FIELD OF THE INVENTION The present invention provides a new protein exhibiting triglyceride hydrolase activity and a process for the preparation of a diglyceride from a triglyceride by means of said protein.

BACKGROUND OF THE INVENTION Animals, seed plants, and fungi commonly store excessive amounts of energy substrates in the form of intracellular triglyceride (TG) deposits. In mammals, TG are stored in adipose tissue providing the primary source of energy during periods of food deprivation. Whole body energy homeostasis depends on the precisely regulated balance of lipid storage and mobilization. Mobilization of stored fat critically depends on the activation of lipolytic enzymes, which degrade adipose TG and release non-esterifϊed fatty acids (FA) into the circulation. Dysregulation of TG-lipolysis in man has been linked to variation in the concentration of circulating FA, an established risk factor for the development of insulin resistance (1-4). During periods of increased energy demand, lipolysis in adipocytes is activated by hormones, such as catecholamines. Hormone interaction with G-protein coupled receptors is followed by increased adenylate cyclase activity, increased cAMP levels, and the activation of cAMP -dependent protein kinase (protein kinase A, PKA) (5). PKA phosphorylates two important targets with established function in lipolysis: hormone-sensitive lipase (HSL), currently the only enzyme known to catabolize adipose tissue TG and perilipin A, an abundant protein located on the surface of lipid droplets. These modifications result in the translocation of HSL from the cytoplasma to the lipid droplet where efficient TG hydrolysis occurs (6). Current models depict HSL as the rate-limiting enzyme in TG mobilization. However, recent observations of HSL knock-out (HSL-ko) mice are inconsistent with predictions of these models: HSL-deficient adipose tissue retains a marked basal and PKA-stimulated lipolytic capacity (7, 8) and HSL-ko mice exhibited normal body weight and were not obese. Instead, these animals exhibited reduced adipose tissue mass (9, 10) due to the downregulation of triglyceride synthesis (10). The accumulation of diglycerides (DG) in various tissues of HSL-ko mice suggests that HSL is actually rate-limiting for the hydrolysis of DG in vivo but not for the catabolism of TG (7). These results imply the existence of one or more unidentified lipase(s) in adipose tissue that preferentially hydrolyze(s) the first ester bond (sn-1 or sn-3) of the TG molecule. SUMMARY OF THE INVENTION It is the object of the invention to provide a process for selectively hydro lyzing triglycerides to diglycerides and to substantially avoid further hydrolysing the diglycerides to monoglycerides and/or free fatty acids. We discovered a novel lipase that is expressed in adipose tissue that fulfills the requirements for an enzymatically active TG-hydrolase that also is expressed at high levels in murine adipose tissue. For the purpose of the present specification we name the novel lipase "adipose triglyceride lipase" (ATGL). The DNA coding for the novel lipase comprises the sequence according to SEQ No. 1 or SEQ No. 2 or variants thereof. Sequence SEQ No. 1 is identical to the coding sequence 203-1717 of NCBI nucleotide entry NM_020376 (gi: 34147340; human). Sequence SEQ No. 2 is identical to the coding sequence 58-1518 of NCBI nucleotide entry AK031609 (gi: 26327464; mouse). Therefore in one aspect the invention is directed to a protein exhibiting triglyceride hydrolase activity comprising a polypeptide strand encoded by (1) the DNA sequence according to SEQ No. 1 or SEQ No. 2, or (2) a DNA sequence which is capable of hybridization under conditions of high stringency to one of the DNA sequences SEQ No. 1 and SEQ No. 2. For conditions of high stringency see Sambrock et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbour Laboratory Press, NY, 1989. In a further aspect the invention is directed to a process for the preparation of a diglyceride from a triglyceride by enzymatically catalyzed hydrolysis of the triglyceride, characterized in that the triglyceride is treated with the novel lipase.

