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Milan Vasak - One of the best experts on this subject based on the ideXlab platform.
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specific reactions of s nitrosothiols with cysteine hydrolases a comparative study between Dimethylargininase 1 and ctp synthetase
Protein Science, 2007Co-Authors: Oliver Braun, Markus Knipp, Serge Chesnov, Milan VasakAbstract:S-Transnitrosation is an important bioregulatory process whereby NO(+) equivalents are transferred between S-nitrosothiols and Cys of target proteins. This reaction proceeds through a common intermediate R-S-N(O(-))-S-R' and it has been proposed that products different from S-nitrosothiols may be formed in protein cavities. Recently, we have reported on the formation of such a product, an N-thiosulfoximide, at the active site of the Cys hydrolase Dimethylargininase-1 (DDAH-1) upon reaction with S-nitroso-l-homocysteine (HcyNO). Here we have addressed the question of whether this novel product can also be formed with the endogenously occurring S-nitrosothiols S-nitroso-l-cysteine (CysNO) and S-nitrosoglutathione (GSNO). Further, to explore the reason responsible for the unique formation of an N-thiosulfoximide in DDAH-1 we have expanded these studies to cytidine triphosphate synthetase (CTPS), which shows a similar active site architecture. ESI-MS and activity measurements showed that the bulky GSNO does not react with both enzymes. In contrast, S-nitrosylation of the active site Cys occurred in DDAH-1 with CysNO and in CTPS with CysNO and HcyNO. Although kinetic analysis indicated that these compounds act as specific irreversible inhibitors, no N-thiosulfoximide was formed. The reasons likely responsible for the absence of the N-thiosulfoximide formation are discussed using molecular models of DDAH-1 and CTPS. In tissue extracts DDAH was inhibited only by HcyNO, with an IC(50) value similar to that of the isolated protein. Biological implications of these studies for the function of both enzymes are discussed.
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Searching for DDAH inhibitors: S-nitroso-L-homocysteine is a chemical lead.
Journal of the American Chemical Society, 2005Co-Authors: Markus Knipp, Oliver Braun, Milan VasakAbstract:The cysteine-hydrolase Dimethylargininase-1 (DDAH-1) is an important regulator of NO production in mammalian tissue for which the availability of an inhibitor for clinics and research would be most appreciated. While studying the effect of the endogenously occurring S-nitroso-l-homocysteine on DDAH-1, an unusual N-thiosulfoximide modification was identified in the active site of the enzyme. Thus, S-nitroso-l-homocysteine in combination with the mechanism proposed herein offers a basis for the rational design of DDAH inhibitors.
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zn ii free Dimethylargininase 1 ddah 1 is inhibited upon specific cys s nitrosylation
Journal of Biological Chemistry, 2003Co-Authors: Markus Knipp, Oliver Braun, Peter Gehrig, Ragna Sack, Milan VasakAbstract:Abstract The endogenous nitric oxide synthase inhibitorsl-N ω-methylarginine andl-N ω,N ω-dimethylarginine are catabolized by the enzyme Dimethylargininase. Dimethylargininase-1 from bovine brain contains one tightly bound Zn(II) coordinated by two cysteine sulfur and two lighter ligands. Activity measurements showed that only the apo-enzyme is active and that the holo-enzyme is activated by zinc removal. In this work, the effect of NO on Dimethylargininase-1 structure and its activity was investigated using 2-(N,N-dimethylamino)-diazenolate-2-oxide as an NO source. The results showed that whereas the holo-form was resistant to S-nitrosylation, the apo-form could be modified. The results of absorption spectroscopy, mass spectrometry, and fluorometric S-NO quantification revealed that two of five cysteine residues reacted with NO yielding cysteine-S-NO. The modification reaction is specific, because by liquid chromatography/mass spectrometry experiments of digested S-NO-Dimethylargininase-1, cysteines 221 and 273 could be identified as cysteine-NO. Because Zn(II) protects the enzyme against nitrosation, it is suggested that both cysteines are involved in metal binding. However, specific cysteine-S-NO formation occurred in the absence of a characteristic sequence motif. Based on a structural model of Dimethylargininase-1, the activation of both cysteines may be accomplished by the close proximity of charged residues in the tertiary structure of the enzyme.
