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Effect of ionic strength on the regulatory properties of 2-oxoglutarate dehydrogenase complex
Biochimie (1992) 74,171-176 © Soci6t6 fran~:aise de biochimie et biologie mol6culaire / Elsevier, Paris
171
Effect of ionic strength on the regulatory properties of 2-oxoglutarate dehydrogenase complex T Pawelczyk*, S Angielski Chair and Department of Clinical Biochemistry Medical Academy, 80-211 Gdansk, Poland
(Received 12 August 1991; accepted 8 November ! 991 )
Summary m The effects of changing ionic strength on the activity of the 2-oxoglutarate dehydrogenase complex from pig kidney cortex were explored. This enzyme complex is found to be influenced in many ways by the ionic strength of the reaction medium. The enzyme shows an optimum activity at 0.1 M ionic strength. Increase in ionic strength from 0.1 M to 0.2 M resulted in a decrease of S~..s for 2-oxoglutarate, and in an increase of Sf~.stbr NAD. Changes in ionic strength over the range of 0.05--0.2 M have little, if any, effect on Sa..s for CoA. The Hill coefficient for 2-oxoglutarate and NAD at 0.2 M ionic strength was 1.0, whereas at 0.05 M ionic strength it was 0.85 and 1.2 for 2-oxoglutarate and NAD, respectively At 0.05 M ionic strength the pH optimum of the enzyme ranges between 7.4-7.6, but at 0.15 M ionic strength the pH optimum shifts to 7.8. The magnitude of inhibition of enzyme activity by ATP is not influenced by changes in ionic strength in the absence of calcium. However, in the presence of Ca2*, increases in ionic strength lower the inhibitory effects of ATP. The Sio.5 for ATP in both presence and absence of Ca2+ was not affected by changes in ionic strength in the range of 0.1-0.2 M. In contrast, the Sao5 for ADP in the absence of Ca 2+ decreases as ionic strength increases. In the presence of calcium and 0.2 M ionic strength ADP has no effect on 2-oxoglutarate dehydrogenase complex activity. 2-oxoglutarate dehydrogenase / ionic strength / pH / ATP / ADP / kidney Introduction The 2-oxoglutarate dehydrogenase complex as an enzyme of the tricarboxylic acid cycle occupies a key position in intermediary metabolism. The mammalian 2-oxoglutarate dehydrogenase complex consists of 3 catalytic components, 2-oxoglutarate dehydrogenase ( E l ) , lipoamide succinyltransferase (E2) and lipoamide dehydrogenase (E3). The enzyme complex contains a core consisting of 24 identical E2 chains to which about 12 identical E l chains (6 dimers) and about 12 identical E3 chains (6 dimers) are non-covalently attached [1-3]. The total molecular weight (Mr) of the complex is approximately 2.7 x 106 [3, 41. In an electron micrograph the enzyme complex appears as a polyhedral structure having a gross diameter of about 260 A [4]. The stabilization of such a quaternary structure involves various interactions such as electrostatic, hydrogen bonding, van der Waals, hydrophobic *Correspondence and reprints. Present address: Graduate Department of Biochemistry, Brandeis University Waltham, MA 02254, USA Abbreviations: It, ionic strength; OGDC, 2-oxoglutarate dehydrogenase complex; S0.5, substrate concentration giving halfmaximal activity; Sa0.s, activator concentration giving halfmaximal stimulation; Si0.5, inhibitor concentration giving half-maximal inhibition; Vm~,true value of maximal activity.
and conformational tension. Thus, changes in the magnitude of any of these contributing interactions may have a substantial effect on the catalytic properties of the enzyme complex. The 2-oxoglutarate dehydrogenase complex is regulated by a number of factors. NADH, succinyI-CoA, ATP and GTP are inhibitory 18, 91; ADP, Ca 2+, Sr2+ and inorganic phosphate are stimulatory [10-14] It has been demonstrated that variations in ionic strength and pH influence electrostatic interactions within a protein molecule [5, 71, and affect the binding of nucleotides and ions [6, 15, 16]. Under normal conditions most mammalian cells maintain constant ionic strength. However, in the kidney some cells function under high osmolality and ionic strength. In addition, intracellular concentration of solutes chnges in response to alterations in the diuretic state 1 19]. Therefore, it is of interest to determine the effect of ionic strength on the catalytic properties of this enzyme complex, particularly in the presence of its effectors.
Materials and methods Materials
HEPES [N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulphonic acid], NAD+, cysteine hydrochloride, Tris ITris(hydroxyethyl)-
172 l
I
I
L,
I
l
100
,t,,) .im
90
lml
80 "m
~m
?0
Results
0
L.
chloride, 0.5 mM-TPP was incubated for 45 min at 0°C before use. The assay reaction was initiated by addition of cofactor (as required from kinetic experiments, see figure legends) after a preincubation period of 45 s at 30°C. Protein was determined according to Lowry et al [17] with bovine serum albumin as a standard. Estimations of the concentrations of conjugate base and conjugate acid for all used buffers were calculated from the Henderson-Hasselbalch equation. The concentrations of free Ca2+ and MgATp2- were calculated using the SPECS program [26]. The results presented are representative for three enzyme preparations.
GO
50 0.00
I
0.05
I
I
0.10
i
l
0.15
i
0.20
*
0.25
Ionic strength (M) Fig 1. Effect of ionic strength on the maximal activity of 2oxoglutarate dehydrogenase complex. Assay conditions for OGDC activity were as described in Materials and methods. Activity of OGDC was measured in the presence of 5 mM 2-oxoglutarate (10 mM 2-oxoglutarate at 0.040.08 ionic strength), 0.1 mM CoA, ! mM NAD, 0.1 mM CaCl, and buffers, TEA/Mops (o), TEA/HEPES (c-0, "Iris/ HEPI~S (:), pH 7.8. 100% represents vm~, at 0.1 M ionic strength.
