SUPERACTIVII./ AND PHASE-SENSITIVITY PHOSPHATASE ENTRAPPED IN RESTEI~SE MICRTX.RS
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1 Vol. 40, No. 3, October 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages SUPERACTIVII./ AND PHASE-SENSITIVITY OF POTATO ACID PHOSPHATASE ENTRAPPED IN RESTEI~SE MICRTX.RS J LALIT~IA AND V M MULIMANI* Department of Biochemistry Gulbarga University, Gulbarga Karnatak State, India. Received August 28, 1996 Summary; Potato acid phosphatase, AcPase ( E.C ) was entrapped in reverse micelles of cationic surfactant cetyltrimethylammonium bromide (CTAB) in isooctane and chloroform (1:1). The activity was studied at different values of Wo = ([water] / [surfactant]). AcPase exhibited superactivity in the reverse micellar system. At very low Wo value, activity was found to be less than that of in buffer and further increase in Wo value enhanced the activity thousand fold. At Wo=50, the activity was enhanced more than twenty fold. The effect of second surfactant, TritonX-100 on superactivity was studeid. There was a slight decrease in overall activity, when 3.33 mm TritonX-100 was added to the above reverse micellar system. Keywords: AcPase, Potato acid phosphatase., PNPP,p-nitrophenyl phosphate., CTAB, Cetyltrimethylammonium bromide., TritonX-100., Superactivity., phase-sensitivity. INTRODUCTION Until recently, the enzyme kinetic reactions were carriedout in buffer media, utilizing free enzymes. The valuable experiments aimed at elucidating the structure of biocatalysts were successful only with the enzymes isolated from the living cells in quite a pure form. However, such "pure' experimental results carried out in vitro cannot be correlated with the conditions of its functions in vivo. Individual components of the cell including enzymes interact with each other (I). Majority of enzymes are located on the surface of the biological membranes or inside them (2). Many of them are bound covalantly or non-covalently to the membrane /96/ / by Academic Press Australia. All rights of reproduction in any form reserved.
2 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL surface loosely and the degree of the binding depends on the concentration of the metabolites. (1-4). Many enzymes upon being withdrawn from their natural environment, partly or completely lose their catalytic activity and their other properties such as specificity, kinetic mechanism of the action and stability (5). The essence of contradiction is that in vivo studies of enzymes are conducted in aqueous buffer. Modelleing of the enzymatic function of biomembranes is successfully carried out by constructing membrane models using amphiphiles such as surfactants, phospholipids at the interfaces of water/oil (6), bilayer lipid membranes (7) and liposomes (8) Since last two decades much attention has been given to the micellar solubilization of enzymes. Reverse micelles are microdroplets of water dispersed in a hydrophobic medium with the help of surfactant, where the surfactant forms a layer between water and bulk organic phase. Reverse micelles are capable of solubilizing a variety of biomolecules in the microwater pool or at the oil-water interface (9). The reverse micellar environment provides an aqueous phase for hydrophilic enzymes, an interface for surfaceactive enzymes and an organic phase for hydrophobic substrates or products (i0). Some enzymes exhibit superactivity when entrapped in reverse micelles and their catalytic activity exceed hundred times more than that of in aqueous buffer (11-13). Superactivity is characteristic of some of membrane active enzymes when entrapped in reverse micellar system (14). Acid phosphatases (E.C ), orthophosphoric monoester phosphohydrolases (acid optimum) catalyses the hydrolysis of phosphoester bonds by releasing the inorganic phosphate at acidic conditions (15). In this article, we report the superactivity of AcPase entrapped in 572
3 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL reverse micelles of CTAB in isooctane/chcl 3 (i:i) and the effect of neutral surfactant TritonX-100 on the superactivity and phase sensitivity. MATERIALS AND METHODS AcPase and CTAB were obtained from Sigma Chemical Company St.Louis, U.S.A. Disodium salt of p-nitrophenyl phosphate (PNPP) was from Fluka, Germany. Isooctane and chloroform were of uv-grade products of spectrochem Pvt Ltd. All buffer components were from Fischer Co Ltd., Bombay. Doubly distilled water was used to prepare buffer solutions. Aqueous buffer used during the assay was 20 mm acetate buffer of ph 4.8. Preparation of reverse micelles : 50 mm CTAB solution in isooctane/chcl 