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structural and functional characterization of the zn ii site in Dimethylargininase 1 ddah 1 from bovine brain zn ii release activates ddah 1
Journal of Biological Chemistry, 2001Co-Authors: Markus Knipp, John M Charnock, C D Garner, Milan VasakAbstract:Abstract l-N ω,N ω-Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is a Zn(II)-containing enzyme that, through hydrolysis of side-chain methylated l-arginines, regulates the activity of nitric-oxide synthase. Herein we report the structural and functional properties of the Zn(II)-binding site in DDAH-1 from bovine brain. Activity measurements of the native and metal-free enzyme have revealed that the endogenously bound Zn(II) inhibits the enzyme. Native DDAH-1 could be fully or partially activated using various concentrations of phosphate, imidazole, histidine, and histamine, a process that is paralleled by the release of Zn(II). The slow activation of the enzyme by the bulky complexing agents EDTA and 1,10-phenantroline suggests that the Zn(II)-binding site is partially buried in the protein structure. The apparent Zn(II)-dissociation constant of 4.2 nm, determined by19F NMR using the chelator 5F-BAPTA (1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid), lies in the range of intracellular free Zn(II) concentrations. These results suggest a regulatory role for the Zn(II)-binding site. The coordination environment of the Zn(II) in DDAH-1 has been examined by Zn K-edge x-ray absorption spectroscopy. The extended x-ray absorption fine structure observed is consistent with Zn(II) being coordinated by 2 S and 2 N (or O) atoms. The biological implications of these findings are discussed.
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Dimethylargininase a nitric oxide regulatory protein in alzheimer disease
Free Radical Biology and Medicine, 1998Co-Authors: Mark A Smith, Markus Knipp, Milan Vasak, Rudy J Castellani, George PerryAbstract:In this study, we show that Dimethylargininase, a zinc protein involved in the regulation of nitric oxide synthase, is specifically elevated in neurons displaying cytoskeletal abnormalities and oxidative stress in Alzheimer disease (AD) while none of this enzyme was found in neurons in age-matched control cases. Seen in the context of earlier studies showing widespread nitric oxide related damage in AD and the role of Dimethylargininase to activate nitric oxide synthetase, through catalytic removal of its endogenous inhibitors, these findings indicate major alterations in nitric oxide regulation in AD. Further, that low levels of zinc specifically inhibit Dimethylargininase may provide a link between the numerous studies showing specific abnormalities in zinc and oxidative stress. Finally, our results provide additional evidence that oxidative stress- and nitric oxide-mediated events play important roles in the pathogenesis of AD.
Markus Knipp - One of the best experts on this subject based on the ideXlab platform.
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specific reactions of s nitrosothiols with cysteine hydrolases a comparative study between Dimethylargininase 1 and ctp synthetase
Protein Science, 2007Co-Authors: Oliver Braun, Markus Knipp, Serge Chesnov, Milan VasakAbstract:S-Transnitrosation is an important bioregulatory process whereby NO(+) equivalents are transferred between S-nitrosothiols and Cys of target proteins. This reaction proceeds through a common intermediate R-S-N(O(-))-S-R' and it has been proposed that products different from S-nitrosothiols may be formed in protein cavities. Recently, we have reported on the formation of such a product, an N-thiosulfoximide, at the active site of the Cys hydrolase Dimethylargininase-1 (DDAH-1) upon reaction with S-nitroso-l-homocysteine (HcyNO). Here we have addressed the question of whether this novel product can also be formed with the endogenously occurring S-nitrosothiols S-nitroso-l-cysteine (CysNO) and S-nitrosoglutathione (GSNO). Further, to explore the reason responsible for the unique formation of an N-thiosulfoximide in DDAH-1 we have expanded these studies to cytidine triphosphate synthetase (CTPS), which shows a similar active site architecture. ESI-MS and activity measurements showed that the bulky GSNO does not react with both enzymes. In contrast, S-nitrosylation of the active site Cys occurred in DDAH-1 with CysNO and in CTPS with CysNO and HcyNO. Although kinetic analysis indicated that these compounds act as specific irreversible inhibitors, no N-thiosulfoximide was formed. The reasons likely responsible for the absence of the N-thiosulfoximide formation are discussed using molecular models of DDAH-1 and CTPS. In tissue extracts DDAH was inhibited only by HcyNO, with an IC(50) value similar to that of the isolated protein. Biological implications of these studies for the function of both enzymes are discussed.
