Absence Of Mn2 Research Articles

Hybrid Mn-peroxidase from the ligninolytic fungus Panus tigrinus 8/18. Isolation, substrate specificity, and catalytic cycle.

Increased manganese concentration during submerged cultivation of the ligninolytic white rot fungus Panus tigrinus 8/18 on N-limited mineral medium resulted in the induction of Mn-peroxidase and laccase. The Mn-peroxidase was purified with the purity factor RZ (A(406)/A(280)) = 4.3. The purified enzyme catalyzed H2O2-dependent oxidation of phenol oxidase substrates (aromatic amines, 2,2;-azinobis-(3-ethylbenzthiazolinesulfonic acid), hydroquinone, 2,6-dimethoxyphenol) without Mn2+, which is not typical for the usual Mn-peroxidases. Guaiacol and 2,4,6-trichlorophenol were not oxidized in the absence of Mn2+. Study of absorption spectra of the intermediates of the catalytic cycle revealed that this peroxidase is able to complete the redox cycle, reducing one-electron oxidized intermediate (Compound II) by Mn2+, as well as by an organic substrate (hydroquinone). This means that the enzyme is a "hybrid" Mn-peroxidase, different from the common Mn-peroxidases from ligninolytic fungi.

Degradation of humic acids by the litter-decomposing basidiomycete Collybia dryophila.

The basidiomycete Collybia dryophila K209, which colonizes forest soil, was found to decompose a natural humic acid isolated from pine-forest litter (LHA) and a synthetic (14)C-labeled humic acid ((14)C-HA) prepared from [U-(14)C]catechol in liquid culture. Degradation resulted in the formation of polar, lower-molecular-mass fulvic acid (FA) and carbon dioxide. HA decomposition was considerably enhanced in the presence of Mn(2+) (200 microM), leading to 75% conversion of LHA and 50% mineralization of (14)C-HA (compared to 60% and 20%, respectively, in the absence of Mn(2+)). There was a strong indication that manganese peroxidase (MnP), the production of which was noticeably increased in Mn(2+)-supplemented cultures, was responsible for this effect. The enzyme was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. During solid-state cultivation, C. dryophila released substantial amounts of water-soluble FA (predominantly of 0.9 kDa molecular mass) from insoluble litter material. The results indicate that basidiomycetes such as C. dryophila which colonize forest litter and soil are involved in humus turnover by their recycling of high-molecular-mass humic substances. Extracellular MnP seems to be a key enzyme in the conversion process.

Isolation of Bacillus subtilis (chungkookjang), a poly-gamma-glutamate producer with high genetic competence.

A bacterium with high poly-gamma-glutamate (PGA) productivity was isolated from the traditional Korean seasoning, Chung-Kook-Jang. This bacterium could be classified as a Bacillus subtilis, but sporulation in culture was infrequent in the absence of Mn2+. It was judged to be a variety of B. subtilis and designated B. subtilis (chungkookjang). L-Glutamate significantly induced PGA production, and highly elongated PGAs were synthesized. The volumetric yield reached 13.5 mg ml(-1) in the presence of 2% L-glutamate. The D-glutamate content was over 50% in every PGA produced under the conditions used. During PGA production, glutamate racemase activity was found in the cells, suggesting that the enzyme is involved in the D-glutamate supply. Molecular sizes of PGAs were changed by the salt concentration in the medium; PGAs with comparatively low molecular masses were produced in culture media containing high concentrations of NaCl. B. subtilis (chungkookjang) harbors no plasmid and is the first B. subtilis strain reported with both naturally high PGA productivity and high genetic competence.

Oxidation of hydroquinones by the versatile ligninolytic peroxidase from Pleurotus eryngii. H2O2 generation and the influence of Mn2+.