Incubation of the 2-oxoglutarate dehydrogenase c o m plex from pig kidney cortex at different buffer concentrations resulted in a change in e n z y m e activity. However, enzyme activity remained the same when sucrose or mannitol were used to create different osmolarity (data not shown). Based on these observations we assumed that the e n z y m e activity is sensitive to changes in ionic strength. The effect of ionic strength
I
II
I
I
I
I
100
~.
ill
I!0
iim
aminomethanel, Mes [2-(N-morpholino)-ethane sulfonic acid], Mops [2-(N-morpholino)-propane-sulphonic acid], EGTA ethylene glycol bis ([3-aminoethyl ether)-N,N3V'3V'-tetraacetic acid, 2-oxoglut,*u'ate acid, CoA, ATE ADP were obteined from Sigma Chemical Co (St Louis, Mo, USA). Thiamine pyrophosphate (TPP) was from Calbiochem Behring. A highly purified preparation o~ p,~ kid,ey cortex 2-oxoglmm'a~ ~.~a~',.,~,,:,::~se complex (12 I~mol/min per rag) was prepared as previously described [14]. All other chemicals were of the purest grade commercially available.
N
'g
q;o
N
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0
~ L
20
Methods
2-Oxoglutarate dehydrogenase complex activity was assayed by continuously measuring the rate of NADH formation at 340 nm and 30°C using a DW Aminco spectrophotometer with recorder. The reaction mixture contained Tris/HEPES buffer, NAD +, CoA, 0.5 mM-thiamine pyrophosphate, 2 mMMgCI2, 2,6 mM cysteine hydrochloride, 2-oxoglutarate and 1.5 ~g of the enzyme. The NAD +, CoA and 2-oxoglutarate were added in concentrations as noted in the figure legends. Ionic strength and pH of the reaction mixture and stock solution were standarized to the required value by varying the concentration of buffer. The effectors studied were added in concentrations as noted in the figure legends. A stock solution containing enzyme complex (0.4 mg/ml) in Tris/I]EPES buffer, 2 mM MgCl2, 2.6 ,nM-cysteine hydro-
0
5.0
.
l
6.2
.
l
G.G
l
l
?.0
7.4
I
7.0
i
I
8.2
i
O.G
pH Fig 2. Effect of pH on the activity of 2-oxoglutarate dehydrogenase complex. Assay conditions for OGDC activity were as described in Materials and methods. Activity of OGDC was measured in the presence of 5 mM 2-oxoglutarate, 0.1 mM CoA, 1 mM NAD, 0.1 mM CalCl2 and TEA/Mes (o), Tris/Mes (z~), Tris/Mops (D), Tris/HEPES (V) buffers. Ionic strength 0.05 M (open symbols), 0.15 M (closed symbols).
173
Table I. Effect of ionic strength on the kinetic constants for
1.0
2-oxoglutarate dehydrogenase complex. Assay conditions for OGDC activity were as described in Materials and methods. Results are mean values from 5 determinations + SD.
0.5
|
|
A
So.5 !
St (M)
2-oxoglurarate a (m~')
NAD b (BM)
CoA c (pM)
0.05
0.65 + 0.06
31 + 3
4.2 + 0.7
0.10
0.43 + 0.07
38 + 6
3.5 + 0.5
0.15
0.27 + 0.03
50 + 5
3.2 + 0.4
0.20
0.16 + 0.04
60 + 4
4.2 + 0.6
0.0
-0.5
J
-1.0 -1.5
aActivity of OGDC was measured in the presence of Tris/HEPES buffer, pH 7.8, 0. I mM CaCI2 and ! mM NAD, 0.1 mM CoA. b5 mM-2-oxoglutarate, 0.1 mM-CoA, c5 mM2-oxoglutarate, 1 mM-NAD. on the enzyme activity was examined using three buffer systems with the results shown in figure 1. The 2-oxoglutarate dehydrogenase complex at pH 7.8 shows a narrow optimum activity at 0.1 M ionic strength. An increase in the ionic strength from 0.1 M to 0.25 M resulted in a 40% decrease in enzyme activity. Previously we have shown that the enzyme from bovine kidney has a pH optimum in the range of 7.4-7.6 when assayed at 0.08 M ionic strength [14]. In the present study we found that the pH optimum for 2-oxoglutarate dehydrogenase complex is dependent on ionic strength (fig 2). At ionic strength of 0.05 M the enzyme has a broad optimum activity in the range of pH 7.4-7.6. While, when the enzyme is assayed at ionic strength 0.15 M, the optimum activity is found to be at pH 7.8 (fig 2). Results summarised in table I show that ionic strength affects the Sos for 2-oxoglutarate and NAD. The S0.5 for 2-oxoglutarate decreases four-fold when ionic strength changes from 0.05 M to 0.2 M. It can be seen from Hill plots of these data (fig 3A) that at low ionic strength the slope is less than unity. The Hill coefficient at 0.05 M ionic strength is 0.85, whereas at 0.2 M ionic strength the Hill coefficient increases to 1.0. As can be seen in table I, an increase in ionic strength from 0.05 to 0.2 M results in a two-fold increase of the S05 for NAD. At 0.05 M ionic strength the Hill coefficient for N A D is 1.2 and decreases to 1.05 at 0.2 M ionic strength (fig 3B). Data presented in table I indicate that changes in ionic strength have no significant effect on the So5 for CoA. It has been found that the Hill coefficient for C o A is the same (1.15) over the range 0.05-0.2 M ionic strength (data not shown).
///
_o
I
i
I
-0.5
0.5
log [ 2-oxoglutarate (mM) ]
|
!
B 1.0
!
0.0
~0
-1.0
-2.0
' -3
' -2
'
' -1
' 0
log [ NAD (mM) ] Fig 3. Effect of ionic strength on the activity of 2-oxoglutarate dehydrogenase complex. Hill plot. Assay conditions for OGDC activity were as described in Materials and methods. Actvity of OGDC was measured in the presence of Tris/HEPES buffer pH 7.8, 0.1 mM CaCI2, 0.1 mM CoA and 1 mM NAD (A), 5 mM 2-oxoglutarate (B). Ionic strength of assay medium was 0.05 M (e), 0.1 M (EJ), 0.15 M (A), 0.2 (o). The correlation coefficients for all Hill plots were greater than 0.98 and the mean value was 0.992.
