Studies on the nucleotide specificity of mitochondrial inner membrane particles (original) (raw)
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The Problem of Cation‐Binding Sites in the Energized Membrane of Intact Mitochondria
European Journal of Biochemistry, 1973
Weak bases, such as the acridine and the aminophenazine dyes, which undergo protonation in the chromophoric group, are actively bound in sonicated fragments. On the other hand strong bases such as the divalent cations, the safranine and the cyanine dyes, which carry a permanent charge, are actively bound in intact mitochondria. In the latter case the process results in stimulation of the respiration, H+ ejection, and a large change in absorbance, which is attributed to a stacking of the dyes. The effects are inhibited or reversed by uncouplers, and insensitive to nigericin.Four alternatives are considered: accumulation in the inner aqueous space; binding to matrix proteins; binding to the outer membrane surface; and binding to sites in the membrane fabric. The view that the energized membrane contains high affinity sites for cationic dyes is supported by: comparison of the spectra of the membrane‐bound and polyanion‐bound dye; competition with divalent cations; osmotic activity; and...
Biochemical Journal, 1992
Under defined conditions liver mitochondria from hypothyroid rats show an apparent lowering of the ADP/O ratio, which can be corrected by addition in vitro of 0.1 nM-tri-iodothyronine (T3). Nicotinamide prevents this restoration by hormone, lowers the ADP/O ratio of euthyroid-rat mitochondria to hypothyroid-rat values and induces T3-sensitivity in euthyroid-rat mitoplasts indistinguishable from that found with hypothyroid-rat preparations. Incorporation into the trichloroacetic-acid insoluble fraction of mitoplasts and hypothyroid-rat mitochondria of radiolabel from [adenine-14C]-NAD+ was stimulated by T3: this stimulation was abolished by nicotinamide. The findings strongly suggest that this incorporation occurs external to the matrix. Confirming the work of others, PAGE of radiolabelled mitoplasts shows alkali-labile modification of a major species of approx. 30 kDa: both nicotinamide and T3 abolish this modification. By contrast, T3 promotes incorporation of label into a single m...
A study of mitochondrial membranes in relation to elementary particles
Journal of cell science, 1967
SUMMARY Elementary particles that commonly have been seen by electron microscopy to be attached by stalks to mitochondrial cristae in negatively stained preparations, were not apparent in similarly stained mitochondria from exponentially growing wild-type Neurospora crassa when these were isolated in sucrose solution containing 1 X io~ 3 M EDTA. However, elementary particles were easily demonstrable in electron micrographs if the mitochondria were isolated without EDTA in the sucrose solution. A biochemical study indicated that both kinds of mito- chondrial preparations, isolated in the presence or absence of EDTA, had about the same capacity for oxidative phosphorylation. Observations on rat-liver mitochondria also suggested that the stalked elementary particles were more easily demonstrated if the preparation was made in the absence of EDTA. It was difficult to demonstrate elementary particles in wild-type Neurospora mitochondria isolated with or without EDTA and subsequently prepared for electron microscopy by spreading on the surface of an aqueous solution of potassium phosphotungstate. Elementary particles could be demonstrated in poky Neurospora mitochondria isolated with EDTA if the mitochondria were spread on the surface of an aqueous solution of phosphotung- state. It was concluded that biochemical functions associated with elementary particles are independent of structural configuration as seen by electron microscopy.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1982
The characteristics of the binding sites for ADP and adenylyl imidodiphosphate have been studied in soluble and particulate F1-ATPase from bovine heart mitochondria. ADP, but not electrochemical gradients, removes the inhibitory effect of adenylyl imidodiphosphate on ATPase activity in coupled submitochondrial particles. In sosuble FI-ATPase, methanol at 20% concentration diminishes the ability of ATP and adenylyl imidodiphosphate to inhibit ATP and ITP hydrolysis; these findings suggest that ADP and adenylyl imidodiphosphate inhibit hydrolysis by acting on the same site. Methanol at 20% stimulates the hydrolytic activity of soluble F1-ATPase, but fails to stimulate significantly the activity of the particulate enzyme, even though in particulate F~-ATPase methanol markedly diminishes the inhibiting action of added ADP and adenylyl imidodiphosphate on ATP and ITP hydrolysis. This is consistent with the idea that in the particulate system there are two inhibitory binding sites for ADP, one accessible to methanol, and another which is inaccessible to methanol; the latter is transitorily occupied by ADP arising from ATP hydrolysis. Indeed, experiments on the effect of ADP on ITP hydrolysis by submitochondrial particles show the existence of two ADP inhibitory sites.
Molecular and Cellular Biochemistry, 1994
Cytosolic proteins as components of the physiological mitochondrial environment were substituted by dextrans added to media normally used for incubation of isolated mitochondria. Under these conditions the volume of the intermembrane space decreases and the contact sites between the both mitochondrial membranes increase drastically. These morphological changes are accompanied by a reduced permeability of the mitochondrial outer compartment for adenine nucleotides as it was shown by extensive kinetic studies of mitochondrial enzymes (oxidative phosphorylation, mi-creatine kinase, mi-adenylate kinase). The decreased permeability of the mitochondrial outer membrane causes increased rate dependent concentration gradients in the micromolar range for adenine nucleotides between the intermembrane space and the extramitochondrial space. Although all metabolites crossing the outer membrane exhibit the same concentration gradients, considerable compartmentations are detectable for ADP only due to its low extramitochondrial concentration. The consequences of ADP-compartmentation in the mitochondrial intermembrane space for ADP-channelling into the mitochondria are discussed. (Mol Cell Biochem 133/134. 85-104, 1994)