The ARG11 gene of Saccharomyces cerevisiae encodes a mitochondrial integral membrane protein required for arginine biosynthesis

Prototype strain MG409 (arg11-1) is a severe arginine bradytroph with greatly reduced ornithine and arginine pools, although all known enzymes required for arginine biosynthesis are functional. To identify the function required for normal arginine production impaired in MG409, we have cloned, sequenced, and performed a first molecular characterization of ARG11. We show that the ARG11 open reading frame encodes a putative 292-residue protein with a predicted molecular mass of 31.5 kDa. Sequence similarities, a tripartite organization, and six potential hydrophobic transmembrane spans suggest that Arg11p belongs to the mitochondrial integral inner membrane carrier family. We have used immuno-Western blotting and hemagglutinin epitope-tagged derivatives of Arg11p, Arg8p (a mitochondrial matrix marker), and Arg3p (a cytosolic marker) to demonstrate that Arg11p is confined to the mitochondria and behaves like an integral membrane protein. A deletion created in ARG11 causes the same arginine-leaky behavior as the original arg11-1 mutation, which yields a premature stop codon at residue 266. Arg11p thus appears to fulfill a partially redundant function requiring its 27 carboxyl-terminal amino acids. As a working hypothesis, we propose that Arg11p participates in the export of matrix-made ornithine into the cytosol.     

Characterization of endothelial cell amino acid transport systems involved in the actions of nitric oxide synthase inhibitors

N omega-Substituted analogues of L-arginine have proven useful as specific inhibitors of nitric oxide formation in various biological systems. In the present study we describe the characteristics of amino acid transporters that mediate uptake of N omega-methyl-L-arginine (L-NMA) and N omega-nitro-L-arginine (L-NNA) into cultured porcine aortic endothelial cells. The transport of L-[14C]NMA showed biphasic kinetics, with Km values of 4 and 368 microM, and was inhibited by L-arginine, L-homoarginine, L-lysine, and L-ornithine but not by L-leucine or L-isoleucine. Similar transport kinetics (Km values of 6 and 609 microM) and substrate specificities were obtained for L-[3H]arginine uptake, indicating that L-arginine and L-NMA are transported by the same system. In contrast to L-arginine and L-NMA transport, uptake of L-[3H]NNA was monophasic (Km = 617 microM) and was inhibited by L-leucine and L-isoleucine but not by L-arginine, L-homoarginine, L-NMA, L-lysine, or L-ornithine. Uptake studies with L-[3H]leucine revealed that the transport of this amino acid occurred in a manner very similar to that of L-[3H]NNA transport, suggesting that the uptake of both compounds may be mediated by the same system. In additional experiments, we determined the effects of L-NMA and L-NNA on the A23187-induced accumulation of intracellular cGMP, to establish to what extent these transport systems are involved in the actions of nitric oxide synthase inhibitors. L-Lysine and L-ornithine, which both inhibited L-NMA uptake, increased the IC50 of L-NMA from 7.8 microM to 57 microM but did not reduce the inhibitory effects of L-NNA. In the presence of L-leucine or L-isoleucine, however, which both inhibited L-NNA uptake, the IC50 of L-NNA was increased from 1.2 microM to 37 microM but the inhibitory actions of L-NMA remained unaffected. These data demonstrate that the endothelial transport systems for L-arginine and L-leucine mediate the biological effects of L-NMA and L-NNA, respectively.     

Arginine activates glycolysis of goat epididymal spermatozoa: an NMR study

The present study explores the mechanism underlying the action of L-arginine on the metabolic activity of spermatozoa. Goat epididymal spermatozoa were incubated with different concentrations of L-arginine to determine its effect on the utilization of glucose, fructose, and pyruvate. NMR techniques have been applied to elucidate the effect of L-arginine, L-lysine, and L-ornithine on the glycolysis of epididymal goat spermatozoa. Whereas 31P NMR has been used to estimate the change of pH in the presence of different concentrations of L-arginine, 13C NMR has been used to estimate the substrate consumption and lactate production. At optimal concentration of L-arginine, the forward metabolic rates have been found to increase by two to three times over control experiments. Arginine is not consumed in these reactions, but acts as an activator. Longitudinal relaxation time (T1) measurements indicate that the guanidino group of L-arginine plays an active role in binding to cells. The amino acid L-lysine is less effective, and L-ornithine is ineffective.     