174 80
,
,
,
Ca 2÷ markedly reduces the effects of nucleotides on the enzyme activity [9, 11 ]. In the present study, we examined the effect of ATP and ADP on 2-oxoglutarate dehydrogenase complex activity at different ionic strengths. Results given in figure 4 show that, in the absence of calcium, inhibition by 2 mM ATP is not dependent upon changes in ionic strength in the range of 0.05-0.2 M. However, in the presence of 0.1 m M Ca2+, ATP becomes less inhibitory as ionic strength is increased from 0.05 M to 0.2 M. In the presence of Ca2+ and at ionic strength 0.2 M, ATP decreases enzyme activity by only ! 8% (fig 4). The concentration of Ca 2÷, Mg2+, CaATP2- and MgATP2- at pH 7.8 and ionic strength 0.1 M was calculated to be 40 gM, 39 laM, 60 laM and 1.61 m M respectively. Changes in ionic strength over the range 0.05-0.2 M resulted in variation of the concentrations of the above species by less than 10%. Results summarised in table II indicate that in the presence of calcium the Sio,.~ for ATP is 0.18 mM and changes in ionic strength in the range 0.05-0.2 M do not affect this parameter. In the absence of Ca2+, the Sio.5 for ATP is slightly lower (0. ! mM) and a change in ionic strength from 0.05 M to 0.1 M results in a two-fold increase in the So5 for ATP. As shown in figure 5, the extent of stimulation by A D P is dependent upon ionic strength and calcium. In the absence of Ca2+, ADP increases the enzyme activity with a maximum effect of 1.8-fold at 0.1 M ionic strength, in contrast, in the presence of Ca2+ the maximal effect of ADP on the enzyme activity is observed at 0.05 M ionic strength (fig 5). At 0.2 M ionic strength and in the presence of Ca2+, A D P has no effect on 2-oxoglutarate dehydrogenase complex activity. Data summarised in table II indicate that in the absence of Ca2+, change in ionic strength from 0.1 to 0.2 M reduces the S%.s for ADP 1.7 fold.
'
70
6O
so
'~
3o
10
0 O. 04
i
I 0.08
i
I 0.12
i
I
i
0.16
I 0.20
Ionic strength ( M ) Fig 4. Effect of ATP on the activity of 2-oxoglutarate dehydrogenase complex at varying ionic strength in the presence or absence of Ca2+. Assay conditions for OGDC activity were as described in Materials and methods. Activity of OGDC was measured in the presence of Tris/HEPES buffer pH 7.8, 0.1 mM CoA, I mM NAD, 2 mM ATP and 0. ! mM CaCl_, (o) or 5 mM EGTA (o). Enzyme was measured at 0.5 mM and 5 mM 2-oxoglutarate in the presence and absence of calcium, respectively. Previous studies have shown that ATP, GTP and ADP affect mammalian 2-oxoglutarate dehydrogenase complex activity [9-111. It has also been reported that
Table !1. Effect of ionic strength and Ca2+ on the inhibition constant for ATP and stimulation constant for ADP. Assay conditions for OGDC activity were as described in Materials and methods, Activity of OGDC was measured in the presence of Tris/HEPES buffer, pH 7,8, 0,1 mM CoA, ! mM NAD, In the presence of Ca2+ (O.ImM CaCI:) the enzyme activity was measured at 0,5 mM 2-oxoglutarate. In the absence of Ca 2+ (5 mM EGTA) the concentration of 2-oxoglutarate was 5 mM. Results are mean values from four determinations + SD. g
S,, ~for ATP . . C a 2+
S%~for ADP + C a 2+
IMl
_ C a 2+
+ C a 2+
ImM!
0.5
0,05 + 0.02
0.18 + 0.03
0.40 + 0.04
0.25 + 0.08
0.10
0.11 + 0.03
0.17 + 0.02
0.50 + 0.05
0.22 + 0.10
0.15
0.10 _+. 0.01
0.18 + 0.03
0.32 + 0.03
ND"
0.20
0.11 + 0.02
0.18 + 0.03
0.30 + 0.05
ND
aND, not determined.
175 Discussion
90
This study has demonstrated that changes in ionic strength cause diverse effects on 2-oxoglutarate dehydrogenase complex activity. Ionic strength over 0. I M markedly reduced maximal activity and S0.5 for 2-oxoglutarate. In contrast, the So.~ for NAD at 0.2 M ionic strength was two-fold higher than at 0.05 M ionic strength. While changes in ionic strength affected little, if any, the S0.5 for CoA. It seems likely that changes in ionic strength mainly affect the activity of El and E3 components. Indeed, the Hill coefficient for 2-oxoglutarate increased from 0.85 at 0.05 M ionic strength to 1.0 at 0.2 M ionic strength. The Hill coefficient for NAD changed from 1.2 to 1.05 during changes in ionic strength from 0.05 M to 0.2 M. In contrast, the observed Hill coefficient for CoA was the same over the range 0.05-0.2 M ionic strength. A Hill coefficient less than unity for 2-oxoglutarate was previously reported by Lawlis and Roche [91 for 2oxoglutarate dehydrogenase complex from bovine kidney. Such a Hill coefficient could be a result of a multiple Km form of the 2-oxoglutarate dehydrogenase component [14] or site-site interactions between protein subunits of this enzyme subunit. Our studies showed that such a Hill coefficient is induced by low ionic strength. It is difficult to estimate intracellular ionic strength, although at least 0.2 M ionic strength can be calculated. At this ionic strength the Hill coefficient for 2-oxoglutarate and NAD was 1.0. Thus, it can be concluded that at a physiological range of ionic strength site-site interactions do not occur between 2oxoglutarate dehydrogenase subunits or between lipoamide dehydrogenase subunits. In our study we examined the effect of ionic strength on the enzyme complex using Tris/HEPES buffer only (see Materials and methods). However, it is possible that in the presence of different electrolytes the enzyme complex may show kinetic properties different from those described above. It has been reported that ions such as K ÷, Na ÷, CI- or HPO42- affect pyruvate dehydrogenase complex activity [ 181. In the absence of calcium (5 mM EGTA) the extent of enzyme inhibition caused by ATP was independent of changes in ionic strength, whereas in the presence of calcium an increase in ionic strength lowered ATP inhibition. In contrast, effect of ADP on the enzyme activity was dependent on ionic strength in both the presence and absence of Ca2+. In addition, the affinity of the enzyme for ATP was not affected by changes in ionic strength. In contrast the S05 for ADP in the absence of Ca2+ decreased slightly during an increase in ionic strength. Thus, the data suggest existence within the enzyme complex of separate classes of binding sites for ATP and ADP.
8O
=
60
~
4o
t_
!
