Oxaluric Acid: a Non-Metabolizable Inducer of the Allantoin Degradative Enzymes in Saccharomyces cerevisiae

Saccharomyces cerevisiae degrades allantoin in five steps to ammonia, CO(2), and glyoxylate. Previously we demonstrated that allophanic acid, the last intermediate of the pathway, was required for induction of all five degradative enzymes. The data presented here indicate that oxaluric acid, an allophanate analogue, is capable of serving as a non-metabolizable inducer. Oxaluric acid brings about a high level of induction even in strains lacking urea carboxylase. Induction observed with parabanic acid was found to result from its spontaneous breakdown to oxaluric acid.     

Metabolite compartmentation in Saccharomyces cerevisiae.

Uninduced cultures of Saccharomyces cerevisiae exhibit high basal levels of allantoinase, allantoicase, and ureidoglycolate hydrolase, the enzymes responsible for degrading allantoin to urea. As a result, these activities increase only 4- to 8-fold upon induction, whereas the urea-degrading enzymes, urea carboxylase and allophanate hydrolase, have very low basal levels and routinely increase 30-fold on induction. Differences in the inducibility of these five enzymes were somewhat surprising because they are all part of the same pathway and have the same inducer, allophanate. Our current studies reconcile these observations. S. cerevisiae normally contained up to 1 mM allantoin sequestered in a cellular organelle, most likely the vacuole. Separation of the large amounts of allantoin and the enzymes that degrade it provide the cell with an efficient nitrogen reserve. On starvation, sequestered allantoin likely becomes accessible to these degradative enzymes. Because they are already present at high levels, the fact that their inducer is considerably removed from the input allantoin is of little consequence. This suggests that at times metabolite compartmentation may play an equal role with enzyme induction in the regulation of allantoin metabolism. Metabolism of arginine, another sequestered metabolite, must be controlled both by induction of arginase and compartmentation because arginine serves both as a reserve nitrogen source and a precursor of protein synthesis. The latter function precludes the existence of high basal levels of arginase.     

Isolation and characterization of mutants that produce the allantoin-degrading enzymes constitutively in Saccharomyces cerevisiae.

Degradation of allantoin, allantoate, or urea by Saccharomyces cerevisiae requires the participation of four enzymes and four transport systems. Production of the four enzymes and one of the active transport systems is inducible; allophanate, the last intermediate of the pathway, functions as the inducer. The involvement of allophanate in the expression of five distinct genes suggested that they might be regulated by a common element. This suggestion is now supported by the isolation of a new class of mutants (dal80). Strains possessing lesions in the DAL80 locus produce the five inducible activities at high, constitutive levels. Comparable constitutive levels of activity were also observed in doubly mutant strains (durl dal80) which are unable to synthesize allophanate. This, with the observation that arginase activity remained at its uninduced, basal level in strains mutated at the DAL80 locus, eliminates internal induction as the basis for constitutive enzyme synthesis. Mutations in dal80 are recessive to wild-type alleles. The DAL80 locus has been located and is not linked to any of the structural genes of the allantoin pathway. Synthesis of the five enzymes produced constitutively in dal80-1-containing mutants remains normally sensitive to nitrogen repression even though the dal80-1 mutation is present. From these observations we conclude that production of the allantoin-degrading enzymes is regulated by the DAL80 gene product and that induction and repression of enzyme synthesis can be cleanly separated mutationally.     

Pleiotropic control of five eucaryotic genes by multiple regulatory elements.

We have previously shown that allophanate acts as an inducer for five structural genes whose products participate in the degradation of allantoin by Saccharomyces cerevisiae. This observation led us to hypothesize that these genes might be controlled in common and to test the hypothesis by searching for mutants unable to induce production of the allantoin-degrading enzymes. Such mutants have been found. These strains grew poorly when provided with any of the allantoin pathway intermediates, but used other nitrogen sources normally. The mutations carried in these strains were recessive to wild-type alleles and complemented mutations in all known loci associated with the allantoin pathway. The locus containing the most thoroughly studied mutation (dal81-1) was not fund to be tightly linked to any of the allantoin pathway structural genes. The low basal levels of allantoin pathway enzymes observed in Dal81- strains remained the same whether or not the inducer was present in the growth medium. However, the levels of enzyme increased moderately when mutants were grown on poor nitrogen sources. From these observations, we conclude that dal81 mutant strains possess a defect in the induction of enzyme synthesis; enzyme production due to relief of nitrogen catabolite repression, however, appears normal. The observed epistatic relationships of mutations in the DAL80 and DAL81 loci suggest that their products may possess a reasonable degree of functional independence.     

