Isolation of Tightly Coupled Mitochondria From Acidic Plant Tissues

T'b is comilitmication emphasizes the nieed for conltin1uouts conitrol of pH when macerating acidic plant tissues for the l)url)ose of isolating tightly coupled mitochondria. A useful device for such control is described. Mitochonidria capable of oxidative but not pl)osphorylative activity have been isolated fromli apple fruits wlhenl either alkaline homogeniates (5) or l)oIyvinylpyrrolidone (PV7P) (1) was used to supl)ress phenolic compounds. However, it was niot until a pH slightly below 7, pluis 0.5 % I'VP, wvere used that phospliorylating, "coupled" miitoclhonldria were obtained froml the l)arenchymatous tissues of both pear andl apple fruits (7, 8). Loomlis and Battaile (6) have sinice slioNvni that PVP is less effective at an ailkalinie pH. Wiskich (9) utilized an isolation medium at pH 7.4, btut containing 1 % PVP, to obtain apple mitochondria with respiratory colntrol ratios (RCR) of somewhat less than 2. Hulmle ct al. (2) reported results similar to those obtainied by Wiskiclh. Tightlty coupled mitoclhondria (RCR's approachilng infiniity) were obtained from pear tissues only wlhen additional precautions were taken to control pH during nmaceration. This was accomplished with the device illustrated in figures 1 and 2. The surface of the stainless steel screen (fig 2) has been grotund slightly to provide shearing edges. Downward pres-