Sung Youl Hong

Identification and characterization of nitric oxide synthase inSalmonella typhimurium

The presence of the nitric oxide synthase (NOS) enzyme fromSalmonella typhimurium (S. typhimurium) was identified by measuring radiolabeled L-[3H]citrulline and NO, and Western blot analysis. NOS was partially purified by both Mono Q ion exchange and Superose 12HR size exclusion column chromatography, sequentially. The molecular weight of NOS was estimated to be 93.3 kDa by Western blot analysis. The enzyme showed a significant dependency on the typical NOS cofactors; an apparent Km for L-arginine of 34.7 mM and maximum activity between 37°C and 43°C. The activity was inhibited by NOS inhibitors such as aminoguanidine and NG,NG-dimethyl-L-arginine. Taken together, partially purified NOS inS. typhimurium is assumed to be a different isoform of mammalian NOSs.

Nitric oxide synthase from bovine pancreas: Purification and characterization

Nitric oxide synthase, NOS (EC.1.14.13.39), was purified from bovine pancreas over 5,500-fold with a 7.6% yield using 30% ammonium sulfate precipitation, and 2′,5′-ADP-agarose and calmodulin-agarose affinity chromatography. The purified bovine pancreatic NOS (bpNOS) showed a single band on SDS-PAGE corresponding to an apparent molecular mass of 160 kDa, whereas it was 320 kDa on non-denaturating gel-filtration. This indicated a homodimeric nature of the enzyme. The specific activity of the purified bpNOS was 31.67 nmol L-citrulline fored/mtn/mg protein and an apparentKm for L-arginine was 15.72 μM. The enzyme activity was dependent on Ca2+ and calmodulin, and to a lesser extent on NADPH, FAD and FMN. H4B was not required as a cofactor for the activity. In an inhibition experiment with L-arginine analogues, NG-nitro-L-arginine (NNA) had the most potent inhibitory effect on bpNOS, and NG, NG′-dimethyl-L-arginine (symmetric; sDMA) did not have any inhibitory effect. Immunohistochemical analysis of the bovine pancreas using brain type NOS antibody (anti-bNOS antibody) revealed that acinar cells showed strong immunoreactivity against the antibody.

Inducible nitric oxide synthase inhibitors fromMelia azedarach var.Japonica

In bioassay-guided search for inducible nitric oxide synthase (iNOS) inhibitory compounds from higher plants of South Korea, two β-carboline alkaloids, 4-methoxy-1-vinyl-β-carboline (1) and 4,8-dimethoxy-1-vinyl-β-carboline (2) have been isolated from the cortex ofMelia azedarach var.japonica. The structures of these compounds were elucidated on the basis of spectroscopic data. Compounds1 and2 showed marked inhibitory activity of iNOS on LPS-and interferon-γ-stimulated RAW 264.7 cells.

A turbidimetric determination of protein by trichloroacetic acid

Based on the turbidimetric response of protein with 50%-trichloroacetic acid (TCA), this study aims to introduce an assay method for protein in solution. The standard procedure consists of mixing equal volume of sample solution (standard or unknown) with 50%-TCA solution and measuring the absorbance at 450 nm after 20 min. The absorbances of the solutions were almost stable over 120 min at room temperature. This assay method is simple, reproducible, and tolerant to many interfering substances. It can detect less amount than 10 μg/ml of bovine serum albumin. The assay method has low protein-to-protein variability over wide range of molecular weight.

An endogenous proteinacious inhibitor in porcine liver for S-adenosyl-L-methionine dependent methylation reactions: identification as oligosaccharide-linked acyl carrier protein.

