THE EFFECTS OF INORGANIC SALTS ON THE STABILITY OF ISOCITRATE DEHYDROGENASE
FROM BEEF (BOS TAURUS) HEART AND LIVER
AHMET BALAMIR and KAYA EMERK Department of Biochemistry, Hacettepe University, Ankara, Turkey
(Received 21 July 1981)
Abstract--1. Distinct differences are established between the two isozymes of NADP÷-dependent isoci- trate dehydrogenase, purified from beef (Bos taurus) heart and liver, with respect to their net electrical charges and stabilization properties in the presence of inorganic salts.
2. Inorganic salts also inhibit the isozymes with different apparent K~ values. 3. Both isozymes are found to be heat labile in the absence and cold labile in the presence of salts. 4. The results indicate that different mechanisms of heat stabilization exist due to different conforma-
tional states which are less active but more stable.
INTRODUCTION
Two types of NADP+-dependen t isocitrate dehydro- genases, ( th reo-D: i soc i t r a t e -NADP + oxido reductase (decorboxylating), EC 1.1.1.42) the heart and the liver type, exist in mammal ian tissues. It is shown that the heart type is p redominant in the mi tochondr ia (Fata- nia & Dalziel, 1980) and the liver type is predominant in the extramitochondr ia l cytoplasm (Carlier & Pan- taloni, 1973) in beef (Bos taurus). Some differences in similarities with respect to heat and pH stability, effect of inhibitors, molecular weights have been found between the two isozymes (Siebert et al., 1956; Islam et al., 1972; MacFar lane et al., 1977; Carlier & Panta- loni, 1973). Several binding and kinetic studies have also been carried out with bo th isozymes (Reynolds et al., 1978; Dalziel et al., 1978; Carlier & Pantaloni , 1976a,b, 1978).
This paper describes the effects of various inorganic salts on the stability and kinetic parameters of isoci- trate dehydrogenases from beef (Bos taurus) heart and liver. The results indicate that different mechanisms of heat stabilization exist, p robably due to different con- formational states i.e. enzyme proteins that are less active but more stable. This prel iminary data may serve to provide more detailed conformat ional studies of the two isozymes.
MATERIALS AND METHODS
NADP +, DL-isocitrate, NADP+-Agarose phenazine- methosulphate and p-nitro blue tetrazolium were obtained from Sigma Chem. Co., U.S.A. Sephadex G-150 and 2',5' ADP-Sepharose were from Pharmacia Fine Chemicals. All other chemicals used were of the highest purity available from Merck and BDH Ltd.
Activity The rate of dehydrogenation and decarboxilation of
o-isocitrate was measured by following the rate of NADPH formation, using a Beckman Model 25 recording
spectrophotometer with an expanded scale. In kinetic studies the reaction was initiated by the addition of the desalted enzyme. Initial rates were calculated from the recordings, using portions, where not more than 10% of the substrate is used. The actual concentration of the sub- strates in the reaction cuvette was calculated from experi- ments, carried out to completion at high enzyme concen- tration. One unit of enzyme activity was defined as the amount of enzyme which catalyzes the formation of 1 #mol of NADPH/min at 37°C under the following conditions: 0.33 mM DL-isocitrate, 1 mM MnC12, 0.1 mM NADP ÷ in 0.1 M Tris-HCl buffer, pH 7.5.
Protein concentrations were determined spectrophoto- metrically at 280nm using an absorbance coefficient of 1.18cm2/mg for heart (MacFarlane et al., 1977) and 1.29 cm2/mg (Carlier & Pantaloni, 1973) for liver isozymes.
Enzyme purification Purification of the beef (Bos taurus) heart enzyme is car-
ried out according to the method of MacFarlane et al. (1977) except for the introduction of a Sephadex G-150 gel filtration step before ion exchange chromatography. Intro- duction of this step gave a 2-fold purification at the ion exchange chromatography, although it did not affect the yields significantly. This isozyme could not be further puri- fied using 2',5' ADP-Sepharose 4B and NADP+-agarose as affinity media. Purification of the beef liver enzyme was carried out according to the method of Carlier & Panta- loni (1973) except for the introduction of a Sephadex G-150 gel filtration step before ion exchange chromatography and omission of the hydroxyapatite treatment. Liver isozyme could not be further purified using the above mentioned affinity media either.
Specific activity for both enzymes is found to be 45 U/rag. This value is somewhat higher than the values reported for the heart (39 U/mg) (MacFarlane et al., 1977) and liver isozymes (17.7 U/rag) (Pantaloni et al., 1973) but this can be due to the assay conditions and temperature.
Desalting of the enzyme was performed using Sephadex G-25 columns equilibrated with 5 mM sodium citrate buffer, pH 7.4. The efficiency of desalting was checked for sulphate using BaCI 2 in 0.1 M HC1 and for citrate enzyma- tically.