,
i
|
,o 10 0 0.04
0.08
0.12
0.16
0.20
Ionic strength ( M )
Fig 5. Effect of ADP on the activity of 2-oxoglutarate dehydrogenase complex at varying ionic strength in the presence of Ca2+. Assay conditions for OGDC activity were as described in Materials and methods. Activity of OGDC was measured in the presence of Tris/HEPES buffer pH 7.8, 0.1 mM CoA, 1 mM NAD, 2 mM ADP and 0.1 mM CaCI, (•) or 5 mM EGTA (o). Enzyme activity was measured at 0.5 mM and 5 2-oxoglutarate in the presence and absence of calcium, respectively.
In conclusion, the 2-oxoglutarate dehydrogenase complex activity is affected by changes in ionic strength. The regulatory properties of the enzyme at high ionic strength are markedly different from those at low ionic strength. This property of the enzyme complex can be important for regulation of cell metabolism especially in the kidney. The kidney medulla cells are cells which are normally exposed to high concentrations of urea and NaCI. It has been found that extracellular concentrations of urea and NaC! in renal papila can reach 1.5 M and 0.45 M respectively [20]. Intracellular osmolality equal to the extracellular osmolality in medulla cells is maintained by high concentrations of betaine, lactate, glycerolphosphocholine, inositol and sorbitol [21-231. Thus, the intracellular ionic strength of these cells can be much higher than in other cells. In addition, it has been reported that the intracellular concentration of these solutes changes in response to alterations in the diuretic state [24, 251.
176 References 1 Ishikawa E, Oliver RM, Reed LJ (1966) ot-Keto acid dehydrogenase complexes. V. Macromolecular organization of pyruvate and ~-ketoglutarate dehydrogenase complexes isolated from beef kidney mitochondria. Proc Nati Acad Sci USA 56, 534-541 2 Tanaka N, Koike K, Hamada M, Otsuka KJ, Suematsu T, Koike M (1972) Mammalian ot-keto acid dehydrogenase complexes. VII. Resolution and reconstitution of the pig heart 2-oxoglutarate dehydrogenase complex. J Biol Chem 247, 4043--4049 3 Koike M, Koike K (1976) Structure, assembly and function of mammalian tx-keto acid dehydrogenasc complexes. Adv Biophys 9, 187-227 4 Hirashima M, Hayakawa T, Koike M (1967) Mammalian ot-keto acid dehydrogenase complexes. III. An improved procedure for the preparation of 2-oxoglutarate dehydrogenase complex from heart muscle. J Biol Chem 242, 902-907 5 Matthew JB, Hanania GIH, Gurd FRN (1979) Electrostatic effects in hemoglobin. Bohr effect and ionic strength dependence of individual groups. Biochemistry 18, 1928-1936 6 Matthew JB, Richards FM (1982) Anion binding and pHdependent electrostatic effects in ribonuclease. Biochemistry 21,4989-4999 7 Matthew JB, Gurd FRN (1986) Calculation of electrostatic interactions in protein. Stalibization and destabilization of protein structure by charge interactions. In: Methods in Enzymology (Hirs CHW, Timasheff SN, eds) voi 130, Academic Press Inc, Orlando, San Diego, New York, Austin, Boston, London, Sydney, Tokyo, Toronto, 413-453 8 Garland PB (1964) Some kinetic properties of pig-heart oxoglutarate dehydrogenase that provide a basis for metabolic control of the enzyme activity and also a stoichiometric assay for coenzyme A in tissue extracts. Biochem J 92.10c 9 Lawlis VB, Roche TE (1981) Regulation of bovine kidney ¢t-ketoglutarate dehydrogenase complex by calcium and adenine nucleotides. Effects on S0.~ for obketoglutarate. Biochemistry 20, 25 ! 2-25 i 8 lO Denton RM, Richards DA, Chin JG (1978) The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex. Biochem J 176, 899-906 i I MCCormack JK, Denton RM (1979) The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenas¢ complex. Biochem J 176, 899-906 12 Lawlis VB, Roche TE (1980) Effect of micromolar Ca2+ on NADH inhibition of bovine kidney ot-ketoglutarate
13
14
15 16 17 18
19 20 21
22 23
24 25 26
dehydrogenase complex and possible role of Ca2+ in signal amplification. Mol Cell Biochem 32, 147-152 Lawlis VB, Roche TE (1981) Inhibition of bovine kidney a-ketoglutarate dehydrogenase complex by reduced nicotynamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition. Biochemistry 20, 2519-2524 Pawelczyk T, Angielski S (1984) Cooperation of Ca 2+ and pH in regulation of the activity of the 2-oxoglutarate dehydrogenase complex and its components from bovine kidney cortex. Acta Biochem Pol 3 l, 289-305 Friend SH, Gurd FRN (1979) Electrostatic stabilization in myoglobin pH dependence of summed electrostatic contributions. Biochemistry 18, 46 ! 2-4619 Hermans J, Scheraga HA (1961) Structural studies of ribonucleases. V. Reversible change of configuration. J Am Chem Soc 83, 3283-3292 Lowry OH, Rosebrough NJ, Fan" AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193, 265-275 Pawelczyk T, Easom RA, OIson MS (1988) The effects of various anions and cations on the regulation of pyruvate dehydrogenase complex activity from pig kidney cortex. Biochem J 253, 819-825 Wolff SD, Balaban RS (1990) Regulation of the predominant renal medullary organic solutes in vivo. Annu Rev Physio152, 727-746 Beck F, Dorge A, Rick R, Thuran K (1984) Intra- and extracellular element concentrations of rat renal papilla in antidiuresis. Kidney Int 25, 397-403 Yancey PH (1988) Osmotic effectors in kidneys of xeric and mesic rodents: corticomedullary distributions and changes with water availability. J Comp Physiol 158, 369-380 Dell RB, Winters RW (1967) Lactate gradients in the kidney of the dog. Am J Physio1213, 301-307 Bagnasco S, Balaban R, Fales HM, Yang YM, Burg M (1986) Predominant osmotically active organic solutes in rat and rabbit renal medullas. J Biol Chem 261, 58725877 Wirthensohn G, Beck FX, Guder WG (1987) Role and regulation of glycerophosphorylcholine in rat renal medulla, Pflugers Arch 409, 411-415 Wolff SD, Balaban RS (1990) Sodium-23 nuclear magnetic resonance imaging of the rabbit kidney in vivo. Am J Physio1257, F675-F681 Fabiato A (1988) Computer programs for calculating total from specified free or free from specified total concentrations in aqueous solutions containing multiple metals and ligands. In: Methods in Enzymology (Reisher S. Fleisher B, eds) vol 157, Academic Press, Inc New York, 378-417
171
Effect of ionic strength on the regulatory properties of 2-oxoglutarate dehydrogenase complex T Pawelczyk*, S Angielski Chair and Department of Clinical Biochemistry Medical Academy, 80-211 Gdansk, Poland