Oxalurate transport in Saccharomyces cerevisiae.

Oxalurate, the gratuitous inducer of the allantoin degradative enzymes, was taken into the cell by an energy-dependent active transport system with an apparent Km of 1.2 mM. Efflux of previously accumulated oxalurate was rapid, with a half-life of about 2 min. The oxalurate uptake system appears to be both constitutively produced and insensitive to nitrogen catabolite repression. The latter observations suggest that failure of oxalurate to bring about induction of allophanate hydrolase in cultures growing under repressive conditions does not result from inducer exclusion, but rather from repression of dur1,2 gene expression.     

Isolation and properties of the cytochrome B-C1 complex from Rhodopseudomonas sphaeroides

A highly purified and active cytochrome $\text{b}$-$\text{c}$₁ complex has been isolated from the chromatophores of the photosynthetic bacteria $\text{Rhodopseudomonas}\text{sphaeroides}$ R-26, through steps of Triton X-100 solubilization, salt fractionation and calcium phosphate column chromatography. The isolated enzyme complex catalyzes fully antimycin A sensitive oxidation of ubiquinol by cytochrome $\text{c}$ with a turnover number of 1500 per minute at 23° based on cytochrome $\text{c}$₁. It contains 8.3 nmoles of cytochromes $\text{b}$ and $\text{c}$₁ per mg protein and shows four polypeptides in the sodium dodecylsulfate polyacrylamide gel electrophoresis.     

The NAD-linked α-glycerophosphate dehydrogenase of trypanosomes

NAD-linked α-glycerophosphate dehydrogenase plays a key role in the α-glycerophosphate cycle of $\text{Trypanosoma brucei}$. The activity in cell lysates was ample for this role. The enzyme was activated by salts (e.g. MgCl₂ or NaCl); it had a broad pH-optimum for the reduction of dihydroxyacetone phosphate centred at pH 7.4, with an apparent K_m of 0.5 mM; and it was weakly bound to particulate components of cell lysates. The enzyme from $\text{T. vivax}$ was similar to that of $\text{T. brucei}$. These trypanosomal enzymes resemble that of the trypanosomatid $\text{Crithidia fasciculata}$, but are rather different from the enzymes of mammals, birds and insects.     

Release of the "ammonia effect" on three catabolic enzymes by NADP-specific glutamate dehydrogenaseless mutations in Saccharomyces cerevisiae

Mutations which inactivate the NADP-glutamate dehydrogenase (anabolic GDHase) pleiotropically release the ammonia inhibition (NH₄⁺ effect) on a number of distinct catabolic activities. In addition to releasing inhibition on several permeability functions (1), these mutations suppress the NH₄⁺ effect on the synthesis of arginase, urea amidolyase and allantoinase. They do not affect the NH₄⁺ effect on the NAD-glutamate dehydrogenase.Two mechanisms of action of these mutations have to be considered, namely a modification of the process of $\text{induction}$ (such as removal of inducer exclusion) and a suppression of $\text{nitrogen catabolite repression}$.     

A novel in vitro method for demonstrating proestrogens. Metabolism of methoxychlor and o,p'DDT by liver microsomes in the presence of uteri and effects on intracellular distribution of estrogen receptors.

A method for demonstrating proestrogens $\text{in}$$\text{vitro}$ has been developed. The method involves the incubation of the potential proestrogen with liver microsomes and NADPH in the presence of rat uteri, followed by examination of the effects of metabolism of the compound on the distribution of uterine estrogen receptor (R) in the cytosol (R_c) and in the nucleus (R_n). Thus, we examined whether DDT derivatives, which possess estrogenic activity $\text{in}$$\text{vivo}$, exhibit pro-estrogenic properties $\text{in}$$\text{vitro}$. Using this method, it appears that methoxychlor is a proestrogen, since the presence of microsomal enzymatic activity is required for methoxychlor to elicit translocation of uterine R_c into the nucleus, namely, the lowering of R_c and elevation of R_n. By contrast, o,p'DDT was active $\text{per}$$\text{se}$ in translocating R_c and did not require the presence of microsomal enzymes for activity.     

