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W K Paik

Publications and source records attributed to W K Paik.

At least 19 recordsLinked to original sources
PubMed1992

Studies on NG-methylarginine derivatives in myelin basic protein from developing and mutant mouse brain.

The amounts of NG-methylarginine derivatives in myelin basic protein (MBP) purified from dysmyelinating mutant and different stages of normal myelinating mouse brains have been studied by using h.p.l.c. with a highly sensitive post-column o-phthaldialdehyde derivative-formation method. All three naturally occurring derivatives (NG-monomethylarginine (MeArg), NGN'G-dimethylarginine [Me2(sym)Arg] and NGNG-dimethylarginine [Me2(asym)Arg]) were found in MBP; however, their relative concentrations varied significantly with the age of the animal. The amounts of MeArg and Me2(sym)Arg in MBP increased as a function of the age of the brain, whereas that of Me2(asym)Arg decreased. MBP from early-myelinating mouse brain was shown to contain a high proportion of Me2(asym)Arg, which was hardly detectable in older brain MBP. This derivative, Me2(asym)Arg, was also absent from MBP embedded in the most compact multilamellar myelin, but was present in MBP in the least compact myelin (P3B). Comparing the extent of total methylation in vivo (sum of all three arginine derivatives), MBP extracted from less-compact myelin (P3A and P3B) showed a level approx. 40% higher than that from compact myelin. MBPs isolated from dysmyelinating mutant mouse brains, such as jimpy (jp/y) and quaking (qk/qk), contained a much higher level of Me2(asym)Arg relative to the other two methyl derivatives and also in comparison with those levels in the mother brain MBP. SDS/PAGE analysis of MBPs extracted from the mutant (both jp/y and qk/qk) as well as young normal (6-13 days old) mouse brains indicated the presence of a high-molecular-mass isoform of MBP (about 32 kDa), but this isoform was not found in adult brains. These results therefore indicate that structural integrity of myelin membrane in which MBP is embedded appears to play a pivotal role in determining the extent and the kind of Me2Arg formation in MBP at the post-translational level.

Animals↗
PubMed1992

Enzymatic methylation of heterogeneous nuclear ribonucleoprotein in isolated liver nuclei.

Protein N-methyltransferase activity has been studied in the rat liver nuclei, using recombinant heterogeneous nuclear ribonucleoprotein particle protein A1 and histone as the methyl acceptors. The hydrolysates of these two enzymatically [methyl-3H]-labeled proteins, however, yielded different patterns of methylated amino acids on HPLC analysis: NG-monomethylarginine (92%) and NG-NG-dimethyl (asymmetric) arginine (6.5%) were the major methylated amino acids identified in the protein A1, whereas epsilon-N-methylated lysine derivatives constituted a predominant portion (71%) of the methylated amino acids in histone. When liver extracts isolated from rats fed a methyl deficient diet were assayed, the methyl accepting activity of protein A1 increased 64% over the control (rats fed normal diet), while that of histone increased 260%. Partial hepatectomy induced a 7.9-fold and 2.3-fold increase in the protein A1 methylase activity after 24 and 48 h of regeneration, respectively. These results, together with the fact that myelin basic protein-specific protein methylase I does not significantly methylate protein A1, indicate the presence of an enzyme in the rat liver nuclei which methylates the protein A1.

Animals↗
PubMed1992

Effect of methyl substitution on protein tertiary structure.

Biological effects caused by the post-translational methylation of certain side chains in proteins has been thought to be due solely to changes in charge, steric relations or hydrophobicity at the site of the methyl group. However, there is increasing evidence that the presence of CH3 can also induce a "global" effect on the protein molecule. Some of the evidence is described in this paper.

Amines↗
PubMed1992

In vivo and in vitro methylation of lysine residues of Euglena gracilis histone H1.

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) from Euglena gracilis [J. Biol. Chem., 260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as epsilon-N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as the in vitro substrate. Presently, histone H1 has been purified from Euglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. The Euglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higher Rf value than the other histones H1 in acid/urea gel electrophoresis. When the Euglena histone H1 was [methyl-3H]-labeled in vitro by a homologous enzyme (one of the two Euglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C18 columns on HPLC, we demonstrated that Euglena histone H1 contains approximately 9 mol% of epsilon-N-methyllysines (1.40, 1.66, and 5.62 mol% for epsilon-N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of epsilon-N-methyllysines in histone H1.

Animals↗
PubMed1991

Enzymic methylation of myelin basic protein in myelin.