Macula densa arginine delivery and uptake in the rat regulates glomerular capillary pressure. Effects of salt intake

These studies tested the hypothesis that delivery and/or cellular uptake of L-arginine limits macula densa nitric oxide generation and actions on tubuloglomerular feedback (TGF) during salt restriction. Maximal TGF responses were assessed from reductions in proximal stop flow pressure during loop of Henle (LH) perfusion at 40 nl/min with artificial tubular fluid containing vehicles or drugs. Orthograde LH perfusion of L-arginine (10[-3] M) reduced maximal TGF significantly in rats adapted to low salt (LS: 7.9+/-0.4-6.3+/-0.4 mmHg; P < 0.05), but not high salt (HS: 5.8+/-0.3-5.9+/-0.3; NS). The effects were stereospecific and prevented by coperfusion with NG-methyl-L-arginine. Microperfusion of L-arginine (10[-3] M) into the peritubular capillaries reduced the maximum TGF response more in nephrons of LS than HS rats (deltaTGF: LS, 32+/-6 vs. HS, 13+/-4%; P < 0.05) and restored a TGF response to luminal perfusion of NG-methyl-L-arginine in LS rats. Coperfusion of nephrons with excess L-lysine or L-homoarginine, which compete with L-arginine for system y+ transport, blocked the fall in proximal stopflow pressure produced by orthograde LH perfusion of L-arginine in LS rats. Reabsorption of [3H]arginine by the perfused loop segment was similar in LS (93+/-2%) and HS (94+/-1%) rats. Coperfusion with excess L-arginine, L-lysine, or L-homoarginine, however, reduced [3H]arginine reabsorption significantly (P < 0.05) more in HS rats than in LS rats. In conclusion, blunting of maximal TGF responses in salt-restricted rats by nephron-derived NO is limited by L-arginine availability and cellular uptake via system y+.     

Measurement of maxillary sinus volume using computerized tomographic images

We have investigated the effects of L-arginine, D-arginine and L-lysine on airway smooth muscle responsiveness to spasmogens in vitro. Both L-arginine and D-arginine (100 mM) significantly reduced the contractile potency and maximal contractile response to histamine but not to methacholine or potassium chloride in guinea-pig epithelium-denuded isolated trachea. Similarly, the contractile response to histamine was significantly reduced by L-arginine (100 mM) in rabbit epithelium-denuded isolated bronchus. The amino acid L-lysine (100 mM) failed to significantly alter the contractile potency of histamine in guinea-pig isolated trachea (P > 0.05). In guinea-pig isolated trachea precontracted with histamine, both L-arginine and D-arginine produced a concentration-dependent relaxation which was not significantly altered by epithelium removal or by the presence of the nitric oxide synthase inhibitor, NG-nitro L-arginine methyl ester (L-NAME; 50 microM). Thus, at very high concentrations, arginine exhibit a non-competitive antagonism of histamine-induced contraction of isolated airway preparations that was independent of the generation of nitric oxide and was not dependent on charge. These observations confirm previous studies of cutaneous permeability responses and of contractile responses of guinea-pig isolated ileal smooth muscle. Taken together, the data suggest that high concentrations of arginine can exert an anti-histamine effect.     

Effects of various amino acids on gastric lesions induced by acetylsalicylic acid (ASA) and gastric secretion in pylorus-ligated rats

The simultaneous oral administration of various amino acids such as L-lysine, L-arginine, L-histidine, L-serine and others at 750, 250 or 83.3 mg/kg in pylorus-ligated rats produced a marked prevention of the gastric mucosal damages caused by oral acetylsalicylic acid (ASA) at 100 mg/kg. In regard with L-lysine and L-arginine, it was assumed that these amino acids might inhibit the ASA-induced gastric lesions through neutralization of acid because of the high alkalinity of these amino acids. In addition, the lesser effect of the hydrochoride salts of these amino acids as compared with the free form on ASA-induced gastric lesions was observed. The other effective amino acids markedly prevented the back diffusion of acid in response to ASA, suggesting as one of the possible mechanisms of lesion formation. However, L-cysteine, which exerted insignificant effect on ASA-induced gastric lesions, also prevented the back diffusion of acid even though the Na+ concentration had not returned to the control level.     

