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Studies on Triacylglycerol Ester Hydrolase from Bat Adipose Tissue

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Studies on Triacylglycerol Ester Hydrolase from Bat Adipose Tissue J. Biosci., Vol.5, Number 1, March 1983, pp. 35–41 © Printed in India. Studies on triacylglycerol ester hydrolase from bat adipose tissue SUBHASH S. PATIL, CHANDA Κ. BHANDARI and VIJAY A. SAWANT Animal Physiology Laboratory, Zoology Department, Shivaji University, Kolhapur 416 004 MS received 20 April 1982; revised 4 October 1982. Abstract. Triacylglycerol ester hydrolase was isolated from bat adipose tissue and characterized. The partially purified enzyme had pH optimum of 8.6 and a Km value of 0.6 mM. The enzyme was denaturated upon freezing and thawing, which was prevented by 25% glycerol. The enzyme was activated by EDTA and NaCl, while it was inhibited by serum and bovine serum albumin. Heparin, sodium fluoride and diisopropyl fluorophosphate had no effect on triacylglycerol ester hydrolase activity. It hydrolyzed triglycerides partially. Triacylglycerol ester hydrolase lost its activity during delipidation but it was reactivated by endogenous lipids and phospholipids, viz. phosphatidyl ethanolamine, phosphatidyl choline and sphingomyelin. The enzyme shows kinetic properties altogether different from lipoprotein lipase and hormone sensitive lipase. Keywords. Bat adipose tissue; triacylglycerol ester hydrolase; bovine serum albumin; phospholipids. Introduction Hormone sensitive lipase [EC 3.1.1.3] and lipoprotein lipase [EC 3.1.1.34] have been extensively studied (Hiromichi and Setsuro, 1974; Mare, 1975; Bolzano, 1977; Setsurp and Yasuyuki, 1977; Anon, 1979). The functional importance of lipase has been shown (Vaughan et al., 1964; Hollenberg, 1965). Matsumura et al. (1976a,b) showed the presence of triglyceride lipase in rat and pig adipose tissue in addition to the occurrence of lipoprotein lipase and hormone sensitive lipase. Triglyceride lipase was active in the absence of serum and was strongly inhibited by bovine serum albumin. It was not affected by NaF and cyclic AMP-dependent protein kinase. Similarly many authors have reported hepatic triglyceride lipases in liver homogenates (Varinkova and Mosinger, 1965) and in plasma membrane, cytosol, microsomes and lysomes (Hayashi and Tappel, 1970; Assmann et al., 1973; Teng and Kaplan, 1974; Debeer et al., 1979). The cellular fractions show alkaline pH optima for lipolytic activity, except the lysosomal preparation, which has an optimum of pH 4 to 6. Very little is known about lipase activity of bat adipose tissue. Geroge and Eapan (1959) assayed lipase in the crude extracts and showed a difference in the levels of the enzyme in brown and yellow adipose tissues of bat. No reports are available on Abbreviations used: BSA, bovine serum albumin; DFP, diisopropyl fiuorophosphate 35 36 Patil et al. the isolation and characterization of lipase from the adipose tissue of bat. In the present studies attempts were made to purify partially and characterize the lipase. The enzyme showed kinetic properties altogether different from hormone sensitive lipase and lipoprotein lipase. Hence the enzyme has been referred to as triacylglycerol ester hydrolase or triglyceride lipase (Matsumura et al., 1976a, b). Materials and methods Materials Diphenyl carbazid was obtained from E. Merk, Dermstat, Germany, Diphenyl carbazone was from Veb Jenpharm Laborchemie, Apolda, Germany. Tris (hydroxymethyl)-aminomethane was from British Drug House Ltd., Poole, England. Bovine serum albumin (BSA), phosphatidyl ethanolamine, phosphatidyl choline, sphingomyelin, cholesterol, lysolecitin, phosphatidyl inositol, phosphatidyl serine, triglycerides and brain extract were obtained from Sigma Chemical Company, St. Louis, Missouri, USA. Diisopropylfluorophosphate (DFP) was from Kotch-Light Laboratories Ltd., Colnbrook , Bucks, England. Other chemicals were of the highest purity commercially available. Methods Purification of triglycerides from olive oil: Triglycerides from olive oil were purified using neutral alumina gel (Jensen et al., 1966). Purity of triglycerides was checked with thin layer chromatography (Sawant and Varute, 1973). Preparation of the substrate emulsion: The emulsion of triglycerides was prepared in gum acacia according to Fraser and Nicol (1966). Other emulsifiers when used were dispersed in appropriate buffer for the preparation of substrate emulsion. Preparation of delipidated tissue powder: The bats, Cynopterus sphinx sphinx were collected from their local natural habitat and were killed by occipital blow. The interscapular brown adipose tissue was dissected out and pooled. The tissue (6.4 g) was homogenized with approximately 10 vol of chilled acetone and the mixture was rapidly filtered through Whatman No. 1 filter paper. The residue was washed with 10 vol of chilled acetone and 10 vol of chilled ethyl ether. The defatted residue of adipose tissue thus obtained weighed one g. It was stored below 0°C. Preparation of enzyme: Delipidated adipose tissue powder was homogenized in 0.1 M Tris-HCl buffer pH 8.6, containing 1 mM DFP, 1 mM EDTA and 