DETAILED DESCRIPTION OF THE INVENTION The following experimental part was undertaken with mouse ATGL, the cDNA of which exhibiting more than 96% homology to human DNA coding for human ATGL. The full length cDNA of ATGL containing the complete ORF was amplified by RT- PCR from total RNA of mouse white adipose tissue and subjected to DNA sequence determination. The nucleotide sequence of mouse ATGL is shown as SEQ No. 2 and exhibits 100% sequence identity to NCBI nucleotide entry AK031609 (gi: 26327464). The 1.460 bp coding sequence specifies a putative protein of 486 amino acids (NCBI accession number BAC27476) with a calculated molecular weight of 53.652 D. Northern blotting analysis of total RNA from various C57B16 mouse tissues revealed that ATGL mRNA is expressed at high levels in white and brown adipose tissue (Figure IA). Weak mRNA signals for ATGL were additionally observed in testis, cardiac muscle and skeletal muscle. During a differentiation time course of murine 3T3-L1 adipocytes, ATGL mRNA expression was first detected 4 days after induction of differentiation and a maximum of expression was obtained at day 6 (Figure IB). This niRNA expression profile is typical for late markers of adipocyte differentiation and closely resembles the expression pattern of HSL mRNA (not shown). To investigate whether ATGL hydrolyzes neutral lipids, His-tagged ATGL was transiently expressed in COS-7 cells using an eukaryotic expression vector. For comparison, COS -7 cells were also transfected with a similar construction expressing His-tagged HSL. Both His-tagged ATGL and HSL protein were detected in the cytosolic supernatant and the membrane pellet fraction of transfected COS cells by Western blotting analysis (Figure 1C). The apparent molecular weights of ATGL and HSL were estimated as 54 kD and 84 kD, respectively. When extracts from transfected cells were preincubated with a fluorescent lipase inhibitor (NBD-HEHP) (11) and subsequently subjected to SDS-PAGE analysis and fluorography, fluorescent signals were observed in positions corresponding to the expected molecular weight of ATGL and HSL (Figure 1C). The fact that the fluorescent probe only reacts with enzymatically active Ser-lipases (11) provided evidence that ATGL is enzymatically active in transfected COS cells. To confirm this, TG-hydrolase activity assays were performed using a radioactively labelled [9,10-3H(N))]-ttiolein substrate (Figure ID). The cytosolic fractions of ATGL transfected COS-7 cells exhibited a marked increase in TG hydrolase activity (3.7-fold compared to LacZ transfected control cells). No enzymatic activities were observed when radioactively labeled retinyl palmitate, cholesteryl oleate or phosphatidylcholine were used as lipid substrates. In accordance with previous data (12, 13), cytosolic fractions of HSL-transfected cells exhibited increased TG hydrolase (4.2-fold), cholesteryl ester hydrolase (23-fold), and retinyl-ester hydrolase (2.3-fold) activities compared to lacZ transfected cells. Thus ATGL possesses triglyceride hydrolase activity, but in contrast to HSL, this enzyme appears to be substrate-specific for TG and does not hydrolyze cholesteryl- or retinyl-ester bonds. To specify the function of ATGL in TG catabolism in comparison to HSL, we determined the relative abundance of lipolytic reaction products after incubation of a [9,10- 3H(N)] -triolein labeled substrate with cytosolic extracts of ATGL or HSL transfected COS-7 cells. Reaction products were separated by TLC and quantitated via scintillation counting of distinct lipid fractions (Figure 2). Compared to control extracts of LacZ transfected cells, extracts from ATGL and HSL-transfected cells contained 7.5 and 10-fold higher activities, respectively (Figure 2A). In the presence of ATGL the accumulation of diacylglycerol (DG) was increased 21 -fold compared to LacZ transfected cells suggesting that the enzyme predominantly hydro lyzed the first ester bond