(Received 12 August 1991; accepted 8 November ! 991 )
Summary m The effects of changing ionic strength on the activity of the 2-oxoglutarate dehydrogenase complex from pig kidney cortex were explored. This enzyme complex is found to be influenced in many ways by the ionic strength of the reaction medium. The enzyme shows an optimum activity at 0.1 M ionic strength. Increase in ionic strength from 0.1 M to 0.2 M resulted in a decrease of S~..s for 2-oxoglutarate, and in an increase of Sf~.stbr NAD. Changes in ionic strength over the range of 0.05--0.2 M have little, if any, effect on Sa..s for CoA. The Hill coefficient for 2-oxoglutarate and NAD at 0.2 M ionic strength was 1.0, whereas at 0.05 M ionic strength it was 0.85 and 1.2 for 2-oxoglutarate and NAD, respectively At 0.05 M ionic strength the pH optimum of the enzyme ranges between 7.4-7.6, but at 0.15 M ionic strength the pH optimum shifts to 7.8. The magnitude of inhibition of enzyme activity by ATP is not influenced by changes in ionic strength in the absence of calcium. However, in the presence of Ca2*, increases in ionic strength lower the inhibitory effects of ATP. The Sio.5 for ATP in both presence and absence of Ca2+ was not affected by changes in ionic strength in the range of 0.1-0.2 M. In contrast, the Sao5 for ADP in the absence of Ca 2+ decreases as ionic strength increases. In the presence of calcium and 0.2 M ionic strength ADP has no effect on 2-oxoglutarate dehydrogenase complex activity. 2-oxoglutarate dehydrogenase / ionic strength / pH / ATP / ADP / kidney Introduction The 2-oxoglutarate dehydrogenase complex as an enzyme of the tricarboxylic acid cycle occupies a key position in intermediary metabolism. The mammalian 2-oxoglutarate dehydrogenase complex consists of 3 catalytic components, 2-oxoglutarate dehydrogenase ( E l ) , lipoamide succinyltransferase (E2) and lipoamide dehydrogenase (E3). The enzyme complex contains a core consisting of 24 identical E2 chains to which about 12 identical E l chains (6 dimers) and about 12 identical E3 chains (6 dimers) are non-covalently attached [1-3]. The total molecular weight (Mr) of the complex is approximately 2.7 x 106 [3, 41. In an electron micrograph the enzyme complex appears as a polyhedral structure having a gross diameter of about 260 A [4]. The stabilization of such a quaternary structure involves various interactions such as electrostatic, hydrogen bonding, van der Waals, hydrophobic *Correspondence and reprints. Present address: Graduate Department of Biochemistry, Brandeis University Waltham, MA 02254, USA Abbreviations: It, ionic strength; OGDC, 2-oxoglutarate dehydrogenase complex; S0.5, substrate concentration giving halfmaximal activity; Sa0.s, activator concentration giving halfmaximal stimulation; Si0.5, inhibitor concentration giving half-maximal inhibition; Vm~,true value of maximal activity.
and conformational tension. Thus, changes in the magnitude of any of these contributing interactions may have a substantial effect on the catalytic properties of the enzyme complex. The 2-oxoglutarate dehydrogenase complex is regulated by a number of factors. NADH, succinyI-CoA, ATP and GTP are inhibitory 18, 91; ADP, Ca 2+, Sr2+ and inorganic phosphate are stimulatory [10-14] It has been demonstrated that variations in ionic strength and pH influence electrostatic interactions within a protein molecule [5, 71, and affect the binding of nucleotides and ions [6, 15, 16]. Under normal conditions most mammalian cells maintain constant ionic strength. However, in the kidney some cells function under high osmolality and ionic strength. In addition, intracellular concentration of solutes chnges in response to alterations in the diuretic state 1 19]. Therefore, it is of interest to determine the effect of ionic strength on the catalytic properties of this enzyme complex, particularly in the presence of its effectors.
Materials and methods Materials
HEPES [N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulphonic acid], NAD+, cysteine hydrochloride, Tris ITris(hydroxyethyl)-
172 l
I
I
L,
I
l
100
,t,,) .im
90
lml
80 "m
~m
?0
Results
0
L.
chloride, 0.5 mM-TPP was incubated for 45 min at 0°C before use. The assay reaction was initiated by addition of cofactor (as required from kinetic experiments, see figure legends) after a preincubation period of 45 s at 30°C. Protein was determined according to Lowry et al [17] with bovine serum albumin as a standard. Estimations of the concentrations of conjugate base and conjugate acid for all used buffers were calculated from the Henderson-Hasselbalch equation. The concentrations of free Ca2+ and MgATp2- were calculated using the SPECS program [26]. The results presented are representative for three enzyme preparations.
GO
50 0.00
I
0.05
I
I
0.10
i
l
0.15
i
0.20
*
0.25
Ionic strength (M) Fig 1. Effect of ionic strength on the maximal activity of 2oxoglutarate dehydrogenase complex. Assay conditions for OGDC activity were as described in Materials and methods. Activity of OGDC was measured in the presence of 5 mM 2-oxoglutarate (10 mM 2-oxoglutarate at 0.040.08 ionic strength), 0.1 mM CoA, ! mM NAD, 0.1 mM CaCl, and buffers, TEA/Mops (o), TEA/HEPES (c-0, "Iris/ HEPI~S (:), pH 7.8. 100% represents vm~, at 0.1 M ionic strength.
Incubation of the 2-oxoglutarate dehydrogenase c o m plex from pig kidney cortex at different buffer concentrations resulted in a change in e n z y m e activity. However, enzyme activity remained the same when sucrose or mannitol were used to create different osmolarity (data not shown). Based on these observations we assumed that the e n z y m e activity is sensitive to changes in ionic strength. The effect of ionic strength
I
II
I
I
I
I
100
~.
ill
I!0
iim
aminomethanel, Mes [2-(N-morpholino)-ethane sulfonic acid], Mops [2-(N-morpholino)-propane-sulphonic acid], EGTA ethylene glycol bis ([3-aminoethyl ether)-N,N3V'3V'-tetraacetic acid, 2-oxoglut,*u'ate acid, CoA, ATE ADP were obteined from Sigma Chemical Co (St Louis, Mo, USA). Thiamine pyrophosphate (TPP) was from Calbiochem Behring. A highly purified preparation o~ p,~ kid,ey cortex 2-oxoglmm'a~ ~.~a~',.,~,,:,::~se complex (12 I~mol/min per rag) was prepared as previously described [14]. All other chemicals were of the purest grade commercially available.
