David N. Parcej

Antibodies specific for distinct Kv subunits unveil a heterooligomeric basis for subtypes of .alpha.-dendrotoxin-sensitive potassium channels in bovine brain

The authentic subunit compositions of neuronal K+ channels purified from bovine brain were analyzed using a monoclonal antibody (mAb 5), reactive exclusively with the Kv1.2 subunit of the latter and polyclonal antibodies specific for fusion proteins containing C-terminal regions of four mammalian Kv proteins. Western blotting of the K+ channels isolated from several brain regions, employing the selective blocker alpha-dendrotoxin (alpha-DTX), revealed the presence in each of four different Kvs. Variable amounts of Kv1.1 and 1.4 subunits were observed in the K+ channels purified from cerebellum, corpus striatum, hippocampus, cerebral cortex, and brain stem; on the other hand, contents of Kv1.6 and 1.2 subunits appeared uniform throughout. Each Kv-specific antibody precipitated a different proportion (anti-Kv1.2 > 1.1 >> 1.6 > 1.4) of the channels detectable with radioiodinated alpha-DTX in every brain region, consistent with a widespread distribution of these oligomeric subtypes. Such heterooligomeric combinations were further documented by the lack of additivity upon their precipitation with a mixture of antibodies to Kv1.1 and Kv1.2; moreover, cross-blotting of the multimers precipitated by mAb 5 showed that they contain all four Kv proteins. Collectively, these findings demonstrate that subtypes of alpha-DTX-susceptible K+ channels are prevalent throughout mammalian brain which are composed of different Kv proteins assembled in complexes, shown previously to also contain auxiliary beta-subunits [Parcej, D. N., Scott, V. E. S., & Dolly, J.O. (1992) Biochemistry 31, 11084-11088].

Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase

Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins.

Structure of Alcohol Oxidase from Pichia pastoris by Cryo-Electron Microscopy.

The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD cofactors. When these yeasts are grown with methanol as the carbon source, AOX forms large crystalline arrays in peroxisomes. We determined the structure of AOX by cryo-electron microscopy at a resolution of 3.4 Å. All residues of the 662-amino acid polypeptide as well as the FAD are well resolved. AOX shows high structural homology to other members of the GMC family of oxidoreductases, which share a conserved FAD binding domain, but have different substrate specificities. The preference of AOX for small alcohols is explained by the presence of conserved bulky aromatic residues near the active site. Compared to the other GMC enzymes, AOX contains a large number of amino acid inserts, the longest being 75 residues. These segments are found at the periphery of the monomer and make extensive inter-subunit contacts which are responsible for the very stable octamer. A short surface helix forms contacts between two octamers, explaining the tendency of AOX to form crystals in the peroxisomes.