of TG (Figure 2B). TLC analysis of DG isomers indicated a strong preference of ATGL for the sn-1 position of TG (not shown). In contrast, lipolysis assays with cytosolic extracts from HSL transfected cells did not result in DG accumulation. The finding of efficient cleavage of DG by HSL observed here is consistent with the previously observed high substrate specificity of HSL for DG (10- fold higher than for TG) (14). Monoglyceride (MG) accumulation was only barely detectable with extracts of ATGL and HSL transfected cells (Figure 2C). From the molar ratios of DG and MG accumulation vs. FA release it can be calculated that -90% of the FA molecules released in the presence ATGL originate from the hydrolysis of TG in the first ester bond. In contrast, in the presence of HSL, most FA originate from all three ester bonds resulting in glycerol formation. Thus, our results demonstrate that ATGL and HSL possess distinctly different substrate-specificities within the lipolytic cascade, suggesting that they might act coordinately in the catabolism of TG. This assumption was confirmed by the product profiles generated in triolein hydrolysis assays using the combined extracts of LacZ, ATGL, or HSL transfected cells (Figure 2D). Relative to extracts from LacZ transfected cells, the acyl-hydrolase activity was increased in equal volume mixtures of HSL/LacZ extracts (4.8-fold), ATGL/LacZ extracts (4-fold) and ATGL/HSL extracts (16-fold). The accumulation of DG was increased 12.5 -fold when LacZ/ ATGL extracts were used and reduced to basal levels with ATGL/HSL extracts (Figure 2E). Although we do not want to be bound to any theory, considering this marked difference in substrate specificity of ATGL and HSL, we think that during the lipolytic breakdown of TG, ATGL is predominantly responsible for the initial step of TG hydrolysis whereas HSL acts to hydrolyze the resulting DG to monoglycerides. These, in turn, are converted to FA and glycerol by monoglyceride lipase (15). This model is supported by a marked cooperative effect observed in the combined presence of ATGL and HSL. As shown in Figure 2D, the total acyl-hydrolase activity in ATGL/HSL containing extracts was nearly 2-fold higher than the sum of the individual activities. To determine whether ATGL is functional also in adipocytes, a recombinant adenovirus encoding the His-tagged full length mouse ATGL cDNA was constructed and used to infect mouse 3T3-L1 adipocytes at day 6 of differentiation. Western blotting analysis of cell-extracts of infected adipocytes revealed expression of His-tagged ATGL at the appropriate molecular weight (Figure 3A). The enzyme was found to be tightly associated with lipid droplets of adipocytes even after extensive purification of the droplets by multiple centrifugation (16). Stimulation of lipolysis by isoproterenol did not affect the localization of the enzyme arguing for a constitutive association of ATGL with lipid droplets in adipocytes. Additionally, ATGL expressing 3T3-L1 cells released higher levels of FA (5 -fold) and glycerol (1.8-fold) compared to LacZ infected cells under basal conditions. After isoproterenol stimulation, FA release was increased by 1.8-fold and glycerol release by 2.9- fold compared to LacZ expressing control cells. Thus overexpression of ATGL in adipocytes can markedly augment both basal and isoproterenol-stimulated lipolysis, indicative for a functional lipase in adipose tissue. In summary, ATGL is a potent TG hydrolase with little or no specificity for DG, cholesteryl ester, retinyl ester and phosphatitylcholine. The mouse enzyme is predominantly expressed in adipose tissue. It is lipid droplet associated and enhances basal and β- adrenergically stimulated FA release. Although the regulatory mechanism for the activation of ATGL remain to be elucidated, these findings suggest that the enzyme is an important component of the lipolytic process and the mobilization of lipid stores in mammals.