N
'g
q;o
N
E
4O
0
~ L
20
Methods
2-Oxoglutarate dehydrogenase complex activity was assayed by continuously measuring the rate of NADH formation at 340 nm and 30°C using a DW Aminco spectrophotometer with recorder. The reaction mixture contained Tris/HEPES buffer, NAD +, CoA, 0.5 mM-thiamine pyrophosphate, 2 mMMgCI2, 2,6 mM cysteine hydrochloride, 2-oxoglutarate and 1.5 ~g of the enzyme. The NAD +, CoA and 2-oxoglutarate were added in concentrations as noted in the figure legends. Ionic strength and pH of the reaction mixture and stock solution were standarized to the required value by varying the concentration of buffer. The effectors studied were added in concentrations as noted in the figure legends. A stock solution containing enzyme complex (0.4 mg/ml) in Tris/I]EPES buffer, 2 mM MgCl2, 2.6 ,nM-cysteine hydro-
0
5.0
.
l
6.2
.
l
G.G
l
l
?.0
7.4
I
7.0
i
I
8.2
i
O.G
pH Fig 2. Effect of pH on the activity of 2-oxoglutarate dehydrogenase complex. Assay conditions for OGDC activity were as described in Materials and methods. Activity of OGDC was measured in the presence of 5 mM 2-oxoglutarate, 0.1 mM CoA, 1 mM NAD, 0.1 mM CalCl2 and TEA/Mes (o), Tris/Mes (z~), Tris/Mops (D), Tris/HEPES (V) buffers. Ionic strength 0.05 M (open symbols), 0.15 M (closed symbols).
173
Table I. Effect of ionic strength on the kinetic constants for
1.0
2-oxoglutarate dehydrogenase complex. Assay conditions for OGDC activity were as described in Materials and methods. Results are mean values from 5 determinations + SD.
0.5
|
|
A
So.5 !
St (M)
2-oxoglurarate a (m~')
NAD b (BM)
CoA c (pM)
0.05
0.65 + 0.06
31 + 3
4.2 + 0.7
0.10
0.43 + 0.07
38 + 6
3.5 + 0.5
0.15
0.27 + 0.03
50 + 5
3.2 + 0.4
0.20
0.16 + 0.04
60 + 4
4.2 + 0.6
0.0
-0.5
J
-1.0 -1.5
aActivity of OGDC was measured in the presence of Tris/HEPES buffer, pH 7.8, 0. I mM CaCI2 and ! mM NAD, 0.1 mM CoA. b5 mM-2-oxoglutarate, 0.1 mM-CoA, c5 mM2-oxoglutarate, 1 mM-NAD. on the enzyme activity was examined using three buffer systems with the results shown in figure 1. The 2-oxoglutarate dehydrogenase complex at pH 7.8 shows a narrow optimum activity at 0.1 M ionic strength. An increase in the ionic strength from 0.1 M to 0.25 M resulted in a 40% decrease in enzyme activity. Previously we have shown that the enzyme from bovine kidney has a pH optimum in the range of 7.4-7.6 when assayed at 0.08 M ionic strength [14]. In the present study we found that the pH optimum for 2-oxoglutarate dehydrogenase complex is dependent on ionic strength (fig 2). At ionic strength of 0.05 M the enzyme has a broad optimum activity in the range of pH 7.4-7.6. While, when the enzyme is assayed at ionic strength 0.15 M, the optimum activity is found to be at pH 7.8 (fig 2). Results summarised in table I show that ionic strength affects the Sos for 2-oxoglutarate and NAD. The S0.5 for 2-oxoglutarate decreases four-fold when ionic strength changes from 0.05 M to 0.2 M. It can be seen from Hill plots of these data (fig 3A) that at low ionic strength the slope is less than unity. The Hill coefficient at 0.05 M ionic strength is 0.85, whereas at 0.2 M ionic strength the Hill coefficient increases to 1.0. As can be seen in table I, an increase in ionic strength from 0.05 to 0.2 M results in a two-fold increase of the S05 for NAD. At 0.05 M ionic strength the Hill coefficient for N A D is 1.2 and decreases to 1.05 at 0.2 M ionic strength (fig 3B). Data presented in table I indicate that changes in ionic strength have no significant effect on the So5 for CoA. It has been found that the Hill coefficient for C o A is the same (1.15) over the range 0.05-0.2 M ionic strength (data not shown).
///
_o
I
i
I
-0.5
0.5
log [ 2-oxoglutarate (mM) ]
|
!
B 1.0
!
0.0
~0
-1.0
-2.0
' -3
' -2
'
' -1
' 0
log [ NAD (mM) ] Fig 3. Effect of ionic strength on the activity of 2-oxoglutarate dehydrogenase complex. Hill plot. Assay conditions for OGDC activity were as described in Materials and methods. Actvity of OGDC was measured in the presence of Tris/HEPES buffer pH 7.8, 0.1 mM CaCI2, 0.1 mM CoA and 1 mM NAD (A), 5 mM 2-oxoglutarate (B). Ionic strength of assay medium was 0.05 M (e), 0.1 M (EJ), 0.15 M (A), 0.2 (o). The correlation coefficients for all Hill plots were greater than 0.98 and the mean value was 0.992.
