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Partial purification and comparison of the kinetic properties of ovine liver Echinococcus granulosus hydatid cyst fluid malate dehydrogenase and the cytoplasmic enzyme from the host liver
Comp. Biochem. Physiol. Vol. 10715,No. 3, pp. 447-451, 1994 © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 03054)491/94 $6.00 + 0.00
Pergamon
Partial purification and comparison of the kinetic properties of ovine liver Echinococcus granulosus hydatid cyst fluid malate dehydrogenase and the cytoplasmic enzyme from the host liver Mahmood Vessal and Nasrin Bambaea-Row Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz, 71345, Iran Ovine fiver Echinocoecusgranulosus hydatid cyst fluid and cytoplasmic healthy ovine fiver malate dehydrogennses were purified 24- and 30-fold by Sephadex G-200 and DEAE-Sephadex chromatography. Both enzymes were eluted with the same elution volume and the same salt concentration from the respective columns. The pH optimum of the enzymes from both sources was 8.4 in either Tris--HC! or barbital buffer. The Km values for oxaloacetate were 0.211 and 0.200 mM for hydatid cyst fluid and healthy ovine fiver enzymes, respectively. The Km values for NADH were 0.220 and 0.213 mM for bydatid cyst fluid and healthy ovine fiver enzymes, respectively. Enzyme from both sources demonstrated similar heat denaturation patterns. Both enzyme preparations were inhibited at high concentrations of either substrate. Neither enzyme was inhibited by para-hydroxymercuribenzoate or fumarate, and both enzyme preparations were specific for NADH as a cofactor. The results are discussed in terms of the possible infiltration of the host enzyme into the cyst fluid. Key worth: Echinococcus granulosus; Hydatid cyst; Malate dehydrogenase.
Comp. Biochem. Physiol. 107B, 447-451, 1994.
Introduction
Hydatid cyst fluid (HCF) is believed to be composed of an ultrafiltrate of the host tissue fluids and the secretions of the germinal epithelium of the cyst (Krvavica et al., 1961). Of the total proteins present in HCF, some are produced by the parasite and others by the host (Agosin and Repetto, 1967). In order to find possible differences in the properties of the parasite and the host enzymes to be used in the development of sound chemotherapy, comparison has been made of the physical and kinetic properties of phosphoglucose isomerases, UTPD-glucose-l-phosphate uridyltransferases and alkaline phosphatases of Echinococcus species and the host liver by several investigators (Faradji et al., 1974; Vessal and Abdolrasulnia, 1976; Sarciron et al., 1987, 1991). Fairly high Correspondence to: M. Vessal, Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz, 71345, Iran. Received 22 June 1993; accepted 30 July 1993. 447
levels of NADP-linked isocitrate dehydrogenase and NAD-dependent malate dehydrogenase enzymes (MDH) have been reported in ovine liver HCF (Vessal and Ghalambor, 1970). A high level of soluble isocitrate dehydrogenase in the serum is considered to be an indication of liver damage (Moss et al., 1986). There are also reports on the presence of several host proteins in cyst fluids and in the patient's sera (Vidor et al., 1987). In this investigation it was thought that perhaps the fairly high levels of soluble malate and isocitrate dehydrogenases in the cyst fluid may be a result of damage to hepatocytes. Therefore, preliminary experiments were set up to partially purify soluble MDH from healthy ovine liver (HOL) and ovine liver Echinococcus granulosus HCF and to compare some of their physical and kinetic properties in order to find a clue as to the probable origin of the hydatid fluid malate dehydrogenase enzyme.
448
Mahmood Vessal and Nasrin Bambaea-Row
Materials and Methods Reagents cis-Oxaloacetic acid (OAA), p-hydroxymer-
24
curibenzoate (pHMB-disodium salt) and fumaric acid (potassium salt) were purchased from BDH Biochemicals (Poole, Dorset, U.K.); Sephadex G-200 and DEAE-Sephadex A-50 from Pharmacia (Uppsala, Sweden); and NADH (disodium salt) from Sigma (St Louis, MO). All other reagents were of highest purity and obtained through other commercial sources.
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Freshly secured sheep liver (100 g) was chilled on ice, cut into small pieces, washed several times with 100raM Tris-HC1 buffer (pH 7.4) and homogenized in I00 ml of the same buffer in a Waring blender for four 2-min cycles. The homogenate was centrifuged at 27,000g for 10min and the supernatant fluid diluted to a protein concentration of about 40 mg/ml with 100 mM Tris-HCl (pH 7.4). This supernatant solution was used as the starting material in the purification of liver MDH.