Material and Methods

cDNA cloning and transient expression of recombinant His-tagged proteins in COS-7 cells and STS-Ll adipocytes. The coding sequenz of ATGL and HSL were amplified by PCR from cDNA prepared from mRNA of mouse white adipose tissue by reverse transcription. The open reading frame, flanked by KpnllXlϊol sites for ATGL and HSL were cloned into the eucaryotic expression vector pcDNA4/HisMax (Invitrogen). Transfection of COS-7 cells was performed with Metafectene™ (Biontex) according to the manufacturer's description. The PCR primers used to generate these probes were as follows. ATGL forward 5'-rGG7MCCGTTCCCGAGGGAGACCAAGTGGA-3', ATGL revers 5 '-CCTCGAGCGC AAGGCGGGAGGCCAGGT-S'. HSL forward 5 '-TGGTA CCT- ATGGATTTACGC ACGATGAC AC A-3 c , HSL revers 5'-CCTCGAGCGTTCAGTGGTGCAGCAGGCG-B' .

Construction of the recombinant adenovirus for ATGL expression (ATGL-Ad) and infection of STS-Ll cells: The recombinant adenovirus coding for mouse ATGL was prepared by cotransfection of the shuttle plasmid pAvCvSv containing the ATGL cDNA and pJM 17 into HEK-293 cells. The 1.65 kb MIu I - CIa I flanked mouse ATGL cDNA fragment (His-tag included) was amplified by PCR from the eucaryotic expression vector pcDNA4/HisMax containing mouse ATGL cDNA and subcloned into MIu I — CIa I digested pAvCvSv. The resulting shuttle plasmid was cotransfected with pJM 17 into HEK-293 cells using the calcium phosphate coprecipitation method. Large scale production of high titer recombinant ATGL-Ad was performed as described elsewhere. 3T3-L1 fibroblasts were cultured in DMEM containing 10% FCS and differentiated using a standard protocol (27). Adipocytes were infected on day 8 of differentiation with a multiplicity of infection (moi) of -400 plaque forming units/cell. For that purpose appropriate pfu were preactivated in DMEM containing 0.5 μg/ml of polylysin for 100 min and afterwards the cells were incubated with this virus suspension for 24 hours. After 24 h the medium was removed and the cells were incubated for further 24 h with complete medium. For most of the experiments, recombinant adenovirus expressing β-galactosidase was used as a control (LacZ-Ad). Subcellular fractionation ofCOS-7 cells. Transfected COS-7 cells were collected by trypsinisation and washed three times with PBS. Cells were disrupted on ice in lysis buffer (0.25 M sucrose, 1 mM EDTA, 1 niM dithiothreitol, 20 μg/ml leupeptin, 2 μg/ml antipain, 1 μg/ml pepstatin, pH 7) by sonication (Virsonic 475). Nuclei and unbroken materials were removed by centrifugation at 1.000 g at 4°C for 15 min to obtain cytoplasmatic extracts. The cytplasmatic extracts were centrifuged at 100.00Og at 40C for one hour to obtain cytosolic extracts and membrane pellets.

Isolation of lipid droplets. 3T3-L1 adipocytes from two 10 cm plates were disrupted in buffer A (20 mM Tricine, pH 7.8, 0.25 M sucrose, 2 mM MgCl2 0.2 mM PMSF) by sonication (Virsonic 475). 6 ml of puffer A were overlaid with 6 ml of buffer B containing 20 mM Hepes (pH 7.4), 100 mM KCl, 2 mM MgCl2, 0,2 mM PMSF and centrifuged for 3 hours at 40.000 rpm at 4°C. The lipid droplets concentrating at the top of the tube were collected and washed several times with buffer B as described (28).

Western analysis. Cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Schleicher & Schuell, Germany). For detection of His-tagged proteins, blots were incubated with 1/10000 diluted Anti-His monoclonal antibody (6xHis, Clonetech). Perilipin was detected using a guinea pig polyclonal antibody against Perilipin A and B (PROGEN). Bound immunoglobulins were detected with a HRP-labeled IgG conjugates (Vector Inc.) and and visualized by ECL detection (ECL plus, Amersham Pharmacia Biotech, Germany) on a Storm Image Analysis system. Quantitation was performed using ImageQuant Software.

