Browsing by Subject "Mitochondrial membranes"
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Item Biochemical Characterizations of the Steroidogenic Acute Regulatory (StAR) Protein(Texas Tech University, 1998-05) King, Steven RobertWhile the first step in the production of steroid hormones in the body is the conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage system (CSCC), the rate-limiting step is the delivery of substrate cholesterol from the outer to the inner mitochondrial membrane and the CSCC. Although it has been known for over three decades that this requires the de novo synthesis of a protein(s) in response to trophic hormone stimulation, the identity of this factor has remained unknown. Our laboratory has recently cloned and characterized a candidate mitochondrial protein that can induce steroid production in the absence of stimulation in transfected MA-10 mouse Leydig tumor cells. As a result, it was named the Steroidogenic Acute Regulatory (StAR) protein. In this work, we showed that all previously described forms of StAR can be generated from a single mRNA, the 37 kDa form of StAR is the precursor form, and that upon stimulation, transcription of StAR is upregulated. StAR was demonstrated to be sufficient to induce steroidogenesis in isolated MA-10 cell mitochondria, in a specific and time- and dose-dependent manner. Together with other lines of evidence, we conclude that StAR is the long-sought acute regulator of steroidogenesis. We have further investigated requirements for StAR function. StAR-induced cholesterol transfer was determined to require ATP hydrolysis and a mitochondrial electrochemical gradient in vitro. Consistent with the former requirement, phosphorylation of StAR at serine 194 in mouse and 195 in human was found to be critical for maximal activity. This phosphorylation appears to occur as the precursor form of StAR is processed by the mitochondria to die 32 kDa intermediate. Disruption of either the electrochemical gradient or the potential across the inner mitochondrial membrane in whole cells resulted in inhibition of steroidogenesis induced by StAR as well as mitochondrial import. Therefore, either import of StAR or another factor. such as mitochondrial calcium, is requisite for stimulation of intermembrane cholesterol delivery to the CSCC. Since we find that StAR can induce cholesterol transfer in nonsteroidogenic cells, the mechanism by which StAR acts is probably conserved through different cell types.Item Characterization of mitochondrial C₁-tetrahydrofolate synthase transcript and protein expression in adult and embryonic mammalian tissues and the role of the mitochondrial one-carbon pathway in the cytoplasmic methyl cycle(2008-12) Pike, Schuyler Todd, 1966-; Appling, Dean RamsayIn eukaryotes, folate-dependent one-carbon (1-C) metabolism is composed of two parallel pathways compartmentalized to either the cytoplasm or mitochondria. In each, 1-C units, carried on tetrahydrofolate (THF), are interconverted by four catalytic activities. Serine hydroxymethyltransferase transfers the 3-carbon of serine to THF forming 5,10-methylene-THF which is oxidized in 3 successive steps to formate via the intermediates, 5,10-methenyl-THF and 10-formyl-THF. Because of the redox potential in each compartment, 1-C flux is thought by most authors to be from formate to serine in the cytosol and in the opposite direction in mitochondria. Transport of serine, glycine and formate across the mitochondrial membranes creates a 1-C cycle. All eukaryotes characterized to date contain a cytoplasmic trifunctional C1-THF synthase possessing 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase and 10-formyl-THF synthetase activities which interconvert the catalytic intermediates between 5,10-methylene-THF and formate. However, despite the observation that adult rat liver mitochondria oxidize serine to formate, no known enzymatic activities correlate with those of cytoplasmic C1-THF synthase. In embryos, a bifunctional protein, containing 5,10-methylene-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase, accounts for two of these activities. But the 10-formyl-THF synthetase activity has no associated enzyme in mitochondria. Reported here is the discovery of a monofunctional homolog of C1-THF synthase in mammalian mitochondria. Characterization of the protein confirms mitochondrial localization and 10-formyl-THF synthetase activity. Likewise, the adult human transcript is present and differs in size and tissue distribution from cytosolic C1-THF synthase. In mouse embryos, the temporal expression of the mRNA starts out relatively low and increases as the embryos age. The spatial distribution of the transcript is ubiquitous but with areas of elevated expression corresponding to proliferative regions within the embryo. The temporal expression pattern of the protein and transcript correspond well. However, mitochondrial flux studies and immunoblotting data suggest that mitochondrial C1-THF synthase is not the rate-limiting enzyme in mitochondria, at least during the mid to later stages of embryogenesis. Additionally, studies modulating the expression of mitochondria 1-C proteins demonstrate the likelihood that most cytoplasmic 1-C units are mitochondrially derived.Item Studies on the cytochrome bc1 complexes of purple non-sulfur photosynthetic bacteria(Texas Tech University, 1992-08) Güner, SaadettinNot available