Data are presented suggesting that intact Neurospora crassa mitochondria contain an enzyme complex incorporating five enzymes necessary for the biosynthesis of isoleucine and valine. The functional integrity and stability of the complex has been shown to be dependent on the metabolic state of the mitochondria. The complex has been solubilized by digitonin and has an approximate molecular weight of 400,000.We have shown previously in this laboratory that isoleucine and valine are synthesized in the mitochondria of Neurospora crassa (1). We have now obtained evidence for the existence of an active enzyme complex that appears to incorporate the isoleucine and valine enzymes, a-acetohydroxy acid synthetase, a-hydroxy, fl-keto acid reductoisomerase, a,#-dihydroxyacid dehydratase, and an aminotransferase, as well as threonine deaminase. This complex is capable of synthesizing valine from pyruvate and isoleucine from threonine and pyruvate. The active complex is not released from isolated mitochondria unless they are first incubated in a medium that contains the necessary substrates for respiration and oxidative phosphorylation. Otherwise only the individual enzymes are solubilized, and these can be separated from one another on Sephadex columns by virtue of the differences in their molecular weights (M. Collins, K. Kiritani, S. Narise, and R. P. Wagner, unpublished results).
MATERIALS AND METHODSPreparation of Mitochondria. Flasks containing one liter of Vogel's minimal medium were inoculated with 6-day-old conidia of Neurospora crassa, strain LSDT(1969)A (2) and incubated for 15.5-16 hr at 250 on a rotary shaker. The harvested mycelium was washed with "(" buffer (a buffer designed to preserve the integrity of the mitochondria during isolation as to respiration, oxidative phosphorylation, and synthesis of valine and isoleucine from pyruvate). The "Q" buffer consists of 0.3 M sucrose, 10 mM potassium phosphate at pH 7.4, 10 mM sodium succinate, 0.15% bovine serum albumin, 50 mM sodium pyruvate, 34 mM 2-mercaptoethanol, 2 mM pyridoxal-5'-phosphate, 0.13 mM thiamine pyrophosphate, 47nM FAD, and 59MM MnSO4. The washed mycelium was ground in an ice-cold mortar with twice its wet weight of acid-washed sand. The ground mycelial preparation was diluted with additional "al" buffer and centrifuged twice for 10 min at 1500 X g. The mitochondria were spun down by centrifugation for 1 hr at 12,000 X g. The pellet was taken up in "a" buffer, and the mitochondria were resuspended in a Teflon grinder to give an adjusted protein concentration of 40 mg/ml. Preincubation. The mitochondrial suspension (40 mg/ml) was divided in two portions, placed in a 30°water bath, and stirred for the time intervals indicated in Results. At zero time, to portion A (7 ml) ( Table 1)