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Searching for DDAH inhibitors: S-nitroso-L-homocysteine is a chemical lead.
Journal of the American Chemical Society, 2005Co-Authors: Markus Knipp, Oliver Braun, Milan VasakAbstract:The cysteine-hydrolase Dimethylargininase-1 (DDAH-1) is an important regulator of NO production in mammalian tissue for which the availability of an inhibitor for clinics and research would be most appreciated. While studying the effect of the endogenously occurring S-nitroso-l-homocysteine on DDAH-1, an unusual N-thiosulfoximide modification was identified in the active site of the enzyme. Thus, S-nitroso-l-homocysteine in combination with the mechanism proposed herein offers a basis for the rational design of DDAH inhibitors.
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zn ii free Dimethylargininase 1 ddah 1 is inhibited upon specific cys s nitrosylation
Journal of Biological Chemistry, 2003Co-Authors: Markus Knipp, Oliver Braun, Peter Gehrig, Ragna Sack, Milan VasakAbstract:Abstract The endogenous nitric oxide synthase inhibitorsl-N ω-methylarginine andl-N ω,N ω-dimethylarginine are catabolized by the enzyme Dimethylargininase. Dimethylargininase-1 from bovine brain contains one tightly bound Zn(II) coordinated by two cysteine sulfur and two lighter ligands. Activity measurements showed that only the apo-enzyme is active and that the holo-enzyme is activated by zinc removal. In this work, the effect of NO on Dimethylargininase-1 structure and its activity was investigated using 2-(N,N-dimethylamino)-diazenolate-2-oxide as an NO source. The results showed that whereas the holo-form was resistant to S-nitrosylation, the apo-form could be modified. The results of absorption spectroscopy, mass spectrometry, and fluorometric S-NO quantification revealed that two of five cysteine residues reacted with NO yielding cysteine-S-NO. The modification reaction is specific, because by liquid chromatography/mass spectrometry experiments of digested S-NO-Dimethylargininase-1, cysteines 221 and 273 could be identified as cysteine-NO. Because Zn(II) protects the enzyme against nitrosation, it is suggested that both cysteines are involved in metal binding. However, specific cysteine-S-NO formation occurred in the absence of a characteristic sequence motif. Based on a structural model of Dimethylargininase-1, the activation of both cysteines may be accomplished by the close proximity of charged residues in the tertiary structure of the enzyme.
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structural and functional characterization of the zn ii site in Dimethylargininase 1 ddah 1 from bovine brain zn ii release activates ddah 1
Journal of Biological Chemistry, 2001Co-Authors: Markus Knipp, John M Charnock, C D Garner, Milan VasakAbstract:Abstract l-N ω,N ω-Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is a Zn(II)-containing enzyme that, through hydrolysis of side-chain methylated l-arginines, regulates the activity of nitric-oxide synthase. Herein we report the structural and functional properties of the Zn(II)-binding site in DDAH-1 from bovine brain. Activity measurements of the native and metal-free enzyme have revealed that the endogenously bound Zn(II) inhibits the enzyme. Native DDAH-1 could be fully or partially activated using various concentrations of phosphate, imidazole, histidine, and histamine, a process that is paralleled by the release of Zn(II). The slow activation of the enzyme by the bulky complexing agents EDTA and 1,10-phenantroline suggests that the Zn(II)-binding site is partially buried in the protein structure. The apparent Zn(II)-dissociation constant of 4.2 nm, determined by19F NMR using the chelator 5F-BAPTA (1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid), lies in the range of intracellular free Zn(II) concentrations. These results suggest a regulatory role for the Zn(II)-binding site. The coordination environment of the Zn(II) in DDAH-1 has been examined by Zn K-edge x-ray absorption spectroscopy. The extended x-ray absorption fine structure observed is consistent with Zn(II) being coordinated by 2 S and 2 N (or O) atoms. The biological implications of these findings are discussed.
