Cyclophorase

HistmricaL The TCA-cycle was discovered almost simultaneously in 1937 by Krebs, and by Martins and Knoop. Green coined the term cyclophorase for the total multienzyme complex of the TCA-cycle and the associated respiratory chain. Early studies with tissue suspensions showed that C from Cl of acetate was found oidy in the 4-carboxyl carbon of 2-oxoglutarate, whereas the syimnetiical metabolism of citrate... 

Glutamic acid is oxidized to completion by the kidney and liver cyclophorase suspensions of Green and co-workers. The oxidation proceeds by way of a-ketoglutarate, and the cyclophorase complex contains the enzymes necessary for the aerobic and anaerobic synthesis of glutamate from a-ketoglutarate and ammonia. The enzyme preparation is specific for L-glutamate D-glutamate appears to exert an inhibitory effect on the oxidation of the L-isomer. Phosphate esterification accompanies the oxidation. 

The oxidation of n-glutamate by the cyclophorase system requires the presence of AMP, Mg++, and Pi. Only with a well-aged cyclophorase preparation is there a stimulation of glutamate oxidation by added DPN. The authors state that the n- lutamic acid oxidase of Euler et al. and Dewan is involved, but that there is a difference in the state of the oxidase in the two systems. In the cyclophorase preparation the enzyme is fully conjugated, whereas in the isolated enzyme the protein is dissociated from the pyridine nucleotide. 

Still and co-w orkers were unable to secure the oxidation of L-aspartic acid with rabbit liver cyclophorase preparations, although they discovered the occurrence of an enzyme in these preparations capable of oxidizing D-aspartate. 

The propionate oxidase can be obtained as a saline extract from acetone-dried, washed liver cyclophorase preparation. This enzyme is very labile and loses its activity within several hours after its preparation even at 5 C. or in the frozen state. The material called a-hydroxy acid racemase is found in the first supernatant fluid of cyclophorase preparations of either liver or kidney. The active substance appears to be a protein fairly stable to elevated temperatures, repeated freezing and thawing, and precipitation by acetone and alcohol. 

Glutamic acid as an intermediate is formed from proline by the action of the cyclophorase system in the absence of AMP and Mg++. Under these conditions the oxidation is incomplete, and its accumulation was demonstrated by tests with the glutamic acid decarboxylase of E. coli, and by the action of chloramine-T, which yielded an equivalent amount of succinic acid. In addition to glutamic acid, a bisulfite-binding intermediate is produced in about equal amount, which may be glutamic semialdehyde. 

There is a. ccTtain amount of evi(l(Mic( that tlu semialdehydo of glutamic acid is the intermediate formy the loss of the 5-araino group of ornithine. This evidence is the isolation of what was presumed to be the 2,4-dinitrophenylhydrazone of the semialdehyde upon partial oxidation of proline by the cyclophorase system. The glutamic acid semialdehyde can be expected to condense readily to pyrroline carboxylic acid, and reduction of this compound would yield proline (Fig. 2). 

Glutamic acid is oxidized to completion by the kidney and liver cyclo-phorase suspensions of Green and co-workers (P). The oxidation is associated with aerobic phosphorylation, and requires the presence of adenosine 5 -phosphate (AMP), Mg++, and P. Since aged preparations are stimulated by DPN+ it appears highly probable that cyclophorase preparations contain L-glutamate dehydrogenase and enzymes of the TCA cycle. 

Taggart and Krakaur 174) determined that both L-proline and hydroxyproline were oxidized by the kidney cyclophorase system of Green. Proline was oxidized completely to CO , H2O, and NHa, whereas hy-drox5rproline was only partially oxidized. If AMP and Mg++ were omitted from the medium, glutamic acid was formed as an intermediate, and also a carbonyl compound not further identified. 

II. CYCLOPHORASE-MITOCHONDRIAL SYSTEM 1. Some General Properties of the System... 

The association of an enzymatic unit with a structural unit has posed a problem of nomenclature. When one speaks of an enzyme the concept of activity is tacit. An enzyme without activity is no longer an enzyme. However, a mitochondrion as such is recognizable by microscopic and other criteria whether or not it shows enzymatic activity. The term mitochondrion is a structural term and provides no information about the catalytic properties of the mitochrondrion. The term cyclophorase is a functional term and refers to the enzymatic activities exhibited by normal, intact mitochondria. To overcome the difficulty of naming a unit which has both a structural and functional aspect, the term cyclophor-ase-mitochondrial system, abbreviated C.M. system, has been recom-... 

In cyclophorase-mitochondrial preparations of kidney, propionate is formed from the terminal 3 carbons of straight-chain fatty acids of the odd series. Crotonic, vinylacetic, and a series of 8-keto acids, all of which are perhaps intermediates of j8-oxidation, are completely oxidized. The oxidase apparently does not distinguish between ct and trans forms of A -unsaturated fatty acids or between the n- and L-forms of j8-hydroxy-acids. The manner in which fatty acids are activated in order to initiate their oxidation is discussed in a later section (VII). 

Propionate, identified by the counter-current distribution technique, arises from isobutyric acid in the cyclophorase system of rabbit kidney. A 3-carbon fragment with the properties of propionate is also formed from the isopropyl group of isobutyrate in rats. Thus, for those acids which have ra = 2, 4, 6,. . . , it may be assumed that a 3-carbon fragment similar to propionate arises from the isopropyl group. 

The "Cyclophorase" System. Besides the electron transport chain and the system for ATP production, the mitochondria also contain the citrate cycle. A few of its enzymes may be found in the cytoplasm, but the intact cycle operates only within the mitochondria. Green has called this multi-enzyme system cyclophorase. It is no accident that the citrate cycle and the respiratory chain are in such close proximity, because the citrate cycle can ojierate continuously only when NADH2 is reoxidized through the respiratory chain. 

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