Formation of H2O2 during the oxidation of three lignin-derived hydroquinones by the ligninolytic versatile peroxidase (VP), produced by the white-rot fungus Pleurotus eryngii, was investigated. VP can oxidize a wide variety of phenols, including hydroquinones, either directly in a manner similar to horseradish peroxidase (HRP), or indirectly through Mn3+ formed from Mn2+ oxidation, in a manner similar to manganese peroxidase (MnP). From several possible buffers (all pH 5), tartrate buffer was selected to study the oxidation of hydroquinones as it did not support the Mn2+-mediated activity of VP in the absence of exogenous H2O2 (unlike glyoxylate and oxalate buffers). In the absence of Mn2+, efficient hydroquinone oxidation by VP was dependent on exogenous H2O2. Under these conditions, semiquinone radicals produced by VP autoxidized to a certain extent producing superoxide anion radical (O2*-) that spontaneously dismutated to H2O2 and O2. The use of this peroxide by VP produced quinone in an amount greater than equimolar to the initial H2O2 (a quinone/H2O2 molar ratio of 1 was only observed under anaerobic conditions). In the presence of Mn2+, exogenous H2O2 was not required for complete oxidation of hydroquinone by VP. Reaction blanks lacking VP revealed H2O2 production due to a slow conversion of hydroquinone into semiquinone radicals (probably via autooxidation catalysed by trace amounts of free metal ions), followed by O2*- production through semiquinone autooxidation and O2*- reduction by Mn2+. This peroxide was used by VP to oxidize hydroquinone that was mainly carried out through Mn2+ oxidation. By comparing the activity of VP to that of MnP and HRP, it was found that the ability of VP and MnP to oxidize Mn2+ greatly increased hydroquinone oxidation efficiency.

Ligninolytic enzyme production in selected sub-tropical white rot fungi under different culture conditions

Lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase activities in selected sub-tropical white rot fungal species from Zimbabwe were determined. The enzyme activities were assayed at varying concentrations of C, N and Mn2+. Manganese peroxidase and laccase activities were the only expressed activities in the fungi under the culture conditions tested. Trametes species, T. cingulata, T. elegans and T. pocas produced the highest manganese peroxidase activities in a medium containing high carbon and low nitrogen conditions. High nitrogen conditions favoured high manganese peroxidase activity in DSPM95, L. velutinus and Irpex spp. High manganese peroxidase activity was notable for T. versicolor when both carbon and nitrogen in the medium were present at high levels. Laccase production by the isolates was highest under conditions of high nitrogen and those conditions with both nitrogen and carbon at high concentration. Mn2+ concentrations between 11-25 ppm gave the highest manganese peroxidase activity compared to a concentration of 40 ppm or when there was no Mn2+ added. Laccase activity was less influenced by Mn2+ levels. While some laccase activity was produced in the absence of Mn2+, the enzyme levels were higher when Mn2+ was added to the culture medium.

Modulation of integrin signal transduction by ILKAP, a protein phosphatase 2C associating with the integrin-linked kinase, ILK1.

ILKAP, a protein serine/threonine (S/T) phosphatase of the PP2C family, was isolated in a yeast two-hybrid screen baited with integrin-linked kinase, ILK1. Association of ILK1 and ILKAP was independent of the catalytic activity of either partner, as assayed in co-precipitation and two-hybrid experiments. Condi tional expression of ILKAP in HEK 293 cells resulted in selective inhibition of ECM- and growth factor-stimulated ILK1 activity, but did not inhibit Raf-1 kinase activity. A catalytic mutant of ILKAP, H154D, did not inhibit ILK1 kinase activity. Two cellular targets of ILK1, glycogen synthase kinase 3 beta (GSK3beta) and protein kinase B (PKB)/AKT, were differentially affected by ILKAP-mediated inhibition of ILK1. Catalytically active, but not mutant ILKAP, strongly inhibited insulin-like growth factor-1-stimulated GSK3beta phosphorylation on Ser9, but did not affect phosphorylation of PKB on Ser473, suggesting that ILKAP selectively affects ILK-mediated GSK3beta signalling. Consistent with this, active, but not H154D mutant or the related PP2Calpha, selectively inhibited transactivation of a Tcf/Lef reporter gene, TOPFlash, in 293 cells. We propose that ILKAP regulates ILK1 activity, targeting ILK1 signalling of Wnt pathway components via modulation of GSK3beta phosphorylation.

