Masumi Kimoto

Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase

1 Dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes the endogenous nitric oxide synthase inhibitors NG‐monomethyl‐L‐arginine and NG, NG‐dimethy‐L‐arginine to citrulline, was identified by Western blotting in rat and human tissue homogenates. 2 S‐2‐amino‐4(3‐methylguanidino)butanoic acid (4124W) inhibited the metabolism of [14C]‐NG‐monomethyl‐L‐arginine to [14C]‐citrulline by rat liver homogenates (IC50 416 ± 66μm; n = 9), human cultured endothelial cells (IC50 250 ± 34 μm; n = 9)and isolated purified dimethylarginine dimethylaminohydrolase. 3 Addition of 4124W to culture medium increased the accumulation of endogenously‐generated NG, NG‐dimethy‐L‐arginine in the supernatant of human cultured endothelial cells from 3.1 ± 0.3 to 5 ± 0.7 μm (n=15; P < 0.005). 4 4124W (1 μm‐1 mM) had no direct effect on endothelial nitric oxide synthase activity but caused endothelium‐dependent contraction of rat aortic rings (1 mM 4124W increased tone by 81.5 ± 9.6% of that caused by phenylephrine 100 nM). This effect was reversed by L‐arginine (100 μm). 4124W reversed endothelium‐dependent relaxation of human saphenous vein (19.2 ± 6.7% reversal of bradykinin‐induced relaxation at 1 mM 4124W). 5 These data suggest that inhibition of dimethylarginine dimethylaminohydrolase increases the intracellular concentration of NG, NG‐dimethyl‐L‐arginine sufficiently to inhibit nitric oxide synthesis. Inhibiting the activity of DDAH may provide an alternative mechanism for inhibition of nitric oxide synthases and changes in the activity of DDAH could contribute to pathophysiological alterations in NO generation.

Occurrence of a new enzyme catalyzing the direct conversion of NG,NG-dimethyl-L-arginine to L-citrulline in rats.

An enzyme concerned with the degradation of NG,NG-dimethyl-L-arginine to L-citrulline was investigated in rats. The enzyme purified from rat kidney catalyzed the direct conversion of NG,NG-dimethyl-L-arginine to L-citrulline with liberation of dimethylamine from the methylated guanidino moiety. The reaction required no co-factor and the maximum activity was obtained at pH 6.5. The enzyme was highly specific for NG,NG-dimethyl-L-arginine.

The synaptic protein UNC-18 is phosphorylated by protein kinase C.

The C. elegans unc-18 encoded protein UNC-18 is implicated in the interactions between synaptic vesicles and presynaptic plasma membrane. To further characterize the neural protein, we investigated the phosphorylation in vitro of the protein expressed in Spodoptera frugiperda Sf21 cells. The UNC-18 protein is selectively phosphorylated by protein kinase C (PKC) but not by casein kinase II and cyclic AMP-dependent protein kinase. The presumed phosphorylation sites determined by manual Edman degradation were serine-2, serine-322, threonine-462 and serine-515, of which the last is highly conserved as a consensus phosphorylation site for PKC in Drosophila and the mammalian homologue. Phosphorylated UNC-18 extracted from C. elegans was also detected, indicating that it has a physiological role in intact nerve terminals. Therefore, the phosphorylation by PKC may play a physiological role in the regulation.

Cloning and sequencing of cDNA encoding 4-aminobenzoate hydroxylase fromAgaricus bisporus

Abstract A cDNA clone encoding 4-aminobenzoate hydroxylase (EC 1.14.13.27) has been isolated using a probe prepared by PCR on the basis of partially determined amino acid sequences of the enzyme. The cDNA contained 1380-base pair open reading frame encoding 460 amino acid residues (Mr 50 974), 14-base pair 5′-untranslated region and 123-base pair 3′-untranslated region including a poly(A) tail of 20 nucleotides. All of the partially determined amino acid sequences were shown to be included in the deduced amino acid sequence. Homology analyses showed that the two regions on the enzyme share other flavoproteins such as salicylate hydroxylase and p-hydroxybenzoate hydroxylase.