The activation of human platelets mediated by two monoclonal antibodies raised against CD9

The inducible human cationic amino acid transporter hCAT-2B was expressed in Xenopus laevis oocytes, and this system was used to test the effect of several NO synthase (NOS) inhibitors and/or L-arginine analogues on L-arginine transport by this y+ carrier. L-NG-Methyl-L-arginine (L-NMA), asymmetrical L-NG, NG-dimethyl-L-arginine (L-ADMA), L-N5-(1-iminoethyl)-ornithine (L-NIO), L-NG-nitro-L-arginine (L-NNA), and L-NG-nitro-L-arginine methyl ester (L-NAME) all inhibited the inducible NOS II extracted from RAW 264.7 macrophages induced with bacterial lipopolysaccharide. L-NMA, L-ADMA, and L-NIO also competed with L-arginine for transport by hCAT-2B, whereas L-NNA and L-NAME did not. The two L-arginine analogues, symmetrical NG, NG-dimethyl-L-arginine (L-SDMA) and alpha-amino-delta-isothioureidovaleric acid (AITV), as well as L-lysine, did not block enzymatic activity of NOS II, but did compete for L-arginine transport mediated by hCAT-2B. L-Lysine and L-SDMA were transported efficiently by hCAT-2B and exchanged against intracellular L-arginine, resulting in an L-arginine depletion of the cells. AITV was a much poorer substrate of hCAT-2B and had only little effect on intracellular L-arginine concentrations. These data indicate that substrate recognition differs markedly between the inducible L-arginine transporter hCAT-2B and the inducible NOS II, with different L-arginine analogues having affinity to only one or both of these proteins.     

Capillary electrophoresis analysis of nitric oxide synthase related metabolites in single identified neurons

Intracellular concentrations of L-citrulline (Cit) and its metabolites are related to nitric oxide synthase (NOS) activity, an enzyme producing the intercellular messenger NO in animal tissues including the nervous system. A capillary electrophoresis system using laser-induced fluorescence detection is described, and methods are developed to monitor the levels of L-arginine (Arg), Cit, and related molecules in identified neurons of the marine slugs, Pleurobranchaea californica and Aplysia californica. The limits of detection for Arg, Cit, L-arginino-succinate, L-ornithine, and L-arginine phosphate range from 50 amol to 17 fmol (5 nM to 17 microM in the neurons under study); these detection limits are significantly lower than actual intracellular levels of the metabolites, allowing the direct assay of single cells. The levels of NOS metabolites in individual neurons varied form 6 (Arg) and 4 mM (Cit) in putative NOS-containing neurons down to < 1 microM (undetectable) levels in many putative NOS-negative cells. The Arg/Cit ratio is independent of cell volume, correlates with NADPH-diaphorase staining, and appears to be a characteristic parameter for the presence of NOS activity in identified neurons.     

L-arginine attenuates platelet reactivity in hypercholesterolemic rabbits

Platelets are capable of producing nitric oxide (NO) through the L-arginine-NO synthase pathway. Acute exposure to supraphysiological concentrations of L-arginine in vitro increases the production of NO by platelets and is associated with an increase in platelet cyclic GMP (cGMP) levels and a reduction in platelet aggregation. The purpose of this study was to determine if chronic oral administration of L-arginine decreases platelet aggregation in hypercholesterolemic animals and to determine if this effect is mediated by the metabolism of L-arginine to NO. Male New Zealand White rabbits were fed normal chow (Con), a 1% cholesterol diet (Chol), or a 1% cholesterol diet supplemented with a sixfold enrichment of dietary L-arginine (Arg) or L-methionine (Met). After 10 weeks, cholesterol levels were equally increased in Chol and Arg animals, whereas plasma arginine levels were doubled in the Arg group. There was no difference in maximum aggregation initiated by ADP (100 mumol/L) between washed platelets from Con, Met, and Chol animals, but aggregation of platelets from Arg animals was significantly decreased (P < .05). In aggregating platelets from Arg animals, cGMP levels were significantly higher than the other groups (P < .05). When platelets were incubated ex vivo with the NO synthase inhibitor NG-monomethyl-L-arginine, the effects of dietary L-arginine were reversed. Chronic dietary supplementation of L-arginine decreases platelet aggregation in hypercholesterolemic rabbits. This effect appears to be due to the metabolism of L-arginine to NO.     

Optimum dietary arginine:lysine ratio for broiler chickens is altered during heat stress in association with changes in intestinal uptake and dietary sodium chloride

1. The effects of varying the dietary arginine:lysine (Arg:Lys) ratio for broiler chickens at thermoneutral and high temperatures was studied in a series of 5 experiments which measured intestinal epithelial transport or evaluated growth and food efficiency with practical diets or diets supplemented with L-arginine free base. 2. The growth studies showed that increasing the Arg:Lys ratio at high temperatures produced consistent improvements in food conversion without any loss in growth. 3. Increasing dietary sodium chloride concentration reduced the Arg:Lys ratio necessary for optimum food conversion. 4. Food conversion responses were improved whether L-arginine free base was used as a dietary supplement in place of an inert filler or practical diets with differing ingredients were used to vary the Arg:Lys ratio. 5. In the presence of an equimolar concentration of lysine the uptake of arginine by the intestinal epithelium of heat-stressed broilers was reduced significantly compared with that of broilers at thermoneutral temperatures. 6. The results indicate that the ideal amino acid balance for broilers varies with ambient temperature.     

