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ATP-dependent protease in bovine adrenal cortex. Tissue specificity, subcellular localization, and partial
Abstract:
Proteolytic activities in bovine adrenocortical mitochondria were investigated using [14C-methyl]casein as a substrate. Washed mitochondria showed a low proteolytic activity at pH 7.5 or 8.2. ATP (5 mM) plus MgCl2 (7.5 mM) stimulated the proteolysis 9 times at pH 8.2. It was further demonstrated unequivocally by various approaches that the ATP-dependent proteolytic activity localizes in mitochondrial matrix. The activity of the solubilized protease was sensitive to N-ethylmaleimide, mersalyl acid, phenylmethylsulfonyl fluoride, o-vanadate, m-vanadate, vanadyl sulfate, and quercetin but not by oligomycin and ouabain. The ATP-dependent proteolytic activity was eluted at the position of 650,000 daltons on an Ultrogel AcA 22 column as a single symmetrical peak. The gel-filtered enzyme showed high specificity to ATP. GTP and UTP partially substituted ATP. ADP, AMP, tripolyphosphate, alpha, beta-methylene ATP, and beta, gamma-methylene ATP had little or no stimulating activity. ATP did not stimulate the activity in the absence of MgCl2. We measured ATP-dependent proteolytic activities in mitochondrial fractions from several tissues in rat and bovine. Adrenal cortex was one of the tissues of highest activity. In addition, we investigated the effect of adrenal atrophy on the ATP-dependent protease activity in rat adrenal. The ATP-dependent protease activity/adrenal decreased by dexamethasone treatment. The extent of the decrease was similar to that of cytochrome oxidase and succinate dehydrogenase, but smaller than that of cytochrome P-450.
Insights
Mitochondrial proteolysis in bovine adrenal cortex is significantly stimulated by adenosine triphosphate (ATP) and magnesium ions (MgCl2). This ATP-dependent protease activity is located in the mitochondrial matrix and shows high specificity for ATP.
Area of Science:
- Biochemistry
- Mitochondrial Biology
- Enzymology
Background:
- Mitochondria possess proteolytic enzymes crucial for protein turnover.
- The role and regulation of mitochondrial proteases, particularly ATP-dependent ones, are not fully understood.
Purpose of the Study:
- To investigate ATP-dependent proteolytic activities within bovine adrenocortical mitochondria.
- To characterize the localization, properties, and specificity of this enzymatic activity.
Main Methods:
- Utilized [14C-methyl]casein as a substrate to measure proteolytic activity.
- Employed differential centrifugation, gel filtration chromatography, and enzyme inhibition assays.
- Investigated activity across various tissues and the effect of dexamethasone treatment on rat adrenal glands.
Main Results:
- Washed mitochondria exhibited low basal proteolytic activity, significantly stimulated (9-fold) by ATP and MgCl2 at pH 8.2.
- The ATP-dependent proteolytic activity was localized to the mitochondrial matrix.
- The solubilized protease was sensitive to various inhibitors but showed high specificity for ATP, with GTP and UTP showing partial substitution.
- Adrenal cortex demonstrated high activity, which decreased with dexamethasone-induced adrenal atrophy.
Conclusions:
- Bovine adrenocortical mitochondria possess a distinct ATP-dependent protease localized in the matrix.
- This protease exhibits specific requirements for ATP and MgCl2 and is modulated by physiological conditions like adrenal atrophy.