Substrate Independence of Molecular Weight of Triphosphopyridine Nucleotide-specific

SUMMARY The techniques of gel filtration and light scattering were used to evaluate a report that the molecular weight of the TPN-specific isocitrate dehydrogenase of pig heart can be influenced by its substrates. The results here presented indicate that the elution position of the enzyme from a column of Sephadex G-150 is not appreciably altered by the addition of isocitrate, manganous ion, TPN, oc-ketoglutarate, or TPNH. The Stokes radius is estimated as 39.3 A. Similarly the weight average molecular weight of the enzyme, as measured by light scattering, does not vary significantly from 58,000 when isocitrate, a-ketoglutarate, metal ion, and the coenzymes are added. Ir is concluded that the pig heart TPN-dependent isocitrate dehydrogenase exists as a single molecular weight species independent of the presence of substrates. Pig heart TPN-specific isocitrate to consist of a single polypeptide with molecular weight approximately nondenaturing as and


SUMMARY
The techniques of gel filtration and light scattering were used to evaluate a report that the molecular weight of the TPN-specific isocitrate dehydrogenase of pig heart can be influenced by its substrates.
The results here presented indicate that the elution position of the enzyme from a column of Sephadex G-150 is not appreciably altered by the addition of isocitrate, manganous ion, TPN, oc-ketoglutarate, or TPNH.
The Stokes radius is estimated as 39.3 A. Similarly the weight average molecular weight of the enzyme, as measured by light scattering, does not vary significantly from 58,000 when isocitrate, a-ketoglutarate, metal ion, and the coenzymes are added. Ir is concluded that the pig heart TPN-dependent isocitrate dehydrogenase exists as a single molecular weight species independent of the presence of substrates.
Pig heart TPN-specific isocitrate dchydrogcnase has been reported to consist of a single polypeptide chain with a molecular weight of approximately 58,000 on the basis of equilibrium celltrifugation studies in a nondenaturing buffer as well as in urea and guanidine hydrochloride solutions, electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate, and gel filtratioll in guanidine hydrochloride (1). However, Kemper and Kaplan (2) have recently reported that the active species catalyzing t.he reduction of TPN is a 30,000 molecular weight monomer, whereas the species catalyzing the oxidation of TPKH is a 120,000 molecular weight' species; these conclusions were drawn from the sedimentation constants measured in the presence of substrates.
The mechanisms of regulation of enzymes which undergo reversible associatiotl-dissociation reactions (3, 4) are quite distinct from those Aaracteristic of a single peptide chain enzyme with an invariant molecular weight. It, therefore, seemed important to assess by independent methods which of these categories best describes the TPS-dependent isocitrate dehydrogenase.
This paper presents gel filtration and light scattering experiments demonstrating that the molecular size of this r'llzymct is not influenced by the addition of its substrates isocit,rate, Tl'S, TPNIZ, cu-ltetoglutarclte, or mangnnous ion. The TPN-specific pig heart isocitrate dehydrogenase was obtained from the 13oehringer Mannheim Corp. and was purified IO-fold by column chromatography on carboxymethylcellulose followed by gel filtration on Sephades G-150 as described previously (5). The resulting preparation is homogeneous in the ultracentrifuge and on cellulose acetate and polyacrglamide gel electrophoresis under mild and denaturing conditions (I, 5). The peak of the elution profile from a gel filtration column reflects the molecular size of the protein. Fig. 1 records the clution position of isocitrate dehydrogenase w-hell filtered in buffer alone, with added isocitrate and manganous ion, with oc-ketoglutarate, MnS04, and TPNH, and with isocitrate, AInSO+ and 'I'PX. The last experiment represents a complete reaction mixture and the enzyme catalyzes the oxidative derarboxylation of isocitrate to its final equilibrium position during the course of its migration through the column. In all of these cases, the buffer contains 107; glycerol which has been cited by Kemper and Kaplan (2) as stabilizing the tetramer. It is notable that the peak positions and the width of the elution profiles shown in Fig with the composition of the buffer and thus no change in the molecular size of the enzyme seems to be induced or stabilized by the addition of these substrates.
The data of Fig. 1 were used to calculat,c a distribution coeflclient, Kd, which is defined as: where Vt is the total volume of the gel bed, V, is the clution volumr of the sample, Vo is the void volunle dctcrmined using dextran blue, and m,, and uy arc the weight and 1)artial specific volume of the gel matrix.
The value of k-d for isocitrate dehydrogenase in buffer alone is 0.216 and does not cahange appreciably in the presence of substrates.
The position of elution from a Sephadex column is determined primarily by the Stokes radius (a) of the protein (6). Using bovine serum albumin (Kd = 0.252; a = 3.70 nm) and cytochrome c (Kd = 0.542; a = 1.74 nm) to calibrate the column, the Stokes radius of isocitrate dehydrogenase was estimated by the method of Ackers (6) to be 3.93 nm. Assuming a molecular weight of 58,000, a frictional ratio (f:fo) of 1.52 may be calculated, indicating that the isocitrate dehydrogenase molecule may be somewhat clongated. It is interesting that when the enzyme is denatured, as is the case during gel filtration in buffers containing 6 M guanidine hydrochloride, it is eluted behind bovine serum albumin and exhibits a molecular weight of 58,000 (1).