A proteinacious inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent transmethylation reactions was purified to homogeneity from porcine liver by size exclusion chromatography and FPLC. The molecular weight of the inhibitor was 12,222 Da. A 7400 Da polypeptide fragment of the purified inhibitor was sequenced by matrix-associated laser desorption ionization; time-of-flight MS, and was found to be identical with the known sequence of spinach acyl carrier protein (ACP). Although the remainder of the molecule was not clearly defined, 1H and H-H correlation of spectroscopy (COSY) NMR analysis revealed the presence of an oligosaccharide with alpha-glycosidic linkage. The purified oligosaccharide-linked ACP inhibited several AdoMet-dependent transmethylation reactions such as protein methylase I and II. S-farnesylcysteine O-methyltransferase, DNA methyltransferase and phospholipid methyltransferase. Protein methylase II was inhibited with a Ki value of 2.4 x 10(-3) M in a mixed inhibition pattern, whereas a well-known competitive product inhibitor S-adenosyl-L-homocysteine (AdoHcy) had Ki value of 6.3 x 10(-6) M. Commercially available active ACP fragments (65-74) and ACP from Escherichia coli had less inhibitory activity toward S-farnesylcysteine O-methyltransferase than the purified inhibitor. The biological significance of this oligosaccharide-linked ACP which has two seemingly unrelated functions (inhibitor for transmethylation and fatty acid biosynthesis) remains to be elucidated.

Changes of nitric oxide synthase activity and free methylarginines contents in regenerating rat liver after partial hepatectomy

In the present study, liver regeneration rate (%) was increased up to 70% 3 days after partial hepatectomy (PH). Nitric oxide synthase (NOS) activity in liver tissue as well as serum nitrite/nitrate content had no timed response, revealing no significant difference between shamoperated and partially hepatectomized rat liver. Contents of free methylarginines in liver tissue were increased biphasically in a time-dependent manner after PH. However, those in serum did not exhibit the same patterns as in liver. Taken together, the results suggest that NG-monomethyl-L-arginine (MMA) and NG, NG-dimethylarginine (DMA) play a role in inhibiting nitric oxide (NO) synthesis in regenerating rat liver because the increase of their contents was synchronized with NOS expression.

Immunoglobulin can be functionally regulated by protein carboxylmethylation in Fc region

Protein carboxylmethylation methylates the free carboxyl groups in various substrate proteins by protein carboxylO-methyltransferase (PCMT) and is one of the post-translational modifications. There have been many studies on protein carboxylmethylation. However, the precise functional role in mammalian systems is unclear. In this study, immunoglobulin, a specific form of γ-globulin, which is a well-known substrate for PCMT, was chosen to investigate the regulatory roles of protein carboxylmethylation in the immune system. It was found that the anti-BSA anibody could be carboxylmethylatedvia spleen PCMT to a level similar to γ-globulin. This carboxylmethylation increased the hydrophobicity of the anti-BSA antibody up to 11.4%, and enhanced the antigen-binding activity of this antibody up to 24.6%. In particular, the Fc region showed a higher methyl accepting capacity with 80% of the whole structure level. According to the amino acid sequence alignment, indeed, 7 aspartic acids and 5 glutamic acids, as potential carboxylmethylation sites, were found to be conserved in the Fc portion in the human, mouse and rabbit. The carboxylmethylation of the anti-BSA antibody was reversibly demethylated under a higher pH and long incubation time. Therefore, these results suggest that protein carboxylmethylation may reversibly regulate the antibody-mediated immunological events via the Fc region.

An endogenous proteinacious inhibitor forS-adenosyl-L-methionine-dependent transmethylation reactions; Identification ofS-adenosylhomocysteine as an integral part

A proteinacious inhibitor with a molecular weight of 1,600 Da which inhibits S-adenosyl-L-methionine-dependent transmethylation reactions was purified from porcine liver to homogeneity by procedures including boiling, Sephadex G-25 column chromatography and repeated HPLC. Employing both Nuclear Magnetic Resonance (NMR) and Fast Atom Bombardment-Mass (FAB-Mass) spectroscopy, S-adenosylhomocysteine was conclusively identified as an integral part of the inhibitor. The purified S-adenosylhomocysteine was competitive with S-adenosyl-L-methionine with Ki value of 6.3×10−6 M towards protein methylase II.