Enzyme assay MDH was assayed by a spectrophotometric procedure (Wacker etal., 1956). The standard assay mixture contained 0.4 ml of the respective enzyme solution and the following ingredients in/~moles in a total volume of 1.1 ml: NADH, 0.30; OAA (freshly prepared), 3.3; Tris-HCl buffer, 40; pH7.4 during purification and pH 8.4 in kinetic studies. Reactions were started by the addition of OAA solution and the decrease in absorbance at 340nm (25°C) was followed spectrophotometrically at different time intervals. Control cuvettes in which water was substituted for OAA were also included. Enzyme unit was expressed as the amount of enzyme protein which caused a decrease of 0.001 units in absorbance (340 nm) per minute per 1.1 ml of the incubation mixture under standard assay conditions at 25°C. The relation between the activity of HCF or HOL enzyme and the time of incubation was linear up to 10 rain. Enzyme activity was linear up to 50 #g of the partially purified HCF pro-
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Preparation of liver homogenates
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Preparation of HCF All procedures were carried out at 0-4°C unless stated otherwise. HCF was prepared as described (Faradji et al., 1974). HCF volume was reduced to 1/75 by ultrafiltration over a UM-2 Diaflo membrane (Amicon Corporation, Lexington, MA) under nitrogen gas. Concentrated HCF was used as the starting fluid in the purification of cyst fluid MDH.
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Fig. I. Sephadex G-200 column chromatograms of HCF and HOL proteins. Chromatography was done as described under Methods. Enzyme activities were detected in peak II (HCF, • • and HOL, (3 0).
tein or up to 15 #g of partially purified HOL protein in 1.1 ml of the incubation mixture. The effect of pH on enzyme activity was observed by using either 40 #moles of Tris-HCl buffer or 40 #moles of barbital buffer (pH 7.4-8.8) per 1.1 ml of the incubation mixture with all other conditions remaining the same. For heat inactivation experiments, the enzyme solutions were incubated at different temperatures (37-65°C) for 2 min prior to enzyme assay. Michaelis constants were calculated from Lineweaver-Burk plots. Protein was determined by the method of Lowry et aL (1951).
Sephadex G-200 chromatography The starting fluid (2 ml) containing from 40 to 43 mg protein was applied to a column of Sephadex G-200 (2.5 x 31 cm) equilibrated with ,
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Fraction Number
Fig. 2. DEAE-Sephadex column chromatogram of HCF and HOL proteins. Chromatography was performed on specified aliquots of the Sephadex G-200 as described under Methods. The HCF enzyme was eluted between 330 and 375 mM NaCl (A A, peak II) and the HOL enzyme was eluted between 310 and 375mM NaCI ((3 O, peak !).
Echinococcus MDH
449
Table !. Purification of hydatid cyst fluid and healthy ovine liver MDH Vol Total enzyme Total protein Specificactivity (ml) (units)* (mg) (units/rag protein) Fraction HCF HOL HCF HOL HCF HOL HCF HOL Starting fluids 50t 10 125,000 56,000 2160 400 57.8 140 Sephadex G-200 40 15 90,000 40,800 480 90 187.5 453 DEAE-Sephadex 5 5 85,000 25,000 60 6 1416.6 4167 *Enzyme unit expressed as the amount of enzyme protein which causes a decrease of 0.001 units in absorbance (340 nm) per rain per 1.1 ml of the incubation mixture under the standard assay conditions. tHCF used in these experiments was concentrated 75-fold. 100 m M T r i s - H C l buffer, p H 7.4. The column was eluted with the same buffer and 4 m l fractions were collected. The protein elution patterns were established by reading the absorbance o f each fraction at 280 nm. Fractions containing enzyme activity were pooled and concentrated by ultrafiltration over a U M - 2 Diaflo membrane. Concentrated enzyme solutions f r o m several Sephadex G-200 columns were pooled and their volumes decreased by ultrafiltration. This final concentrated solution was used for the further purification o f the enzyme in the next step.
each in 200 ml o f the same buffer at p H 7.6. Fractions (4ml) were collected and protein elution patterns were established by reading the absorbances at 280 nm. The solutions f r o m fractions having enzyme activity were pooled and concentrated as before to a final volume o f 5 ml. Concentrated enzyme solutions from several D E A E - S e p h a d e x columns were pooled, further concentrated and stored at - 20°C prior to their use in kinetic studies. The enzyme preparation from both sources was stable for m o r e than a m o n t h when stored at this temperature.