Reaction of ATGL and HSL with the fluorescent lipase inhibitor NBD-HEHP. Transfected COS-7 cells were washed twice with PBS, scraped into lysis buffer (0.25 M sucrose, 1 mM EDTA, 1 mM dithioerythritol, 20 μg/ml leupeptin, 2 μg/ml antipain, 1 μg/ml pepstatin) and disrupted on ice by sonication. Nuclei and unbroken materials were removed by centrifugation at 1.000 g at 40C for 15 min to obtain cytoplasmatic extracts. 50 μg of protein was incubated with 1 nmol fluorescently labelled lipase inhibitor O-((6-(7-Nitrobenz-2-oxa- l,3-diazol-4-yl)amino)hexanoyl)aminoethyl-0-(n-hexyl)phospho nic acid p-nitrophenyl ester (NBD-HEHP) (29) and 1 mM Triton X-100 (especially purified for membrane research, Hofmann LaRoche) at 37°C for 2 hours under shaking. Protein was precipitated with 10% TCA for Ih on ice, washed with acetone and separated by 10% SDS-PAGE. Gels were fixed in 10% ethanol and 7% acetic acid. Fluorescence was detected with a BioRad FX Pro Laserscanner (excitation 488 nm, emission 530 nm). Northern analysis. The cDNA probe for northern blot analysis of mouse ATGL was prepared by RT-PCR by use of first-strand cDNA from mouse fat mRNA. The PCR primers used to generate this probe were as follows: forward 5'-TGGAACATCTCATTCGCTGG-S', revers 5'-AATGCCGCCATCCACATAG-B'. Total RNA was isolated from various mouse tissues using the TRI Reagent procedure according to manufacturer's protocol (Molecular Research Center, Karlsruhe, Germany). Specific mRNAs were detected using standard Northern blotting techniques with 10 μg total RNA. 32P-labeled probes for hybridization were generated using random priming. Northern blots were visualized by exposure to a Phosphorlmager Screen (Apbiotech, Freiburg, Germany) and analyzed using ImageQuant Software.

Assay for TG lipase, cholesteryl esterase, retinyl esterase and phospholipase activity. For determination of lipase activity 0.1 ml of cytosolic extracts and 0.1 ml substrate were incubated in a water bath at 37 °C for 60 min. The reaction was terminated by adding 3.25 ml of methanol/chloroform/heptane (10:9:7) and 1 ml of 0.1 M potassium carbonate, 0.1 M boric acid, pH 10.5. After centrifugation (800 g, 20 min) the radioactivity in 1 ml of the upper phase was determined by liquid scintillation counting. Neutral lipase activity was measured in 50 mM potassium phosphate buffer, pH 7.0 and 2.5% defatted BSA. The substrate for neutral TG lipase activity contained 33 nmol triolein/assay with [9,10-3H(N)]-triolein (40.000 cpm/nmol, NEN Life Science Products) as radioactive tracer for COS-7 cells and 167 nmol/assay for 3T3-L1 adipocytes (7300 cpm/nmol). The substrates for cholesteryl esterase and retinyl esterase activity contained 10 nmol/assay of cholesteryl oleate or retinyl palmitate and the corresponding tracers cholesteryl [9,10-3H]-oleate or retinyl [9,10-3H(N)]-palmitate (50.000 cpm/nmol). For determination of phospholipase activity in cytosolic extracts the substrate contained 20 nmol/assay phosphatidylcholine and [dipalmitoyl-l-14C]- phosphatidylcholine (12.000 cpm/nmol). All substrates were prepared by sonication (Virsonic 475) essantially as described (30). For investigation of DG formation in the in vitro assay the reaction was terminated by adding 1 ml of CHC13/Methanol (2:1) containing oleic acid (10 μg/ml) and standards for mono- and dioleine (sn-1.2 and sn-1.3; Sigma). The mixture was vortexed vigorously three times over a period of 15 min. After centrifugation (4000 g, 10 min), 0.5 ml of the lower phase was collected and evaporated under nitrogen. The lipid pellet was dissolved in chloroform and loaded onto a TLC plate (Merck Silica gel 60). The TLC was developed with chloroform/acetone/acetic acid (96:4:1) as solvent. The lipids were visualized with iodine vapor and the bands corresponding to mono-, di-, trioleine and oleic acid were cut out. The comigrating radioactivity was determined by liquid scintillation counting. Determination of FA and glycerol release from 3T3-L1 adipocytes. Cells were incubated in DMEM medium (GIBCO) containing 2% fatty acid free BSA (Sigma) with or without 10 μM isoproterenol (Sigma) at 37 °C. Aliquots of the medium were collected and investigated for the FFA and glycerol content by using commercial kits (WAKO).

