Rat colon ornithine and arginine metabolism: coordinated effects after proliferative stimuli.

Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway, a highly regulated pathway in which activity increases during rapid growth. Other enzymes also metabolize ornithine, and in hepatomas, rate of growth correlates with decreased activity of these other enzymes, which thus channels more ornithine to polyamine biosynthesis. Ornithine is produced from arginase cleavage of arginine, which also serves as the precursor for nitric oxide production. To study whether short-term coordination of ornithine and arginine metabolism exists in rat colon, ODC, ornithine aminotransferase (OAT), arginase, ornithine, arginine, and polyamine levels were measured after two stimuli (refeeding and/or deoxycholate exposure) known to synergistically induce ODC activity. Increased ODC activity was accompanied by increased putrescine levels, whereas OAT and arginase activity were reduced by either treatment, accompanied by an increase in both arginine and ornithine levels. These results indicate a rapid reciprocal change in ODC, OAT, and arginase activity in response to refeeding or deoxycholate. The accompanying increases in ornithine and arginine concentration are likely to contribute to increased flux through the polyamine and nitric oxide biosynthetic pathways in vivo.

The effect of a low protein diet on the response of rat colonic and hepatic ornithine decarboxylase activity to sodium deoxycholate and thioacetamide treatment.

Adult male Sprague-Dawley rats fed ad libitum AIN-76A diet with 5% casein for 10 days had the same basal level of colonic ornithine decarboxylase (ODC) as rats fed 20% casein, but showed a higher level following induction of the colonic enzyme by sodium deoxycholate. Total colonic ODC activity was more responsive than holo-ODC activity to the regimen of induction, which suggested to us that functional (i.e., holoenzyme) levels of the decarboxylase may not be as sensitive to dietary modulation as total enzyme levels. Contrary to the results observed for the colon, the basal level of hepatic ODC was decreased in rats fed 5% casein, and the level following induction of the hepatic enzyme by thioacetamide was also diminished compared with induced enzyme levels in animals fed 20% casein. Additionally, there was no difference in the degree of response between holoenzyme and total hepatic enzyme to dietary treatment and a regimen of enzyme induction.

Ornithine decarboxylase induction in rat colon: synergistic effects of intrarectal instillation of sodium deoxycholate and starvation-refeeding.

Starvation-refeeding, intrarectal instillation of the suspected colon tumor promoter sodium deoxycholate (NaDOC), and a combination of the treatments were compared for their effects on ornithine decarboxylase (ODC) activity in the colon of male Sprague-Dawley rats. Starvation (48 hours) and refeeding (12 hours) led to a fivefold increase in ODC levels compared to ad libitum-fed controls, while NaDOC instillation led to a threefold rise. The combination of the two treatments gave a synergistic 16-fold increase over controls. The synergism observed in colon may indicate that the two treatments used act via different mechanisms to induce ODC, possibly by an increase in general macromolecular synthesis after starvation-refeeding and a specific increase in ODC synthesis after NaDOC treatment. Since this starvation-refeeding regimen is quite similar to the "starve and gorge" feeding pattern exhibited by pair-fed control animals, the use of pair-fed controls may not be appropriate for examining either ODC levels or processes, such as tumor promotion, which may be linked to ODC levels. The synergistic enhancement of tumor promoter-related ODC induction by a dietary pattern (rather than a dietary component) suggests a new area for investigation of potential nutrition-cancer interactions.

Purification and properties of ornithine decarboxylase from Physarum polycephalum.

Ornithine decarboxylase (EC 4.1.1.17) has been purified 3,500-fold from the plasmodia of Physarum polycephalum. The purified material exhibited a Km for ornithine of 0.6 mM and Vmax of 20 mumol of CO2 formed per min/mg at 30 degrees C (62 mumol/min/mg at 37 degrees C). It migrated as a single protein and activity species on high pressure liquid chromatography (TSK-3000) in 0.15 M NaCl (Mr = 80,000) and in native gels containing 5, 6.5, 8, and 9.5% acrylamide. A single protein band (Mr = 43,000) was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Activity was lost upon incubation with alpha-difluoromethyl[5-14C] ornithine, and the inactivated material appeared as a single Mr = 43,000 14C-band on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels. The decarboxylase activity was specific for ornithine and was pyridoxal-P-dependent. The Km for pyridoxal-P (10 microM) was identical with the Kd for pyridoxal-P binding determined from the quenching of protein fluorescence (lambda ex = 282 nm, lambda em = 350 nm, maximal quenching 81%). Using specific antibody obtained from rabbit hyperimmune serum as a probe, an Mr = 43,000 immunoreactive species was detected on nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels of plasmodial homogenates and all pooled purification fractions, but no higher molecular weight cross-reactive material was detected.

Effects of acute and chronic protein deprivation on ornithine decarboxylase levels in rat liver and colon.

The effects of protein intake on the levels of total and holo-ornithine decarboxylase (ODC) in colon and liver have been examined in male Sprague-Dawley rats fed varying amounts of casein in a defined purified diet. Pair-feeding was used to eliminate effects of food intake per se. In the colon, both total and holo-ODC activity increased in animals fed a low-protein diet (1% casein). In the liver, on the other hand, both total and holo-ODC activity decreased with protein deprivation. The observed changes in both colon and liver appeared to be maximal within several days after institution of the dietary regimen. Refeeding with a high-protein diet (20% casein) led to a dramatic rise in ODC activity in the liver in animals previously fed a low-protein diet. These results may be important in sorting out the effects of diet on the tumorigenic process.

Some effects of indole on the interaction of amino acids with tryptophanase.

