• BMB Reports
• /
• v.32 no.3
• /
• pp.299-305
• /
• 1999

Protein carboxyl O-methyltransferase (PCMT) catalyzes the transfer of a methyl group from Sadenosyl-L-methionine to free carboxyl groups of methyl-accepting substrate proteins. Two isozymes were separated by DEAE-Sephacel chromatography from porcine brain cytosol and designated PCMT I and II. Isozymes I and II were further purified by adenosyl homocysteine-Sepharose 4B and Superose HR 12 chromatography. The molecular weights of the purified PCMT I and II were determined by mass spectrometry to be 20,138 Da and 25,574 Da, respectively. The two enzymes displayed different isoelectric points; 7.9 for PCMT I and 5.3 for PCMT II. Isozymes I and II exhibited similar substrate specificities when tested with various methyl-accepting proteins. Myelin basic protein, a component of myelinated neurons, was found to be an excellent methyl-accepting substrate for both PCMT isozymes with different $K_m$ values, $21.1\;{\mu}M$ for PCMT I and $10.6\;{\mu}M$ for PCMT II. The PCMT activity and methyl-accepting capacity displayed similar distribution in the various brain regions with an exception of the lower values in the cerebellum. The overall distribution may relate to a general function of protein repair by PCMT in the brain.

• Journal of The Korean Society of Inherited Metabolic disease
• /
• v.16 no.1
• /
• pp.1-9
• /
• 2016

Newborn screening (NBS) is important if early intervention is effective in a disorder and if there are sensitive and specific biochemical markers to detect disorder. Methionine is a useful marker to detect abnormal methionine-homocysteine metabolism, especially homocystinuria which needs urgent medical intervention. However, hypermethioninemia could occur in other metabolic disorder including liver disease, tyrosinemia type I, methionine adenosyltransferase (MAT) I/III deficiency, glycine N-methyltransferase (GNMT) deficiency, or adenosylhomocysteine hydrolase deficiency. However, experience with NBS for homocystinurias and methylation disorders is limited. Especially, MAT I/III deficiency which is the most common cause of persistent hypermethioninemia have two inheritance, autosomal recessive (AR) and autosomal dominant (AD), and their clinical manifestation is different between AR and AD. Here, author reviewed recent articles of guideline and proposed guideline for homocystinuria and methylation disorder.

• Asian Pacific Journal of Cancer Prevention
• /
• v.17 no.7
• /
• pp.3213-3222
• /
• 2016

Background: Methyl donor status influences DNA stability and DNA methylation although little is known about effects on DNA methyltransferases. The aim of this study was to determine whether methyl-donor status influences DNA methyltransferase (Dnmt) gene expression in cervical cancer cells, and if so, whether there are associated effects on global DNA methylation. Materials and Methods: The human cervical cancer cell line, C4-II, was grown in complete medium and medium depleted of folate (F-M+) and folate and methionine (F-M-). Growth rate, intracellular folate, intracellular methionine and homocysteine in the extracellular medium were measured to validate the cancer cell model of methyl donor depletion. Dnmt expression was measured by qRT-PCR using relative quantification and global DNA methylation was measured using a flow cytometric method. Results: Intracellular folate and methionine concentrations were significantly reduced after growth in depleted media. Growth rate was also reduced in response to methyl donor depletion. Extracellular homocysteine was raised compared with controls, indicating disturbance to the methyl cycle. Combined folate and methionine depletion led to a significant down-regulation of Dnmt3a and Dnmt3b; this was associated with an 18% reduction in global DNA methylation compared with controls. Effects of folate and methionine depletion on Dnmt3a and 3b expression were reversed by transferring depleted cells to complete medium. Conclusions: Methyl donor status can evidently influence expression of Dnmts in cervical cancer cells, which is associated with DNA global hypomethylation. Effects on Dnmt expression are reversible, suggesting reversible modulating effects of dietary methyl donor intake on gene expression, which may be relevant for cancer progression.

• Nutrition Research and Practice
• /
• v.17 no.4
• /
• pp.597-615
• /
• 2023

Healthy aging can be defined as an extended lifespan and health span. Nutrition has been regarded as an important factor in healthy aging, because nutrients, bioactive food components, and diets have demonstrated beneficial effects on aging hallmarks such as oxidative stress, mitochondrial function, apoptosis and autophagy, genomic stability, and immune function. Nutrition also plays a role in epigenetic regulation of gene expression, and DNA methylation is the most extensively investigated epigenetic phenomenon in aging. Interestingly, age-associated DNA methylation can be modulated by one-carbon metabolism or inhibition of DNA methyltransferases. One-carbon metabolism ultimately controls the balance between the universal methyl donor S-adenosylmethionine and the methyltransferase inhibitor S-adenosylhomocysteine. Water-soluble B-vitamins such as folate, vitamin B6, and vitamin B12 serve as coenzymes for multiple steps in one-carbon metabolism, whereas methionine, choline, betaine, and serine act as methyl donors. Thus, these one-carbon nutrients can modify age-associated DNA methylation and subsequently alter the age-associated physiologic and pathologic processes. We cannot elude aging per se but we may at least change age-associated DNA methylation, which could mitigate age-associated diseases and disorders.

