Active Mitochondria Do Not Produce Ethylene

Abstract

Although numerous reports on ethylene evolution from subcellular preparations have appeared (2,3,4,5, 8,9,11,18,19,20), some of these works have been criticized by Burg and Burg (1) and by Meigh (17). and the question whether or not mitochondria produce ethylene remains unclear. It is known that ethvlene can be evolved nonenzymically from methionine and its derivatives, mediated either bv jCu2'-ascorbate-HOO, (10) or by 23). Enzvmic systems of ethylene production were described recently by Mapson and Wardale ('13), Ku et al. (7), and Meheriuk and Spencer (14,15,16 (12). The fruits were washed with distilled water and chilled (0-40) from 1 to 4 hours before processing by the method of Ku et al. (6). The flesh of the fruit wall was carefully removed, avoiding the inclusion of any skin, seeds, or the jellv-like tissue around the seeds.For preparation of mitochondria, the fruit tissue was immersed in a buffer containing 0.4 M sucrose, 50 mM tris-HCl, 2 mm MgCl,, 10 mM KCI, 5 mMi EDTA, and BSA (bovine serum albumin, 0.5 mg per ml), and was cut with a stainless steel razor blade into very small pieces. The pH was kept within the range of 7.0 to 7.2 by dropwise addition of 1 N KOH during cutting. The tissue was strained through 8 layers of cheesecloth and centrifuged at 2500 X g for 15 minutes to give fraction 1. The stupernatant was then centrifuged at 11,000 X g for 20 minutes to give the mitochondrial fraction, and the sediments of both fractions were each 1 Tlis investigation was supported in part by a research grant (UIO0102) from the National Center for Urban and Industrial Health, United States Public Health Service.washed once with a medium containing 0 4 M sucrose, 10 nmM KH,PO4, 10 mm KCI, and BSA (0.5 mg per ml) at pH 7.4. Electron micrographs show that fraction I contains nuclei, grana, plastids, small vesicles, mitochondria, and unidentified organelles and fragments, while the mitochondrial fraction is a homogeneous preparation of relatively uninjured mitochondria (6). Fraction I and the mitochondrial fraction were each suspended in a known volunme of buffer, containing 0.25 M sucrose, 10 mm KH.,PO4, 0.5 mg per ml BSA, 10 mm KCI, pH 7.4. and dialyzed against the same buffer for 2 hours, changing the buffer once.For a preparation of soluble enzymnes, the flesh was honmogenized with a mininmal volume of 0.1 M K.)HPO4 in a Waring Blendor, squeezed through cheesecloth, and centrifuged at 30.000 X g for 20 minutes. Tlle supernatant was brought to 90 % saturation wvith ammonium sulfate, and the protein was collected by centrifugation, redissolved in a ninimlnal volumiie of 50 ni-m tris-HCl ( pH 7.6), and dialyzed against 5 mnI tris-HiCl (pH 8.0) for 18 hours with 5 changes of buffer. The dialysate was centrifuged, and the supernatant was used as the enzynme solution.A standard incubation mixture contained 1 ,umole of substrate, cofactors (see tables), and a known volume of particle suspension or enzyme solution, made to 1.0 or 2.0 ml in a 25 ml Erlenmeyer flask fitted with a rubber serum cap. The flasks were in...

Ethylene formation in plant mitochondria. Dependence on the transport of 1‐aminocyclopropane‐1‐carboxylic acid across the membrane

“…The possibility that mitochondria are in-volved in ethylene production received considerable attention in the past (Ratn Chandra and Spencer 1962, Mehetiuk and Spencer 1964, 1967a,b, Ku and Pratt 1968. However, these studies often produeed contradicting results; tnethionine as well as other preeursors were used, yielding an insignificant amount of ethylene in the purified mitoehondrial preparations (Ku and Pratt 1968). In addition, the reaction was reported to be temperature-insensitive (Spencer and Meheriuk 1963).…”