3 (1:1) was prepared. A calculated amount of buffered substrate PNPP, was injected into the above solution and a volume of 3 ml was taken for the assay of enzyme activity. Final water content depending on the Wo value was adjusted by adding a required amount of buffer before injecting the AcPase solution. Micelles of mixed surfactants CTAB with TritonX-100 were prepared by adding known amount of TritonX-100 to CTAB/isoocatne/CHCl 3 system. The Wo values were calculated for the final surfactant concentration as described earlier. Assay of AcPase activity~ The activity measurements were made spectrophotometrically in Perkin Elmar 2 UV-Visible spectrophotometer following the initial steady state rate of liberation of p-nitrophenol at 340 nm for 5 minutes (16), using PNPP as substrate. The reaction was initiated by adding 10 microliter of buffered enzyme stock to a micellar solution containing known amount of substrate and buffer. The molar concentration of surfactant and Wo were calculated for the resulting final volume. The molar absorbance ~ for p-nitrophenol liberated was determined as a function of concentration of all components of the media and the ph of buffer. RESULTS AND DISCUSSION AcPase showed an increase in catalytic activity when entrapped in reverse micelles of CTAB in isooctane/chcl 3 (i:i). At very lower Wo (=5), AcPase exhibited less activity since at lower hydration degrees, the empty micelles are too small for the whole enzyme molecule to be entrapped and this is accompanied by conformational changes in the protein globule causing reduction in catalytic activity (13-16). There is gradual increase in activity with 573
4 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL increase in Wo value upto ii, further increase in Wo value enhanced the activity more than thousand fold (Figure I). At Wo=50, there was fold enhancement in activity. The catalytic activities are different for enzymes solubilized in micelles of each type. Martinek et al (17) and Klyachko et al (18) have discussed extensively about the key research problems in micellar enzymology and its relationship to enzyme membranology. Several enzymes are known to exhibit "superactivity" when entrapped in reverse micelles including u-chymotrypsin, alcohol dehydrogenase, pancreatic lipase and alkaline phosphatase (9). The increase in catalytic activity of AcPase by huner~d fold is one of the most stricking phenomenon exhibited by superactive enzymes entrapped in reverse micelles. The catalytic activity of peroxidase was reported to be more than i00 times and about 60 times greater for laccase in reverse micelles than in buffer solutions (ii). Acid phosphatase showed 300 times greater activity when entrapped in reverse micelles of lecithin / octane / methanol system (15). Several factors influence the catalytic activity of enzyme entrapped in reverse micelles. The activity mainly depends on the degree of forced contacts of protein molecules with the surface layer and physical properties of the surrounding microenvironment (9). The phase transitions of the types, reverse micelles <--> reversed hexagonal structure or reversed hexagonal <--> lamellar structure excists in the ternary system (i.e. oil/surfactant/water system) and the phase transitions evokes a sharp reversible changes in the enzymatic activity. The phase transition in the medium of enzymatic reaction was revealed earlier in case of luciferase (19), and peroxidase (20). This phenomenon is called as "phase 574
5 Vol. 40, No. 3, ] 996 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL 70 6"5 I 6"0 / V X I0' I 2"C 1"5 i I' III I I I I 0!0? Wo I CTAB/isooctane/CHCI 3 II CTAB/isooctane/CHCI3= + 3"SXI()3M TritonX-IO0 III Activity in buffer Figure. I Catalytic activity of potato acid phosphatase in reverse micelles entrapped sensitivity" of the enzymes under study, which is due to the interaction of enzymes with the surfactant matrics through "anchore' regions on their molecular surface (21). These anchore regions formed by lipid molecules for luciferase or sugar residues for peroxidase (22) and laccase (23) are involved structurally in enzyme binding to biological membranes in vivo and these enzymes inturn are found to be membrane active ones (24). The sugar residues are the anchore regions in AcPase and are involved in binding with the biological mambrane (25). Our results are also supported by the comparitive investigation of native and modified u-chymotrypsin to correlate membrane-activity and phase 575
