Function beef heart

The general composition of the complex in all systems studied so far is also universal they always contain two h-type cytochromes, one cytochrome of c type and a high potential Fe-S protein (the Rieske protein, so called after its discoverer in Complex III of the respiratory chain of beef heart mitochondria). In addition to these functions in electron transfer, the h/cj complexes also play a role in energy transduction, since they represent an essential part of the proton translocating apparatus of photosynthetic electron transfer chains. 

Succinate dehydrogenase is structurally and functionally very closely related to the fumarate reductase from E. coli, indeed the catalytic subcomplex SdhAB lacking the membrane anchors can be obtained from beef heart mitochondria and other sources. SdhAB can be adsorbed at a PGE electrode, although it has proven difficult to obtain films that have a sufficiently high coverage to observe non-tumover signals. Even so, some interesting catalytic activity is revealed, as shown in Fig. 4-8. 

Figure 39. Circular dichroism spectra of heavy beef heart mitochondria in KCI as a function of sonication, which causes the membrane aggregates to decrease in size. As the size of the aggregate decreases, there is a progressive increase in the magnitude of the elliptiiaty and a shifting of the location of the negative extremum to 222 nm. These changes are due to particle size-dependent distortions in the CD spectra. Most pronounced is the effect on the magnitude of the bands, including the 222 nm band. Reproduced, with permission, from [105].

Early studies, in vitro, were able to show that unconjugated bilirubin was a potent uncoupler of oxidadve phosphorylation (Day, 1954 Brown and Waters, 1958 Zetterstrom and Emster, 1956). These studies and others were performed in mitochondria from a variety of tissues including beef heart, rat liver, and rat brain. The results were consistent regardless of the tissue used. Biliverdin did not show these toxic effects (Brown and Watas, 1958). A variety of further studies examined the effects of bilirubin on other enzyme systems in vitro, and the results were laigely consistent with the concept that bilirubin affects mitochondrial function Karp, 1979 Schenka- et al., 1986). 

In rat liver most of the cellular mono(ADP-ribosylated) proteins are associated with the mitochondrial fraction (1). Two mono(ADP-ribosyl)ating systems have been described in mitochondria, one in die soluble (matrix) fraction (2, 3), the other in submitochondrial particles (SMP, inverted inner membrane vesicles) (3, 4). The ADP-ribosylated matrix protein has a molecular mass of 100 kDa and appears to consist of two major subunits of equal mass. In SMP of both rat liver (4) and beef heart (3), there is one major acceptor protein for mono(ADP-ribose), which migrates with an apparent molecular mass of 30 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Mono(ADP-ribosylation) of the acceptor protein of beef heart SMP was suggested to occur non-enzymicaUy (3). In rat liver SMP, ADP-ribosylation of the 30 kDa protein most probably occurs at an arginine residue, and is readily reversible in the presence of ATP (4). The characteristics of this ADP-ribosylation reaction, i.e. protein specificity and sensitivity to ATP, together widi the observation that intramitochondrial hydrolysis of NAD(P)+ is accompanied by release of Ca + from mitochondria suggests a functional link between mitochondrial protein ADP-ribosylation and Ca2+ release (5,6). 

Several lines of evidence suggest that ADP-ribosylation in rat liver SMP can be due to nonenzymatic covalent protein modification with free ADP-ribose previously formed by enzymatic hydrolysis of NAD+. A similar reaction sequence has been proposed (3) for the mono(ADP-ribosylation) of a 30 kDa acceptor protein in beef heart SMP. The covalent modification with free ADP-ribose has the same specificity with respect to the acceptor protein as with NAD+. It is not a Schiff base adduct. Although possibly nonenzymatic, the specific mono(ADP-ribosylation) of the 30 kDa protein may fiiUfiU a physiological function in intact mitochondria. 

Nucleoside diphosphate kinases exhibit activity over a wide pH range, but usually have optima at or near pH 7. They require the presence of divalent metals for activity, but have a low specificity in this respect, since Mg +, Mn +, Ca +, Co +, and to a lesser extent, Ni + and Zn +, may satisfy this requirement. Magnesium is evidently the physiological ion serving this enzyme s requirement for a divalent ion. The function of magnesium ion in the nucleoside diphosphate kinase reaction is apparent in the work of Colomb et al. (27), who have shown that for the enzyme from beef heart mitochondria, MgATP, but not free ATP, serves as the phosphate donor. Further, free ADP was shown to be preferred over MgADP as the phosphate acceptor. 

Cardiolipin may function as a hapten and is the active principle of beef heart extracts in the complement fixation reaction with serum from syphilitic patients. 

Cytochrome C oxidase catalyzes the transfer of electrons from cytochrome C to molecular oxygen and is one of the best investigated intrinsic membrane proteins. The beef-heart enzyme can be purified in an almost lipid-free form and can be functionally reconstituted by incorporation into different lipid systems since the natural lipid composition is usually not required for reconstitution of an active enzyme (see Fig. 9). 

Many iron-sulfur centers are known to function in the mitochondrial chain. At least five different iron-sulfur centers have been characterized in beef-heart mitochondrial complex I (Albracht et al, 1977). Complex II contains two to three different iron-sulfur centers (Ohnishi et al, 1974a Beinert et al, 1975), whereas complex III contains one iron-sulfur center (Rieske et al, 1964 Orme-Johnson et al, 1974). Complex II exhibits an ESR signal in the oxidized state (Ohnishi et al, 1974b). The ESR spectra and redox properties are similar to those of the signal of the HiPIP from Chromatiwn vinosum. This signal therefore probably occurs for the [4Fe-4S]1-(i- 2-) cluster. The number of iron atoms per center is not known for the other iron-sulfur centers in the respiratory chain. 

Studies with yeast, heart, and brain have shown that concentrations of intermediates within the glycolytic pathway often follow an oscillating function. Continuous spectrophotometric recording techniques for determining the NAD" /NADH ratio in cell-free extracts first revealed oscillations of the NADH level in these systems. These studies then led to the discovery of glycolytic oscillations in yeast cell and cell-free extracts, beef heart extracts, rat skeletal muscle extracts, and in ascites tumor cells, with concentrations of intermediates varying in the range between 10 and 10 M (Chance et al., 1973). 

The numbers calculated for the apparent Km(ADP) values, determined in the presence of 1 mM MgCl2 from Lineweaver-Burk plots revealed through their linear appearance that the velocity of the reaction was a function of ADP concentrations. This implies only one active absorption site for ADP on the enzyme by the substrate Mg-ADP as it has been concluded for spinach chloroplasts by Murakami and Strotmann (1978). The adenylate kinases were sensitive to AMP which functioned as an product inhibitor, as expressed by the obtained K (AMP) values, and ADP exhibited a substrate inhibition effect indicated by the Ki(ADP) values obtained (Table 2). DAPP likewise functioned as a competitive inhibitor in rather low concentrations (Table 2) in contrast to its action in beef heart mitochondria (Tomasselli and Noda, 1980). 

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