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Dimethylargininase a nitric oxide regulatory protein in alzheimer disease
Free Radical Biology and Medicine, 1998Co-Authors: Mark A Smith, Markus Knipp, Milan Vasak, Rudy J Castellani, George PerryAbstract:In this study, we show that Dimethylargininase, a zinc protein involved in the regulation of nitric oxide synthase, is specifically elevated in neurons displaying cytoskeletal abnormalities and oxidative stress in Alzheimer disease (AD) while none of this enzyme was found in neurons in age-matched control cases. Seen in the context of earlier studies showing widespread nitric oxide related damage in AD and the role of Dimethylargininase to activate nitric oxide synthetase, through catalytic removal of its endogenous inhibitors, these findings indicate major alterations in nitric oxide regulation in AD. Further, that low levels of zinc specifically inhibit Dimethylargininase may provide a link between the numerous studies showing specific abnormalities in zinc and oxidative stress. Finally, our results provide additional evidence that oxidative stress- and nitric oxide-mediated events play important roles in the pathogenesis of AD.
M Kimoto - One of the best experts on this subject based on the ideXlab platform.
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Asymmetrical dimethylarginine, an endogenous nitric oxide synthase inhibitor, in experimental hypertension.
Hypertension (Dallas Tex. : 1979), 1997Co-Authors: H Matsuoka, S Itoh, M Kimoto, K Kohno, O Tamai, Y Wada, H Yasukawa, G Iwami, S Okuda, T ImaizumiAbstract:NG,NG-dimethyl-L-arginine (ADMA) is an endogenously synthesized nitric oxide (NO) synthase inhibitor which has potent pressor/vasoconstrictor effects. Dimethylargininase metabolizes ADMA to L-citrulline and plays a key role in determining the in vivo levels of ADMA. To investigate the role of ADMA in the pathogenesis of hypertension, we measured 24-hour urinary excretion of ADMA (UADMA) and nitrate/nitrite (NOx) in Dahl salt-sensitive hypertensive rats and spontaneously hypertensive rats (SHR). In Dahl salt-resistant rats, high-salt diet (8% NaCl) did not increase blood pressure and increased urinary NOx (P < .01) without changes in UADMA compared with low-salt diet (0.3% NaCl). In contrast, in Dahl salt-sensitive rats, high-salt diet increased blood pressure (P < .01), did not change urinary NOx excretion, and increased UADMA (P < .01). There was a significant (r = .65, P < .01) correlation between UADMA and the level of blood pressure in Dahl salt-sensitive rats. Plasma levels of NOx and ADMA and renal Dimethylargininase content were comparable among them. These results may suggest that in Dahl salt-resistant rats, blood pressure is kept constant during high-salt intake, possibly due to the compensatory increased production of NO, and that in Dahl salt-sensitive rats, high-salt intake increases the production of ADMA, attenuates the compensatory increases in NO, and increases blood pressure. These results also suggest that the systemic production of ADMA is not dependent on renal Dimethylargininase. SHR had significantly greater urinary NOx excretion (P < .05) and smaller UADMA than Wistar-Kyoto rats (P < .05), and UADMA was inversely correlated with their mean arterial pressure (r =.64, P < .05). In conclusion. ADMA, independently of the renal Dimethylargininase content, may play a role in the pathogenesis in Dahl salt-sensitive hypertensive rats but not in SHR.
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Asymmetrical Dimethylarginine, an Endogenous Nitric Oxide Synthase Inhibitor, in Experimental Hypertension
Hypertension, 1997Co-Authors: H Matsuoka, S Itoh, M Kimoto, K Kohno, O Tamai, Y Wada, H Yasukawa, G Iwami, S Okuda, T ImaizumiAbstract:N G , N G -dimethyl-l-arginine (ADMA) is an endogenously synthesized nitric oxide (NO) synthase inhibitor which has potent pressor/vasoconstrictor effects. Dimethylargininase metabolizes ADMA to l-citrulline and plays a key role in determining the in vivo levels of ADMA. To investigate the role of ADMA in the pathogenesis of hypertension, we measured 24-hour urinary excretion of ADMA (UADMA) and nitrate/nitrite (NOx) in Dahl salt-sensitive hypertensive rats and spontaneously hypertensive rats (SHR). In Dahl salt-resistant rats, high-salt diet (8% NaCl) did not increase blood pressure and increased urinary NOx ( P P P r =.65, P P P r =.64, P
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N ^ G ,N ^ G -Dimethyl-l-arginine, a dominant precursor of endogenous dimethylamine in rats
Amino Acids, 1994Co-Authors: M Kimoto, H. Tsuji, T. OgawaAbstract:The metabolic significance of N ^ G ,N ^ G -dimethyl- l -arginine (DMA) as a precursor of endogenous dimethylamine (DMN) in rats was examined in connection with the wide distribution and active operation of Dimethylargininase (EC3.5.3.18) in rat tissues (Kimoto et al., 1993). When [methyl-^14C]DMA was administered intraperitoneally to rats, the radioactive DMN was detected in various tissues as a major radioactive metabolite one hour after injection, and about 65% of the radioactivity administered was recovered in the first 12-h urine as DMN. In the case of the [^14C] DMN-injected rats, almost all the radioactivity was excreted in the 12-h urine as DMN, except for a negligible amount of radioactivity found in urea. The time-dependent decrease in the specific radioactivity of DMA and DMN in urine showed that Dimethylargininase was significantly involved in the in vivo formation of DMN by the hydrolytic cleavage of DMA released from methylated proteins and that DMA is a dominant precursor of endogenous DMN in rats.