Urea is a product of ureidoglycolate degradation in chickpea. Purification and characterization of the ureidoglycolate urea-lyase.

A ureidoglycolate-degrading activity was analyzed in different organs of chickpea (Cicer arietinum). Activity was detected in all the tissues analyzed, but highest levels of specific activity were found in pods, from which it has been purified and characterized. This is the first ureidoglycolate-degrading activity that has been purified to homogeneity from any photosynthetic organism. Only one ureidoglycolate-degrading activity was found during the purification. The enzyme was purified 1,500-fold, and specific activity for the pure enzyme was 8.6 units mg(-1), which corresponds with a turnover number of 1,600 min(-1). The native enzyme has a molecular mass of 180 kD and consists of six identical or similar-sized subunits of 31 kD each. The enzyme exhibited hyperbolic, Michaelian kinetics for (-) ureidoglycolate with K(m) values of 6 and 10 microM in the presence or absence of Mn(2+), respectively. Optimum pH was between 7 and 8 and maximum activity was found at temperatures above 70 degrees C, the enzyme being extremely stable and resistant to heat denaturation. The activity was inhibited by EDTA and enhanced by several bivalent cations, thus suggesting that the enzyme is a metalloprotein. This enzyme has been characterized as a ureidoglycolate urea-lyase (EC 4.3.2.3), which catalyzes the degradation of (-) ureidoglycolate to glyoxylate and urea. This is the first time that such an activity is detected in plant tissues. A possible function for this activity and its implications in the context of nitrogen mobilization in legume plants is also discussed.

Oxalic acid production by Aspergillus niger: an oxalate-non-producing mutant produces citric acid at pH 5 and in the presence of manganese.

The external pH appeared to be the main factor governing oxalic acid production by Aspergillus niger. A glucose-oxidase-negative mutant produced substantial amounts of oxalic acid as long as the pH of the culture was 3 or higher. When pH was decreased below 2, no oxalic acid was formed. The activity of oxaloacetate acetylhydrolase (OAH), the enzyme believed to be responsible for oxalate formation in A. niger, correlated with oxalate production. OAH was purified from A. niger and characterized. OAH cleaves oxaloacetate to oxalate and acetate, but A. niger never accumulated any acetate in the culture broth. Since an A. niger acuA mutant, which lacks acetyl-CoA synthase, did produce some acetate, wild-type A. niger is apparently able to catabolize acetate sufficiently fast to prevent its production. An A. niger mutant, prtF28, previously isolated in a screen for strains deficient in extracellular protease expression, was shown here to be oxalate non-producing. The prtF28 mutant lacked OAH, implying that OAH is the only enzyme involved in oxalate production in A. niger. In a traditional citric acid fermentation low pH and absence of Mn2+ are prerequisites. Remarkably, a strain lacking both glucose oxidase (goxC) and OAH (prtF) produced citric acid from sugar substrates in a regular synthetic medium at pH 5 and under these conditions production was completely insensitive to Mn2+.

A conformational switch at the 3' end of a plant virus RNA regulates viral replication.

