/pdf/polyoxometalates-in-medicine-3giyx6h3ls.pdf

Polyoxometalates in Medicine

Publisher Summary This chapter discusses techniques for the preparation and characterization of mitochondrial and submitochondrial fractions from liver and liver-derived tissues. The chapter represents the most detailed account of preparative procedures available for mitochondria and submitochondrial particles of rat liver. The chapter initially summarizes several methods for isolating rat liver mitochondria. Some of these methods yield type-1 mitochondria (partially purified but intact), and some yield type-2 mitochondria (highly purified). The two most common tests used to determine the quality of mitochondrial and submitochondrial preparations involve an examination of their respiratory characteristics in a small chamber equipped with an oxygen electrode, and an examination of their structural and morphological features under the electron microscope. The chapter presents flow diagrams that include many of the methods to obtain an overall view of a given procedure. The methodology involved in preparing mitochondria for evaluation in the electron microscope is summarized here, together with a few statements about what each step involves and why it is carried out.

Preparation and characterization of mitochondria and submitochondrial particles of rat liver and liver-derived tissues.

Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli.

A simple procedure for the purification of Mg2+-stimulated ATPase of Escherichia coli by fractionation with poly(ethylene glycols) and gel filtration is described. The enzyme restores ATPase-linked reactions to membrane preparations lacking these activities. Five different polypeptides (alpha, beta, gamma, delta, epsilon) are observed in sodium dodecyl sulfate electrophoresis. Freezing in salt solutions splits the enzyme complex into subunits which do not possess any catalytic activity. The presence of different subunits is confirmed by electrophoretic and immunological methods. The active enzyme complex can be reconstituted by decreasing the ionic strength in the dissociated sample. Temperature, pH, protein concentration, and the presence of substrate are each important determinants of the rate and extent of reconstitution. The dissociated enzyme has been separated by ion-exchange chromatography into two major fragments. Fragment IA has a molecular weight of about 100000 and contains the alpha, gamma, and epsilon polypeptides. The minor fragment, IB, has about the same molecular weight but contains, besides alpha, gamma, and epsilon, the delta polypeptide. Fragment II, with a molecular weight of about 52000, appears to be identical with the beta polypeptide. ATPase activity can be reconstituted from fragments IA and II, whereas the capacity of the ATPase to drive energy-dependent processes in depleted membrane vesicles is only restored after incubation of these two fractions with fraction IB, which contains the delta subunit.

ATPase of Escherichia coli: purification, dissociation, and reconstitution of the active complex from the isolated subunits.

Subunit composition, function, and spatial arrangement in the Ca2+-and Mg2+-activated adenosine triphosphatases of Escherichia coli and Salmonella typhimurium.

William A. Catterall

Papers

Adenosine triphosphatase from rat liver mitochondria. III. Subunit composition

Effects of phosphotungstic acid and silicotungstic acid on respiration and integrity of rat liver mitochondria.

Contribution of ATP synthesis from endogenous substrates to the oligomycin-sensitive ADP-ATP exchange activity of rat liver mitoplasts.