An inducible allantoinase and the specific toxicity of parabanic acid on allantoin utilization in aRhizobium species

ARhizobium sp. (strain NC 92) has been shown to be capable of utilizing uric acid, allantoin, allantoate, urea, and oxaluric acid as sole nitrogen sources. Allantoinase is repressible by NH ₄ ⁺ and inducible by allantoin and, less efficiently, by uric acid, oxaluric acid, and allophanate, but not by urea or parabanic acid. This allantoinase (purified 50-fold to homogeneity) is of 166 Kd M.W., is optimally active at pH 7.5, has a K_m of 4.16 mM and no requirement for sulfhydryl groups or metal ions, and is competitively inhibited by acetohydroxamate (K_i 9 mM). Parabanic acid is nontoxic toRhizobium NC 92 on inorganic N and is highly toxic to growth on allantoin N. Growth inhibition is reversed by supplemented allantoin, and suggestive evidence indicates that NC 92 metabolizes allantoin via the pathway: allantoin allantoate urea NH₃; allophanate is not an intermediate herein. Analysis of allantoinase induction indicates that the mandatory structural requirement is for a free urea moiety in an inducing molecule.     

Evidence for the glycoprotein nature of the inducer of sexuality in Volvox

The inducer of sexuality in $\text{Volvox carteri}$ binds to the saccharide binding site of concanavalin A. Its activity show heterogeneous and relatively low mobility during electrophoresis on sodium dodecyl sulfate polyacrylamide gels. These and other findings support the view that the inducer of sexuality is a glycoprotein.     

Crotoxin effects on Torpedo californica cholinergic excitable vesicles and the role of its phospholipase A activity

The phospholipolytic neurotoxin from $\text{Crotalus}\text{durissus}\text{terrificus}$, crotoxin, is able to produce a dose- and time-dependent block of carbachol-stimulated ²²Na efflux from pre-loaded $\text{Torpedo}\text{californica}$ excitable vesicles. The blocking activity is dependent on calcium and is abolished by chemical modification with p-bromophenacyl bromide. The isolated basic subunit, crotoxin B, produces an identical block, whereas the isolated acidic subunit, crotoxin A, has no detectable effect. Neither crotoxin nor crotoxin B antagonizes the binding of $\text{[}{}^{125}\text{I]-α-bungarotoxin}$ to purified acetylcholine receptor, although, at high concentrations, they antagonize its binding to acetylcholine receptor-rich membrane fragments. Certain phospholipase A₂ enzymes and the fatty acid products of their digestion can mimic the crotoxin action. It is therefore suggested that, although considered a pre-synaptic neurotoxin, crotoxin can have $\text{in}\text{vitro}$ post-synaptic effects, possibly mediated by its endogeneous phospholipase A₂ activity.     

Calmodulin-like activity in the soluble fraction of Escherichia coli

A heat-stable factor with properties similar to those of calmodulin was found in the fraction containing Ca²⁺-dependent cyclic AMP phosphodiesterase of $\text{Escherichia}\text{coli}$. The factor activated such enzymes as cyclic nucleotide phosphodiesterase of bovine brain, (Ca²⁺,Mg²⁺)ATPase of human erythrocyte menbrane and myosin light chain kinase of rabbit myometrium in a Ca²⁺-dependent fashion with an apparent K_a of 5 × 10^?5$\text{M}$. The factor and brain calmodulin had no effect on the phosphodiesterase of $\text{E.}\text{coli}$. It may be concluded that calmodulin or a calmodulin-like protein occurs in prokaryotes.     

Induction of heat shock proteins and thermotolerance by ethanol in Saccharomyces cerevisiae

The temperature sensitivity of $\text{Saccharomyces}\text{cerevisiae}$ and the conditions of moderate heat pretreatment required to induce thermotolerance are established. Ethanol is identified as an inducer of heat shock proteins and an inducer of thermotolerance.     

Genetic expression of aryl hydrocarbon hydroxylase activity in the mouse: Distinction between the “responsive” homozygote and heterozygote at the Ah locus

β-Napththoflavone administration induces certain monooxygenase activities, such as aryl hydrocarbon (benzo[a]pyrene) hydroxylase, and cytochrome P₁-450 formation in the “responsive” C57BL/6 and C3H/He inbred mouse strains, whereas these changes are absent or relatively small in the so-called nonresponsive $\text{DBA}\text{2}$ inbred strain. Dose-response curves—with the use of large numbers of animals of the same age and sex and with either β-naphthoflavone or the much more potent 2,3,7,8-tetrachlorodibenzo-p-dioxin as inducer—reveal a small, but statistically significant, difference in the hydroxylase induction between the $\text{C}\text{57}\text{BL}\text{6}\text{J}$ homozygote and the ($\text{C}\text{57}\text{BL}\text{6}\text{J}$)($\text{DBA}\text{2}\text{J}$)F₁ heterozygote in liver, kidney, bowel, and lung. The (C3H/HeJ)($\text{DBA}\text{2}\text{J}$)F₁ heterozygote displays additive inheritance in each of these same tissues.     