3' untranslated regions of alfamo- and ilar-virus RNAs fold into a series of stem-loop structures to which the coat protein binds with high affinity. This binding plays a role in initiation of infection ('genome activation') and has been thought to substitute for a tRNA-like structure that is found at the 3' termini of related plant viruses. We propose the existence of an alternative conformation of the 3' ends of alfamo- and ilar-virus RNAs, including a pseudoknot. Based on (i) phylogenetic comparisons, (ii) in vivo and in vitro functional analyses of mutants in which the pseudoknot has been disrupted or restored by compensatory mutations, (iii) competition experiments between coat protein and viral replicase, and (iv) investigation of the effect of magnesium, we demonstrate that this pseudoknot is required for replication of alfalfa mosaic virus. This conformation resembles the tRNA-like structure of the related bromo- and cucumo-viruses. A low but specific interaction with yeast CCA-adding enzyme was found. The existence of two mutually exclusive conformations for the 3' termini of alfamo- and ilar-virus RNAs could enable the virus to switch from translation to replication and vice versa. The role of coat protein in this modulation and in genome activation is discussed.

Phosphoenolpyruvate carboxykinase is involved in the decarboxylation of aspartate in the bundle sheath of maize

We recently showed that maize (Zea mays L.) leaves contain appreciable amounts of phosphoenolpyruvate carboxykinase (PEPCK; R.P. Walker, R.M. Acheson, L.I. Tecsi, R.C. Leegood [1997] Aust J Plant Physiol 24: 459-468). In the present study, we investigated the role of PEPCK in C4 photosynthesis in maize. PEPCK activity and protein were enriched in extracts from bundle-sheath (BS) strands compared with whole-leaf extracts. Decarboxylation of [4-14C]aspartate (Asp) by BS strands was dependent on the presence of 2-oxoglutarate and Mn2+, was stimulated by ATP, was inhibited by the PEPCK-specific inhibitor 3-mercaptopicolinic acid, and was independent of illumination. The principal product of Asp metabolism was phosphoenolpyruvate, whereas pyruvate was a minor product. Decarboxylation of [4-14C]malate was stimulated severalfold by Asp and 3-phosphoglycerate, was only slightly reduced in the absence of Mn2+ or in the presence of 3-mercaptopicolinic acid, and was light dependent. Our data show that decarboxylation of Asp and malate in BS cells of maize occurs via two different pathways: Whereas malate is mainly decarboxylated by NADP-malic enzyme, decarboxylation of Asp is dependent on the activity of PEPCK.

Identification of Two Uridine Binding Domain Peptides of the UDP-Glucose-Binding Site of Rabbit Muscle Glycogenin

Glycogenin, the autoglucosyltransferase that initiates thede novobiosynthesis of glycogen, photoaffinity labeled with [β32P]5-azido-UDP-glucose. The photoinsertion of the azidouridine derivative showed activating ultraviolet light dependency, saturation effects, and inhibition by UDP-glucose, thus demonstrating the specificity of the interaction. In the absence of Mn2+, the requirement for the catalytic activity of glycogenin, the photolabeling decreased by 70%. Competitive binding experiments indicated that the pyrophosphate or a phosphate was the moiety of UDP-glucose implicated in the strongest interaction at the binding site. Proteolytic digestion of photolabeled glycogenin resulted in the identification of two labeled fragments, 89–143 and 168–233, that carried the uridine binding sites. This is the first report of the region of glycogenin that harbors the UDP-glucose-binding domain.

Light-induced Mn2+ influx in Limulus ventral photoreceptors.

In contrast to insect species, light-activated influx of divalent ions into Limulus ventral photoreceptors has proven difficult to demonstrate. We used the quench of the fluorescent indicator dye, fura-2, to measure Mn2+ influx. Limulus ventral photoreceptors were injected with fura-2 and excited at 360 nm. When the photoreceptors were bathed in 1 mmol.l-1 Mn2+, an approximately 1% per 10 s decline in the fura-2 fluorescence during intervals between 50-ms flashes was taken as a measure of Mn2+ entry in darkness. Fluorescence decline during 10-s flashes was used to monitor Mn2+ entry during the photoresponse. During the 10-s flashes we observed a small rapid decline of the fura-2 fluorescence even in the absence of Mn2+. This reflected a contamination of the fluorescence signal arising from light-induced release of intracellular calcium stores. A subsequent slower decline in fluorescence during the 10-s flash, amounting to approximately 9% per 10 s, was only observed in the presence of extracellular Mn2+ and was attributed to Mn2+ influx. This light-activated influx was not through voltage-gated calcium channels since it persisted under voltage clamp, was not stimulated by depolarizing current injections, nor blocked by NiCl2. Depletion of internal calcium stores by cyclopiazonic acid treatment did not accelerate Mn2+ influx.