6 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL sensitivity. Where native ~-chymotrypsin which was unable to bind to membrane was converted to the membrane active form by chemical hydrophobization with stearic acid residues (26). The neutral surfactant, TritonX-100 is used to release the membrane active/mambrane bound enzymes (27) was added to the CTAB reverse micelles to study its effect on superactivity and phase- sensitivity of AcPase. A decrease in overall activity of in micelles of mixed surfactants than in simple micelles was observed. The activity in mixed micelles interns was greater than in buffer. Katiyar and De Takas have reported the superactivity of glucose-6-phosphate dehydrogenase from yeast solubilized in a reverse micelles of mixed surfactants viz., AOT and TritonX-45 in n-heptane (28), and the maximum activity obtained was 233 times greater than that of activity in aqueous buffer at Wo=55.6. TritonX-lO0 decreases the hydrophobicity of reverse micelles and at higher concentrations was shown to destroy the reverse micelles (29). Addition of aqueous buffer i.e. increase in Wo value enhanced the AcPase activity and there is little decrease in overall activity when TritonX-100 was added to the media due to changes in microenvironment of reverse micellar water core. AcPase exhibited superactivity in reverse micelles of CTAB. More than thousand time enhancement in activity was observed at higher Wo values. AcPase is found to be a membrane bound enzyme and exhibited phase-sensitivity. The enzyme activity in reverse micelles is not only due to the nature of enzyme investigated but also depends on the type / types of surfactants used in preparation of reverse micelles. This is evidenced by the effect of added TritonX-100 to the CTAB reverse micelles. TritonX
7 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL decreased the overall activity and phase sensitivity of AcPase in reverse micelles. These experiments will help to understand the mechanism of enzyme catalysis in reverse micelles. Acknowledgements: One of the author ( J L ) is greatful to the Council of Scientific and Industrial Research, India for financial assistance. REFERENCES i. Wombacher.H. (1983) Mol.Cell.Biochem.56, Fendler, J.H. (1982) in 'Membrane mimetic chemistry' pp John Wiley publication, New York. 3. Werner.M., Garrett,C., Chin,A. and Klempner,L. (1982) Clin. Chem, 28, Masters,C.J. (1981) CRC crit Rev Biochem, 11, Barbaric, S.and Luisi, P.L. (1981) J.Am.Chem. Soc. 103, May,S.W and Li.N.N (1977) in Biomedical applications of immobilized enzymes (Chang T.M S ed) pp Plenum press, New York, 7. Darsozon,A. (!983) J.Bioenerg.Biomembry, 5, Eytan,G.D. (1982) Biochim.Biophys.Acta. 694, Martinek,., Levashov, A.V., Klyachko,N., Khmelnitsky, Y.U. and Beregin,I. (1986) Eur.J.Bioche. 155, Creagh,A.L., Prausnitz,J.M. and Blanch,H.W. (1993) Enzyme. Microb.Technol, 15, ii Pshezhehsky,A.V.,Klyachko,N.L. and Levashov,A.V. (1987) in Proc. Symp. Chemical Physics of enzymes catalysis. Tallin. Estonian SSR, p Levashov,A.V., Klyachko,N.L. and Martinek,K. (1980) Trans. Bioorganicheskya.Khimiya. 7, Kabanov,A.V., Levashov, A.V., Klyachko,N.L., Namyotkin,S.N., Psezhetsky,A.Y.and Martinak,K.(1988) J.Ther.Biol. 133, Walde,P., Peng,Q., Fadnavis,N.W., Baltistel,E. and Luisi,P.L. (1988) Eur.J.Biochem, 173, Ostrovsky,V.S. (1986) in Acid phosphatase in physicochemical problems of enzyme catalysis (Russ), Moscow Nauk press. 16.Nametkin,S.N., Kabanov,A.V., Klyachko,N.L. and Levashov,A.V. (1991) Bioorg.Khim, 17, Belongova,E.I., Brovko,L.Yu., Ugarova,N.N., Klyachko,N.L., Levashov,A.V., Martinek,K. and Berezin,I.V.(1983) Dokl Akad Nauk, SSSR. 273, Klyachko,N.L., Levashov,A.V., Pshezhetsky,A.V., Bogdanova,N.G. and Martinek, K. (1986) Eur. J.Biochem. 161, Ugarova,N.N., Dukhovich,A.F., Shvets,S.V., Philippova,N.Yu. and Berezin,I.V. (1987) Bioche.Biophys.Acta. 921, Welinder,K.G.and Smillie,L.B.(1972) Can.J.Biochem, 50, Reinhammar,B. (1984) in Laccase, in copper proteins and copper enzymes,vol.iii pp.l-36. (Lontie,R ed), CRC press, Florida, Edited by Lontie,R, pp
8 Vol. 40, No. 3, ] 996 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL 24.Martinek,K., Klyachko,N.L., Kabanov,A.V., Khmelnitsky,Yu.L. and Levashove,A.V.(1989) Biochim.Biophys.Acta, 981, Pshezhetsky,A.V.,Klyachko,N.L., Levashov,A.V. and Martinek,K. (1990) Biocatalysis, 4, ,Kabanov,A.V., Nametkin,S.N., Levashov,A.V. and Martinek,K. (1985) Biol.Membrany (Russ) 2, Nicholas.C.Price and Lewis.Stevens (1989) in Fundamentals of enzymology,second ed.p398,oxford University press,oxford New York. 28.Katiyar,S.S. and De Takas.(1990) Biochem.Int. 20, Kabanov,A.V., Nametkin,S.N., Klyachko,N.L. and Levashov,A.V. (1991) FEBS, 278,
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