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N (G) ,N (G) -Dimethyl-L-arginine, a dominant precursor of endogenous dimethylamine in rats.
Amino Acids, 1994Co-Authors: M Kimoto, H. Tsuji, T. OgawaAbstract:The metabolic significance ofNG,NG-dimethyl-l-arginine (DMA) as a precursor of endogenous dimethylamine (DMN) in rats was examined in connection with the wide distribution and active operation of Dimethylargininase (EC3.5.3.18) in rat tissues (Kimoto et al., 1993). When [methyl-14C]DMA was administered intraperitoneally to rats, the radioactive DMN was detected in various tissues as a major radioactive metabolite one hour after injection, and about 65% of the radioactivity administered was recovered in the first 12-h urine as DMN. In the case of the [14C] DMN-injected rats, almost all the radioactivity was excreted in the 12-h urine as DMN, except for a negligible amount of radioactivity found in urea. The time-dependent decrease in the specific radioactivity of DMA and DMN in urine showed that Dimethylargininase was significantly involved in thein vivo formation of DMN by the hydrolytic cleavage of DMA released from methylated proteins and that DMA is a dominant precursor of endogenous DMN in rats.
Oliver Braun - One of the best experts on this subject based on the ideXlab platform.
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specific reactions of s nitrosothiols with cysteine hydrolases a comparative study between Dimethylargininase 1 and ctp synthetase
Protein Science, 2007Co-Authors: Oliver Braun, Markus Knipp, Serge Chesnov, Milan VasakAbstract:S-Transnitrosation is an important bioregulatory process whereby NO(+) equivalents are transferred between S-nitrosothiols and Cys of target proteins. This reaction proceeds through a common intermediate R-S-N(O(-))-S-R' and it has been proposed that products different from S-nitrosothiols may be formed in protein cavities. Recently, we have reported on the formation of such a product, an N-thiosulfoximide, at the active site of the Cys hydrolase Dimethylargininase-1 (DDAH-1) upon reaction with S-nitroso-l-homocysteine (HcyNO). Here we have addressed the question of whether this novel product can also be formed with the endogenously occurring S-nitrosothiols S-nitroso-l-cysteine (CysNO) and S-nitrosoglutathione (GSNO). Further, to explore the reason responsible for the unique formation of an N-thiosulfoximide in DDAH-1 we have expanded these studies to cytidine triphosphate synthetase (CTPS), which shows a similar active site architecture. ESI-MS and activity measurements showed that the bulky GSNO does not react with both enzymes. In contrast, S-nitrosylation of the active site Cys occurred in DDAH-1 with CysNO and in CTPS with CysNO and HcyNO. Although kinetic analysis indicated that these compounds act as specific irreversible inhibitors, no N-thiosulfoximide was formed. The reasons likely responsible for the absence of the N-thiosulfoximide formation are discussed using molecular models of DDAH-1 and CTPS. In tissue extracts DDAH was inhibited only by HcyNO, with an IC(50) value similar to that of the isolated protein. Biological implications of these studies for the function of both enzymes are discussed.
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Searching for DDAH inhibitors: S-nitroso-L-homocysteine is a chemical lead.
Journal of the American Chemical Society, 2005Co-Authors: Markus Knipp, Oliver Braun, Milan VasakAbstract:The cysteine-hydrolase Dimethylargininase-1 (DDAH-1) is an important regulator of NO production in mammalian tissue for which the availability of an inhibitor for clinics and research would be most appreciated. While studying the effect of the endogenously occurring S-nitroso-l-homocysteine on DDAH-1, an unusual N-thiosulfoximide modification was identified in the active site of the enzyme. Thus, S-nitroso-l-homocysteine in combination with the mechanism proposed herein offers a basis for the rational design of DDAH inhibitors.