Although indole is a potent inhibitor (KI = 0.01 mM) of pyruvate formation from substrates of tryptophanase (EC 4.1.99.1, from Escherichia coli), we could not detect binding of indole to free tryptophanase (KD greater than 1.0 mM). However, indole, skatole, and toluene increased the affinity of tryptophanase for certain inhibitory amino acids. Binding of amino acids with small side chains (e.g. Ala, Gly) was increased, but there was little or no effect on the binding of amino acids with bulky side chains (e.g. norvaline, ethionine). These effects were quantitated by using changes in the absorption spectra of the enzyme . amino acid complexes. Indole decreases the absorbance obtainable at 500 nm for amino acids with small hydrophobic side chains (L-Ala, Gly), increases this absorbance for amino acids with small polar side chains (beta-cyano-L-alanine), and does not change the spectra of tryptophanase complexes with amino acids with bulky side chains, i.e. amino acids whose binding affinities are unaffected by indole. These spectral differences are interpreted in terms of an effect of bound indole (or side chain binding) on the partitioning of the bound amino acid between catalytic forms of the enzyme. The data indicate that substrate-induced conformational changes occur at the enzyme active site that generate a high affinity indole-binding site during catalytic turnover of tryptophanase and are important in the catalytic functioning of the enzyme. These changes also explain reproducible differences in KI values observed previously for amino acids in different assay systems used for steady state kinetic inhibition studies. The optimal conditions for the growth of E. coli for tryptophanase production are outlined, together with a procedure for purification of holotryptophanase.

Essential arginine residues in tryptophanase from Escherichia coli.

Tryptophanase from Escherichia coli B/1t7-A is inactivated by the arginine-specific reagent, phenylglyoxal, in potassium phosphate buffer at pH 7.8 AND 25 degrees. Apo- and holoenzyme are inactivated at the same rate, and inactivation of both is correlated with modification of 2 arginine residues/tryptophanase monomer. Substrate analogs having a carboxyl group protect the holoenzyme against both inactivation and arginine modification but have no effect on the inactivation or modification of the apoenzyme. Phenylglyoxal-modified apotryptophanase retains the capacity to bind the coenzyme, pyridoxal-P, but the spectrum of this reconstituted species differs from that of native holotryptophanase. Neither this reconstituted species nor the phenyglyoxal-modified holoenzyme shows the 500 nm absorption characteristic of the native enzyme when substrates are added. These results demonstrate a requirement for specific arginine residues for substrate binding and are discussed in the context of the known conformational and spectal forms of tryptophanase with regard to a possible role for arginine residues in formation of a catalytically effective enzyme-pyridoxal-P complex.

D-Serine dehydratase from Escherichia coli. Essential arginine residue at the pyridoxal 5'-phosphate binding site.

D-Serine apodehydratase from Escherichia coli is rapidly inactivated by butanedione in K+ borate buffer or by phenylglyoxal in K+ phosphate buffer at pH 8, 25 degrees. Pyridoxal-P protects against the inactivation. Modification of the apoenzyme abolishes its ability to bind the cofactor, pyridoxal-P, but the apparent Km for the substrate, D-serine, is not altered. The concentration dependence of the rate of butanedione inactivation in K+ borate buffer indicates that it is a two-step process with one butanedione bound per molecule of apoenzyme to give an inactive complex; half-maximal rate of inactivation is obtained at 37 mM butanedione. Butanedione inactivation is fully reversed following removal of excess reagent and borate. Similar studies with [14C]phenylglyoxal show that in the presence of pyridoxal-P at least 2 arginine residues may be modified without loss of activity. In the absence of pyridoxal-P modification of a single additional arginine residue results in loss of activity. Results with both inactivating reagents thus demonstrate that a critical arginine residue participates in binding of the coenzyme, pyridoxal-P. The stoichiometry of phenylglyoxal incorporation into the enzyme is different in the presence and absence of borate. Under both conditions incorporated phenylglyoxal is slowly lost on dialysis at neutral pH. A possible explanation of these effects is discussed.

Rabbit liver pyridoxamine (pyridoxine) 5'-phosphate oxidase. Purification and properties.

Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC 1.4.3.5) has been purified 2000-fold from rabbit liver. The enzyme preparation migrates as a single protein and activity band on analytical disc gels containing 4,7, or 9 percent acrylamide, and as a single protein band on sodium dodecyl sulfate acrylamide gels. The oxidase is, therefore, homogeneous by these criteria. The pure enzyme catalyzes the following reactions in the presence of FMN: (See journal for formula). These activities copurify in the ratio of 1:1:1. Apparent K-m values are 10 muM for pyridoxamine-P, 30 muM for pyridoxine-P, and 40 nM for FMN. Apparent K-m values for N-(phosphopyridoxyl)amines range from 3.1 times 10-5 M to 1.6 times 10-3 M. The dissociation constant for FMN binding, determined by quenching of protein fluorescence, is 20 nM. The pH optima for all three types of substrates are broad, with maxima near pH 9. The pH dependence of FMN binding, measured by quenching of flavin fluorescence, has the same shape as the substrate activity profile. The holoenzyme has absorption maxima red-shifted from those of FMN to 380 nm and 448 nm, and exhibits spectral changes typical of flavoproteins upon reduction with dithionite. Its oxidation-reduction potential at pH 7 in phosphate buffer is -0.131 volt. The native enzyme has a molecular weight of 54,000 and is made up of two possibly identical polypeptide chains with molecular weights of 27,000. The applicability of proposed mechanisms of flavin catalysis to this flavoprotein is discussed.