• Journal of Life Science
• /
• v.17 no.4 s.84
• /
• pp.522-528
• /
• 2007

In the present investigation, we studied the modulating effects of caffeic acid, chlorogenic acid, and (-)-epigallocatechin-3-gallate(EGCG) on the methylation status of promoter regions of cell cycle regulator, p16, in human breast cancer T-47D cells. We demonstrated that treatment of T-47D cells with caffeic acid, chlorogenic acid, or EGCG partially inhibited the methylation status of the promoter regions of p16 genes determined by methylation-specific PCR. In contrast, unmethylated p16 genes were increased with the treatment of T-47D cells with $20{\mu}M$ of caffeic acid or chlorogenic acid for 6 days. Treatment of T-47D cells with 5, 20 or $50{\mu}M$ of EGCG increased the unmethylation status of p16 gene up to 100%, and the methylation-specific bands of this gene were decreased up to 50% in a concentration-dependent manner. The finding of present study demonstrated that coffee polyphenols and EGCG have strong inhibitory effects of the cellular DNA methylation process through increased formation of S-adenosyl-homocysteine(SAH) during the catechol-O-methyltransferase (COMT)- mediated O-methylation of these dietary chemicals or an direct inhibition of the DNA methyltransferases. In conclusion, various dietary polyphenols could reverse the methylation status of p16 gene in human breast T-47D cells.

• Proceedings of the Korean Society for Applied Microbiology Conference
• /
• 1986.12a
• /
• pp.505.1-505
• /
• 1986

The gene for iso-1-cytochrome c for Saccharomyces cerevisiae was recloned into a pSP65 vector containing an active bacteriophage SP6 promoter. The iso-1-cytochrome c gene was cloned as an 856 bp Xho 1-Hind III fragment. When the resulting plasmid was digested at the Hind 111 site 279 bases downstream from the termination codon of the gene and transcribed in vitro using SP6 RNA polymerase, full length transcripts were produced. The SP6 iso-1-cytochrome c mRNA was translated using a rabbit reticulocyte lysate system and the protein products analyzed on SDS polyacrylamide gels. One major band was detected by autofluorography. This band was found to have a molecular weight of 12,000 Da and coincided with the Coomassie staining band of apocytochrome c from S. cerebisiae. The product was also shown to be identical with that of standard yeast apocytochrome c on an isoelectric focusing gel. The in vitro synthesized iso-a-cytochrome c was methylated by adding partially purified S-adenosyl-L-methionine . protein-lysine N-methyltransferase (Protein methylase III; EC 2.1.1.43) from S. cerevisiae along with S-adenosyl-L-methionine to the in vitro translation mixtures. The methylation was shown to be inhibited by the addition of the methylase inhibitor S-adenosyl-L-homocysteine or the protein synthesis inhibitor pu omycin. The methyl derivatives in the protein were identified as $\varepsilon$-N-mono, di and trimethyllysine by amino acid analysis. The molar ratio of methyl groups incorporated to that of cytochrome c molecules synthesized showed that 23% of the translated cytochrome c molecules were methylated by protein methylase III.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.7
• /
• pp.1266-1271
• /
• 2017

Lactobacillus fermentum strain JDFM216, isolated from a Korean infant feces sample, possesses the ability to enhance the longevity and immune response of a Caenorhabditis elegans host. To explore the characteristics of strain JDFM216 at the genetic level, we performed whole-genome sequencing using the PacBio system. The circular draft genome has a total length of 2,076,427 bp and a total of 2,682 encoding sequences were identified. Five phylogenetically featured genes possibly related to the longevity and immune response of the host were identified in L. fermentum strain JDFM216. These genes encode UDP-N-acetylglucosamine 1-carboxyvinyltransferase (E.C. 2.5.1.7), ErfK/YbiS/YcfS/YnhG family protein, site-specific recombinase XerD, homocysteine S-methyltransferase (E.C. 2.1.1.10), and aspartate-ammonia ligase (E.C. 6.3.1.1), which are involved in peptidoglycan synthesis and amino acid metabolism in the gut environment. Our findings on the genetic background of L. fermentum strain JDFM216 and its potential candidate genes for host longevity and immune response provide new insight for the application of this strain in the food industry as newly isolated functional probiotic.

• YAKHAK HOEJI
• /
• v.35 no.6
• /
• pp.473-482
• /
• 1991

Protein methylase II (S-adenosyl-L-methionine:protein carboxyl-O-methyltransferase; EC 2.1.1.24., PM II) was purified from chicken pancreas by subcellular fractionation, DEAE-cellulose chromatography, QAE-Sephadex A-50 chromatography, Sephadex G-75 chromatography, and Sephadex G-75 rechromatography. The purified PM II gave a single band upon polyarcrylamide gel electrophoresis both in the presence of SDS and in Tris glycine buffer without SDS. The pI value of purified PM II was identified as 5.7 on isoelectric focusing gel. Properties and activities of PM II were studied and the following results were obtained. 1) PM II from chicken pancreas was purified approximately 221-fold with a yield of 1.3%. 2) The purified PM II appear constituted of a single polypeptide chain of a molecular weight 46,800 daltons. 3) Hemoglobin exhibited the highest of methyl-accepting activity among the substrates tested. 4) The purified PM II has a $K_m$ of $4.67{\times}10^{-6}M$ and a $V_{max}$ of 37.5 pmoles of $methyl-^{14}C/min./mg$ enzyme for $SAM^{-14}CH_3$ as methyl donor in the presence of histone type II-As. 5) It is found that S-adenosyl-L-homocysteine is a competitive inhibitor for PM II with $K_i$ value of $3.23{\times}10^{-5}M$.