Kinetics and Mechanism of the Reactions of Superoxochromium(III) Ion with Biological Thiols

The kinetics of the oxidation of three biological thiols (l-cysteine, glutathione, and dl-penicillamine) to their sulfinic and sulfonic acid derivatives by CrOO2+ in aqueous perchloric acid and in the presence of 2-propanol have been studied spectrophotometrically with the aid of the initial-rates method. The kinetic order of the oxidant is 2, whereas that of the reductant is not defined. The acidity of the medium has a slight effect on the initial rates (acid catalysis for both l-cysteine and dl-penicillamine and base catalysis for glutathione). An increase of the ionic strength leads to a rise of the initial rate for both l-cysteine and dl-penicillamine, whereas the initial rate for glutathione is insensitive to the ionic strength. The reactions are inhibited by both 2-propanol and dissolved O2 and catalyzed by Mn2+, whereas Ce3+ has almost no effect on them. At low 2-propanol concentration and in the absence of Mn2+ the initial rate vs temperature plots have a minimum at around 20 °C, whereas in the presence of either concentrated 2-propanol or Mn2+ the Arrhenius law is fulfilled. A single mechanism is proposed for the three reactions involving a CrOO2+/thiol complex, CrOOH2+, and CrO2+ as intermediates. The bimolecular rate constants for the reactions of the intermediate CrO2+ with l-cysteine and dl-penicillamine at 25.0 °C have been obtained (around 103 M-1 s-1 in both cases). Some kinetic data for the decomposition of CrOO2+ in the absence of thiol are also given.

Solvent Extraction of Water-Soluble Calixarenes and Their Metal Complexes with Trioctylmethylammonium Chloride

Abstract Water-soluble hydroxycalix[n]arene-p-sulfonates, 1nn−(n=4,6 and 8) in an aqueous solution were extracted efficiently with trioctylmethylammonium chloride (capriquat), TMA+Cl−, into chloroform in the presence and absence of Mn2+. The extracted species for 144− was estimated to be the ionic associates, such as 14(TMA)4 at pH 4.6 and 14(TMA)5 at pH 9.1. By the present 144−-TMA+ extraction system, Mn2+ was separated from Mg2+,Al3+, Ca2+, Sc3+, Cr3+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Zr4+, Ln3+ (except Ce3+), Hf4+ and UO22+ at pH 8.0.

The effect of Mn2+ on ethanol production from lactose using Kluyveromyces marxianus IMB3 immobilized in magnetically responsive matrices

The thermotolerant, ethanol producing yeast strain, K. marxianus IMB3 was immobilized in calcium alginate containing magnetically responsive Fe3O4 particles. In these studies the β-galactosidase derived from K. marxianus IMB3 was immobilized onto the Fe3O4 particles prior to inclusion into the alginate matrix. Ethanol production by the immobilized microorganism in the presence of Fe3O4 reached a maximum of 16 g/L on 40 g/L lactose whereas prior immobilization of the enzyme to the particles and inclusion into the alginate matrix increased ethanol production to a maximum concentration of 18 g/L. When Mn2+ was incorporated into fermentations containing the immobilized enzyme in the alginate matrix, ethanol production increased further to a maximum concentration of 20 g/L. In addition, the behaviour of the magnetically responsive biocatalyst containing the co-immobilized enzyme was examined in a batch-fed system in the presence and absence of Mn2+.

