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Biomedical subjects

D E Griffiths

Publications and source records attributed to D E Griffiths.

At least 19 recordsLinked to original sources
PubMed1996

Interaction of dibutyltin-3-hydroxyflavone bromide with the 16 kDa proteolipid indicates the disposition of proton translocation sites of the vacuolar ATPase.

The organotin complex dibutyltin-3-hydroxyflavone bromide [Bu2Sn(of)Br] has been shown to bind to the 16 kDa proteolipid of Nephrops norvegicus, either in the form of the native protein or after heterologous expression in Saccharomyces and assembly into a hybrid vacuolar H(+)-ATPase. Titration of Bu2Sn(of)Br against the 16 kDa proteolipid results in a marked fluorescence enhancement, consistent with binding to a single affinity site on the protein. Vacuolar ATPase-dependent ATP hydrolysis was also inhibited by Bu2Sn(of)Br, with the inhibition constant correlating well with dissociation constants determined for binding of Bu2Sn(of)Br complex to the proteolipid. The fluorescence enhancement produced by interaction of probe with proteolipid can be back-titrated by dicyclohexylcarbodiimide (DCCD), which covalently modifies Glu140 on helix-4 of the polypeptide. Expression of a mutant proteolipid in which Glu140 was changed to a glycine resulted in assembly of a vacuolar ATPase which was inactive in proton pumping and which had reduced ATPase activity. Co-expression studies with this mutant and wild-type proteolipids suggest that proton pumping can only occur in a vacuolar ATPase containing exclusively wild-type proteolipid. The fluorescent enhancement of affinity of Bu2Sn(of)Br for the mutant proteolipid was not significantly altered, with the organotin complex having no effect on residual ATPase activity. Interaction of the probe with mutant proteolipid was unaffected by DCCD. These data suggest an overlap in the binding sites of organotin and DCCD, and have implications for the organization and structure of proton-translocating pathways in the facuolar H(+)-ATPase.

Adenosine Triphosphate↗
PubMed1996

Use of electrospray ionization mass spectrometry and tandem mass spectrometry to study binding of F0 inhibitors to ceroid lipofuscinosis protein, a model system for subunit c of mitochondrial ATP synthase.

Ceroid lipofuscinosis protein (CLP), the major accumulating protein in several forms of ceroid lipofuscinosis, has an amino acid sequence that is identical to that of the F0 subunit c of normal bovine ATP synthase. Electrospray ionization mass spectrometry (ESI-MS) has shown that ovine CLP and normal bovine F0 subunit c are identical, including a 42 mass unit post-translational modification. Although the identity and the location of this modification have not been fully established in both species, CLP can be used as a convenient and a unique source of subunit c for studies of F0 inhibitor interactions by ESI-MS analysis. Analysis of mixtures of CLP incubated with several known F0 inhibitors showed that N, N'-dicyclohexylcarbodiimide and organotins bind covalently to CLP but interactions with oligomycin and venturicidin were not observed. The sulphydryl inhibitors, 2,3-dimethoxy-5-methyl-1,4,-benzoquinone (UQ0) and N-ethyl maleimide (NEM) were also shown to bind covalently to the protein. The binding stoichiometry and the relative rate of reaction were then determined for each inhibitor. Tandem mass spectrometry experiments performed on the [M+5H]5+ ion of the intact CLP and of the complexes UQ0-CLP and NEM-CLP allowed the identification of 80% of the CLP sequence and revealed that UQ0 and NEM are both bound to cysteine-64. This work shows the exceptional utility of ESI-MS in studies of the interaction of CLP with a range of inhibitors which are applicable to studies of the F0 component of ATP synthase.

Adenosine Triphosphatases↗
PubMed1994

Fo membrane domain of ATP synthase from bovine heart mitochondria: purification, subunit composition, and reconstitution with F1-ATPase.

The Fo membrane domain of the F1Fo-ATP synthase complex has been purified from bovine heart mitochondria. The purification procedure involves the removal of peripheral membrane proteins, including F1-ATPase, from submitochondrial particles with guanidine hydrochloride, followed by extraction of Fo and other membrane proteins from the stripped membranes in the presence of the detergent n-dodecyl beta-D-maltoside. Fo was then purified by ion-exchange and dye ligand chromatography in the presence of the same detergent. Approximately 15 mg of pure Fo was recovered from 1.8 g of mitochondrial membrane protein. The purified Fo is a complex of nine different polypeptides. They are subunits a, b, c, d, e, F6, and A6L characterized before in F1Fo-ATPase preparations, and two new hitherto undetected subunits, named f and g. The sequences of subunits f and g have been determined. They are not related significantly to any known protein, but subunit f appears to contain a membrane-spanning alpha-helix. Proteins f and g are also present in approximately stoichiometric amounts in a highly purified preparation of intact F1Fo-ATPase, and so it is concluded that they are authentic subunits of the bovine enzyme with unknown functions. Dibutyltin 3-hydroxyflavone, an inhibitor of F1Fo-ATPase, also binds to the purified Fo in detergent and competes for binding with venturicidin. In the presence of F1 and OSCP, the purified Fo was reassembled into the intact F1Fo-ATPase complex. Therefore, this procedure provides a relatively abundant source of pure and functional Fo that is suitable for structural analysis.

