Sucrose Density Gradient Distribution of Mitochondrial Protein and Enzymes from Preclimacteric and Climacteric Pears

Abstract

Suimnary. Mlitochondrial fractions isolated fronm pears (Pyrus coknimmnis L.) at the climacteric minimum alnd peak were sulbjected to sucrose densitv gradient centrifugation. The distribution of protein and specific activities of 3 enzvmles from this mitochondrial fraction were investigated.Cytochrome oxidase specific activity remained associated with the particulate fraction and increased slightly during the period in which respiration of the whole fruit reached its climacteric peak. Catalase and acid phosphatase specific activity was associated with 1)oth the particulate and the least dense region of the gradient and decreased with postharvest ripening.Evidence for several differences between the subcellular behavior of catalase and acid phosphatase from pear tissue compared to their counterparts isolated from mammalian cells is discussed. A general shift of maximum specific enzymic activities and protein distribution to lighter regions of the density gradient occurs with ripening, suggestive of diminution in size or density of intracellular particles.Several studies relating mnitochoiidrial activity wvith the climacteric sequence in ripening fruit have been discussed by Biale (3) and Hulme (11). A recent report (12) indicates that the respiratory activitv of mitochondria remains high, well past the climacteric peak. Bain and Mercer (1) have further shown bv electron microscopy that mitochondrial structures remain virtually unaltered tbrough the ripening period.Nonetheless, the stress of senescence may be expected to prodtuce some cytoplasmic adjustments as in the case of placental cells of ripening tomatoes (7). In a preceeding study (17), a decrease in yield of nmitochondria was noted coincident with the climacteric peak and immediate post-climacteric phase of ripening pears.Since the particles of the mitochondrial fraction are known to play an important role in intracellular metabolism, a study on the effect of ripening on several enzymes from these organelles appeared to be of value. The sucrose density gradient centrifugation techniquie was utilized to further characterize the mitochondrial fraction.

Isolation and comparative proteomic analysis of mitochondria from the pulp of ripening citrus fruit

“…The activities of several mitochondrial enzymes were also assessed in mitochondrial isolates from banana 20 , lemon 21 , and mango 22 . The introduction of density gradient centrifugation using sucrose 23 or Percoll 24 greatly enhanced the purity of mitochondria isolated from fruits, making it possible to characterize the mitochondrial proteomes of various fruit crops. Subsequently, proteomic analysis revealed that oxidative damage and carbonylation modification of mitochondrial proteins are widely involved in the regulation of fruit ripening and senescence [25][26][27][28] .…”

Microbodies-Peroxisomes and Glyoxysomes

“…Micros copically they are present in tobacco stem and ca llus tissue (49), in oat coleoptile (49,159,176), and in etiolated leaf tissue (P. J. Gruber & E. Newcomb, personal communication). The detection of ca talase activity on sucrose gradients of particles suggests that microbodies are present in mung bean hypocotyl (129), oat coleoptiles (176), and ripening pears (107 ). Microbodies are present in th e developing Sym plocarpus spadix (10; Ph .…”