NAD⁺-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

Abstract

The NAD'-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP'-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD' and NADP'-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD -dependent isocitrate dehydrogenase showed sigmoidal kinetics in response to isocitrate (So.s = 0.3 mM). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD+ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD+ (Km = 0.2 mM). NADH was a competitive inhibitor (Ki = 0.2 mM) and, unexpectedly, NADPH was a noncompetitive inhibitor (Ki = 0.3 mM). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.

Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii1

“…Therefore, the enzyme in its native form appears to be an octamer, but from our data we cannot determine if it is composed of identical or different subunits. The octameric structure and the native molecular mass value are consistent with those reported for the NAD-IDH from bovine heart (333 kD; Giorgio et al, 1970) and from P. blakesleeanus (338 kD; Alvarez-Villafañ e et al, 1996), and for the matrix (300 kD; McIntosh and Oliver, 1992) and membrane forms (320 kD; McIntosh, 1997) from pea. McIntosh and Oliver (1992) reported the correlation between the loss of activity during subsequent purification steps and a change in the native molecular mass of the protein from pea.…”

“…2B). According to the purification method described here, isolation of the mitochondrial fraction was not required to purify the NAD-IDH from C. reinhardtii, as was reported previously for the plant enzyme (Tezuka and Laties, 1983;McIntosh and Oliver, 1992). In contrast to the NAD-IDH from pea, where 35% to 50% of the activity was found tightly associated with the membrane (McIntosh, 1997), no NAD-IDH activity was detected in the membrane pellet obtained in the crude extract step after solubilization with 0.5% Triton X-100.…”

“…In addition, the NADP-IDH preparation can be further resolved in two different isoenzymes by affinity chromatography on Blue-Sepharose (Martínez-Rivas and Vega, 1994). The main problem in achieving complete purification of the NAD-IDH from photosynthetic organisms has been its low stability (McIntosh and Oliver, 1992). To stabilize the NAD-IDH activity from C. reinhardtii, we found that it was critical to keep ethylene glycol in the buffer after elution from the Phenyl-Sepharose chromatography step.…”

“…However, partial purifications have been reported from pea (Cox and Davies, 1967;Cox, 1969;McIntosh and Oliver, 1992), potato (Laties, 1983), spruce (Cornu et al, 1996), and the eukaryotic microalga Chlamydomonas reinhardtii (Martínez-Rivas and Vega, 1994). Matrix and membrane forms of the enzyme have been detected in potato (Tezuka and Laties, 1983) and pea (McIntosh, 1997).…”

“…Matrix and membrane forms of the enzyme have been detected in potato (Tezuka and Laties, 1983) and pea (McIntosh, 1997). Although it is an allosteric enzyme that exhibits sigmoidal kinetics with respect to isocitrate (Cox and Davies, 1967;McIntosh and Oliver, 1992) and is activated in vitro by ABA (Tezuka et al, 1990), the regulatory importance of NAD-IDH in photosynthetic organisms is still under debate.…”