Effect of 1-aminoproline on methionine metabolism in rats

Abstract By the intraperitoneal injection of 1-aminoproline and l -[U- 14 C]methionine, three unidentified radioactive compounds appeared abnormally in rat urine together with large amounts of radioactive l -cystathionine. One of them was identified as S -[ l -2-(acetylamino)-2-carboxyethyl]- l -homocysteine and the other two compounds were proved to be the diastereoisomers of l -cystathionine sulfoxide. These observations indicate that the disturbance of methionine metabolism by 1-aminoproline resulted in the accumulation of l -cystathionine and its novel derivatives.

Preparation and characterization of monoclonal antibodies against 4-aminobenzoate hydroxylase from Agaricus bisporus

A monoclonal antibody (mAb, A) recognizing the FAD-binding domain of 4-aminobenzoate hydroxylase (4-aminobenzoate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating, decarboxylating), EC 1.14.13.27) from Agaricus bisporus, a common edible mushroom, had been produced (Tsuji, H., Ogawa, T., Bando, N., Kimoto, M. and Sasaoka, K. (1990) J. Biol. Chem. 265, 16064-16067). In the present study, three other mAbs (B1, B2 and B3) against the enzyme have been further prepared in order to facilitate the structural characterization of the enzyme. The three new mAbs immunoblotted the enzyme. The four mAbs, including A, were specific for different epitopes on the enzyme. B1 and B2 immunoprecipitated the apoenzyme and the immunoprecipitation was inhibited in the presence of FAD, whereas B3 failed to immunoprecipitate the apoenzyme in the absence or presence of FAD. B1 and B2 competed with FAD for the binding to the apoenzyme. These findings show that B1 and B2 recognize the FAD-binding domain of the enzyme in analogy with A. The immunoblotting analyses of the peptides obtained from the enzyme by digestion with lysyl endopeptidase (EC 3.4.21.50) provided useful knowledge as to the location of the epitopes to the mAbs on the enzyme, suggesting that the FAD-binding domain of the enzyme can be located and characterized by detailed investigations on the location of the epitopes.

N-acetyl conjugates of basic amino acids newly identified in rat urine

Abstract Ninhydrin-negative conjugates of basic amino acids were isolated from rat urine and were characterized. The following conjugates of basic amino acids are the compounds newly identified in animal urine specimens, N α -acetyl- N π -methylhistidine, N α -( N -acetyl-β-alanyl)histidine ( N -acetylcarnosine), N α -acetyl- N G ,N ′G -dimethylarginine, N α -acetyl- N G ,N G -dimethylarginine, and N α -acetyl- N ϵ ,N ϵ ,N ϵ -trimethyllysine.

A convenient method for the determination of the optical configuration of cystathionine

Abstract A convenient and sensitive method was developed for the determination of the optical configuration of cystathionine in microscale. The technique consists of the reductive desulfuration of cystathionine to alanine and α-amino-n-butyric acid with Raney nickel and the chromatographic resolution of the diastereoisomeric l -leucyl dipeptides of both the desulfuration products. Examples used for cystathionine isolated from natural sources, a commercial product, and a synthetic specimen were presented.

N G ,N G -Dimethyl-l-arginine, a dominant precursor of endogenous dimethylamine in rats

SummaryThe 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.

A Novel Aminotransferase Concerned with the Metabolism of Dimethylarginines in Rats

A novel aminotransferase concerned with the metabolism of N G , N G - and N G , Ns G -dimethyl-L-arginines was purified from rat kidney. The enzyme catalyzed effectively transamination between N ω -substituted L-arginines and pyruvate. The enzyme had a molecular weight of 108,000 and consisted of two subunits with identical molecular weight of 52,000. The absorption maxima of the enzyme were at 278, 335 and 415 nm. The enzyme activity was inhibited by carbonyl reagents, and the inhibition was restored by the addition of PLP. These facts indicated the enzyme to be PLP-dependent.