DEAE-Sephadex chromatography
Results
F o u r milliliters o f H C F Sephadex G-200 fraction (12 mg protein/ml) or 5 ml o f H O L Sephadex G-200 fraction ( 6 m g protein/ml) were applied to a column o f D E A E - S e p h a d e x (2.5 x 31 cm) equilibrated with 5 m M T r i s - H C l buffer, p H 7.6 containing 3 0 0 m M N a C l and l m M E D T A (disodium salt). The column was first eluted with the same buffer, then the elution was continued with a linear concentration gradient o f N a C l between 300 and 600 m M N a C l
Sephadex G-200 column c h r o m a t o g r a m s o f concentrated H C F and H O L h o m o g e n a t e are shown in Fig. 1. Peak II from both sources (fractions 22-35 for H C F and 25--40 for H O L ) contained M D H activity. The D E A E - S e p h a d e x column c h r o m a t o g r a m s from both sources are shown in Fig. 2. H C F enzyme was eluted between 330 and 375 m M NaC1, while the H O L enzyme was eluted between 310 and 3 7 5 m M NaCI. A s u m m a r y o f the results obtained for the partial purification o f H C F and H O L enzymes is shown in Table 1. As seen, the specific activities o f the concentrated H C F and the H O L
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Fig. 3. MDH activity as a function of pH. Assays were done in duplicate according to standard conditions, except that pH was varied as indicated. Enzyme sources were either 48 #g of HCF DEAE-Sephadex fraction (A /X and • • ) or 12#g of HOL DEAE-Sephadex fraction (O O and • •). The buffers were either 40/~m01es of Tris-HCl buffer (A A and O O) or the same amount of barbital buffer ( • • and • • ) per incubation mixture. CBPB
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Fig. 4. Double reciprocal plots of the initial reaction velocity (110)vs oxaloacetate concentration (OAA) for HCF ( Q - O) and for healthy ovine liver (O O) enzymes. The buffer was 40/z moles of Tris-HC1 (pH 8.4) per 1.I ml of the incubation mixtures. Enzyme sources were as in Fig. 3. The concentration of NADH was as standard assay conditions and OAA concentration was varied as indicated. Results are averages of duplicate determinations.
450
Mahmood Vessal and Nasrin Bambaea-Row
As noted from Figs 4 and 5, both enzymes were inhibited at high concentrations of either Y.6.8 xlO-3* 1.45x lO-°X O A A or N A D H . Neither H C F nor H O L enzymes were inhibited by p H M B when up to 3 . 6 m M of this compound was included in the incubation mixtures. Although inhibition of this enzyme by p H M B from pig heart has been reported (Wolfe and Neilands, 1956), some M D H enzymes such as that studied from Haemophilus influenza are [-o.] not inhibited by sulfhydryl reagents (Yoon and Fig. 5. Double reciprocalplots of the initial reaction velocity Anderson, 1988). The enzymes from H C F or (V0) vs NADH concentration for HCF (O O) and for H O L were also not inhibited by fumarate when HOL (O 0) enzymes.Assayswere done in duplicate as up to 10 m M potassium fumarate was included in Fig. 4, except that OAA concentration was 0.407 #moles per incubation mixture and NADH was varied as indicated. in incubation mixtures. The partially purified enzyme from either source was absolutely specific for N A D H , and substitution of equimolar concentrations of enzymes increased about 24- and 30-fold, re- N A D P H for N A D H resulted in complete lack spectively. of the activity of either enzyme. The effect of p H on the velocity of M D H Heat denaturation patterns of partially reaction from both sources is shown in Fig. 3. ptirified enzyme from H C F or H O L are shown As indicated, the p H optimum of the enzyme in Fig. 6. As can be seen, two fairly identifical from both sources was found to be around 8.4 patterns were obtained for the enzyme from using either Tris-HC1 or barbital buffer. both sources. Both enzymes were completely The Lineweaver-Burk plots, showing the stable when heated for 2 min at various temeffects of O A A concentration on the activity of peratures up to 50°C, but gradually lost activity the respective enzymes, are shown in Fig. 4. The at higher temperatures. However, neither enKm values for O A A calculated from the zyme was completely inactive even after heating equation of the regression lines were 0.211 and for 2 min at 65°C. 0.200 m M for H C F and H O L enzymes, respectively. Discussion Figure 5 shows the Lineweaver-Burk plots for the effects of N A D H concentration on the It was demonstrated that malate dehydrogenrate