Alpha-difluoromethylornithine (DFMO) as a potent arginase activity inhibitor in human colon carcinoma cells

Alpha-difluoromethylornithine (DFMO) is commonly used as a specific ornithine decarboxylase (ODC, EC4.1.1.17) irreversible inhibitor. ODC is the enzyme responsible for polyamine biosynthesis, which has been shown to be strictly necessary for cell proliferation. In HT-29 Glc-/+ cells, L-arginine is the major precursor of these molecules through the sequential actions of arginase, which leads to L-ornithine generation and ODC. L-ornithine, a substrate for ODC, retroinhibits arginase. Since DFMO is an ornithine analogue, we searched for a direct effect of this agent upon arginase. The flux of L-arginine through arginase in intact cells was inhibited by 51+/-11% by 10 mM of DFMO whereas 10 mM of L-valine, a known potent arginase inhibitor, inhibited this flux by 73+/-6%. DFMO equilibrated between extracellular and intercellular spaces and, when used at 10-mM concentration, was without effect on L-arginine net uptake. Measurement of arginase activity in HT-29 cell homogenates with increasing concentrations of DFMO and L-arginine led to an inhibition with a calculated Ki (inhibitory constant) equal to 3.9+/-1.0 mM. L-ornithine was less effective than DFMO in inhibiting arginase activity. Bovine liver arginase, used as another source of the enzyme, was also severely inhibited by DFMO. The inhibitory effect of DFMO upon arginase, one step upstream of the ODC reaction in the metabolic conversion of L-arginine to polyamines, is of potential physiological importance, since it could alter the production of ornithine and thus its metabolism in pathways other than the ODC pathway.     

The efflux of lysine from the basolateral membrane of human cultured intestinal cells (Caco-2) occurs by different mechanisms depending on the extracellular availability of amino acids

The efflux of the nutritionally essential amino acid, L-lysine from the basolateral (BL) membrane was characterized in human cultured intestinal cells (Caco-2) grown and differentiated on permeable filter supports. Cells were loaded by incubating with 3H-lysine from the apical (AP) side in the absence of sodium (substituted with choline) in the BL medium; under these conditions, cells accumulated lysine in the intracellular soluble pool to 10- to 20-fold the extracellular concentration. L-Lysine efflux in the BL medium was then followed, and initial rates of efflux were calculated under different experimental conditions. L-Lysine efflux exhibited a strong energy dependence. The presence of an inwardly directed gradient of sodium or lithium stimulated lysine efflux; ouabain reduced efflux in both sodium- and lithium-containing medium. When zwitterionic or cationic amino acids were added to the BL medium, L-lysine efflux was strongly stimulated. The most efficient trans-stimulating amino acids were L-leucine > L-methionine = L-ornithine = L-arginine. In the presence of trans-stimulating amino acids in the BL medium, L-lysine efflux exhibited energy independence and was not affected by the presence of a sodium gradient. In addition, the sensitivity, of efflux to N-ethylmaleimide was different in the absence or in the presence of amino acids in the BL medium. These results suggest that different mechanisms may operate in the BL efflux of L-lysine from human intestinal epithelial cells, depending on the extracellular availability of other amino acids, to guarantee optimal bioavailability of this essential amino acid both in the postprandial absorptive period and between meals.     

L-arginine depletion inhibits glomerular nitric oxide synthesis and exacerbates rat nephrotoxic nephritis