Ethanol production at 45°C by alginate-immobilized Kluyveromyces marxianus IMB3 during growth on lactose-containing media

The thermotolerant yeast strain Kluyveromyces marxianus IMB3 was immobilized in calcium alginate and this was used in batch-fed reactor systems to convert lactose (4 g/l) to ethanol. Production of ethanol by the free and immobilized biocatalyst in the presence and absence of Mn2+ was compared. In systems containing the free microorganism in the presence and absence of Mn2+, ethanol increased to a maximum of 8 g/l within 40 hours with no significant difference in production by both systems. Ethanol production by the immobilized system in the absence of Mn2+ increased to a maximum of 13 g/l within 40 hours and then decreased to 9 g/l within 80 hours. Ethanol production by the immobilized system in the presence of Mn2+ increased to 14 g/l within 60 hours and this decreased to 13 g/l at 80 hours. When all systems were re-fed at 80 hours, ethanol production by systems containing the free biocatalyst increased to a maximum of 3 g/l while the immobilized system in the presence of Mn2+ increased to a maximum of 12 g/l. Subsequent experiments involving re-feeding the system at shorter time intervals demonstrated that ethanol production by the immobilized system on lactose-containing media at 45 °C was far superior to ethanol production by the free biocatalyst.

Luminescence of Ca2+‐activated photoprotein obelin initiated by NaOCl and MnCl2

The luminescence of obelin is initiated by NaOCl in a reaction mixture containing no calcium. The addition of Mn2+ enhances the light emission > 300-fold. Sodium azide and histidine, as singlet oxygen quenchers, inhibit NaOCl-activated obelin luminescence in the presence or absence of Mn2+. This suggests that the addition of NaOCl to the mixture causes singlet oxygen formation (stimulated by Mn2+ ions), and singlet oxygen initiates the light-emitting reaction.

Interaction of type IV collagen with the isolated integrins alpha 1 beta 1 and alpha 2 beta 1.

The triple-helical cyanogen-bromide-derived fragment CB3[IV] of collagen IV, located 100 nm from the N-terminus of the molecule, contains the binding sites for the integrins alpha 1 beta 1 and alpha 2 beta 1. To investigate the interaction of these integrins and collagen IV, we performed solid-phase and inhibition assays using as receptor isolated alpha 1 beta 1 and alpha 2 beta 1. The ligands used were the binding-site-bearing trimeric peptide CB3[IV] and its shorter tryptic fragments F1-F4. Using titration curves, in which the binding of soluble receptors to coated ligands and the binding of soluble ligands to coated receptors were analyzed, the binding sites for alpha 1 beta 1 and alpha 2 beta 1 were in different but adjacent areas of CB3[IV]. Triple-helical conformation and distinct primary structures were required for the interaction. Dissociation constants (Kd), for the affinity of integrins for collagen IV, were determined in the 1-nM range in the presence of Mn2+ and Mg2+. In the absence of Mn2+, the Kd values indicated a 30-60-fold decrease in the affinities, which for alpha 2 beta 1 was further reduced by adding Ca2+. In the presence of Ca2+ and Mg2+ the affinity of collagen IV for alpha 1 beta 1 was four-times higher than for alpha 2 beta 1.

Inhibitor-2 functions like a chaperone to fold three expressed isoforms of mammalian protein phosphatase-1 into a conformation with the specificity and regulatory properties of the native enzyme.