174 80
,
,
,
Ca 2÷ markedly reduces the effects of nucleotides on the enzyme activity [9, 11 ]. In the present study, we examined the effect of ATP and ADP on 2-oxoglutarate dehydrogenase complex activity at different ionic strengths. Results given in figure 4 show that, in the absence of calcium, inhibition by 2 mM ATP is not dependent upon changes in ionic strength in the range of 0.05-0.2 M. However, in the presence of 0.1 m M Ca2+, ATP becomes less inhibitory as ionic strength is increased from 0.05 M to 0.2 M. In the presence of Ca2+ and at ionic strength 0.2 M, ATP decreases enzyme activity by only ! 8% (fig 4). The concentration of Ca 2÷, Mg2+, CaATP2- and MgATP2- at pH 7.8 and ionic strength 0.1 M was calculated to be 40 gM, 39 laM, 60 laM and 1.61 m M respectively. Changes in ionic strength over the range 0.05-0.2 M resulted in variation of the concentrations of the above species by less than 10%. Results summarised in table II indicate that in the presence of calcium the Sio,.~ for ATP is 0.18 mM and changes in ionic strength in the range 0.05-0.2 M do not affect this parameter. In the absence of Ca2+, the Sio.5 for ATP is slightly lower (0. ! mM) and a change in ionic strength from 0.05 M to 0.1 M results in a two-fold increase in the So5 for ATP. As shown in figure 5, the extent of stimulation by A D P is dependent upon ionic strength and calcium. In the absence of Ca2+, ADP increases the enzyme activity with a maximum effect of 1.8-fold at 0.1 M ionic strength, in contrast, in the presence of Ca2+ the maximal effect of ADP on the enzyme activity is observed at 0.05 M ionic strength (fig 5). At 0.2 M ionic strength and in the presence of Ca2+, A D P has no effect on 2-oxoglutarate dehydrogenase complex activity. Data summarised in table II indicate that in the absence of Ca2+, change in ionic strength from 0.1 to 0.2 M reduces the S%.s for ADP 1.7 fold.
'
70
6O
so
'~
3o
10
0 O. 04
i
I 0.08
i
I 0.12
i
I
i
0.16
I 0.20
Ionic strength ( M ) Fig 4. Effect of ATP on the activity of 2-oxoglutarate dehydrogenase complex at varying ionic strength in the presence or absence of Ca2+. Assay conditions for OGDC activity were as described in Materials and methods. Activity of OGDC was measured in the presence of Tris/HEPES buffer pH 7.8, 0.1 mM CoA, I mM NAD, 2 mM ATP and 0. ! mM CaCl_, (o) or 5 mM EGTA (o). Enzyme was measured at 0.5 mM and 5 mM 2-oxoglutarate in the presence and absence of calcium, respectively. Previous studies have shown that ATP, GTP and ADP affect mammalian 2-oxoglutarate dehydrogenase complex activity [9-111. It has also been reported that
Table !1. Effect of ionic strength and Ca2+ on the inhibition constant for ATP and stimulation constant for ADP. Assay conditions for OGDC activity were as described in Materials and methods, Activity of OGDC was measured in the presence of Tris/HEPES buffer, pH 7,8, 0,1 mM CoA, ! mM NAD, In the presence of Ca2+ (O.ImM CaCI:) the enzyme activity was measured at 0,5 mM 2-oxoglutarate. In the absence of Ca 2+ (5 mM EGTA) the concentration of 2-oxoglutarate was 5 mM. Results are mean values from four determinations + SD. g
S,, ~for ATP . . C a 2+
S%~for ADP + C a 2+
IMl
_ C a 2+
+ C a 2+
ImM!
0.5
0,05 + 0.02
0.18 + 0.03
0.40 + 0.04
0.25 + 0.08
0.10
0.11 + 0.03
0.17 + 0.02
0.50 + 0.05
0.22 + 0.10
0.15
0.10 _+. 0.01
0.18 + 0.03
0.32 + 0.03
ND"
0.20
0.11 + 0.02
0.18 + 0.03
0.30 + 0.05
ND
aND, not determined.
175 Discussion
90
This study has demonstrated that changes in ionic strength cause diverse effects on 2-oxoglutarate dehydrogenase complex activity. Ionic strength over 0. I M markedly reduced maximal activity and S0.5 for 2-oxoglutarate. In contrast, the So.~ for NAD at 0.2 M ionic strength was two-fold higher than at 0.05 M ionic strength. While changes in ionic strength affected little, if any, the S0.5 for CoA. It seems likely that changes in ionic strength mainly affect the activity of El and E3 components. Indeed, the Hill coefficient for 2-oxoglutarate increased from 0.85 at 0.05 M ionic strength to 1.0 at 0.2 M ionic strength. The Hill coefficient for NAD changed from 1.2 to 1.05 during changes in ionic strength from 0.05 M to 0.2 M. In contrast, the observed Hill coefficient for CoA was the same over the range 0.05-0.2 M ionic strength. A Hill coefficient less than unity for 2-oxoglutarate was previously reported by Lawlis and Roche [91 for 2oxoglutarate dehydrogenase complex from bovine kidney. Such a Hill coefficient could be a result of a multiple Km form of the 2-oxoglutarate dehydrogenase component [14] or site-site interactions between protein subunits of this enzyme subunit. Our studies showed that such a Hill coefficient is induced by low ionic strength. It is difficult to estimate intracellular ionic strength, although at least 0.2 M ionic strength can be calculated. At this ionic strength the Hill coefficient for 2-oxoglutarate and NAD was 1.0. Thus, it can be concluded that at a physiological range of ionic strength site-site interactions do not occur between 2oxoglutarate dehydrogenase subunits or between lipoamide dehydrogenase subunits. In our study we examined the effect of ionic strength on the enzyme complex using Tris/HEPES buffer only (see Materials and methods). However, it is possible that in the presence of different electrolytes the enzyme complex may show kinetic properties different from those described above. It has been reported that ions such as K ÷, Na ÷, CI- or HPO42- affect pyruvate dehydrogenase complex activity [ 181. In the absence of calcium (5 mM EGTA) the extent of enzyme inhibition caused by ATP was independent of changes in ionic strength, whereas in the presence of calcium an increase in ionic strength lowered ATP inhibition. In contrast, effect of ADP on the enzyme activity was dependent on ionic strength in both the presence and absence of Ca2+. In addition, the affinity of the enzyme for ATP was not affected by changes in ionic strength. In contrast the S05 for ADP in the absence of Ca2+ decreased slightly during an increase in ionic strength. Thus, the data suggest existence within the enzyme complex of separate classes of binding sites for ATP and ADP.
8O
=
60
~
4o
t_
!
,
i
|
,o 10 0 0.04
0.08
0.12
0.16
0.20
Ionic strength ( M )
Fig 5. Effect of ADP on the activity of 2-oxoglutarate dehydrogenase complex at varying ionic strength in the presence of Ca2+. Assay conditions for OGDC activity were as described in Materials and methods. Activity of OGDC was measured in the presence of Tris/HEPES buffer pH 7.8, 0.1 mM CoA, 1 mM NAD, 2 mM ADP and 0.1 mM CaCI, (•) or 5 mM EGTA (o). Enzyme activity was measured at 0.5 mM and 5 2-oxoglutarate in the presence and absence of calcium, respectively.
