Kinetic studies on the O-methylation of dopamine by human brain membrane-bound catechol O-methyltransferase

Translation in vitro of membrane-bound polyribosomal mRNAs from rat brain has shown several to be developmentally regulated [Hall & Lim (1981) Biochem. J. 196, 327-336]. Here we describe the isolation and characterization of cDNAs corresponding to two such brain mRNAs. One cDNA (M444) hybrid-selected a 0.95 kb mRNA directing the synthesis in vitro of a 21 kDa pI-6.3 polypeptide, which was processed in vitro by microsomal membranes. A second cDNA (M1622) hybridized to a 2.2 kb mRNA directing the synthesis of a 55 kDa pI-5.8 polypeptide. Both mRNAs were specific to membrane-bound polyribosomes. Restriction maps of the corresponding genomic DNA sequences are consistent with both being single copy. The two mRNAs were present in astrocytic and neuronal cultures, but not in liver or spleen or in neuroblastoma or glioma cells. The two mRNAs were differently regulated during brain development. In the developing forebrain there was a gradual and sustained increase in M444 mRNA during the first 3 weeks post partum, whereas M1622 mRNA appeared earlier and showed no further increase after day 10. In the cerebellum the developmental increase in M444 mRNA was biphasic. After a small initial increase there was a decrease in this mRNA at day 10, coincident with high amounts of M1622 mRNA. This was followed by a second, larger, increase in M444 mRNA, when amounts of M1622 mRNA were constant. The contrasting changes in these two mRNAs in the developing cerebellum are of particular interest, since they occur during an intensive period of cell proliferation, migration and altering neural connectivity. As these mRNAs are specific to differentiated neural tissue, they represent useful molecular markers for studying brain differentiation.

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Cryo-EM structure and kinetics reveal electron transfer by 2D diffusion of cytochrome c in the yeast III-IV respiratory supercomplex

10.1101/2020.11.27.401935 ◽

2020 ◽

Author(s):

Agnes Moe ◽

Justin Di Trani ◽

John L. Rubinstein ◽

Peter Brzezinski

Keyword(s):

Electron Transfer ◽

Respiratory Chain ◽

Supramolecular Assembly ◽

Kinetic Studies ◽

Water Soluble ◽

Complex Iii ◽

Surface Patch ◽

Cellular Respiration ◽

Oxidoreductase Activity ◽

Membrane Bound

AbstractEnergy conversion in aerobic organisms involves an electron current from low-potential donors, such as NADH and succinate, to dioxygen through the membrane-bound respiratory chain. Electron transfer is coupled to transmembrane proton transport that maintains the electrochemical proton gradient used to produce ATP and drive other cellular processes. Electrons are transferred between respiratory complexes III and IV (CIII and CIV) by water-soluble cyt. c. In S. cerevisiae and some other organisms, these complexes assemble into larger CIII2CIV1/2 supercomplexes, the functional significance of which has remained enigmatic. In this work, we measured the kinetics of the S. cerevisiae supercomplex’s cyt.c-mediated QH2:O2 oxidoreductase activity under various conditions. The data indicate that the electronic link between CIII and CIV is confined to the surface of the supercomplex. Cryo-EM structures of the supercomplex with cyt. c reveal distinct states where the positively-charged cyt. c is bound either to CIII or CIV, or resides at intermediate positions. Collectively, the structural and kinetic data indicate that cyt. c travels along a negatively-charged surface patch of the supercomplex. Thus, rather than enhancing electron-transfer rates by decreasing the distance cyt. c must diffuse in 3D, formation of the CIII2CIV1/2 supercomplex facilitates electron transfer by 2D diffusion of cyt. c. This mechanism enables the CIII2CIV1/2 supercomplex to increase QH2:O2 oxidoreductase activity and suggests a possible regulatory role for supercomplex formation in the respiratory chain.Significance StatementIn the last steps of food oxidation in living organisms, electrons are transferred to oxygen through the membrane-bound respiratory chain. This electron transfer is mediated by mobile carriers such as membrane-bound quinone and water-soluble cyt. c. The latter transfers electrons from respiratory complex III to IV. In yeast these complexes assemble into III2IV1/2 supercomplexes, but their role has remained enigmatic. This study establishes a functional role for this supramolecular assembly in the mitochondrial membrane. We used cryo-EM and kinetic studies to show that cyt. c shuttles electrons by sliding along the surface of III2IV1/2 (2D diffusion). The structural arrangement into III2IV1/2 supercomplexes suggests a mechanism to regulate cellular respiration.

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Energization of the transport systems for arabinose and comparison with galactose transport in Escherichia coli

Biochemical Journal ◽

10.1042/bj2000611 ◽

1981 ◽

Vol 200 (3) ◽

pp. 611-627 ◽

Cited By ~ 50

Author(s):

K R Daruwalla ◽

A T Paxton ◽

P J Henderson

Keyword(s):

Escherichia Coli ◽

Transport System ◽

Membrane Vesicles ◽

Kinetic Studies ◽

Transport Systems ◽

Protonmotive Force ◽

Alkaline Ph ◽

Ph Change ◽

Intact Cells ◽

Membrane Bound

1. Strains of Escherichia coli were obtained containing either the AraE or the AraF transport system for arabinose. AraE+,AraF- strains effected energized accumulation and displayed an arabinose-evoked alkaline pH change indicative of arabinose-H+ symport. In contrast, AraE-,AraF+ strains accumulated arabinose but did not display H+ symport. 2. The ability of different sugars and their derivatives to elicit sugar-H+ symport in AraE+ strains was examined. Only L-arabinose and D-fucose were good substrates, and arabinose was the only inducer. 3. Membrane vesicles prepared from an AraE+,AraF+ strain accumulated the sugar, energized most efficiently by the respiratory substrates ascorbate + phenazine methosulphate. Addition of arabinose or fucose to an anaerobic suspension of membrane vesicles caused an alkaline pH change indicative or sugar-H+ symport on the membrane-bound transport system. 4. Kinetic studies and the effects of arsenate and uncoupling agents in intact cells and membrane vesicles gave further evidence that AraE is a low-affinity membrane-bound sugar-H+ symport system and that AraF is a binding-protein-dependent high-affinity system that does not require a transmembrane protonmotive force for energization. 5. The interpretation of these results is that arabinose transport into E. coli is energized by an electrochemical gradient of protons (AraE system) or by phosphate bond energy (AraF system). 6. In batch cultures the rates of growth and carbon cell yields on arabinose were lower in AraE-,AraF+ strains than in AraE+,AraF- or AraE+,AraF+ strains. The AraF system was more susceptible to catabolite repression than was the AraE system. 7. The properties of the two transport systems for arabinose are compared with those of the genetically and biochemically distinct transport systems for galactose, GalP and MglP. It appears that AraE is analogous to GalP, and AraF to MglP.

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