Nitric oxide (NO) synthesis is induced in glomeruli in glomerulonephritis; its role in the pathogenesis of glomerular injury is unknown. Interpretation of its role using the currently available analogues of L-arginine as in vivo inhibitors of NO is complicated by their lack of specificity for inducible NO synthase (iNOS). As NO synthesis by iNOS depends on extracellular L-arginine, we have here examined effects of L-arginine depletion on glomerular NO synthesis and the course of accelerated nephrotoxic nephritis (NTN). Arginase, which converts L-arginine to urea and L-ornithine, was used to achieve L-arginine depletion. A single dose of i.v. arginase produced complete depletion of plasma arginine for four hours. Two forms of NTN were induced in preimmunised rats by nephrotoxic globulin: (1) the systemic form of the model by intravenous nephrotoxic globulin; or (2) the unilateral form of model by left kidney perfusion with nephrotoxic globulin, which avoids the complications of systemic administration of nephrotoxic globulin. Arginase reduced plasma arginine levels and the synthesis of nitrite (the stable end-product of NO) by NTN glomeruli (95% inhibition). Proteinuria was exacerbated. There was no effect on early (24 hr) leukocyte infiltration. In the systemic form of the model arginine depletion by i.v. arginase increased glomerular thrombosis at 24 hours, and the severity of histological changes at four days, accompanied by systemic hypertension. In the unilateral form of the model, where i.v. arginase did not induce hypertension, there was no increase in thrombosis or histological severity of nephritis. These results show that arginine depletion, which inhibits glomerular NO synthesis in NTN, leads to increased proteinuria. Where injury is severe, or accompanied by systemic hypertension, the disease is further exacerbated by glomerular thrombosis. These results suggest that NO has an important role in limiting acute glomerular injury.     

Fluctuation of the first loop facing the matrix of the mitochondrial ADP/ATP carrier deduced from intermolecular cross-linking of Cys56 residues by bifunctional dimaleimides

The effects of six thiol-specific cross-linker dimaleimides, in which the distance of the two maleimide groups ranged from 7.7 to 16. 8 A, on bovine heart mitochondria were studied at pH 7.2 and 0 degrees C. None of the dimaleimides affected mitochondrial proteins, but they caused significantly specific intermolecular cross-linking of the 30 kDa ADP/ATP carrier in submitochondrial particles. All the cross-links were found to be formed specifically between two Cys56 residues in the first loop facing the matrix, as we observed previously in intermolecular disulfide bridge formation catalyzed by copper o-phenanthroline [Majima, E., Ikawa, K., Takeda, M., Hashimoto, M., Shinohara, Y., and Terada, H. (1995) J.Biol. Chem. 270, 29548-29554]. The dimerization was dependent on the cross-linking span of the dimaleimides, being maximum with the dimaleimide having a span of about 12 A. Cross-linking took place in the m-state carrier, but not in the c-state carrier, and inhibited ADP transport via the ADP/ATP carrier. We suggest that a pair of first loops with Cys56 residues in the dimer form of the m-state carrier fluctuates widely with a most probable distance between them of about 12 A, and that this fluctuation modulates the transport activity of the ADP/ATP carrier.     

Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor

A cDNA encoding a thyrotropin-releasing hormone (TRH) receptor expressed in the pituitary was previously cloned (De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) Biochem. J. 284, 891-899; De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) J. Biol. Chem. 267, 25703-25708; Duthie, S. M., Taylor, P. L., Anderson, J., Cook, J., and Eidne, K. A. (1993) Mol. Cell Endocrinol. 95, R11-R15). We now describe the isolation of a rat cDNA encoding a novel subtype of TRH receptor (termed TRHR2) displaying an overall homology of 50% to the pituitary TRH receptor. Introduction of TRHR2 cDNA in HEK-293 cells resulted in expression of high affinity TRH binding with a different pharmacological profile than the pituitary TRH receptor. De novo expressed receptors were functional and resulted in stimulation of calcium transient as assessed by fluorometric imaging plate reader analysis. The message for TRHR2 was exclusive to central nervous system tissues as judged by Northern blot analysis. Studies of the expression of TRHR-2 message by in situ hybridization revealed a pattern of expression remarkably distinct (present in spinothalamic tract, spinal cord dorsal horn) from that of the pituitary TRH receptor (present in hypothalamus, and ventral horn of the spinal cord, anterior pituitary). Therefore, we have identified a novel, pharmacologically distinct receptor for thyrotropin-releasing hormone that appears to be more restricted to the central nervous system particularly to the sensory neurons of spinothalamic tract and spinal cord dorsal horn, which may account for the sensory antinociceptive actions of TRH.     