Extracts of interferon-treated cells can inhibit reticulocyte lysate protein synthesis.

Rumen bacteria retained methanogenic activity when stored at ?60° under H₂. This activity, which resides in $\text{Methanobacterium ruminantium}$ and $\text{Methanobacterium mobilis}$, is not lost when the cells are broken, as has been suggested. Unlike in $\text{Methanosarcina barkerii}$ and $\text{Methanobacterium M.o.H.}$, in rumen bacteria methanogenic enzymes are not soluble but readily precipitated at 15,000 g. Methane was synthesized from tetrahydrofolate derivatives but at slower rates than from CO₂. From the data, it was not possible to determine if methyl- and methylene tetrahydrofolate were oxidized to CO₂ prior to reduction to CH₄. In room light, CH₃-B₁₂ was reduced to CH₄ non-enzymatically in the presence of protein. When the reactions were carried out in the dark, very little CH₄ was formed from CH₃-B₁₂ by rumen bacterial enzymes. The cell-free particulate fraction did not require added ATP for methanogenesis but showed an absolute requirement for H₂.     

Glycine activation of PEP carboxylase from monocotyledoneous C4 plants

Phosphoenolpyruvate carboxylase from $\text{Zea}\text{mays}$ leaves was found to be activated by L-glycine and inhibited by maleic acid, but was not affected by the effectors for the bacterial enzymes. The activating effect of L-glycine was observed with all the enzymes from leaves of several monocotyledoneous C₄ plants, while the enzymes from dicotyledoneous C₄ plants and mono- and dicotyledoneous C₃ plants were not activated by L-glycine. Maleic acid inhibited the enzyme activities of all the higher plants tested.     

The induction of allophanate lyase during the vegetative cell cycle in light-synchronized cultures of Chlamydomonas reinhardi

Allophanate lyase can be induced by urea or acetamied 20–40-fold within 4 h in NH₄⁺-deprived cultures of Chlamydomonas reinhardi. In light-synchronized cultures, allophanate lyase induction appeared to be limited to the light phase of the cell cycle, provided that culture samples were induced under ongoing illumination conditions (i.e. light induction of light phase cells and dark induction of dark phase cells). However, when culture samples were induced under constant light conditions this cell cycle pattern was abolished. Light was found to be required for allophanate lyase induction and this was shown to be due, in part, to light requirement for inducer uptake. The relationship between allophanate lyase induction and gametogenesis is discussed.     

Parallel activation of cyclic AMP phosphodiesterase and cyclic AMP-dependent protein kinase in two human gut adenocarcinoma cells (HT 29 and HRT 18) in culture,by vasoactive intestinal peptide (VIP) and other effectors activating the cyclic AMP system

Vasoactive intestinal peptide (VIP), secretin, catecholamines and prostaglandin E₁ (PGE₁) in the presence of a cyclic nucleotide phosphodiesterase inhibitor stimulate the accumulation of cyclic AMP in two colorectal carcinoma cell lines (HT 29 and HRT 18) with subsequent activation of the cyclic AMP-dependent protein kinases. In HT 29 cells incubated without phosphodiesterase inhibitor, 10^?9 M VIP promotes a rapid and specific activation of the low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ cyclic AMP phosphodiesterase (1.7-fold); at 25°C the effect is maintained for more than 15 min, while at 37°C the activity returns to basal value within 15 min. As shown by dose-response studies, VIP is by far the most effective inducer ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 4 · 10}{}^{\mathrm{?10}}\text{M}$) of the cyclic AMP phosphodiesterase activity; partial activation of the enzyme is obtained by 3 · 10^?7 M secretin, 10^?5 M isoproterenol and 10^?5 M PGE₁; PGE₂ and epinephrine are without effect. In HRT 18 cells VIP is less active ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 2 · 10}{}^{\mathrm{?9}}\text{M}$) whereas 10^?6 M PGE₁, 10^?6 M PGE₂ and 10^?5 M epinephrine are potent inducers of the phosphodiesterase activity. The positive cell response to dibutyryl-cyclic AMP further indicates that cyclic AMP is a mediator in the phosphodiesterase activation process. The incubation kinetics and dose response effects of the various agonists on the cyclic AMP-dependent protein kinase activity determined for both cell types in the same conditions show a striking similarity to those of phosphodiesterase. Thus coordinate regulation of both enzymes by cyclic AMP was observed in all incubation conditions.