Rackers reaction

The mitochondrial complex that carries out ATP synthesis is called ATP synthase or sometimes FjFo-ATPase (for the reverse reaction it catalyzes). ATP synthase was observed in early electron micrographs of submitochondrial particles (prepared by sonication of inner membrane preparations) as round, 8.5-nm-diameter projections or particles on the inner membrane (Figure 21.23). In micrographs of native mitochondria, the projections appear on the matrixfacing surface of the inner membrane. Mild agitation removes the particles from isolated membrane preparations, and the isolated spherical particles catalyze ATP hydrolysis, the reverse reaction of the ATP synthase. Stripped of these particles, the membranes can still carry out electron transfer but cannot synthesize ATP. In one of the first reconstitution experiments with membrane proteins, Efraim Racker showed that adding the particles back to stripped membranes restored electron transfer-dependent ATP synthesis. 

Racker et al. have developed an interesting new combinatorial method for the synthesis of [l,4]oxazepin-7-ones (eg 139, R = Ph) from aldehydes and a-amino alcohols with the Baylis-Hillman reaction being a key step . 

The reversibility of reaction (8) has not been observed as yet (H20, M3, R4) and may be attributed to other systems (M2), such as assumed by Racker (R2), the oxidation of GSH to GSSG being coupled to the reduction of homocystine under control of a transhydrogenase. 

The reaction catalyzed by KDO 8-phosphate synthetase (reaction 2, Scheme 35) was first observed by Levin and Racker9 in extracts from Pseudomonas aeruginosa (see Scheme 1), and later by Ghalambor and Heath29 in extracts from Escherichia coli 0111 B4 and J-5. In the initial experiments of Levin and Racker,135 the fate of D-ribose 5-phosphate in crude bacterial extracts was studied, and the KDO 8-phosphate discovered by the authors is really derived from D-ribose 5-phosphate by three, sequential, enzyme-catalyzed reactions (see Scheme 36). 

These circumstances became apparent to the authors when they attempted to study the formation of KDO 8-phosphate as catalyzed by purified bacterial extracts. These extracts did not catalyze the formation of KDO 8-phosphate from D-ribose 5-phosphate, but required D-arabinose 5-phosphate as the substrate Heath and Ghalambor29 showed that the KDO 8-phosphate synthetase reaction, observed in Pseudomonas extracts by Levin and Racker, is also catalyzed by extracts from Escherichia coli strains 0 111 B4 and J-5. Rick and Osborn136 showed that the KDO 8-phosphate synthetase from a Salmonella typhimurium mutant conditionally defective in cell-wall synthesis had a KM of 6 mM as compared to a KM of 170 pM for the enzyme from wild-type cells. 

The synthesis of ATP is catalyzed by the enzyme ATP synthase (or FiFq-ATP synthase) the Fj portion of this enzyme was first isolated by Racker and coworkers in 1960 [4]. ATP synthase is present in abundance in the membranes of animal mitochondria, plant chloroplasts, bacteria and other organisms. ATP synthesized by our ATP synthase is transported out of mitochondria and used for the function of muscle, brain, nerve, liver and other tissues, for active transport, and for synthesizing myriad compounds needed by the cell. Since the pool of adenosine phosphates in the body is limited, the use of ATP must be continually compensated by its synthesis, and an active person synthesizes his own body weight of ATP every day. The synthesis of ATP is the most prevalent chemical reaction in the body [5]. This is indeed a very important reaction. How exactly does it occur ... 

Figure 8 shows a scheme of the reaction cycle of copper ATPases, assuming that they work by a mechanism analogous to that of Ca - or Na+, K+-ATPases. To pump ions, the enzyme must cycle between a state with a high-affinity copper-binding site accessible from only one side of the membrane and a low-affinity state in which the copper cavity is accessible from the other side of the membrane. The high- and low-affinity forms of P-type ATPases were initially named Ei and E2 by Racker (1980) and for many years these ATPases were called E]E2-ATPases, until they were renamed P-type by Pedersen and Carafoli (1987a). 

It has been shown by Ragan and Racker (36) that phospholipids are necessary for the ubiquinone, but not the ferricyanide, reductase activity of complex I. Thus, removal of about 50% of complex I lipids by extraction with cholate under special conditions resulted in a reversible loss of ubiquinone reductase activity without affecting the ferricyanide reductase activity. More phosphatidylcholine and phosphatidylethanolamine were removed by this procedure than cardiolipin. Readdition of either phosphatidylcholine or phosphatidylethanolamine restored considerable rotenone-sensitive ubiquinone-1 reductase activity, which was further augmented when small amounts of cardiolipin were also added. Using preparations depleted of ubiquinone-10 by pentane extraction, these authors have also shown that enzyme-bound ubiquinone-10 is not necessary for the reduction of added ubliquinone-1 by complex I or the inhibition of this reaction by rotenone. 

