Respiration-Linked Contraction in Corn Mitochondrial

Respiration-linked contraction of corn mitochondria is not noticeably reduced by low, uncoupling concentr*tions of dinitrophenol. However, if a oontraction/respiration ratio is calculated, the contraction proves to be uncoupled. Previous statements that contraction cannot be uncoupled from respiration are in error. The uncoupling of c-ontraction is oonsistent with the concept that dinitrophenol attacks a primary non.phosphoryla-ted high energy intermediate (I,.X). It is proposed thot this intermediate is linked to some oontracti-le mechanism such that the degree of contraction reflects the level of intermediate. One of the more perplexing aspects of respiration-linked contraction in swollen mitochondria is the ineffeotiveness of uncoupling agents on the process. Our observations on 'corn mitochondria (2, 18, 19, 21) show 'that only high concentrations of uncoupler (e.g. 100-500 FM DN'P2) wi,ll accelerate spontaneous swelling and reduce contraction. In a phosphorylating medium, isutch concenftrations alimost eliminate oxidative phosphorylation and strongly inhibit the respiration. Low concentrations of uncoupler (e.g. 10-30 KM DNP) which are adequate 'to depress P/O ratios, accelerate ATPase, release acceptorless respiration, and reduce Ca24 + Pi transport, have little or no effect on respirationlinked contraction. The same in(senisitivity is fouindl with animal mitochondria (1,4, 7). We proposed that contraction is closely linked to respiration and cannot be reduced by uncoupling agents un'less they depress respiration (21). When evidence was obtained supporting our contention that contraction is associated wiith energy conservation, presumably as I-X, the hypothetical nonphosphorylated high energy intermediate, a scheme was presented relating contraction to -the level of Ir--(13). Recenitly we have re-examined the action of DNP on contraction and find our previoous statements to be in error. Dinitrophenol does uncouple contraction if expressed on a respiration b)asis comparable to the P/O ratio. Furthermore, net contraction can be reduced by DN'P w'hille the respiration rate is stilil very high. The findings 'Supported by grants from the Atomic Energy -Commi,ssion (AT/11-1/790) and the Office of Salinc WVater, Department of Interior (14-01-0X1-1088). 2 Abbreviation,s: DNP) 2,4-dinitrophenol. BSA) bovine serum albtmin. are in accord with the classical view that DNP attacks a non-iphosphorylated intermediate produced at coupling sites. Methods and Materials Mitochondria were isolated from 3 day etiolated corn shoots as previously descriibed (13), but with the omission of ADP during washing. Measuirements of light absorbancy and of oxygen were made in parallel on separate but identical solultionts (13), or by s9imultaneously recording light absorption and oxygen 'levels in a 'specially constructed and thermostated cell at 250. Results were the same wi'th both method's. Vessel contents are given wi'th the figures.