ISOLATION AND PHYSIOLOGICAL-PROPERTIES OF INTACT MITOCHONDRIA FROM BELL PEPPER (CAPSICUM-ANNUM) FRUIT

A protocol for isolating mitochondria from bell pepper (Capsicum annum , var. 'Jupiter') fruit by differential centrifugation and purificatio n on a discontinuous 2-step Percoll gradient: was developed based on m odifications of techniques employed for other plant tissues. The mitoc hondrial fraction banded at the interface of the two Percoll concentra tions, and was well separated from thylakoid fragments. Percoll-purifi ed mitochondria exhibited high oxidative and phosphorylative capacitie s with high respiratory control and ADP/O ratios. Catalase activity de creased during purification of mitochondria, with levels associated wi th purified mitochondria representing only about 3% of those present i n crude mitochondrial fractions. The specific activity of cytochrome o xidase increased during purification. Membrane competence of the purif ied mitochondria was about 90%, and the typical double-membrane system remained otherwise intact as revealed in electron micrographs. Mitoch ondrial ATPase (alpha-subunit) and alternative oxidase proteins were d etected at typical molecular-mass values, providing additional evidenc e for the maintenance of mitochondrial membrane integrity during the p urification process. Bell pepper fruit mitochondria are physiologicall y competent in vitro and should prove useful in studies of the respira tory metabolism of fruit ripening and senescence.