Fumarase. Fumarase is determined by measuring the conversion to malate to fumarate. This assay was used in the opposite direction by Racker. The mixture contains, in a volume of 1.3 ml, 8.0 x 10 phosphate buffer (adjusted to pH 7.5), 4.0mM dithiothreitol, 8.0mM sodium malate, and 2 to 25 fxg of protein. The reaction is initiated with malate and is followed at 240 nm. The molar extinction coefficient of fumarate is 2.6 X lO cm /mole. 

That lactic acid is the end product of anaerobic glycolysis in muscle tissue has been known for all of this century (Fig. 1). Cell-free extracts able to catalyze the oxidation of lactate to pyruvate were first obtained in 1932 (5). Warburg (6) and von Euler (7) and their colleagues discovered the above reaction [Eq. (1)] and associated it with the chemical properties of a coenzyme. Racker (8) demonstrated in 1950 that the forward reaction also involved the release of a proton. The first purified enzyme was reported by Straub (9) in 1940, while the first micrographs of LDH crystals were shown by Kubowitz and Ott (10). 

The enzyme transketolase (Racker et al., 1953 Horecker et al., 1953) mediates the removal of carbon atoms 1 and 2 of the ketose phosphate such as fructose-6-phosphate, forming a thiamine pyro-phosphate-glycolaldehyde compound (XII) with the two-carbon piece and producing at the same time an aldose phosphate with two fewer carbon atoms in its chain [Reaction (16)]. 

Venkataraman and Racker 9, 10) subsequently found that a stable transaldolase-dihydroxyacetone complex was formed as an intermediate in the transaldolase-catalyzed reaction this was confirmed by Horecker and his co-workers (4, 11) with crystalline transaldolase. Horecker found that the purified complex had a very high affinity for erythrose-4-P incubation of the complex with 10 Af erythrose-4-P, a concentration which could be attained through the action of transketolase on fructose-6-P, resulted in the ready formation of sedoheptulose-7-P, as shown in Fig. 6-3. The glyceraldehyde-3-P could then react with the transketolase-... 

If the scheme outlined in Fig. 1-21 were applicable to each of the coupling stages, elucidation of the coupling mechanism would be well on its way. Unfortunately, all investigators do not share that optimism, and Racker, who studied the coupling mechanism of mitochondria at the second and third sites of phosphorylation, has emphasized the complexity of the sequence of energy-conserving reactions. 

Kagawa, Y., Kandrach, A., Racker, E. Partial resolution of the enzymes catalyzing oxidative phosphorylation XXVI. Specificity of phospholipids required for energy transfer reactions. J. biol. Chem. 248, 676-684 (1973)... 

Racker to be caused by two pyridine nucleotide dehydrogenases alcohol dehydrogenase and aldehyde dehydrogenase. The product of the oxidation is acetate, and the reaction has not been reversed. A similar... 

E. Racker Have you tried to find out whether glutamylcysteine or glycine inhibits the exchange reaction ... 

E. Racker This lack of inhibition of the exchange reaction would rule out both possibilities you have suggested as mechanisms ... 

E. Racker We didn t measure the whole spectrum but we know that there is a marked interfering reaction at 305 m/x. 

E. Racker It is quite jslear that DPN is not involved directly in the glyoxylase reaction. I tried to point out in the talk that this is of particular interest. Glyoxylase may represent a primitive mechanism of thiol-ester formation which does not involve nucleotide participation and it would therefore be very valuable to know more about the detailed mechanisms. On the other hand, it is quite true that once lactoyl glutathione is formed, secondary nucleotide-linked reactions may take place. 

E. Racker With purified preparation of glyoxalases we obtained the zinc salt of D-lactic acid which was not oxidized by muscle lactic dehydrogenase. I may add t o this point that synthetic preparation of n, L-lactoyl glutathione which Dr. Wieland has sent us was split to completion by glyoxalase II, which means that both d- and L-lactic acid can be formed and therefore the specificity of the over-all reaction must be due to glyoxalase I. 

The biosynthesis of deoxyribose (2-deoxy-j>-erylhro-pentose) has not been demonstrated in plant tissue. Other deoxysugars have been synthesized through the action of the enzyme aldolase working on dihydroxy-acetone phosphate and the appropriate aldehyde. Racker (76) found that Escherichia coli extracts catalyzed the following reaction ... 

---