Three isoforms of mammalian protein phosphatase-1 (PP1 alpha, PP1 beta and PP1 gamma) were expressed in Escherichia coli and purified to near homogeneity. The activities of all isoforms towards phosphorylase, phosphorylase kinase and myosin and their sensitivities to inhibitor-2 were similar to the native PP1 catalytic subunit (PP1C) isolated from vertebrate tissues. Like PP1C, they each formed a complex with the glycogen-targetting(G) subunit which directs PP1C to glycogen particles in skeletal muscle. However, other properties differed strikingly from native PP1C. The expressed isoforms were 100-600-fold less sensitive to inhibitor-1, 3-5-fold less sensitive to okadaic acid, 5-100-fold less sensitive to microcystin-LR and approximately 20-fold more active in dephosphorylating histone H1 than native PP1C. Although PP1 gamma (like PP1C) was active in the absence of Mn2+, expressed PP1 alpha and PP1 beta were completely dependent on Mn2+ for activity. PP1 beta, like PP1C, interacted with the myofibrillar-targetting(M) complexes from skeletal-muscle and smooth-muscle producing species with enhanced myosin-phosphatase activity, whereas expressed PP1 alpha and PP1 gamma did not. The expressed isoforms of PP1 combined with inhibitor-2 to form an inactive complex (PP1I) that could be reactivated by the glycogen-synthase-kinase-3(GSK3)-catalysed phosphorylation of inhibitor-2. This procedure transformed the properties of all three expressed isoforms to those of native PP1C. Their sensitivities to inhibitor-1, okadaic acid and microcystin-LR were increased greatly, their histone-phosphatase activities decreased and the activities of PP1 alpha and PP1 beta became independent of Mn2+. Furthermore PP1 alpha and PP1 gamma now interacted with the M complexes in a similar manner to PP1 beta and PP1C. Conversely, incubation of native PP1C with 50 mM NaF caused conversion to a Mn(2+)-dependent form with properties similar to those of the expressed isozymes. The G subunit from skeletal muscle or the M complex from smooth muscle could displace PP1C from activated PP1I, but not inactive PP1I, to form G-subunit/PP1C and M-complex/PP1C heterodimeric complexes. Inhibitor-2 was also found to be essential for the reactivation of PP1C from 6 M guanidinium chloride in the absence of Mn2+. Taken together, the results suggest that inhibitor-2 is critical for the correct folding of nascent PP1C polypeptides, that its function is similar to that of a molecular chaperone and that it acts as a cytosolic reservoir of PP1C molecules which can be directed to the required subcellular locations following the synthesis of specific targetting subunits.

New kinetic parameters for rat liver arginase measured at near-physiological steady-state concentrations of arginine and Mn2+.

A cytosolic cell-free system from rat liver containing the last three enzymes of the urea cycle, a number of cofactors and the substrates aspartate and citrulline was shown to synthesize urea at near-physiological rates ranging between 0.40 and 1.25 mumol/min per g of liver. This system was used to determine the kinetic parameters for arginase. With saturating amounts of Mn2+ (30 microM), arginine remained at a steady-state concentration of 5-35 microM depending on the aspartate and citrulline supply. Vmax. at micromolar arginine concentrations was between 1.10 and 1.25 mumol/min per g of liver, the K0.5 (arginine) between 6.0 and 6.5 microM and positive co-operativity was observed (Hill coefficient 2). Omission of Mn2+ caused a significant accumulation of arginine during the incubation, suggesting a regulatory effect of arginase. Under these conditions, Vmax. was 1.10-1.65 mumol/min per g of liver and the Km (arginine) increased up to 14.4-21.1 microM. The apparent Ka for Mn2+ in the presence of physiological concentrations of ATP, Mg2+ and arginine was calculated to be maximally 8 microM. Initial-velocity experiments with millimolar arginine concentrations as the direct substrate gave the following results, which are in good agreement with literature data. In the absence of Mn2+, Vmax. was 71.3 mumol/min per g of liver and the Km (arginine) 1.58 mM. With 30 microM-Mn2+, Vmax. was 69.4 mumol/min per g of liver and the Km (arginine) decreased to 0.94 mM. On the basis of our results, we propose the presence of high-affinity and low-affinity sites for arginine on rat liver arginase and postulate that alterations in arginase activity arising from changes in the concentration of arginine and of the cofactor Mn2+ may contribute to the regulation of ureagenesis in vivo.