In conclusion, the 2-oxoglutarate dehydrogenase complex activity is affected by changes in ionic strength. The regulatory properties of the enzyme at high ionic strength are markedly different from those at low ionic strength. This property of the enzyme complex can be important for regulation of cell metabolism especially in the kidney. The kidney medulla cells are cells which are normally exposed to high concentrations of urea and NaCI. It has been found that extracellular concentrations of urea and NaC! in renal papila can reach 1.5 M and 0.45 M respectively [20]. Intracellular osmolality equal to the extracellular osmolality in medulla cells is maintained by high concentrations of betaine, lactate, glycerolphosphocholine, inositol and sorbitol [21-231. Thus, the intracellular ionic strength of these cells can be much higher than in other cells. In addition, it has been reported that the intracellular concentration of these solutes changes in response to alterations in the diuretic state [24, 251.
176 References 1 Ishikawa E, Oliver RM, Reed LJ (1966) ot-Keto acid dehydrogenase complexes. V. Macromolecular organization of pyruvate and ~-ketoglutarate dehydrogenase complexes isolated from beef kidney mitochondria. Proc Nati Acad Sci USA 56, 534-541 2 Tanaka N, Koike K, Hamada M, Otsuka KJ, Suematsu T, Koike M (1972) Mammalian ot-keto acid dehydrogenase complexes. VII. Resolution and reconstitution of the pig heart 2-oxoglutarate dehydrogenase complex. J Biol Chem 247, 4043--4049 3 Koike M, Koike K (1976) Structure, assembly and function of mammalian tx-keto acid dehydrogenasc complexes. Adv Biophys 9, 187-227 4 Hirashima M, Hayakawa T, Koike M (1967) Mammalian ot-keto acid dehydrogenase complexes. III. An improved procedure for the preparation of 2-oxoglutarate dehydrogenase complex from heart muscle. J Biol Chem 242, 902-907 5 Matthew JB, Hanania GIH, Gurd FRN (1979) Electrostatic effects in hemoglobin. Bohr effect and ionic strength dependence of individual groups. Biochemistry 18, 1928-1936 6 Matthew JB, Richards FM (1982) Anion binding and pHdependent electrostatic effects in ribonuclease. Biochemistry 21,4989-4999 7 Matthew JB, Gurd FRN (1986) Calculation of electrostatic interactions in protein. Stalibization and destabilization of protein structure by charge interactions. In: Methods in Enzymology (Hirs CHW, Timasheff SN, eds) voi 130, Academic Press Inc, Orlando, San Diego, New York, Austin, Boston, London, Sydney, Tokyo, Toronto, 413-453 8 Garland PB (1964) Some kinetic properties of pig-heart oxoglutarate dehydrogenase that provide a basis for metabolic control of the enzyme activity and also a stoichiometric assay for coenzyme A in tissue extracts. Biochem J 92.10c 9 Lawlis VB, Roche TE (1981) Regulation of bovine kidney ¢t-ketoglutarate dehydrogenase complex by calcium and adenine nucleotides. Effects on S0.~ for obketoglutarate. Biochemistry 20, 25 ! 2-25 i 8 lO Denton RM, Richards DA, Chin JG (1978) The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex. Biochem J 176, 899-906 i I MCCormack JK, Denton RM (1979) The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenas¢ complex. Biochem J 176, 899-906 12 Lawlis VB, Roche TE (1980) Effect of micromolar Ca2+ on NADH inhibition of bovine kidney ot-ketoglutarate
13
14
15 16 17 18
19 20 21
22 23
24 25 26
dehydrogenase complex and possible role of Ca2+ in signal amplification. Mol Cell Biochem 32, 147-152 Lawlis VB, Roche TE (1981) Inhibition of bovine kidney a-ketoglutarate dehydrogenase complex by reduced nicotynamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition. Biochemistry 20, 2519-2524 Pawelczyk T, Angielski S (1984) Cooperation of Ca 2+ and pH in regulation of the activity of the 2-oxoglutarate dehydrogenase complex and its components from bovine kidney cortex. Acta Biochem Pol 3 l, 289-305 Friend SH, Gurd FRN (1979) Electrostatic stabilization in myoglobin pH dependence of summed electrostatic contributions. Biochemistry 18, 46 ! 2-4619 Hermans J, Scheraga HA (1961) Structural studies of ribonucleases. V. Reversible change of configuration. J Am Chem Soc 83, 3283-3292 Lowry OH, Rosebrough NJ, Fan" AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193, 265-275 Pawelczyk T, Easom RA, OIson MS (1988) The effects of various anions and cations on the regulation of pyruvate dehydrogenase complex activity from pig kidney cortex. Biochem J 253, 819-825 Wolff SD, Balaban RS (1990) Regulation of the predominant renal medullary organic solutes in vivo. Annu Rev Physio152, 727-746 Beck F, Dorge A, Rick R, Thuran K (1984) Intra- and extracellular element concentrations of rat renal papilla in antidiuresis. Kidney Int 25, 397-403 Yancey PH (1988) Osmotic effectors in kidneys of xeric and mesic rodents: corticomedullary distributions and changes with water availability. J Comp Physiol 158, 369-380 Dell RB, Winters RW (1967) Lactate gradients in the kidney of the dog. Am J Physio1213, 301-307 Bagnasco S, Balaban R, Fales HM, Yang YM, Burg M (1986) Predominant osmotically active organic solutes in rat and rabbit renal medullas. J Biol Chem 261, 58725877 Wirthensohn G, Beck FX, Guder WG (1987) Role and regulation of glycerophosphorylcholine in rat renal medulla, Pflugers Arch 409, 411-415 Wolff SD, Balaban RS (1990) Sodium-23 nuclear magnetic resonance imaging of the rabbit kidney in vivo. Am J Physio1257, F675-F681 Fabiato A (1988) Computer programs for calculating total from specified free or free from specified total concentrations in aqueous solutions containing multiple metals and ligands. In: Methods in Enzymology (Reisher S. Fleisher B, eds) vol 157, Academic Press, Inc New York, 378-417