Detailed description of Figures 1-3

Figure 1. Northern blot analysis of ATGL mRNA expression in various mouse tissues and (A) during adipocyte conversion of 3T3-L1 cells (B) . lOμg of total RNA from fasted mice or 3T3 cells were subjected to Northern blot analysis and detected with a specific P- labeled ATGL DNA probe. The acidic ribosomal protein PO was used as a control. 3T3-L1 cells were induced to differentiate into adipocytes two days after confluence (day 0) using a standard differentiation protocol (24). (C) Western blot analysis of His-tagged ATGL and HSL and reaction of the proteins with the fluorescent lipase inhibitor NBD-HEFIP. Transient transfection of COS-7 cells was performed using the eukaryotic expression vector pcDNA4/HisMax (Invitrogen) coding for His-tagged full-length cDNA of ATGL or HSL. The His-tagged proteins were detected by immunoblotting in cytosolic extracts (100.00Og supernatant) and in the membrane fraction (100.00Og pellet). Blots were incubated with Anti- His monoclonal antibody and HRP-anti-mouse IgG conjugate and visualized by ECL detection. For the reaction with NBD-HEHP, cytoplasmic extracts were incubated with 1 nmol fluorescently labeled lipase inhibitor and 1 mM Triton X-IOO at 37°C for 2 hours under shaking. Subsequently, the samples were subjected to SDS-PAGE and labeled proteins were visualized by a BioRad FX Pro Laserscanner. (D) Enzymatic activity and substrate specifity of ATGL. Cytosolic extracts of COS-7 cells expressing His-tagged ATGL, HSL or b- galactosidase (LacZ) were assayed for lipase activity using substrates containing radiolabeled triolein, cholesteryl oleate, retinyl palmitate or phosphatidylcholine. Experiments were performed in triplicate. Data are presented as mean ± S. D. and are representative for at least three independent experiments.

Figure 2. Role of ATGL within the triglyceride hydrolysis cascade. Cytosolic extracts of COS-7 cells, transiently transfected with His-tagged LacZ, ATGL or HSL, were incubated with triolein containing [9,10-3H(N)]-triolein as radioactive tracer. Lipids were extracted and separated by TLC using CHCl3/aceton/acetic acid (96/4/1) as mobile phase. Lipids were visualized with iodine vapor and the radioactivity comigrating with MG, DG, TG and FA standards was determined by liquid scintillation counting. (A) Total acyl-hydrolase activity (FA). (B) Accumulation of DG. (C) Accumulation of MG. (D) Effect of combined activity of ATGL and HSL on TG hydrolase activity. Cytosolic extracts of COS cells expressing LacZ were mixed 1 : 1 with extracts from cells expressing ATGL or HSL (ATGL/LacZ and HSL/LacZ) and compared to extracts prepared from a mixture of ATGL and HSL expressing cells (ATGL/HSL). (E) Effect of combined activity of ATGL and HSL on DG accumulation. All experiments were performed in triplicate. Data are presented as mean ± S.D. and are representative for three independent experiments.

