Kwang Sook Park

Determination of methylated amino acids in human serum by high-performance liquid chromatography

Employing high-performance liquid chromatography with isocratic elution using a two-column system (mu Bondapak C18 and mu Bondapak CN) and phenyl isothiocyanate as a fluorogenic reagent, NG-monomethylarginine (MMA). NG-dimethylarginines, and epsilon-N-trimethyllysine (TML) can be quatitatively separated from human serum samples. The recoveries of these amino acids were over 90%. It was observed that the serum concentrations of MMA, DMA and TML were significantly elevated in sera obtained from patients suffering from diabetes mellitus, hepatitis or hyperthyroidism, particularly the last condition.

Studies on in vitro S-methylation of naturally occurring thiol compounds with N-methyl-N-nitrosourea and methyl methanesulfonate

N-Methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS) were found to rapidly methylate glutathione (GSH) in vitro yielding S-methyl glutathione, as verified and quantitated by high-performance liquid chromatography and thin-layer chromatography. Formation of S-methylcysteine in the acid-hydrolyzate of the methylated GSH further confirmed the formation of S-methyl glutathione. Other naturally occurring thiol compounds such as cysteine and homocysteine were also methylated by MNU. The observed pH dependency of GSH methylation by MNU suggests that the sulfide anion form of the thiol may represent the favored methyl acceptor. The high reactivity of GSH toward MNU and MMS may be of biological significance in that it could compete with macromolecular cellular components as a target for alkylation.

HPLC analysis of methylated amino acids: Methylated amino acids on HPLC

Various naturally occuring, methylated amino acid derivatives were resolved on high performance liquid chromatography (HPLC), usingo-phthaldialdehyde as a fluorogenic reagent.We separated ε-N-monomethyllysine, ε-N-dimethyllysine, and ε-N-acetyllysine from lysine derivatives. NG-Monomethylarginine and NG-dimethylarginine were separated from arginine derivatives. However, ε-N-monomethyllysine and ε-N-trimethyllysine, NG, NG-dimethylarginine and NG, NG-dimethylarginine were not resolved under the conditions employed. S-Methylmethionine, S-methylcysteine, and 1-N-methylhistidine or 3-N-methylhistidine were clearly separated from their reference amino acids, even though 1-N-methyl- and 3-N-methylhistidine could not be separated.

Effect of enzymatic methylation of apocytochrome c on holocytochrome c formation and proteolysis

1. Methylation of the lysine at residue 72 of yeast apocytochrome c increases its import into mitochondria. 2. Using methylated and unmethylated apocytochrome c as substrate and intact yeast mitochondria and a solubilized mitochondrial fraction as a source of cytochrome c heme lyase, the results show that the methylation state of the apoprotein has no significant effect on its conversion to holoprotein. 3. The above result suggests that the import mechanism is separate from the heme-attaching activity. 4. Unmethylated apocytochrome c was less resistant to a yeast homogenate fraction that methylated apocytochrome c, suggesting that methylation of apocytochrome c alters the conformation of the whole protein.

Histone binding to isolated rat liver nuclei

Calf thymus histone H3 bound irreversibly to the isolated rat liver nuclei. The rate and extent of binding was a function of the incubation period and the concentration of both H3 and nuclei, but independent of the temperature. The binding was saturable and was inhibited by simultaneous presence of various histones. Approximately 94% of the bound H3 was associated with nuclear membrane fraction.