Myelin fractions with different degrees of compaction were isolated from bovine brain, and post-translational methylation of membrane-associated proteins was studied. When the purified myelin-basic-protein-specific protein methylase I and S-adenosyl-L-[methyl-14C]methionine were added exogenously, the most compact myelin fraction exhibited higher methyl-accepting activity than the less compact dense fractions. The methylated protein was identified as myelin basic protein (18.4 kDa) exclusively among the several myelin proteins from all membrane fractions, by SDS/PAGE/radioautography of methyl-14C-labelled membrane proteins. The methyl-14C-labelled amino acid residue in the basic protein was identified by h.p.l.c. as NG-methylarginine, indicating the high degree of specificity for the arginine residue as well as the myelin basic protein in the intact myelin membranes. The possibility of a charge alteration of myelin basic protein resulting from its arginine methylation was investigated by using the purified component 1 of myelin basic protein. The methylated component was shown to be less cationic than the unmethylated component by Bio-Rex 70 cation-exchange chromatography, since the former preceded the latter. However, in the presence of the denaturant (guanidinium chloride), the two species were co-eluted, indicating that the charge difference between methylated and unmethylated myelin basic protein can only be shown under the renatured condition.

Amino Acids↗
PubMed1991

An enzyme-linked immunosorbent assay for myelin basic protein-specific protein methylase I.

A sandwich enzyme-linked immunosorbent assay (ELISA) has been developed to determine myelin basic protein (MBP)-specific protein methylase I. Rabbit immunoglobulin anti-bovine MBP-specific protein methylase I, purified by Sepharose-A affinity chromatography, was utilized as the primary antibodies, while the same antibodies which had been conjugated to peroxidase were employed as the indicator antibodies. This assay method was about 280 times more sensitive than the conventional trichloracetic acid (TCA) precipitation method. Employing the ELISA, the level of MBP-specific protein methylase I during mouse brain development was examined; the peak level of the methylase was shown to be at 16th postnatal day, indicating temporal correlation with myelination. Among several species of brains examined, human showed the highest and carp the least amount of MBP-specific protein methylase I; 6.33 micrograms and 0.33 micrograms per mg of brain cytosol protein, respectively. Dysmyelinating jimpy hemizygous mouse brain showed the immunoreactive MBP-specific protein methylase only 60% that of the control at 20 days of age. The high sensitivity of the method together with the fact that MBP-specific protein methylase is present in human cerebrospinal fluid suggests a possible clinical application of this method for evaluating demyelinating disorders.

Animals↗
PubMed1990

Substrate specificity for myelin basic protein-specific protein methylase I.

The substrate specificity of bovine brain myelin basic protein (MBP)-specific protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), which methylates arginine residues of protein, has been studied using various MBPs, several synthetic peptides and heterogeneous nuclear ribonucleoprotein complex protein (hnRNP). (1) Among MBPs from different species of brain, the carp MBP was found to be the best substrate for MBP-specific protein methylase I. This high degree of methyl acceptability is most likely due to the fact that carp MBP is not in vivo methylated at the arginine residue (Deibler, G.E. and Martenson, R.E. (1973) J. Biol. Chem. 248, 2387-2391) and that the methylatable amino acid sequence is present in this protein. (2) In order to study the minimum chain length of MBP polypeptide which functions as the methyl acceptor, several synthetic polypeptides whose sequences are identical to the region surrounding the residue 107 of bovine MBP (the in vivo methylation site) were synthesized. It was found that the hexapeptide, Gly-Lys-Gly-Arg-Gly-Leu (corresponding to residues 104-109 of bovine MBP), was the shortest methyl accepting peptide, while the tetrapeptide, Gly-Arg-Gly-Leu (corresponding to residues 106-109) was inactive as a substrate. (3) hnRNP protein is known to contain methylarginine at residue 193 (Williams, K.R., Stone, K.L., LoPresti, M.B., Merrill, B.M. and Plank, S.R. (1985) Proc. Natl. Acad. Sci. USA 82, 5666-5670) which is post-translationally modified. Thus, the RNP protein overproduced in Escherichia coli and therefore did not contain methylarginine was examined for its methyl acceptability. It was found that neither MBP-specific nor histone-specific protein methylase I could methylate this methylarginine-less RNP protein. This suggests a possible existence of a distinct protein methylase I specific for this nuclear protein.

Amino Acid Sequence↗
PubMed1989

Studies on protein methyltransferase in human cerebrospinal fluid.