Figure 3. Cellular localization, lipolytic activity and antibody-directed inhibition of ATGL in adipocytes. (A) A recombinant adenovirus coding for His-tagged ATGL (ATGL- Ad) was used to infect adipocytes on day 8 after induction of differentiation and experiments were performed 2 days after infection. (16). Cells were cultured in DMEM medium (GIBCO) containing 2% fatty acid free BSA (Sigma) in the absence or in the presence of isoproterenol (10 μM at 37°C for two hours) as indicated (+ iso) prior to harvesting cells or medium. Western blot analysis of ATGL in the cytoplasmic fraction (10 μg of total protein) and in isolated lipid droplets (2 μg of total protein) of adipocytes using an anti-His monoclonal antibody. Purification of lipid droplets was monitored by the enrichment of perilipin ^70- fold) using a rabbit polyclonal antibody against perilipin A and B (Progen). (B) Fluorescent photograph of 3T3-L1 adipocytes transfected with GFP-ATGL. GFP-ATGL was introduced transiently in cells on day 8 after induction of differentiation and photographs were taken 2 days after infection. (C) Glycerol and FA release from ATGL-Ad infected adipocytes were measured in aliquots of culture medium using commercially available kits (WAKO). Recombinant adenovirus expressing β-galactosidase (LacZ) was used as a control. Experiments were performed in triplicate. Data are presented as mean + S.D. and are representative for three experiments. (D) Inhibiton of cytosolic acyl hydrolase activity in WAT and BAT by a polyclonal antibody against mouse ATGL (ATGL-IgG) using [9,10- 3H(N)]-labeled triolein as substrate. The activity in cytosolic extracts of wild-type and HSL- ko mice was determined either in the presence of rabbit non-immune IgG (NI-IgG) or ATGL- IgG. Data are presented as mean ± S.D. of three single mice for each group and are representative for two experiments.

Determination of TG hydrolase activity Neutral TG lipase activity was measured, with triolein as substrate containing [9,10- 3H(N)]-triolein (NEN Life Science Products) as radioactive tracer. The substrate for TG lipase activity was prepared by sonication (Virsonic 475) exactly as describeded by Holm et α/.(30) . Cells were disrupted on ice in lysis buffer (0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, 20 μg/ml leupeptin, 2 μg/ml antipain, 1 μg/ml pepstatin, pH 7) by sonication (Virsonic 475). The cytosolic infranatants were obtained after centrifugation at 1000,000 g, at 4 0C for 60 min. The reaction was performed in a water bath at 37 0C for 60 min with 0.1 ml substrate and 0.1 ml infranatant. The reaction was terminated by adding 3.25 ml of methanol/chloroform/heptane (10:9:7) and 1 ml of 0.1 M potassium carbonate, 0.1 M boric acid, pH 10.5. After centrifugation (800 g, 20 min) the radioactivity in 1 ml of the upper phase was determined by liquid scintillation counting.

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29. Oskolkova, O.V., R. Saf, E. Zenzmaier, and A. Hermetter. 2003. Fluorescent organophosphonates as inhibitors of microbial lipases. Chem Phys Lipids 125: 103-14. 30. Holm, C. and T. Osterlund. 1999. Hormone-sensitive lipase and neutral cholesteryl ester lipase, in Lipase and Phospholipase Protocols, M.Doolittle, K.Reue, Eds. (Humana Press, Totowa, New Jersey) 109, chap.l l. atgtttcc ccgcgagaag acgtggaaca tctcgttcgc gggctgcggc ttcctcggcg tctactacgt cggcgtggcc tcctgcctcc gcgagcacgc gcccttcctg gtggccaacg ccacgcacat ctacggcgcc tcggccgggg cgctcacggc cacggcgctg gtcaccgggg tctgcctggg tgaggctggt gccaagttca ttgaggtatc taaagaggcc cggaagcggt tcctgggccc cctgcacccc tccttcaacc tggtaaagat catccgcagt ttcctgctga aggtcctgcc tgctgatagc catgagcatg ccagtgggcg cctgggcatc tccctgaccc gcgtgtcaga cggcgagaat gtcattatat cccacttcaa ctccaaggac gagctcatcc aggccaatgt ctgcagcggt ttcatccccg tgtactgtgg gctcatccct ccctccctcc agggggtgcg ctacgtggat ggtggcattt cagacaacct gccactctat gagcttaaga acaccatcac agtgtccccc ttctcgggcg agagtgacat ctgtccgcag gacagctcca ccaacatcca cgagctgcgg gtcaccaaca ccagcatcca gttcaacctg cgcaacctct accgcctctc caaggccctc ttcccgccgg agcccctggt gctgcgagag atgtgcaagc agggataccg ggatggcctg cgctttctgc agcggaacgg cctcctgaac cggcccaacc ccttgctggc gttgcccccc gcccgccccc acggcccaga ggacaaggac caggcagtgg agagcgccca agcggaggat tactcgcagc tgccgggaga agatcacatc ctggagcacc tgcccgcccg gctcaatgag gccctgctgg aggcctgcgt ggagcccacg gacctgctga ccaccctctc caacatgctg cctgtgcgtc tggccacggc catgatggtg ccctacacgc tgccgctgga gagcgctctg tccttcacσa tccgcttgct ggagtggctg cccgacgttc ccgaggacat ccggtggatg aaggagcaga cgggcagcat ctgccagtac c'tggtgatgc gcgccaagag gaagctgggc aggcacctgc cctccaggct gccggagcag gtggagctgc gccgcgtcca gtcgctgccg tccgtgccgc tgtcctgcgc' cgcctacaga gaggcactgc ccggctggat gcgcaacaac ctctcgctgg gggacgcgct ggccaagtgg gaggagtgcc agcgccagct gctgctcggc ctcttctgca ccaacgtggc ctftcccgccc gaagctctgc gcatgcgcgc acccgccgac ccggctcccg cccccgcgga cccagcatcc ccgcagcacc agctggccgg gcctgccccc ttgctgagca cccctgctcc cgaggcccgg cccgtgatcg gggccctggg gctgtga<