Morphological observations on liposomes bearing covalently bound protein: studies with freeze-fracture and cryo electron microscopy and small angle X-ray scattering techniques

HT-29 cells, originating from a human colon carcinoma, can proliferate in standard culture conditions with an absolute requirement for polyamines. The major precursor provided in the culture medium for polyamine biosynthesis is L-arginine. L-Arginine conversion to L-ornithine by arginase is followed by stepwise conversion of this latter amino acid to putrescine, spermidine and spermine. The aim of the present work was to document the consequences of a total inhibition of L-arginine flux through arginase, resulting in a decreased L-ornithine availability, on HT-29 cell proliferation and polyamine metabolism. L-Valine, a known arginase inhibitor, when used at a high concentration, i.e., 100 mM, inhibits L-arginine flux through arginase almost totally. The addition in the culture medium of 100 mM L-valine or 50 mM NaCl used to mimic the L-valine induced increase in medium osmolality both reduced equally cellular growth. Cell viability, protein synthesis or oxidative metabolism measured in isolated cells were unaffected by the L-valine treatment, suggesting that decreased proliferation was not associated with an acute toxic effect of this aminoacid, but was rather due to the increase in the medium osmolality. L-Valine treated cells displayed an altered polyamine metabolism when compared with control cells grown in the absence of the amino acid. After 4 days of treatment with 100 mM L-valine, L-ornithine flux through ornithine decarboxylase was significantly higher as well as putrescine and spermidine cellular uptakes in treated cells. However, the changes in polyamine metabolism led to similar polyamine cell contents in untreated and L-valine treated cells. In conclusion, we propose that the observed alterations of polyamine metabolism may reflect an adaptative response of HT-29 cells to the presence of L-valine which contribute together with the low amount of L-ornithine present in the culture medium to polyamine homeostasis.     

Binding of phenylphosphocholine-carrier conjugates to the combining site of antibodies maintains a conformation of the hapten

The structural basis of the binding of phenylphosphocholine haptens to antibodies was studied. This was done by preparing antibodies and testing binding to conjugates of phenylphosphocholine. The choice of haptens was made in order to evaluate the contribution of the carrier to binding, and its effect on hapten conformation in the active site. Thus, phosphocholine (PC) was diazophenyl-linked to tyrosine or histidine as single amino acid carriers and to tripeptides or octapeptides containing tyrosine or histidine as central amino acids to which PC was attached. Relative affinity was assessed by inhibition enzyme-linked immunosorbent assay (ELISA) and binding constants were determined by fluorescence quenching. Fluorinated haptens were used to determine the kinetics of binding using 19F nuclear magnetic resonance. The transferred nuclear Overhauser effect was used to characterize conformation of the bound hapten. We had previously shown that nitrophenylphosphocholine unlinked to carrier is bound in the active site as a bent structure [Bruderer, U., Peyton, D. H., Barbar, E., Fellman, J. H., & Rittenberg, M. B. (1992) Biochemistry 31, 584-589]. We show here that this same bent conformation is retained in the active site regardless of the neighboring carrier or the conformation of the hapten in the unbound conjugate. The presence of the carrier residues in the bound state does, however, influence affinity.     

Purification and kinetic properties of Mus booduga (Gray) hepatic arginase

Hepatic L-arginase from the Mus booduga (Gray) was purified and its kinetic characteristics were investigated. The enzyme was not adsorbed on DEAE-cellulose, but was retained on CM-cellulose column at pH 7.2. The Michaelis-Menten constant was 8.3 mM for L-arginine and was independent of pH in the range of 7.5-10.5. L-arginine concentrations as high as 0.4 M did not exert substrate inhibition in the pH range 7.4-10.0. Manganese was required at a concentration of 0.05 M for full activation of the enzyme. L-ornithine and L-lysine inhibited the enzyme competitively with inhibitory constants of 1.9 mM and 3.7 mM respectively. Several properties of the L-arginase from Mus booduga clearly identify it as an enzyme similar to ureotelic basic arginases from mammalian liver.     

The effect of amino acids and amino acid derivatives on cell proliferation

The effect of certain amino acids and amino acid derivatives on cell proliferation have been studied in the author's Institute for more than 25 years. The optically active forms of arginine, lysine, aspartic acid and glutamic acid influence the growth of transplantable rat tumors. L-arginine, D-lysine, L-aspartic acid and D-glutamic acid promoted; D-aspartic acid, L-glutamic acid, D-arginine and L-lysine inhibited tumour growth. E-amino trimethyl-lysine (TML) stimulated cell proliferation in various cell systems (bone marrow, small intestine, cultured lymphocytes). When administered simultaneously with high doses of Cyclophosphamide, Vincristin or Doxorubicin to tumour-bearing mice, TML decreased the toxicity of the antitumour drugs, resulting in a higher rate of survivors. L-leucine methyl ester caused cell death of mouse peritoneal macrophages by inducing disruption of macrophages.