of M D H reaction from both sources. The ases from hydatid cyst fluid and liver cytosol Km values for N A D H calculated from the were eluted at approximately the same volume equation of the regression lines were 0.220 from Sephadex G-200 columns (Fig. 1). It was and 0.213 m M for H C F and H O L enzymes, also shown that the enzyme from both sources respectively. was eluted between 310 and 375 m M NaCI when subjected to ion-exchange chromatography on D E A E - S e p h a d e x (Fig. 2). The results of these IOO , , . , . . . . . . . . . two experiments provide evidence as to the similarity of the molecular weights and the 80 charges of malate dehydrogenases of H C F and HOL. The enzyme from both sources showed a ~.o p H optimum of 8.4 in either Tris-HCl or barbital buffer (Fig. 3). The Km values for the effect of O A A on enzyme activities from H C F and H O L were reported to be 0.211 and 0.200 m M 2O (Fig. 4), while the corresponding values for N A D H were found to be 0.220 and 0.213 mM, respectively (Fig. 5). In addition, similar heat 35 40 45 50 55 60 65 70 Temperature ( °C ) denaturation patterns (Fig. 6), and similar substrate inhibition patterns (Figs 4 and 5) were Fig. 6. Thermal denaturation of MDH from HCF ( O - O) and HOL (0 0). Assays were done in duplicate observed for the enzyme from both sources. under standard conditions at pH 8.4 except that OAA and Also, both enzymes were found to be specific for NADH concentrations were 0.407 and 0.275 #moles per N A D H and were not inhibited by p H M B or 1.1 ml incubation mixture. The DEAE-Sephadex fractions containing MDH were heated for 2 rain at the indicated fumarate. Based on chromatographic behaviors and on temperatures prior to their assay. The activity of the enzyme kept at 37°C for 2 min was considered as 100%. the similar kinetic properties of these two °" 0_~ ; . 5 , , , ~ - 3 . ,,0 ',,0-`x . . . .
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enzymes, it is suggested that ovine liver hydatid cyst malate dehydrogenase may be among those proteins of the cyst fluid which has its origin in the host tissue (Vidor et al., 1987) and appears in the cyst fluid as a result of damage to hepatocytes. References Agosin M. and Repetto Y. (1967) Studies on the metabolism of Echinococcus granulosus. IX. Protein synthesis in scolices. Exp. Parasitol. 21, 195-208. Faradji B., Vessal M. and Ghalambor M. A. (1974) Partial purification and properties of ovine liver hydatid cyst fluid and healthy ovine liver phosphoglucose isomerases. Clin. Chim. Acta 53, 299-304. Krvavica S., Martincic T. and Asaj R. (1961) Metabolizam aminokiselina had nekih parasita. II. Aminokiseline fi hidatidnoj tekucini i germinativnom epitelu ehinokoka. Helminthol. Abstr. 30, 369. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. BioL Chem. 193, 265-275. Moss D. W., Henderson A. R. and Kachmar J. F. (1986) Enzymes. In Textbook o f Clinical Chemistry (Edited by Tietz N. W.), pp. 619-774. Saunders, Philadelphia.
451
Sarciron M. E., Azzar G., Persat F., Petavy A. F. and Got R. (1987) A comparative study of UTP-D-glucose-l-phosphate uridyl transferase in the cysts of Echinococcus multilocularis and the livers of infected and control Meriones unguiculatus. MoL Biochem. Parasitol. 23, 25-29. Sarciron M. E., Hamoud W., Azzar G. and Petavy A. F. (! 991) Alkaline phosphatase from Echinococcus multilocularis : purification and characterization. Comp. Biochem. Physiol. IOOB,253-258. Vessal M. and Abdolrasulnia R. (1976) Partial purification and properties of ovine liver Echinococcus granulosus protoscolices phosphoglucose isomerase. Clin. Chim. Acta 68, 59-65. Vessal M. and Ghalambor M. A. (1970) Studies on the enzymes of hydatid cyst fluid. Quantitative determination of enzymes present in Echinococcus granulosus cyst fluid. Israel J. Med. Sci. 6, 383-387. Vidor E., Piens M. A. and Garin J. P. (1987) Host serum protein levels in cysts of human hydatidosis. Trans. R. Soc. Trop. Med. Hyg. 81, 669-671. Wacker W., Ulmer D. and Vallee B. (1956) Metaloenzymes and myocardial infarction. II. Malic and lactic dehydrogenase activities and zinc concentrations in serum. New Engl. J. Med. 255, 499-456. Wolfe R. C. and Neilands J. B. (1956) Some molecular and kinetic properties of heart malic dehydrogenase. J. Biol. Chem. 221, 61-69. Yoon H. and Anderson B. M. (1988) Kinetic studies of Haemophilus influenzae malate dehydrogenase. Biochim. Biophys. Acta 955, 10-18.