Gel filtration is a relatively slow process and yields infornlation on the size of the molecule which has been in contact with the substrates for a period of hours. In contrast, the complemclltary technique of light scattering provides the possibility of observing directly within minutes the effect on the weight average molecular weight of the addition of substrates.
Iluffer and substrate solutions were filtered separately.
The I)rot,ein concentration was determined from the absorbance at 280 nm (5) using aliquots of the enzyme solutions after filtration.
The measured refractive index of the solvent was 1.341. The molccular weight was derived from the following equation (7). IIowever, since UC/'? did not vary over the protein concentr:ttion range used (0.17 to 0.75 mg per ml), the last term was neglected.
The weight average ~iloleculnr \wights for isocitratc drhydrogenase as determined by light scattering are recorded irl Table I. In the absence of substrates (Line 1) the enzyme exhibits a molecular weight of 63,300, which agrees reason:lblJ n-cl1 with the value of 58,000 measmwl by equilibrium crntrifugatioll ill buffer (5). The ma~~ganous cwmplex of t,ribusic isocaitratc has been shown to be the substrate for this enzyme (9) and to bind in the absence of pyridinc nucleotidc (10). The addition of isocitrate and MnS04 (Line 2) to the enzyme solution does not significantly change the measured molecular weight.
When TPN is added (Line 3), oxidative decarboxylation of isocitrate procceds; however, no marked decrease in the molecular weight is noted either immediately after addition or after 30 rnin, when the wnction has reached equilibriurn.
The presence of higher concentrations of a-kctoglutarate and TPNH (Lines 4 to 6) produces no large increase in the molecular weight; in fact, the molecular weight observed was remarkably constant throughout these experimcllts.
An average of all of the listed values is 58,400, which is essent,ially identical with the reported determinations made in the analytical ultracentrifuge under both mild and denaturing conditions in the absence of substrates (1,5).
These results lead to the conclusion that the TPN-dependent isocitrate dehydrogcnase exist,s as a single molecular weight species whether or not substrates are lwcsent.
The postulation of a~1 active 30,000 molecular weight species (2) is highly irnprobable on chemical grounds.
On the basis of a molecular weight of 58,000 it has been sl~own that the enzyme binds 1 mole of isocitratc, ot-ketoglutarate, and mallganous ion (10, 11) ; catalytic activity is lost as the result of modification of a single mcthionyl residue (5); and only one MI,-terminal alanine has been identified in the Edmnn reaction (12). Furthermore, the number of peptides detected in a "fingerprint" obtained by paper chromatography and electrophoresis of tryptic digests of the enzyme is consistent with the csistence of a single polypeptide cllaill of molecular weight 58,000 (1). Xo evidence suggesting the formation of a 120,000 molecular weight species of this cnzymc has been found in the present investigation.
If the cwzyme existed in a monomer-dimer-tetramer equilibrium with preferential binding of isocitratc by the monomer and of cr-ketoglutarate by the tetramcr, nonlinear Scatchard plots for the binding of these substrates would have been anticipated (3,13). 011 the contrary, lincw Scatcahard plots have been ol)served for both isocitrate and oc-ketoglutarate (11). The apparent conflict between the conclusions of this paper regarding the lack of influence of substrates on the molecular weight of isocitrate dehydrogrnnse and the report of Kemper and Kaplan (2) is difficult to resolve. These authors apparently used the procedure of Cohen and Mire (14) in which a thin lamella of enzyme solution is layered onto a substrate solution in a rotating ultracentrifuge cell. The analysis by this method assunws that the substrate is present at saturating concentrations throughout the rnzyme band. Cohen and Mire caution that the failure to achieve these conditions may yield an apparent sedimentation constant that is erroneously high. Since the Michaelis constants for a-ketoglutaratc and carbon dioxide arc several orders of magnitude higher than that for isocitrate, adequate conditions would be less readily maintained in the reductive carboxylation reaction.
In fact, it is difficult to see how saturation with respect to carbon dioxide could be achieved under the conditions of ultracentrifugation.
The details of the experiments of Kemper and Kaplan have not yet appeared, making it impossible to evaluate their methods.
Most mammalian tissues have been shown to contain two isocitrute dehydrogenasrs, :I l>l'X;-sljecific enzyme located primarily in the mitochondrin and a TPPu'-dependent enzyme found both in the mitochondria and the cytoplasm (15). The two enzymes catalyze similar renct,ions, but differ in several structural and functional characteristics including the actual form of isocitrate which functions as the substrate (9, 16), the molecular by guest on March 24, 2020 http://www.jbc.org/