Hyang Woo Lee

Nonenzymatic acetylation of histones with acetyl-CoA

Abstract 1. When purified calf thymus histones were incubated with [1- 14 C]acetyl-CoA and treated to remove the acid-soluble fraction with trichloroacetic acid, a large amount of radioactivity still remained in the 15 % trichloroacetic acid-insoluble precipitate. 2. This incorporation of radioactivity into the protein did not seem to be enzymatic, since it took 60 min boiling of the histone to diminish only about 60 % of the incorporation. The boiled histones were eluted faster than the “native” histone on Sephadex G-50 column in 0.02 M HCl, suggesting a change in the tertiary structure of the proteins. Furthermore, polylysine was efficiently acetylated. 3. The reaction was dependent on pH, period of incubation, ionic strength and ionic species. The activation energy for the reaction was 7.5 kcal. 4. All of the various histones were acetylated, but slightly lysine-rich histone was acetylated the most. Among various other proteins and polypeptides tested, only polylysine and polyarginine were highly acetylated. 5. At maximum acetylation, one out of every 120 amino acid residues in histone was acetylated. Amino acid analysis of proteolytic enzyme digests of [ acetyl - 14 C]histone and [ acetyl - 14 C]polylysine revealed that practically all of the incorporated radioactivity was as e - N -acetyllysine. The presence of e - N -[ acetyl - 14 C]acetyllysine was also confirmed by paper chromatography.

Protein methylation during the development of rat brain

Summary The activities of protein methylase I and III are high in the fetal rat brain and decrease rapidly after birth. On the other hand, protein methylase II activity increases slowly during the first 10 days of life and rapidly thereafter. Within 40 days after birth, the levels of all three protein methylase activities reach values corresponding to those of the adult brain. Results obtained for in vivo protein methylation indicate that the H 2 SO 4 -soluble protein is methylated at high rate in fetal brain; this rate then decreases rapidly after birth. Therefore, protein methylation might be involved in the development of the central nervous system.

Histone methylation during hepatic regeneration in rat

The activity of protein methylase I (S-adenosylmethionine : protein-arginine methyltransferase) increases biphasically during hepatic regeneration of rat; reaching the first peak at the second day and the second peak at the fourth day. However, this pattern of enzyme activity does not necessarily correlate with the rate of Me-14C incorporation into NG,NG-dimethylarginine in histones. The protein methylase II (S-adenosylmethionine : protein-carboxyl methyltransferase) activity has a single peak at the 4th day after partial hepatectomy. On the other hand, the activity of protein methylase III (S-adenosylmethionine : protein-lysine methyltransferase) increases biphasically with peaks at the 2nd and 4th day of regeneration. Furthermore, this change of protein methylase III activity coincides with that amount found in histone as well as with the rate of Me-14C incorporation into e-N-monomethyllysine and e-N-dimethyllysine in histone. Under the present conditions, the peak of DNA synthesis was found at the 2nd day of hepatic regeneration. Therefore, histone methylation is not a late event.

Non-enzymatic methylation of proteins with S-adenosyl-L-methionine

It is now well established that various side chains of some proteins are methylated in vivo [ 1,2] These protein methylation reactions are highly specific. Thus, protein methylase I (S-adenosylmethionine: protein-arginine methyltransferase; EC 2.1 .I 23) methylates the guanidino group of arginine residues [3], protein methylase II (S-adenosylmethionine: protein-carboxyl me thyltransferase; EC 2.1.1.24) methylates free carboxyl group of aspartyl and glutamyl residues [4], and protein methylase III (S-adenosylmethionine: protein-lysine methyltransferase; EC 2.1.1.25) methylates the e-amino group of lysine residues. We report here some experimental results which indicate that these enzymecatalyzed protein methylation reactions can occur in the absence of enzyme with S-adenosyl-Lmethionine as methyl donor.

Chapter 5. Purification and Characterization of Protein Methylase I (S-Adenosylmethionine: Protein-Arginine Methyltransferase; EC 2.1.1.23) From Calf Brain

Publisher Summary The chapter discusses an enzyme that has been originally isolated from calf thymus and found to be located primarily in the cytosol. Analysis of endogenous methylated proteins shows that mainly histones are methylated. The enzyme is found in various organs of the rat and is especially elevated in brain, thymus, testis, and spleen. The products of histone methylation by protein methylase I can be identified as N G -mono, N G , N G -di-, and N G , N rG -dimethylarginin. Incorporation of S adenosyl- L [methyl- 14 C] methionine into histone can be measured under conditions (pH 7.2) III favorable for methylation of the guanidino group of arginine residues. Methylation of lysine residues by protein methylase III is negligible at pH 7.2. This chapter discusses the purification procedures where the initial steps are with minor modification. The final enzyme preparation is free of other protein methylases (II and III).

Enzymatic hydrolysis of histones in rat kidney microsomes

Abstract The microsomal fraction of rat kidney contains an enzyme which hydrolyzes basic proteins such as histones and protamine. However, the enzyme is present in all the organs of the rat to some degree. The enzyme is most active toward protamine and histones, but slightly active to ribonuclease. Albumin and globulin are completely resistant to the action of the enzyme. The optimum pH was found to be around 8 – 9, depending on the proteins used as substrate. Evidence indicates that the enzyme is not one of the cathepsins.

Enzymology of Protein Methylation: Recent Development

The presence of e-N-methyllysine was first observed in Salmonella typhimurium in 1959. Since this discovery the ubiquitous occurrence of protein methylation in nature has been well established (Paik & Kim, 1980). It involves N-methylation of lysine, arginine, histidine, alanine, proline, and glutamine, O-methylation of glutamic and aspartic acid, and S-methylation of methionine and cysteine (Paik & Kim, 1985). As shown in Table I, methylated amino acids occur in highly specialized proteins such as histones, flagella protein, myosin, actin, ribosomal proteins, opsin, EFlα(Tu), HnRNP protein, HMG-1 and HMG-2 protein, fungal and plant cytochrome c, myelin basic protein, porcine heart citrate synthase, heat-shock proteins, nucleolar protein, ferredoxin, wheat α-amylase, and calmodulin.

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.