Pergamon
Partial purification and comparison of the kinetic properties of ovine liver Echinococcus granulosus hydatid cyst fluid malate dehydrogenase and the cytoplasmic enzyme from the host liver Mahmood Vessal and Nasrin Bambaea-Row Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz, 71345, Iran Ovine fiver Echinocoecusgranulosus hydatid cyst fluid and cytoplasmic healthy ovine fiver malate dehydrogennses were purified 24- and 30-fold by Sephadex G-200 and DEAE-Sephadex chromatography. Both enzymes were eluted with the same elution volume and the same salt concentration from the respective columns. The pH optimum of the enzymes from both sources was 8.4 in either Tris--HC! or barbital buffer. The Km values for oxaloacetate were 0.211 and 0.200 mM for hydatid cyst fluid and healthy ovine fiver enzymes, respectively. The Km values for NADH were 0.220 and 0.213 mM for bydatid cyst fluid and healthy ovine fiver enzymes, respectively. Enzyme from both sources demonstrated similar heat denaturation patterns. Both enzyme preparations were inhibited at high concentrations of either substrate. Neither enzyme was inhibited by para-hydroxymercuribenzoate or fumarate, and both enzyme preparations were specific for NADH as a cofactor. The results are discussed in terms of the possible infiltration of the host enzyme into the cyst fluid. Key worth: Echinococcus granulosus; Hydatid cyst; Malate dehydrogenase.
Comp. Biochem. Physiol. 107B, 447-451, 1994.
Introduction
Hydatid cyst fluid (HCF) is believed to be composed of an ultrafiltrate of the host tissue fluids and the secretions of the germinal epithelium of the cyst (Krvavica et al., 1961). Of the total proteins present in HCF, some are produced by the parasite and others by the host (Agosin and Repetto, 1967). In order to find possible differences in the properties of the parasite and the host enzymes to be used in the development of sound chemotherapy, comparison has been made of the physical and kinetic properties of phosphoglucose isomerases, UTPD-glucose-l-phosphate uridyltransferases and alkaline phosphatases of Echinococcus species and the host liver by several investigators (Faradji et al., 1974; Vessal and Abdolrasulnia, 1976; Sarciron et al., 1987, 1991). Fairly high Correspondence to: M. Vessal, Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz, 71345, Iran. Received 22 June 1993; accepted 30 July 1993. 447
levels of NADP-linked isocitrate dehydrogenase and NAD-dependent malate dehydrogenase enzymes (MDH) have been reported in ovine liver HCF (Vessal and Ghalambor, 1970). A high level of soluble isocitrate dehydrogenase in the serum is considered to be an indication of liver damage (Moss et al., 1986). There are also reports on the presence of several host proteins in cyst fluids and in the patient's sera (Vidor et al., 1987). In this investigation it was thought that perhaps the fairly high levels of soluble malate and isocitrate dehydrogenases in the cyst fluid may be a result of damage to hepatocytes. Therefore, preliminary experiments were set up to partially purify soluble MDH from healthy ovine liver (HOL) and ovine liver Echinococcus granulosus HCF and to compare some of their physical and kinetic properties in order to find a clue as to the probable origin of the hydatid fluid malate dehydrogenase enzyme.
448
Mahmood Vessal and Nasrin Bambaea-Row
Materials and Methods Reagents cis-Oxaloacetic acid (OAA), p-hydroxymer-
24
curibenzoate (pHMB-disodium salt) and fumaric acid (potassium salt) were purchased from BDH Biochemicals (Poole, Dorset, U.K.); Sephadex G-200 and DEAE-Sephadex A-50 from Pharmacia (Uppsala, Sweden); and NADH (disodium salt) from Sigma (St Louis, MO). All other reagents were of highest purity and obtained through other commercial sources.
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Freshly secured sheep liver (100 g) was chilled on ice, cut into small pieces, washed several times with 100raM Tris-HC1 buffer (pH 7.4) and homogenized in I00 ml of the same buffer in a Waring blender for four 2-min cycles. The homogenate was centrifuged at 27,000g for 10min and the supernatant fluid diluted to a protein concentration of about 40 mg/ml with 100 mM Tris-HCl (pH 7.4). This supernatant solution was used as the starting material in the purification of liver MDH.
