Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.3.14 (ATP synthase)
 7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The removal of tightly bound nucleotides from mitochondrial F1-ATPase was found to affect the inhibition by ADP and chemical reactivity toward 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-C1) and sulfhydryl reagents. Preincubation of nucleotide-depleted F1 with 40 microM ADP in the presence of ethylenediaminetetraacetic acid (EDTA) resulted in a 51% inhibition of the steady-state level of ATPase activity whereas only a 25% inhibition was observed for native F1. Both partially inhibited states of the enzyme could be reversed by the subsequent addition of ATP. Measurement of [14C]ADP binding to nucleotide-depleted F1 in the presence of EDTA reveals three equivalent ADP binding sites with a Kd of 0.45 microM, and a fourth site of lower affinity. The sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) were found to inhibit the ATPase activity of nucleotide-depleted F1 but not native F1 or nucleotide-depleted F1 in the presence of ADP or ATP. Polyacrylamide gel electrophoresis of nucleotide-depleted F1 labeled with [14C]NEM gave a 2-fold increase in incorporation into the (alpha + beta) subunits and a 7-fold increase in label in the gamma subunit after 90 min compared to when ADP was present during the reaction. ADP binding to the noncatalytic sites enhanced the rate of inhibition of nucleotide-depleted F1 by NBD-C1 about 2-fold while retarding the subsequent intramolecular transfer from an essential phenol group to an amino group about 2.8-fold. The results suggest a conformational change in F1 caused by changes in nucleotide--protein interaction at the noncatalytic sites.
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PMID:Changes in chemical properties of mitochondrial adenosinetriphosphatase upon removal of tightly bound nucleotides. 622 55

Protein import into mitochondria involves several components of the mitochondrial outer and inner membranes as well as molecular chaperones located inside mitochondria. Here, we have investigated the effect of sulfhydryl group reagents on import of the in vitro transcribed/translated precursor of the F1 beta subunit of the ATP synthase (pF1 beta) into Solanum tuberosum mitochondria. We have used a reducing agent, dithiothreitol (DTT), a membrane-permeant alkylating agent, N-ethylmaleimide (NEM), a non-permeant alkylating agent, 3-(N-maleimidopropionyl)biocytin (MPB), an SH-group specific agent and cross-linker 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) as well as an oxidizing cross-linker, copper sulfate. DTT stimulated the mitochondrial protein import, whereas NEM, MPB, DTNB and Cu2+ were inhibitory. Inhibition by Cu2+ could be reversed by addition of DTT. The efficiency of inhibition was higher in energized mitochondrial than in non-energized. We have dissected the effect of the SH-group reagents on binding, unfolding and transport of the precursor into mitochondria. Our results demonstrated that the inhibitory effect of NEM, DTNB and Cu2+ on the efficiency of import was not due to the interaction of the SH-group reagents with import receptors. Modification of pF1 beta with NEM prior to the import resulted in stimulation of import, whereas DTNB and Cu2+ were inhibitory. NEM, MPB, DTNB and Cu2+ inhibited import of the NEM-modified pF1 beta into intact mitochondria. Import of pF1 beta through a receptor-independent bypass-route as well as import into mitoplasts were sensitive to DTT, NEM, MPB, DTNB and Cu2+ in a similar manner as import into mitochondria. As MPB does not cross the inner membrane, these results indicated that redox and conformational status of SH groups located on the outer surface of the inner mitochondrial membrane were essential for protein import.
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PMID:Mitochondrial protein import: modification of sulfhydryl groups of the inner mitochondrial membrane import machinery in Solanum tuberosum inhibits protein import. 942 1

Active F1-ATPase prepared from pig heart mitochondria can react with about 2 mol of DTNB (5,5'-dithiobis-2-nitrobenzoic acid) or CPDS (6,6'-dithiodinicotinic acid). The reactivity of these thiol reagents decreases if ATP is absent or if F1-ATPase has been submitted to thermal treatment that increases the specific activity without eliminating any contaminating protein. Affinity chromatography on a Sepharose-DTNB column has shown that the thermal treatment of F1-ATPase induces a conformational change of the enzyme that completely prevents it from being retained on the column while the normal active enzyme can be specifically bound to the Sepharose-DTNB column. A comparative study of the thiols of F1-ATPase reacting with CPDS measured by spectrophotometric estimation of the thione released from CPDS and by [14C]CPDS binding to F1-ATPase suggests involvement of a vicinal dithiol in active F1-ATPase. After CPDS reaction, this vicinal dithiol may become an internal disulfide bridge.
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PMID:Vicinal dithiol in pig heart mitochondrial F1-ATPase related to thermal or ATP-dependent conformational changes. 1826 21