Effect of enzymatic methylation of proteins on their isoelectric points

Enzymatic methylation of arginine and lysine residues of several cytochromec and lysine residue of calmodulin always resulted in lowering of their respective isoelectric points (pI). Employing cytochromesc derived from various sources, we examined a possible relationship between the degree of amino acid sequence degeneracy and the magnitude of change in the pI values by enzymatic methylation, and found that there was no correlation between these two parameters. By constructing space-filling models of oligopeptide fragments adjacent to the potential methylation sites, we have noted that not all the methylatable residues are able to form hydrogen bonds prior to the methylation. Two preparations of yeast apocytochromec, one chemically prepared by removing heme from holocytochromec and the other by translating yeast iso-1-cytochromec mRNAin vitro, exhibited slightly higher Stokes radii than the homologous holocytochromec, indicating relatively “relaxed or open” conformation of the protein. However, when thein vitro synthesized methylated apocytochromec was compared with the unmethylated counter-part, the Stokes radius of the latter was found to be larger.

Site-specificity of histone HL methylation by two H1-specific protein-lysine N-methyltransferases from Euglena gracilis

1. The histone H1 fractions from rat spleen and liver were used as substrates for two H1-specific protein-lysine N-methyltransferases, V-A and V-B (protein methylase III) from Euglena gracilis. 2. When the enzymatically [methyl-3H]labeled H1 fractions were resolved by two-dimensional gel electrophoresis, four subtypes were found to be methylated (H1b, H1c, H1d and H1e). Both enzymes methylated H1c and H1b to approximately the same extent; H1d and H1e were methylated preferentially by enzyme V-B and V-A, respectively. 3. Histone H1c, [methyl-3H]labeled by the methyltransferase V-A, which had been digested by arginine-specific protease (Arg C protease), showed a single radioactive peptide on HPLC, indicating methylation site specificity of the enzyme. 4. Arg C protease-digestion of [methyl-3H]labeled H1c labeled by methyltransferase V-B indicated that this enzyme methylated two sites on the histone molecule. 5. The histone H1c methylation sites of these two enzymes did not overlap, indicating the two enzymes have different site specificity. 6. In combination with the other results, this suggests that the two enzymes serve discrete purposes, possibly involving the presumed different actions of histone H1 subtypes.

Interrelationship between nuclear histone binding and cell proliferation

1. Binding of non-enzymatically [methyl-14C]-labeled histone H3 to nuclei isolated from young and old rat livers, regenerating rat liver, and tumor cells has been investigated. 2. Scatchard plot analysis indicated that various cell types had different binding capacity and different dissociation constant (Kd). 3. Nuclei isolated from younger rats had fewer binding sites and lower Kd (or higher Ka) values for [methyl-14C]H3 than those from older rats. 4. Fewer binding sites and lower Kd values were also observed with nuclei isolated from the maximally regenerating liver (24 hr after partial hepatectomy) and the fast-growing ascites tumor and Novikoff hepatomas. 5. These results strongly suggest that the number of binding sites and affinity of histone H3 for nuclei appears to be correlated with the degree of cell proliferation. 6. Fractionation of the [methyl-14C]H3 bound nuclei into nuclear membrane and nucleoplasm demonstrates that approx. 94% of radioactivity is associated with the former in which less than 6% of DNA is found, whereas 94% of total DNA is found in nucleoplasm. 7. This suggests that the binding of [methyl-14C]H3 to nuclei is independent of DNA present in each fraction.

Effect of Enzymatic Methylation on the Import of in Vitro Synthesized Apocytochrome C into Mitochondria

Little more than a dozen protein posttranslational side-chain modification reactions have been identified. One such reaction, protein methylation, occurs ubiquitously in nature, in organisms ranging from prokaryotic to eukaryotic. The reaction involves N-methylation of lysine, arginine, histidine, alanine, proline and glutamine, O-methylesterifi-cation of glutamic and aspartic acid, and S-methylation of cysteine and methionine (1–3).

CytochromeC methylation: Current knowledge of its biological significance

The yeast cytochromec gene has been recloned, and the resulting cytochromec mRNA has been translated in rabbit reticulocyte lysate translation system. The newly synthesized apocytochromec could be methylated by exogenously added cytochromec-lysine N-methyltransferase. Enzymatic methylation ofin vitro synthesized apocytochromec was found to facilitate specifically its import into mitochondria of yeast, but not of rat liver.