Adenosine Triphosphatases↗
PubMed1992

Organotin-flavone complexes: a new class of fluorescent probes for F1F0ATPase.

Fluorescent 5-coordinate organotin-flavone complexes of 3-hydroxy-flavone (Hof) and 3,5,7,2',4',-pentahydroxyflavone (morin) are good inhibitors of mitochondrial F1F0ATPase but do not inhibit F1-ATPase and they have been examined as possible fluorescent probes of F1F0ATPase. R2SnX (morin) complexes exhibit low fluorescence enhancement on binding to mitochondrial membranes with no displacement by equimolar tributyltin. In contrast R2SnX (of) complexes exhibit high fluorescence enhancement whose extent is variable and is displacable by equimolar tributyltin. Fluorescence enhancement by R2SnX (of) complexes correlates with the ATPase I50 values. Dialkyltin-3-hydroxy flavone, R2SnX(of), complexes act as a new class of fluorescent probes which titrate the F0 segment of F1F0ATPase.

Animals↗
PubMed1991

Characterization of ATP12, a yeast nuclear gene required for the assembly of the mitochondrial F1-ATPase.

Mitochondrial F1-ATPase is an oligomeric enzyme composed of five distinct subunit polypeptides. The alpha and beta subunits make up the bulk of protein mass of F1. In Saccharomyces cerevisiae both subunits are synthesized as precursors with amino-terminal targeting signals that are removed upon translocation of the proteins to the matrix compartment. Recently, two different complementation groups (G13, G57), consisting of yeast nuclear mutants with defective F1, have been described. Biochemical analyses indicate that the mutational block in both groups of mutants affects a critical step needed for the assembly of the alpha and beta subunits into the F1 oligomer after their transport into mitochondria. In this study the ATP12 gene representative of the nuclear respiratory-deficient mutant of S. cerevisiae (pet) complementation group G57 has been cloned and the encoded product partially characterized. The ATP12 reading frame is 975 base pairs long and codes for a protein of Mr = 36,587. The ATP12 protein is not homologous to the subunits of F1 whose sequences are known, nor does it exhibit significant primary structure similarity to any known protein. In vitro import assays indicate that ATP12 protein is synthesized as a precursor approximately 3 kDa larger than the mature protein. The mitochondrial localization of the protein has been confirmed by Western blot analysis of mitochondrial proteins with an antibody against a hybrid protein expressed from a trpE-ATP12 fusion. Fractionation of mitochondria indicates further that the ATP12 protein is either a minor component of the matrix compartment or is weakly bound to the matrix side of the inner membrane. The molecular weight of the native protein, estimated from its sedimentation properties in sucrose gradients, is at least two times larger than the monomer. This suggests that the ATP12 protein is probably part of a larger complex.

Amino Acid Sequence↗
PubMed1991

Changes in amplitude and latency of the P300 component of the auditory evoked potential with sedative and anaesthetic concentrations of nitrous oxide.

The P300 component of the auditory evoked response was recorded from six subjects whilst they listened via headphones to a series of clicks which were interrupted unpredictably by a tone burst. They were instructed to press a button as quickly as possible after hearing the tone whilst breathing first air and then a series of increasing concentrations of nitrous oxide. Both the amplitude and the latency of the P300 changed in a dose-dependent manner with nitrous oxide, as did minimum reaction time. At nitrous oxide concentrations which prevented recall of any events that occurred whilst breathing the gas, four subjects continued to respond to the tone by pressing the button. In three subjects, the P300 wave was still detectable with a nitrous oxide concentration at which the task was no longer performed. These results show that there is retention of the ability to perform a reaction time task when there is a complete loss of recall of the task. There may be some recognition of an auditory stimulus, as manifest by a P300 wave, albeit reduced greatly in amplitude, in the absence of a motor response to it. The P300, therefore, merits investigation as a tool for studying conscious awareness under anaesthesia.

Anesthesia Recovery Period↗
PubMed1988

DNA sequence analysis of the Olir2-76 and Ossr1-92 alleles of the Oli-2 region of the yeast Saccharomyces cerevisiae. Analysis of related amino-acid substitutions and protein-antibiotic interaction.

Petite deletion mapping helped to generate a fine-structure genetic map of the Oli-2 region of the mitochondrial genome of Saccharomyces cerevisiae. Here we report the DNA sequence analysis of the Oli-2 region from two drug-resistant alleles (Olir2-76 and Ossr1-92) which are located in the gene for subunit-6 of mitochondrial ATPase, in agreement with their genetic locations on the mitochondrial genome. An analysis of the corresponding amino-acid substitutions is also presented in the context of protein-antibiotic interactions.

Adenosine Triphosphatases↗