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zn ii free Dimethylargininase 1 ddah 1 is inhibited upon specific cys s nitrosylation
Journal of Biological Chemistry, 2003Co-Authors: Markus Knipp, Oliver Braun, Peter Gehrig, Ragna Sack, Milan VasakAbstract:Abstract The endogenous nitric oxide synthase inhibitorsl-N ω-methylarginine andl-N ω,N ω-dimethylarginine are catabolized by the enzyme Dimethylargininase. Dimethylargininase-1 from bovine brain contains one tightly bound Zn(II) coordinated by two cysteine sulfur and two lighter ligands. Activity measurements showed that only the apo-enzyme is active and that the holo-enzyme is activated by zinc removal. In this work, the effect of NO on Dimethylargininase-1 structure and its activity was investigated using 2-(N,N-dimethylamino)-diazenolate-2-oxide as an NO source. The results showed that whereas the holo-form was resistant to S-nitrosylation, the apo-form could be modified. The results of absorption spectroscopy, mass spectrometry, and fluorometric S-NO quantification revealed that two of five cysteine residues reacted with NO yielding cysteine-S-NO. The modification reaction is specific, because by liquid chromatography/mass spectrometry experiments of digested S-NO-Dimethylargininase-1, cysteines 221 and 273 could be identified as cysteine-NO. Because Zn(II) protects the enzyme against nitrosation, it is suggested that both cysteines are involved in metal binding. However, specific cysteine-S-NO formation occurred in the absence of a characteristic sequence motif. Based on a structural model of Dimethylargininase-1, the activation of both cysteines may be accomplished by the close proximity of charged residues in the tertiary structure of the enzyme.
T. Ogawa - One of the best experts on this subject based on the ideXlab platform.
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N ^ G ,N ^ G -Dimethyl-l-arginine, a dominant precursor of endogenous dimethylamine in rats
Amino Acids, 1994Co-Authors: M Kimoto, H. Tsuji, T. OgawaAbstract:The metabolic significance of N ^ G ,N ^ G -dimethyl- l -arginine (DMA) as a precursor of endogenous dimethylamine (DMN) in rats was examined in connection with the wide distribution and active operation of Dimethylargininase (EC3.5.3.18) in rat tissues (Kimoto et al., 1993). When [methyl-^14C]DMA was administered intraperitoneally to rats, the radioactive DMN was detected in various tissues as a major radioactive metabolite one hour after injection, and about 65% of the radioactivity administered was recovered in the first 12-h urine as DMN. In the case of the [^14C] DMN-injected rats, almost all the radioactivity was excreted in the 12-h urine as DMN, except for a negligible amount of radioactivity found in urea. The time-dependent decrease in the specific radioactivity of DMA and DMN in urine showed that Dimethylargininase was significantly involved in the in vivo formation of DMN by the hydrolytic cleavage of DMA released from methylated proteins and that DMA is a dominant precursor of endogenous DMN in rats.
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N (G) ,N (G) -Dimethyl-L-arginine, a dominant precursor of endogenous dimethylamine in rats.
Amino Acids, 1994Co-Authors: M Kimoto, H. Tsuji, T. OgawaAbstract:The metabolic significance ofNG,NG-dimethyl-l-arginine (DMA) as a precursor of endogenous dimethylamine (DMN) in rats was examined in connection with the wide distribution and active operation of Dimethylargininase (EC3.5.3.18) in rat tissues (Kimoto et al., 1993). When [methyl-14C]DMA was administered intraperitoneally to rats, the radioactive DMN was detected in various tissues as a major radioactive metabolite one hour after injection, and about 65% of the radioactivity administered was recovered in the first 12-h urine as DMN. In the case of the [14C] DMN-injected rats, almost all the radioactivity was excreted in the 12-h urine as DMN, except for a negligible amount of radioactivity found in urea. The time-dependent decrease in the specific radioactivity of DMA and DMN in urine showed that Dimethylargininase was significantly involved in thein vivo formation of DMN by the hydrolytic cleavage of DMA released from methylated proteins and that DMA is a dominant precursor of endogenous DMN in rats.