Enzyme assay MDH was assayed by a spectrophotometric procedure (Wacker etal., 1956). The standard assay mixture contained 0.4 ml of the respective enzyme solution and the following ingredients in/~moles in a total volume of 1.1 ml: NADH, 0.30; OAA (freshly prepared), 3.3; Tris-HCl buffer, 40; pH7.4 during purification and pH 8.4 in kinetic studies. Reactions were started by the addition of OAA solution and the decrease in absorbance at 340nm (25°C) was followed spectrophotometrically at different time intervals. Control cuvettes in which water was substituted for OAA were also included. Enzyme unit was expressed as the amount of enzyme protein which caused a decrease of 0.001 units in absorbance (340 nm) per minute per 1.1 ml of the incubation mixture under standard assay conditions at 25°C. The relation between the activity of HCF or HOL enzyme and the time of incubation was linear up to 10 rain. Enzyme activity was linear up to 50 #g of the partially purified HCF pro-
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Preparation of liver homogenates
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Preparation of HCF All procedures were carried out at 0-4°C unless stated otherwise. HCF was prepared as described (Faradji et al., 1974). HCF volume was reduced to 1/75 by ultrafiltration over a UM-2 Diaflo membrane (Amicon Corporation, Lexington, MA) under nitrogen gas. Concentrated HCF was used as the starting fluid in the purification of cyst fluid MDH.
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Fraction Number
Fig. I. Sephadex G-200 column chromatograms of HCF and HOL proteins. Chromatography was done as described under Methods. Enzyme activities were detected in peak II (HCF, • • and HOL, (3 0).
tein or up to 15 #g of partially purified HOL protein in 1.1 ml of the incubation mixture. The effect of pH on enzyme activity was observed by using either 40 #moles of Tris-HCl buffer or 40 #moles of barbital buffer (pH 7.4-8.8) per 1.1 ml of the incubation mixture with all other conditions remaining the same. For heat inactivation experiments, the enzyme solutions were incubated at different temperatures (37-65°C) for 2 min prior to enzyme assay. Michaelis constants were calculated from Lineweaver-Burk plots. Protein was determined by the method of Lowry et aL (1951).
Sephadex G-200 chromatography The starting fluid (2 ml) containing from 40 to 43 mg protein was applied to a column of Sephadex G-200 (2.5 x 31 cm) equilibrated with ,
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0.4
0.35
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20
30
40
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60
70
Fraction Number
Fig. 2. DEAE-Sephadex column chromatogram of HCF and HOL proteins. Chromatography was performed on specified aliquots of the Sephadex G-200 as described under Methods. The HCF enzyme was eluted between 330 and 375 mM NaCl (A A, peak II) and the HOL enzyme was eluted between 310 and 375mM NaCI ((3 O, peak !).
Echinococcus MDH
449
Table !. Purification of hydatid cyst fluid and healthy ovine liver MDH Vol Total enzyme Total protein Specificactivity (ml) (units)* (mg) (units/rag protein) Fraction HCF HOL HCF HOL HCF HOL HCF HOL Starting fluids 50t 10 125,000 56,000 2160 400 57.8 140 Sephadex G-200 40 15 90,000 40,800 480 90 187.5 453 DEAE-Sephadex 5 5 85,000 25,000 60 6 1416.6 4167 *Enzyme unit expressed as the amount of enzyme protein which causes a decrease of 0.001 units in absorbance (340 nm) per rain per 1.1 ml of the incubation mixture under the standard assay conditions. tHCF used in these experiments was concentrated 75-fold. 100 m M T r i s - H C l buffer, p H 7.4. The column was eluted with the same buffer and 4 m l fractions were collected. The protein elution patterns were established by reading the absorbance o f each fraction at 280 nm. Fractions containing enzyme activity were pooled and concentrated by ultrafiltration over a U M - 2 Diaflo membrane. Concentrated enzyme solutions f r o m several Sephadex G-200 columns were pooled and their volumes decreased by ultrafiltration. This final concentrated solution was used for the further purification o f the enzyme in the next step.
each in 200 ml o f the same buffer at p H 7.6. Fractions (4ml) were collected and protein elution patterns were established by reading the absorbances at 280 nm. The solutions f r o m fractions having enzyme activity were pooled and concentrated as before to a final volume o f 5 ml. Concentrated enzyme solutions from several D E A E - S e p h a d e x columns were pooled, further concentrated and stored at - 20°C prior to their use in kinetic studies. The enzyme preparation from both sources was stable for m o r e than a m o n t h when stored at this temperature.
