presence of both alternative DDC mRNA splice variants (neuronal and nonneuronal) in human placenta. Cloning of the coding region of the DDC cDNA into the pTrcHisA expression vector led to the production of the enzymatically active recombinant protein. The obtained recombinant enzyme specific activity values were in good agreement with the results obtained for the purified enzyme from human kidney. The availability of active recombinant human DDC could provide information leading to the better understanding of the enzyme's structure and substrate specificity, as well as its regulation and involvement in pathological conditions.     

Effect of Interferon on Deoxyribonuclease Induction in Chick Fibroblast Cultures Infected with Cowpox Virus

An exonuclease which degrades native deoxyribonucleic acid at pH 9.2 was induced in chick embryo fibroblast cultures and in human amnion cells by infection with cowpox virus. Highly purified chick embryo interferon suppressed the induction of the enzyme in the homologous cell system but not in the human amnion cell cultures. "Mock" interferon prepared from uninfected chicken eggs and purified in the same manner as biologically active interferon preparations had no effect on the induction of the enzyme.     

Purification and properties of steroid sulfatase from human placenta.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.