By A. Pullman (auth.), J. W. Gorrod, O. Albano, E. Ferrari, S. Papa (eds.)
cytochemical suggestions (ICC) which offer an invaluable adjunct to investigations via immunoblotting. a specific good thing about a cytochemical method of the investiga tion of mitochondrial issues is that it permits the mosaic distribution of sure of those defects to be detected, while the tissue homogeniza tion concerned with traditional enzyme assays or immunoblotting precludes this. yet another benefit of MEA or ICC is that in simple terms small quantities of tissue are wanted, that is vital considering that a few of the affected sufferers are babies or young children. the most target of this communica tion is to stipulate ways that those suggestions can be utilized within the analysis and additional research of mitochondrial problems. Reference might be made not just to these events within which MEA and ICC supply benefits over average enzyme asays and immunoblotting but in addition to contexts during which the opposite applies. four. 2 fabrics and techniques Muscle biopsies for cytochemical research have been snap-frozen utilizing isopentane cooled to - 150°C in liquid nitrogen. Samples have been saved in heat-sealed polythene packets within the vapour section of liquid nitrogen packing containers. four. 2. 1 Microphotometric enzyme assays Frozen sections eight Jlm thick have been reduce utilizing a Reichert-J ung Frigocut cryostat microtome outfitted with motor-driven slicing motion to keep up maximal reproducibility of part thickness. Sections have been picked up on microscope slides and air-dried for 15 min at room temperature.
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Amino acid sequence analysis was carried out as reported in Papa et al. (1989) and Zanotti et al. (1988). same effect when added to F,-depleted particles supplemented with purified F, . Fig. 2 shows a titration of the inhibitory effect exerted by addition of the purified inhibitor protein on the ATPase activity and proton conductivity of depleted particles. Treatment of the isolated inhibitor protein with ethoxyformic anhydride (EFA) under conditions resulting in modification of one out of the five histidine residues of this protein (Guerrieri et al.
132,985-90. W. et al. j. , 164, 517-23. , Capozza, G. et al. , 237, 9-14. N. GADA LETA, V. PETRUZZELLA, M. RENIS, F. FRACASSO AND P. CANTATORE Department of Biochemistry and Molecular Biology, University of Bari and CSMME, Bari, Italy Mammalian mitochondrial DNA is a small, double-stranded, circular molecule of about 16 500 bp which carries the information for part of the inner mitochondrial membrane respiratory complexes and for its own protein synthesis machinery (Cantatore and Saccone, 1987).
A lower content of respiratory units in the mitochondria of old animals has been suggested by Abu-Erreish and Sanadi (1978). , 1990). A consistent reduction of Col mRNA in the synaptic mitochondria of 28-month-old rat cerebral hemispheres was observed. As a further step in this study, we decided to measure the cellular level of the mRNA for a nuclear coded cytochrome c oxidase subunit, in adult and senescent rat. The quantification was performed by hybridizing in solution RNA extracted from the cytoplasm of the cerebral hemispheres of adult and senescent rat with a human probe for the nuclear coded subunit IV.