Extractability and Intracellular Localisation of Urea Cycle Enzym es from Rat Liver

Abstract

1. Urea cycle enzymes and ornithine ketoacid transaminase were extracted from rat liver using various buffer systems and different procedures for the mechanical disintegration of the cells. Of the buffers tested, phosphate and glutathipne gave optimal results, whereas significant differences were found when other extracting media were used: activities of ornithine ketoacid transaminase and ornithine carbamyl transferase were significantly decreased in triethanolamine-and in tris-[hydroxymethyl]-amiiiomethane (tris)-buffer when compared with phosphate. Arginase activity remains unchanged in phosphate and in tris-buffer, but it decreased significantly in triethanolamine-buffer. With the exception of ornithine ketoacid transaminase, no significant increase of enzyme activities were found when livers were extracted with 0.1 % cetylammonium bromide and distilled water respectively. Carbamylphosphate synthetase from rat liver was inactive in the presence of phosphate ions as was ornithine ketoacid transaminase when glutathione (1 mmol/1 GSH) was added to the extracting medium. With the exception of Carbamylphosphate synthetase, all other enzymes tested showed significantly higher activities in liver homogenates than in 100 000 g supernatants. 2. Optimal extraction of urea cycle enzymes from small specimens (e. g. liver biopsies) could be achieved by disintegrating the tissue in an micro all-glass homogenizer, followed by ultrasonic treatment. The use of cetylammonium bromide and the reextractipn of bioptic specimens could be avoided. In contrast, larger tissue samples (500 mg and more) must be reextracted for the complete elution of urea cycle enzymes. 3. Urea cycle enzymes and ornithine ketoacid transaminase were differentiated according to the intracellular localisation by the method of fractional tissue extraction. It could be demonstrated that ornithine ketoacid transaminase, Carbamylphosphate synthetase and ornithine carbamyl transferase were of mitochondrial origin, whereas arginino'succinate lyase activity was found exclusively in the extramitochondrial cell compartment. Arginase activity could be measured mainly extramitochondrially, most probably bound to subcellular structures. However it could not be excluded that at least a small amount of this enzyme was of mitochondrial origin.

Mitochondrial Factors in the Cell Nucleus

“…Mitochondria were discovered during the nineteenth century. However, their association with biochemical reactions reported previously—the tricarboxylic acid cycle (TCA), fatty acid oxidation, OXPHOS, and part of the urea cycle, occurred a century later, with the development of cell-fractionation techniques [ 1 , 2 ]. Subsequently, the characterization of most biochemical pathways led to the recognition of mitochondria as the organelles housing the above processes, in addition to other key reactions in essential pathways, such as heme-group [ 3 ] and iron-sulphur-cluster syntheses [ 4 ], steroidogenesis [ 5 ], and the one-carbon cycle [ 6 ], which are essential for thymine synthesis from uracil and, therefore, for DNA biosynthesis.…”

Untersuchung sub-mitochondrialer Proteinverteilungen in unbehandelten und apoptotischen humanen Zellen mittels STED-Mikroskopie

“…Hierzu gehört die ß-Oxidation von Fettsäuren, der Harnstoffzyklus, die Ketonund Häm-Biosynthese, die Bildung von Eisen-Schwefel-Zentren und der Aminosäure-Metabolismus (Kennedy und Lehninger, 1949;Craddock und Simpson, 1961;Maier et al, 1974;Scheffler, 1999;Bartlett und Eaton, 2004;Levi und Rovida, 2009;Lill, 2009). Ihre herausragende Rolle besteht aber in der Erzeugung des universellen biologischen Energieäquivalents ATP.…”

Untersuchung der Heterogenität submitochondrialer Proteinverteilungen mit hochauflösender Mikroskopie

“…Mitochondrien sind von entscheidender Bedeutung für die Energiebereitstellung innerhalb eukaryotischer Zellen (McBride et al 2006). Darüber hinaus besitzen sie wichtige Funktionen bei einer Reihe zellulärer Prozesse wie Stoffwechselvorgängen und Ionenhomöostase (Kennedy et al 1950, Artom et al 1951, Birt et al 1960, Craddock et al 1961, Maier et al 1974, Scheffler 2001. Das…”

“…Mitochondrien sind die "Kraftwerke" der Zellen und produzieren einen Großteil des zellulären Energieäquivalents Adenosintriphosphat (ATP) durch die Oxidation energiereicher Kohlenhydratmoleküle. Ferner spielen sie auch eine entscheidende Rolle bei einer Vielzahl weiterer zellulärer Prozesse, wie zum Beispiel der Apoptose (Kroemer 1998), der Ionenhomöostase, den Reaktionen des Fettsäurestoffwechsels (Kennedy et al 1950), den Reaktionen des Harnstoffzyklus (Maier et al 1974), der Biosynthese zellulärer Metabolite (Artom et al 1951, Birt et al 1960, Craddock et al 1961, Scheffler 2001) sowie der Zellalterung (Navarro et al 2007). Mitochondrien liegen in Zellen aller eukaryotischer Lebewesen (außer Metamonada) vor, darunter Tiere, Pflanzen, Algen, Pilze und Protozoen, jedoch nicht in Prokaryoten.…”