SEQ No . 1 atg ttcccgaggg agaccaagtg gaacatctca ttcgctggct gcggcttcct cggggtctac cacattggcg tggcctcctg cctccgtgag cacgcgccct tcctggtggc caacgccact cacatctacg gagcctcggc aggggcgctc accgccacag cgctggtcac, tggggcctgc ctgggtgaag caggtgccaa cattattgag gtgtccaagg aggcccggaa gcggttcctg ggtcct ctgc atccctcctt caacctggtg aagaccatcc gtggctgtct actaaagacc ctgcctgctg attgccatga gcgcgccaat ggacgcctgg gcatctccct gactcgtgtt tcagacggag agaacgtcat catatcccac tttagctcca aggatgagct catccaggcc aatgtctgca gcacatttat cccggtgtac tgtggcctca ttcctcctac cctccaaggg gtgcgctatg tggatggcgg catttcagac aacttgccac tttatgagct gaagaatacc atcacagtgt ccccattctc aggcgagagt gacatctgcc ctcaggacag ctccaccaac atccacgagc ttcgcgtcac caacaccagc" atccagttca accttcgcaa tctctaccgc ctctcgaagg ctctcttccc gccagagccc atggtcctcc gagagatgtg caaacagggc tacagagatg gacttcgatt ccttaggagg aatggcctac tgaaccaacc caaccctttg ctggcactgc ccccagttgt cccccaggaa gaggatgcag aggaagctgc tgtggtggag gagagggctg gagaggagga tcaattgcag ccttatagaa aagatcgaat tctagagcac ctgcctgcca gactcaatga ggccctgctg gaggcctgtg tggaaccaaa ggacctgatg accacccttt ccaacatgct accagtgcgc ctggcaacgg ccatgatggt gccctatact ctgccgctgg agagtgcagt gtccttcacc atccgcttgt tggagtggct gcctgatgtc cctgaagata tccggtggat gaaagagcag acgggtagca tctgccagta tctggtgatg agggccaaga ggaaattggg tgaccatctg ccttccagac tgtctgagca ggtggaactg cgacgtgccc agtctctgcc ctctgtgcca ctgtcttgcg ccacctacag tgaggcccta cccaactggg tacgaaacaa cctctcactg ggggacgcgc tggccaagtg ggaagaatgc cagcgtcagc tactgctggg tctcttctgc accaatgtgg ccttcccgcc ggatgccttg cgcatgcgcg cacctgccag ccccactgcc gcagatcctg ccaccccaca ggatccacct ggcctcccgc cttgctga

SEQ No . 2