Bremsstrahlung production by potassium-40 in muscle

Current evidence favors a cycling receptor model for the import of peroxisomal matrix proteins. The yeast Pex14 protein together with Pex13p and Pex17p form the docking subcomplex at the peroxisomal membrane and interact in this cycle with both soluble import receptors Pex5p and Pex7p. In a first step of a structurefunction analysis of Saccharomyces cerevisiae Pex14p, we mapped its binding sites with both receptors. Using the yeast two-hybrid system and pull-down assays, we showed that Pex5p directly interacts with two separate regions of ScPex14p, amino acid residues 1-58 and 235-308. The latter binding site at the C terminus of ScPex14p overlaps with a binding site of Pex7p at amino acid residues 235-325. The functional assessment of these two binding sites of ScPex14p with the peroxisomal targeting signal receptors indicates that they have distinct roles. Deletion of the N-terminal 58 amino acids caused a partial defect of matrix protein import in pex14⌬ cells expressing the Pex14-(59 -341)-p fragment; however, it did not lead to a pex phenotype. In contrast, truncation of the C-terminal 106 amino acids of ScPex14p completely blocked this process. On the basis of these and other published data, we propose that the C terminus of Pex14p contains the actual docking site and discuss the possibility that the N terminus could be involved in a Pex5p-Pex14p association inside the peroxisomal membrane.

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“…Enzyme Assays-Catalase (EC 1.11.1.6) and cytochrome c oxidase (EC 1.9.3.1) were assayed according to published procedures (25)(26)(27)(28).…”

Section: Methodsmentioning

confidence: 99%

Yeast Pex14p Possesses Two Functionally Distinct Pex5p and One Pex7p Binding Sites

Niederhoff

Meindl-Beinker

Kerssen

et al. 2005

Journal of Biological Chemistry

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“…Mitochondria were purified by the procedure of Graham (40). Mitochondria were resuspended in the storage buffer with two modifications: no potassium phosphate was added, and the solution contained 5% Me 2 SO, which acts as a cryoprotectant (41).…”

Section: Methodsmentioning

confidence: 99%

Glucose 6-Phosphate Release of Wild-type and Mutant Human Brain Hexokinases from Mitochondria

Skaff

Kim

Tsai

et al. 2005

Journal of Biological Chemistry

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One molecule of glucose 6-phosphate inhibits brain hexokinase (HKI) with high affinity by binding to either one of two sites located in distinct halves of the enzyme. In addition to potent inhibition, glucose 6-phosphate releases HKI from the outer leaflet of mitochondria; however, the site of glucose 6-phosphate association responsible for the release of HKI is unclear. The incorporation of a C-terminal polyhistidine tag on HKI facilitates the rapid purification of recombinant enzyme from Escherichia coli. The tagged construct has N-formyl methionine as its first residue and has mitochondrial association properties comparable with native brain hexokinases. Release of wild-type and mutant hexokinases from mitochondria by glucose 6-phosphate follow equilibrium models, which explain the release phenomenon as the repartitioning of ligand-bound HKI between solution and the membrane. Mutations that block the binding of glucose 6-phosphate to the C-terminal half of HKI have little or no effect on the glucose 6-phosphate release. In contrast, mutations that block glucose 6-phosphate binding to the N-terminal half require ϳ7-fold higher concentrations of glucose 6-phosphate for the release of HKI. Results here implicate a primary role for the glucose 6-phosphate binding site at the N-terminal half of HKI in the release mechanism.Brain tissue relies almost exclusively on glucose as its source of energy. Approximately 25% of circulating blood glucose and 20% of consumed oxygen is utilized by the brain, an organ that constitutes less than 2% of the human body mass (1). Although four isozymes of hexokinase (ATP:D-hexose 6-phosphotransferase (2.7

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“…The accuracy of the derived TBK is of course critically dependent on the assumed value of the ratio 40 K:TBK. While other workers have paid careful consideration to the accuracy and reproducibility of their measurement techniques, a review of the literature shows that the assumed 40 K:TBK ratio varies from 27.33 to 31.31 Bq g −1 of potassium (Burkinshaw 1967, Ross and Morris 1968, Havlik 1970, Young et al 1975, Manocha and Mohindra 1976, Holtzman 1977, Graham 1983, HMSO 1985-6, Lykken et al 1987, Lan and Weng 1989, Fenwick et al 1991. This paper presents independent measurements and calculations of this ratio in an attempt to reduce the uncertainty in the accepted figure.…”

Section: Introductionmentioning

confidence: 96%