DEAE-Sephadex chromatography
Results
F o u r milliliters o f H C F Sephadex G-200 fraction (12 mg protein/ml) or 5 ml o f H O L Sephadex G-200 fraction ( 6 m g protein/ml) were applied to a column o f D E A E - S e p h a d e x (2.5 x 31 cm) equilibrated with 5 m M T r i s - H C l buffer, p H 7.6 containing 3 0 0 m M N a C l and l m M E D T A (disodium salt). The column was first eluted with the same buffer, then the elution was continued with a linear concentration gradient o f N a C l between 300 and 600 m M N a C l
Sephadex G-200 column c h r o m a t o g r a m s o f concentrated H C F and H O L h o m o g e n a t e are shown in Fig. 1. Peak II from both sources (fractions 22-35 for H C F and 25--40 for H O L ) contained M D H activity. The D E A E - S e p h a d e x column c h r o m a t o g r a m s from both sources are shown in Fig. 2. H C F enzyme was eluted between 330 and 375 m M NaC1, while the H O L enzyme was eluted between 310 and 3 7 5 m M NaCI. A s u m m a r y o f the results obtained for the partial purification o f H C F and H O L enzymes is shown in Table 1. As seen, the specific activities o f the concentrated H C F and the H O L
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Fig. 3. MDH activity as a function of pH. Assays were done in duplicate according to standard conditions, except that pH was varied as indicated. Enzyme sources were either 48 #g of HCF DEAE-Sephadex fraction (A /X and • • ) or 12#g of HOL DEAE-Sephadex fraction (O O and • •). The buffers were either 40/~m01es of Tris-HCl buffer (A A and O O) or the same amount of barbital buffer ( • • and • • ) per incubation mixture. CBPB
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Fig. 4. Double reciprocal plots of the initial reaction velocity (110)vs oxaloacetate concentration (OAA) for HCF ( Q - O) and for healthy ovine liver (O O) enzymes. The buffer was 40/z moles of Tris-HC1 (pH 8.4) per 1.I ml of the incubation mixtures. Enzyme sources were as in Fig. 3. The concentration of NADH was as standard assay conditions and OAA concentration was varied as indicated. Results are averages of duplicate determinations.
450
Mahmood Vessal and Nasrin Bambaea-Row
As noted from Figs 4 and 5, both enzymes were inhibited at high concentrations of either Y.6.8 xlO-3* 1.45x lO-°X O A A or N A D H . Neither H C F nor H O L enzymes were inhibited by p H M B when up to 3 . 6 m M of this compound was included in the incubation mixtures. Although inhibition of this enzyme by p H M B from pig heart has been reported (Wolfe and Neilands, 1956), some M D H enzymes such as that studied from Haemophilus influenza are [-o.] not inhibited by sulfhydryl reagents (Yoon and Fig. 5. Double reciprocalplots of the initial reaction velocity Anderson, 1988). The enzymes from H C F or (V0) vs NADH concentration for HCF (O O) and for H O L were also not inhibited by fumarate when HOL (O 0) enzymes.Assayswere done in duplicate as up to 10 m M potassium fumarate was included in Fig. 4, except that OAA concentration was 0.407 #moles per incubation mixture and NADH was varied as indicated. in incubation mixtures. The partially purified enzyme from either source was absolutely specific for N A D H , and substitution of equimolar concentrations of enzymes increased about 24- and 30-fold, re- N A D P H for N A D H resulted in complete lack spectively. of the activity of either enzyme. The effect of p H on the velocity of M D H Heat denaturation patterns of partially reaction from both sources is shown in Fig. 3. ptirified enzyme from H C F or H O L are shown As indicated, the p H optimum of the enzyme in Fig. 6. As can be seen, two fairly identifical from both sources was found to be around 8.4 patterns were obtained for the enzyme from using either Tris-HC1 or barbital buffer. both sources. Both enzymes were completely The Lineweaver-Burk plots, showing the stable when heated for 2 min at various temeffects of O A A concentration on the activity of peratures up to 50°C, but gradually lost activity the respective enzymes, are shown in Fig. 4. The at higher temperatures. However, neither enKm values for O A A calculated from the zyme was completely inactive even after heating equation of the regression lines were 0.211 and for 2 min at 65°C. 0.200 m M for H C F and H O L enzymes, respectively. Discussion Figure 5 shows the Lineweaver-Burk plots for the effects of N A D H concentration on the It was demonstrated that malate dehydrogenrate of M D H reaction from both sources. The ases from hydatid