25% glycerol. The homogenate was agitated in a metabolic shaker at the rate of 240 strokes per min with 4 cm amplitude for 60 min at 20°C. It was then centrifuged for 30 min at 10000 g. The clear supernatant was removed and stored below 0°C. But lipase denatures upon freezing and thawing. Extraction of the enzyme in a medium containing 25% glycerol prevented denaturation and made it possible to store the enzyme at 0-4°C. This enzyme preparation can be stored for several weeks without loss of its activity. Triacylglycerol ester hydrolase 37 Enzyme assay: The lipase was assayed by the method of Hayashi and Tappel (1970) except for free fatty acids. The assay system contained 0.25 ml of substrate dispersed in gum acacia, 1.0 m. of 0.1 Μ Tris-HCl buffer pH 8.6, and suitably diluted enzyme sample in a total volume of 1.5 ml. The final substrated concentra- tion was 0.6 mM. Incubations were carried out in a metabolic shaker with continuous shaking at the rate of 160 strokes per min with 4 cm amplitude for 10 min at 37°C. At the end of the incubation the liberated fatty acids were measured colorimetrically according to Itaya (1977). Protein determination: Protein was estimated by the method of Lowry et al. (1951). The protein in wet adipose tissue was measured according to Tornqvist and Belfrage (1976). Esterase assay: Esterase was assayed with p-nitrophenyl acetate as a substrate, as described by Egelrud and Olivercrona (1974). Enzyme unit: One unit of lipase was defined as the amount of enzyme, which produced one n mol of free fatty acids per minute under appropriate assay conditions (Matsumura et al., 1967a, b). Results The levels of lipase activity in wet adipose tissue from different locations are shown in table 1. Table 1. Levels of triacylglycerol ester hydrolase in adipose tissue from different locations in bat. Values in the paranthesis represent the number of animals used for the study. Bat adipose tissue lipase was denatured upon freezing and thawing, and the denaturation can be prevented by 25% glycerol (Giudicelli and Boyer, 1973). Then the homogenate of adipose tissue in Tris-HCl buffer pH 8.6, containing 25% glycerol was incubated for 30 min at 50°C and the lipase activity was increased by about 35%. The lipase activity was found in the clear supernatant after centrifuga- tion of the homogenate at 10,000 g for 30 min. No activity was associated with the , 38 Patil et al. lipid fraction. However lipase activity was found in the fat layer, if the homogenate was not incubated at 50°C in the presence of 25% glycerol. Characterization of lipase activity Lipase activity in the homogenate of wet adipose tissue and acetone powder resuspension showed a broad pH optimum, while the enzyme extracted as described in the experimental section had a sharp pH optimum at 8.6. The triglyceride lipase activity was maximal at 37°C. The delipidated tissue powder resuspended in Tris-HCl buffer pH 8.6, agitated for 1 h, centrifuged at 10000 g for 30 min yielded a stable enzyme preparation. The increase in the rate of reaction at pH 8.6 measured as a function of increasing amount of substrate, triglyceride emulsion was apparently maximal at 6 mM. However above 6 mM the velocity was decreased. The Km was estimated to be 0.6 mM. The insoluble proteins present in acetone powder resuspension might interfere with the triacylglycerol ester hydrolase activity similar to the extraneously added starch particles. Effect of NaCl, BSA, serum, DFP, heparin, NaF and Na2EDTA The lipase was assayed in the presence of 0.2, 0.4, 0.6, 0.8 and 1.0 Μ NaCl. The enzyme was stimulated 1.5 fold beyond 0.4 Μ concentration. The enzyme was markedly inhibited by serum (table 2) and BSA (table 3), while DFP, heparin and Table 2. Inhibition of triacylglycerol ester hydrolase activity by serum. Table 3. Inhibition of triacylglycerol ester hydrolase by bovine serum albumin (BSA). Triacylglycerol ester hydrolase 39 NaF had no effect on lipase activity. The activity in the presence of 20, 40, 60, 120 m M EDTA was 133, 170, 200, 218% of the activity in its absence. Effect of emulsifiers Gum acacia, glycerol, BSA, tween 80 and ethanol were used as emulsifying agents for the preparation of substrate emulsion to ascertain their effect on the activity of lipase. It was observed that gum acacia, glycerol and ethanol are suitable emulsi- fiers to prepare the substrate emulsion required for bat adipose tissue lipase. Effect of lipids Emulsions of sphingomyelin, phosphatidyl choline, phosphatidyl ethanol amine, phosphatidyl serine, phosphatidyl inositol, phosphatide acid, triglycerides, brain extract, glycolipid, gangliosides, cholesterol, cholesterol ester and endogenous lipids were prepared separately using an ultrasonic oscillator. A mixture containing 1 mg delipidated tissue powder and 5 μΜ of lipids were incubated at 37°C for 30 min. In these mixtures phosphatidyl ethanolamine, phosphatidyl choline, sphingomyelin and endogenous lipids enhanced the lipase activity (table 4), while Table 4. Effect of phospholipids on triacylglycerol ester hydrolase activity in delipidated tissue powder. Each value is the mean of three expts.
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