RESULTS

Elution Profile

Affinity chromatography using Matrex Red A Gel allowed us to successfully purify Drosophilamelanogaster IDH 340-fold (Table 1).

Table 1: Purification of Drosophila melanogaster IDH

Sample	Volume (mL)	Activity*	Fold Purification
Crude Homog	50	0.003942	1
65% Pellet	6.6	0.0059	1.5
Concentrate	0.25	1.3503	342

* Micromoles of NADP⁺ reduced per minute per microgram of protein.

After pumping the 45-65% pellet preparation over the column and washing the column, two peaks of IDH activity were identified (Figure 5). The first peak is the result of the total amount of IDH greatly exceeding the columnís capacity to bind the enzyme. The second peak represents the elution of the bound enzyme with the NADP⁺/ isocitrate gradient wash. The three fractions in this second peak containing maximal IDH activity were pooled, concentrated and used to determine the parameters of IDH. Those fractions containing IDH that did not bind (the first peak) were pooled, and after the column had been recharged, pumped back onto the column in order to ensure that two forms of IDH, one capable of binding to the matrex and one not, did not exist in our preparation. Additional samples of purified IDH were recovered by repeating the gradient wash.

Figure 5: Characteristic elution profile of Matrex Red A Column.

Substrate Kinetics

The K_m for isocitrate was estimated to be 29.8 ± 7.9 µM, while the K_m for coenzyme was estimated to be 1.05 ± 0.1 µM (Table 2). These K_m values for isocitrate and NADP⁺ are consistent with previous values for Drosophila melanogaster determined by Williamson, et al., (1980).

Table 2: Kinetics of substrate utilization for Drosophila melanogaster IDH

Substrate	K_m (µM)	N*
Isocitrate	33.4 ± 1.6	5
NADP⁺	1.05 ± 0.1	4

*N = number of determinations

Divalent Metal Ions

Our data confirm that Drosophila IDH requires the presence of divalent metal ions for proper catalytic function (Table 3). When no additional metals were added to the assay, a low level of activity was observed due to contaminating metal ions in the buffer ingredients. The addition of EDTA (2 mM) to the reaction mixture reduced this activity to zero. When magnesium (2 mM) was added to the basic reaction mixture, activity was enhanced 3.5-fold. When manganese (2 mM) was added to the basic reaction mixture, activity was enhanced 5.2-fold.

Table 3: Divalent metal requirements of Drosophila melanogaster IDH

Assay	Mean Activity / µL
No added metals	1.86 X 10^-3
EDTA, 2 mM	0
Magnesium, 2 mM	6.56 X 10^-3
Manganese, 2 mM	10.08 X 10^-3

Polyacrylamide Gel Electrophoresis

Three bands of NADP⁺-dependent isocitrate dehydrogenase activity were observed after staining a native gel for IDH activity (Figure 6A). The molecular sizes of these three bands were estimated to be 103, 97, and 90 kDa. Similarly, three protein bands, corresponding to the three bands of IDH activity, were observed in that half of the gel stained with Coomassie Blue (Figure 6B). In addition, two minor protein bands with approximate molecular weights of 200 kDa and 150 kDa were detected in the Coomassie staining process. In a second trial, the three forms of IDH were determined to have molecular weights of 109, 100, and 94 kDa.

Figure 6: A: Native polyacrylamide (10%) gel electrophoresis of Drosophila IDH stained ibidem for activity. Lane 1 was loaded with 10 uL of pre-stained high range standards (molecular weights in kDa). Lane 2 was empty. Lane 3 was loaded with 7.25 ug of protein. B: Native polyacrylamide (10%) gel electrophoresis of Drosophila IDH stained with Coomassie Brilliant Blue R for proteins. Lane 1 was loaded with 10 uL of pre-stained high range standards. Lane 2 was loaded with 7.25 ug of protein. Lane 3 was loaded with 10.875 ug of protein. Lane 4 was loaded with 14.5 ug of protein.

SDS-PAGE indicated that IDH is a heterodimer. On a 7.5% gel, two subunits of different sizes were observed, one estimated to be 65 and the other 51 kDa. On a 10% gel, the subunits were determined to have molecular sizes of 70 and 57 kDa (Figure 7). No other protein bands were detected on either of these SDS gels. The above values are slightly larger than, but consistent with, those reported by Williamson, et al., (60 kDa and 50 kDa, 1980).

Figure 7: SDS polyacrylamide (10%) gel electrophoresis of Drosophila IDH. Lane 1 was loaded with 10 ul of pre-stained low range protein standards (molecular weights in kDa). Lane 2 was loaded with 1.813 ug of protein. Lane 3 was loaded with 3.625 ug of protein. Lane 4 was loaded with 5.438 ug of protein. Lane 5 was loaded with 7.25 ug of protein.

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