cyst fluid and liver cytosol Km values for N A D H calculated from the were eluted at approximately the same volume equation of the regression lines were 0.220 from Sephadex G-200 columns (Fig. 1). It was and 0.213 m M for H C F and H O L enzymes, also shown that the enzyme from both sources respectively. was eluted between 310 and 375 m M NaCI when subjected to ion-exchange chromatography on D E A E - S e p h a d e x (Fig. 2). The results of these IOO , , . , . . . . . . . . . two experiments provide evidence as to the similarity of the molecular weights and the 80 charges of malate dehydrogenases of H C F and HOL. The enzyme from both sources showed a ~.o p H optimum of 8.4 in either Tris-HCl or barbital buffer (Fig. 3). The Km values for the effect of O A A on enzyme activities from H C F and H O L were reported to be 0.211 and 0.200 m M 2O (Fig. 4), while the corresponding values for N A D H were found to be 0.220 and 0.213 mM, respectively (Fig. 5). In addition, similar heat 35 40 45 50 55 60 65 70 Temperature ( °C ) denaturation patterns (Fig. 6), and similar substrate inhibition patterns (Figs 4 and 5) were Fig. 6. Thermal denaturation of MDH from HCF ( O - O) and HOL (0 0). Assays were done in duplicate observed for the enzyme from both sources. under standard conditions at pH 8.4 except that OAA and Also, both enzymes were found to be specific for NADH concentrations were 0.407 and 0.275 #moles per N A D H and were not inhibited by p H M B or 1.1 ml incubation mixture. The DEAE-Sephadex fractions containing MDH were heated for 2 rain at the indicated fumarate. Based on chromatographic behaviors and on temperatures prior to their assay. The activity of the enzyme kept at 37°C for 2 min was considered as 100%. the similar kinetic properties of these two °" 0_~ ; . 5 , , , ~ - 3 . ,,0 ',,0-`x . . . .
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,
].,~60 40
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Echinococcus MDH
enzymes, it is suggested that ovine liver hydatid cyst malate dehydrogenase may be among those proteins of the cyst fluid which has its origin in the host tissue (Vidor et al., 1987) and appears in the cyst fluid as a result of damage to hepatocytes. References Agosin M. and Repetto Y. (1967) Studies on the metabolism of Echinococcus granulosus. IX. Protein synthesis in scolices. Exp. Parasitol. 21, 195-208. Faradji B., Vessal M. and Ghalambor M. A. (1974) Partial purification and properties of ovine liver hydatid cyst fluid and healthy ovine liver phosphoglucose isomerases. Clin. Chim. Acta 53, 299-304. Krvavica S., Martincic T. and Asaj R. (1961) Metabolizam aminokiselina had nekih parasita. II. Aminokiseline fi hidatidnoj tekucini i germinativnom epitelu ehinokoka. Helminthol. Abstr. 30, 369. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. BioL Chem. 193, 265-275. Moss D. W., Henderson A. R. and Kachmar J. F. (1986) Enzymes. In Textbook o f Clinical Chemistry (Edited by Tietz N. W.), pp. 619-774. Saunders, Philadelphia.
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Sarciron M. E., Azzar G., Persat F., Petavy A. F. and Got R. (1987) A comparative study of UTP-D-glucose-l-phosphate uridyl transferase in the cysts of Echinococcus multilocularis and the livers of infected and control Meriones unguiculatus. MoL Biochem. Parasitol. 23, 25-29. Sarciron M. E., Hamoud W., Azzar G. and Petavy A. F. (! 991) Alkaline phosphatase from Echinococcus multilocularis : purification and characterization. Comp. Biochem. Physiol. IOOB,253-258. Vessal M. and Abdolrasulnia R. (1976) Partial purification and properties of ovine liver Echinococcus granulosus protoscolices phosphoglucose isomerase. Clin. Chim. Acta 68, 59-65. Vessal M. and Ghalambor M. A. (1970) Studies on the enzymes of hydatid cyst fluid. Quantitative determination of enzymes present in Echinococcus granulosus cyst fluid. Israel J. Med. Sci. 6, 383-387. Vidor E., Piens M. A. and Garin J. P. (1987) Host serum protein levels in cysts of human hydatidosis. Trans. R. Soc. Trop. Med. Hyg. 81, 669-671. Wacker W., Ulmer D. and Vallee B. (1956) Metaloenzymes and myocardial infarction. II. Malic and lactic dehydrogenase activities and zinc concentrations in serum. New Engl. J. Med. 255, 499-456. Wolfe R. C. and Neilands J. B. (1956) Some molecular and kinetic properties of heart malic dehydrogenase. J. Biol. Chem. 221, 61-69. Yoon H. and Anderson B. M. (1988) Kinetic studies of Haemophilus influenzae malate dehydrogenase. Biochim. Biophys. Acta 955, 10-18.