489
490 AHMET BALAMIR and KAVA EMERK
Heat stability experiments were carried out with the desalted enzyme solutions brought to the desired salt con- centration using saturated solutions of salts at pH 7. Twenty ~d samples were removed at different times after incubation at the indicated temperatures. These samples were diluted at least 100 fold into the assay mixture.
Gel electrophoresis ~md activity stainin9
Polyacrylamide gel electrophoresis experiments were carried on 7% acrylamide gels, according to the method of Davis (1964). For activity staining, a modification of Hen- derson's method was used (1968).
RESULTS AND DISCUSSION
Distinct charge differences were observed between the isozymes during the purification procedures. The results of binding to anion and cation exchangers are summarized in Table 1. The pI of the heart enzyme is estimated to be between 7.4 and 8.3 based on its be- haviour on gel electrophoresis (Ry = 0.03 at pH 8.3: see Fig. 1), its strong binding to CM cellulose and inability to b ind to DEAE at pH 7.4. The pI of liver enzyme is estimated to be equal to or less than 5, based on the same criteria (R I = 0.27 at pH 6.1 (Car- lier & Pantaloni , 1973) and R I = 0.42 at pH 8.3: Fig. 1).
Amino acid composit ions reported earlier for heart and liver isozymes from pig, bovine and human tissues indicate the presence of higher glutamyl + as- partyl residues than lysyl + histidyl + arginyl resi- dues per molecule (Seelig & Colman, 1978). The apparent discrepancy between the composi t ion and the pI proposed, may be at t r ibuted to the amide con- tent for the heart enzyme.
The heat stability of the purified heart and liver isozymes at two different temperatures is shown in Fig. 2. The difference is more pronounced at 50°C. The heart isozyme loses 9 2 ~ of its initial activity, while the liver isozyme remains 100~o active at the end of 5 rain. It is interesting to note that glycerol at 20~o (v/v) does not have any protective effect on either enzymes, since Islam et al. (1972) found that glycerol protects the rat isozymes against heat inactivation. The differences in heat stability are better demon-
Table 1. Binding of heart and liver IDH's to ion exchange media at different pH's
Ion exchanger Heart Liver
DEAE Sephadex A-50 - +* pH = 7.4
DEAE Sephadex A-50 pH = 6.1
CM Sephadex C-50 pH = 6.5
CMSephadex C-50 pH = 5.8
+
+
* Positive sign indicates binding. All columns were equilibrated with 5 mM
trisodium citrate, 5raM MgSO4, l m M EI)TA at the indicated pH.
a b
Fig. I. Polyacrylamide gel electrophoresis of NADP +- dependent isocitrate dehydrogenase of beef heart (a) and liver (b). The protein was run on 7% acrylamide gels con- taining 1/38 bis-acrylamide at pH = 8.3 in Tris-glycine buffer, at 5 mA per gel. Each get contains approx. 60 ~g of protein. Activity was stained using a medium, 2.5 mM DL- isocitrate, 0.2 mM NADP+, 0.01% p-nitro blue tetrazolium and 0.0033°~; pheanzine methosulphate in 0.05 M Tris HC1 buffer pH = 7.4, for 20 min at room temperature and fixed
in 7°,i acetic acid solution.
strated by the addi t ion of inorganic salts into the incubat ion mixtures. The effect of ammon ium sul- phate on heat denatura t ion of heart and liver iso- zymes is shown in Fig. 3. The liver isozyme is stabil-
g
I00
80
60
40
20
~. • ,~'
h i t
I 0 @0 3 0 4 0 5 0 6 0 7 0
T i m e (min i
Fig. 2. Difference in heat stability of heart and liver IDH's. The isozymes were incubated at 37 and 50'C at a protein concentration of 0.25 mg/ml. The assay medium contained ; 0.33 mM DL-isocitrate~ 0.1 mM NADP*, 1 mM McCI2 in 0.1 Tris-HCl pH = 7.5. The incubation medium contained 5 mM sodium citrate, 20{'J;, glycerol. 1 mM EDTA at a pH of 7.5, 20/d samples were diluted into the assay mixture of 3 ml at indicated time intervals. Rates are expressed as !L, of original (zero) time. [] Heart IDH at 50 C: • Liver IDH at 50C: O Heart IDH at 37'C: • Liver 1DH at 37 C.
