Amino acid-activating enzymes liver

Experimentally, C14-aminoacyl sRNA was incubated with rat liver microsomes or ribosomes, GTP, various fractions obtained from the nonparticulate portion of rat liver homogenates, and buffered salt-sucrose medium in a total volume of approximately 2 ml. (6-10). The C14-aminoacyl sRNA was prepared by the phenol-extraction procedure from the pH 5 amino acid-activating enzymes, fraction of rat liver after incubation with C14-L-amino acids (9, 13). C14-leucyl sRNA (approximately 1000 c.p.m.), having a specific radioactivity of approximately 55,000 c.p.m. per mg. of RNA, and containing a complement of endogenous, unlabeled, bound amino acids, was used in most of these studies. The microsomes were sedimented from the post-mitochondrial supernatant at 104,000 x g (10) and the ribosomes were prepared from them by extraction with deoxycholate (16). 

Many of the amino acids originally tested by Krebs were racemic mixtures. When naturally occurring L-amino acids became available the oxidase was found to be sterically restricted to the unnatural, D series. [D-serine occurs in worms free and as D-phosphoryl lombricine (Ennor, 1959)]. It could not therefore be the enzyme used in the liver to release NH3 in amino acid metabolism. D-amino acid oxidase was shown by Warburg and Christian (1938) to be a flavoprotein with FAD as its prosthetic group. A few years later Green found an L-amino acid oxidase in liver. It was however limited in its specificity for amino acid substrates and not very active—characteristics which again precluded its central role in deamination. 

The high substrate specificity of these plant enzymes is in contrast to the lower specificities of enzymes from animal sources, and from certain microorganisms. The bovine liver enzyme shows activity with a number of 2-0X0 and a-amino acids (Frieden, 1963b), the activity in each case being markedly lower than activities with the true substrates, however. The NADP-linked GDH of Neurospora crassa (Brown ef al., 1974) will also react with several 2-oxo and a-amino acids, and enzymes from bovine liver, Neurospora, and Bacillus thurngiensis (Borris and Aronsen, 1969) also show alanine dehydrogenase activity. 

Secondly, one wants a structure which is a good acceptor for an acyl group but a poor one for a phosphoryl group. This could be an SH, a reactive OH, or the NH of an imidazole or amide group. Among the known vitamins and cofactors there are, of course, several with the necessary structural requirements outlined above. The first to come to mind is CoA, but so far it has not been possible to demonstrate a CoA requirement for amino acid incorporation into mammalian or plant microsomes. Nevertheless this vitamin seems to be essential for the incorporation of amino acids into the proteins of hen oviducts (56) and it does, of course, the job of displacing activated fatty acids from their activating enzymes (170). Vitamin Bi2 also fits the structural requirements and it has, indeed, been claimed to be essential for amino acid activation and subsequent incorporation into rat liver microsomes (i07, 178, 179), but this requirement has not yet been confirmed by other authors (180,181). 

The pig serum amidase is not active upon iS-L-aspartamido-lV-acelyi-D-glucosamine bonds in intact ovalbumin or orasomucoid ( i-acid glycoprotein) or in appropriate derivatives of the dipeptides aspan ne-threonine or threonine-asparagine (hlsddno cl al., 1966), and while the rat liver amidase may be somewhat more tolerant of perhaps another appended amino acid, the enzyme would not attack ovalbumin yoopeptides bearing several amino acid residues. 

This is a group of hormones, mostly steroid in nature, that have not yet been extensively studied in regard to their effects on liver XDH. Results with one (hydrocortisone) are shown in Figure 2. As can be seen, total activity is increased about 3-fold (yet specific activity is only doubled since this hormone causes an increase in liver size). Preliminary results indicate that levels of PNP are unaffected, which distinguishes these effects from those in Group A. Since cortisone is known to cause increased levels of liver tyrosine transaminase (9), coordinate control of XDH and amino acid degrading enzymes is a possibility. 

Beyond pharmaceutical screening activity developed on aminothiazoles derivatives, some studies at the molecular level were performed. Thus 2-aminothiazole was shown to inhibit thiamine biosynthesis (941). Nrridazole (419) affects iron metabohsm (850). The dehydrase for 5-aminolevulinic acid of mouse liver is inhibited by 2-amino-4-(iS-hydroxy-ethyl)thiazole (420) (942) (Scheme 239). l-Phenyl-3-(2-thiazolyl)thiourea (421) is a dopamine fS-hydroxylase inhibitor (943). Compound 422 inhibits the enzyme activity of 3, 5 -nucleotide phosphodiesterase (944). The oxalate salt of 423, an analog of levamisole 424 (945) (Scheme 240),... 

Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. (Note that alanine is the a-amino acid counterpart of the a-keto acid, pyruvate.) Furthermore, liver pyruvate kinase is regulated by covalent modification. Flormones such as glucagon activate a cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme. The phos-phorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher for PEP, so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the pathway (to be described in Chapter 23), instead... 

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