THE RESPIRATORY ENZYMES OF DIAPAUSING SILKWORM PUPAE: A NEW INTERPRETATION OF CARBON MONOXIDE-INSENSITIVE RESPIRATION

Abstract
1. To characterize the respiratory enzyme chain that functions during diapause, the respiration of diapausing pupae of the Cecropia, Cynthia, Promethea and Polyphemus silkworms was measured in the presence of specific mixtures of oxygen, nitrogen and carbon monoxide, after injection of various metabolic inhibitors and after injury. 2. Pupal respiration is at best only slightly inhibited by carbon monoxide and is often stimulated. Whatever CO-sensitivity there is occurs only in pupae with high basal metabolic rates. Moreover, when respiration is accelerated by injecting dinitrophenol (DNP), or by injury, this evokes an enhanced sensitivity to carbon monoxide. Indeed, it appears that the fraction of respiration sensitive to carbon monoxide is a function of the rate of oxygen consumption of the silkworm at all stages. 3. Reducing external oxygen tension to 2% fails to inhibit oxygen consumption, but increases markedly the CO-sensitivity of pupal respiration. Thus low oxygen tensions seem to unmask CO-sensitivity. 4. Pupal respiration is insensitive to azide concentrations as high as 5 x 10-4M. However, the azide-sensitivity, like the CO-sensitivity, increases markedly when pupal respiration is stimulated by DNP or injury. 5. Antimycin A at a concentration of 10-6M inhibits less than one-third of normal pupal respiration or DNP-stimulated respiration. Compared to other organisms diapausing pupae are resistant to this inhibitor of the cytochrome c reductase system. 6. Dinitrophenol at a concentration of 5 x 10-4M stimulates pupal respiration an average of 12-fold and as much as 16-fold. These are among the largest DNP-stimulations ever recorded. Although pupae with high basal metabolic rates are less stimulated proportionately by DNP than are pupae with low basal metabolic rates, they develop a greater over-all respiration under the influence of DNP. 7. Dinitrophenol-stimulated respiration is inhibited by carbon monoxide. The higher the DNP-stimulated respiration, the greater the inhibition by carbon monoxide. From this and other evidence it appears very likely that DNP accelerates the turnover of one or several components of the respiratory chain while having a lesser effect on cytochrome oxidase. 8. Dinitrophenol delays the appearance of injury-stimulated respiration, suggesting that the development of this increased respiration requires phosphate bond energy. Furthermore, exposure to carbon monoxide causes the death of injured pupae indicating that injury respiration is obligatory and involves the synthesis of new respiratory components. 9. Newly molted pupae not yet firmly in diapause do not respond to wounding with an injury respiration and their respiration is sensitive to carbon monoxide. These findings are correlated with their high respiratory rate. 10. The modes of action of the several inhibitors within diapausing, injured, and developing insects are considered in detail and a new explanation is proposed to account for the CO-, azide-, and cyanide-insensitivity of pupal respiration. 11. It is concluded that the insensitivity of diapausing pupae to inhibitors of cytochrome oxidase results from an excess of this enzyme over its functional requirements in the pupal respiratory chain. This concept is examined in detail and found to be theoretically sound. Evidence is presented that the limiting link in the respiratory chain is cytochrome c. Thus, contrary to earlier conceptions, it appears that cytochrome oxidase is the principal terminal oxidase during diapause as well as during all the other stages of the life history, and that the CO-insensitivity of pupal respiration stems from a great excess of cytochrome oxidase relative to cytochrome c. 12. The increased CO-and azide-sensitivity of pupal respiration after injection of DNP or injury results from an increase in the saturation of cytochrome oxidase provoked on the one hand by an increase in the turnover rate of cytochrome c, and on the other by the synthesis of cytochrome c. 13. It is suggested that the CO-insensitivity of the respiration of other organisms may be the result of an excess of cytochrome oxidase relative to some other components of the respiratory chain.