Stability of isocitrate dehydrogenases 491
120
I00
'~ 80
60
4O
20
,o 20 ~o ,'o 5'0 6o io do 9o ,ao T i m e (rain)
Fig. 3. Heat stability of heart and liver isozymes at 50°C, in the presence of (NH4)2SO 4. The enzymes, desalted on Sephadex G-25 in 5 mM sodium citrate, 1 mM EDTA pH = 7.5 were incubated at 50°C, at the indicated salt concentration provided by the addition of saturated salt solutions. The experiment is carried out as described in the legend of Fig. 2. • Heart IDH; No ammonium sulphate; • Liver IDH; No ammonium sulphate; • Heart IDH; 10~o ammonium sulphate; [] Heart IDH; 20~o ammonium
ized at 10~o ammonium sulphate (w/v), whereas the heart isozyme is stabilized at 40~. A similar effect was observed with sodium sulphate but at lower concen- trations (Fig. 4). The effects of NaC1 and NH4CI are illustrated in Fig. 5. The liver enzyme is not only stabilized but also activated by the inorganic salts except NH4C1 (Figs 2-5). No activation is observed however for the heart enzyme. There is a distinct dif- ference between the heat stability of isozymes at 50°C in the presence of NaC1 (Fig. 5). The liver isozyme is totally stable in 5~o NaCI for 90min whereas the heart isozyme loses 92~ of its activity in 20 min even in the presence of saturated NaC1. On the other hand
NH4C1 enhances the thermal denaturation of the heart isozyme, but it has no effect on the liver isozyme at 50°C. Similar results are obtained at 30°C, although longer time intervals were necessary to obtain the same degree of inactivation.
At 0°C however, all the above mentioned inorganic salts enhance the inactivation of both isozymes. The remaining activity at the end of 48hr (for heart enzyme) and after 96 hr (for liver enzyme) at 0°C are listed in Table 2. The results suggest that stabilization of the liver isozyme is due to a general salt effect at 30 and 50°C. The enzyme appears to be heat-labile in the absence and cold-labile in the presence of salts. The
"° f ',i[f
,'o ~o ~'o ~o ~o 60 7'0 80 9'0
T i m e (ra in)
Fig. 4. Heat stability of heart and liver isozymes at 50°C in the presence of Na2SO 4. The experiment is carried out as described in the legend of Fig. 3. • Heart IDH; 1~o Na2SO4; • Heart IDH; 5~ Na2SO4;
effect o f N H 4 + is exceptional since it does not protect the enzyme against heat denaturation. The heart enzyme can only be stabilized by sulphate ions against thermal denaturation and NH4 + ions enhance the inactivation at all temperatures tested. The specific stabilizing effect of sulphate ions may be due to the formation of an ionic structure between this ion and the heart isozyme, which is rich in positively charged side groups.
The apparent Km values for Mn 2+ ions, isocitrate, and N A D P + were determined from double reciprocal plots. The K,, values for Mn 2 + ions in the presence of 0.1raM De-isocitrate and 0.05mM N A D P + are 2.1~M and 1.2#M for heart and liver isozymes re- spectively. The K,, for D-isocitrate at saturating N A D P + concentration, was found to be 0.7 and 2 #M for heart and liver isozymes respectively. K,, values for N A D P + at saturating isocitrate are 6.8 and 8.0/~M for heart and liver enzymes respectively. The
Table 3. Inhibition of heart and liver IDH's by inorganic salts
Apparent Ki values, calculated from plots of }~, inhibition vs concentration are given in ionic strength, where 500 inhibition is obtained. The assay medium contained 50/~M o-isocitrate, 50#M NADP + and 1 mM MnC1 z in 0.01 M Tris-HC1 pH = 7.5. Enzyme concentration = 0.1 mg/ml.
K,, values for both substrates compare favourably with values cited in literature for isozymes from differ- ent species (Carlier & Pantaloni, 1973; Seelig & Col- man, 1977; Plaut et al., 1975; Ingebretsen & Sanner, 1976).
In addition to their stabilizing action the inorganic salts have an inhibitory effect on the isozymes in the range of 500-1000 mM. The data in Table 3 indicate that ammonium chloride inhibits the enzymes at lower ionic strength than the other salts. It is interest- ing to note that liver isocitrate dehydrogenase is in- hibited by all salts at lower concentrations than the heart isozyme, which might indicate a relationship between inhibition and stabilization, i.e. the macro- molecule at the inhibited conformation is heat resist- ant. The inorganic salts may be changing the inner hydration of the native protein molecule and convert- ing it to an inactive conformation, which is heat stable. Since ammonium sulphate is found to stabilize both isozymes, the stabilizing effect of sulphate appears to be stronger than the inactivating effect of ammonium ions.
To elucidate the differences in the mechanism for the conformational changes between the two iso- zymes, further mechanistic and structural work is still necessary.
Table 2. Cold lability of heart and liver IDH's in the presence of inorganic salts: the enzyme solutions (100/ag/ml) were incubated at 0cC with inorganic salts. The samples were diluted 100 fold into the assay mixtures. Enzyme activity was measured as described in the
legend of Fig. 2
Sample }~ remaining activity*
NH4C1 (0.5 M1NaCI (1 M) Na2SO4 (0.5 M)
Heart IDH 1.6 25.2 81.1 (After 48 hr)
Liver IDH 11.8 41.5 70.3 (After 96 hr)
* The values are percentages of activity of desalted enzyme at the end of the indicated periods. The desalted enzymes did not lose more than 20% of the original activity (activity at zero time), within these periods.
Stability of isocitrate dehydrogenases 493
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