Protein methyltransferases, rich in most mammalian brains, were studied in human cerebrospinal fluid (CSF). Among several well-characterized groups of methyltransferases, protein methylase I (S-adenosylmethionine:protein-arginine N-methyltransferase, EC 2.1.1.23) was found in significant amounts in human CSF samples. Both myelin basic protein (MBP) -specific and histone-specific protein methylase I activities were observed, the latter being generally higher in most CSF. S-Adenosyl-L-homocysteine, a potent product inhibitor for the methyltransferase, inhibited approximately 90% of MBP-specific protein methylase I activity at a concentration of 1 mM. The optimum pH of the MBP-specific protein methylase I was found to be around 7.2. Identity of exogenously added MBP as the methylated substrate for CSF enzyme was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An amino acid analysis of the [methyl-3H]protein hydrolysate showed two major radioactive peaks cochromatographing with monomethyl- and dimethyl (symmetric)-arginine. Human CSF contained relatively high endogenous protein methylase I activity (activity measured without added substrate protein): The endogenous substrate can be immunoprecipitated by antibody raised against calf brain MBP. Finally, CSF from several neurological patients were analyzed for protein methylase I, and the results are presented.

Humans↗
PubMed1989

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.

Apoproteins↗
PubMed1989

Site-specificity of histone H1 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.

Amino Acids↗
PubMed1989

Cytochrome c methylation.

In this review, protein methylation is outlined in general terms, highlighting the major amino acids that are methylated and some of the proteins in which they are found. The majority of the review examines the methylation of cytochrome c at Lys-77 of lower eukaryotes as a possible model for methylation studies. Early work involving the purification and characterization of the methyltransferase responsible for this methylation indicated cytochrome c was methylated posttranslationally, yet prior to import into the mitochondria. Methylation in vitro occurred only at the in vivo methylation site and only on cytochrome c. Later studies using in vitro translated apocytochrome c revealed that methylated, as compared with unmethylated, apocytochrome c was imported preferentially into yeast, but not rat liver, mitochondria. Efforts to discover the reasons for this preference have shown that methylation of apocytochrome c dramatically lowers its isoelectric point (against a predicted increase) and decrease its Stokes radius. A possible mechanism for these differences involving the disruption of hydrogen bonds is presented here with space-filling models. Finally, the in vivo significance of this modification is also discussed.

Animals↗
PubMed1988

Purification and molecular identification of two protein methylases I from calf brain. Myelin basic protein- and histone-specific enzyme.

Two different molecular species of protein methylases I (S-adenosylmethionine:protein-arginine N-methyltransferase, EC 2.1.1.23), one specific for myelin basic protein (MBP) and the other for histone, have been purified from calf brain to near homogeneity, as discerned by nondenaturing polyacrylamide gel electrophoresis. Although both methylases share some common properties, such as utilization of S-adenosyl-L-methionine as the methyl donor and methylation of protein-bound arginine residues, they are distinctly different from each other in molecular weight and in catalytic, as well as the immunological, properties. The MBP-specific protein methylase I (approximately 500 kDa) methylates MBP preferentially (Km = 2 X 10(-7) M) and histone to a much lesser extent (Km = 1 X 10(-4) M), while the histone-specific methylase I (approximately 275 kDa) methylates histone only. Both methylases exhibit two major subunit bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: 100 and 72 kDa for the MBP-specific and 110 and 75 kDa for the histone-specific. At 0.5 mM p-chloromercuribenzoate, about 50% of the MBP-specific enzyme remained as active, while most of the histone-specific enzyme activity was lost. In 2 mM guanidine HCl, approximately 90% of the former enzyme activity remained while nearly complete inactivation of the latter enzyme was observed. The enzymes also exhibited quite different inactivation profiles toward high temperature (45-65 degrees C); MBP-enzyme was stable up to 50 degrees C and was rapidly inactivated at higher temperatures with an inflection point at about 57 degrees C. However, under the identical conditions, histone-enzyme was inactivated progressively and linearly in the same temperature range. Finally, Western immunoblot analysis of polyclonal antibodies directed against either enzyme exhibited no cross-reactivity with the other.

Animals↗
PubMed1988

Effect of enzymatic methylation of yeast iso-1-cytochrome c on its isoelectric point.

Yeast iso-1- unmethylated and methylated apocytochrome c were synthesized in vitro by translating yeast cytochrome c mRNA, and by subsequently methylating the protein product. Unmethylated and methylated iso-1-holocytochrome c were extracted from Saccharomyces cerevisiae. By employing a column isoelectrofocusing technique, the pI values of these proteins were determined. The pI values of unmethylated and methylated apocytochrome c were found to be 9.60 and 8.70, respectively, with a difference of 0.90 pI unit. On the other hand, the pI values of unmethylated and methylated holocytochrome c were 9.72 and 9.68, respectively, with a difference of 0.04 unit. Therefore, although the pI values of both apo- and holocytochrome c decreased by methylation, methylation of apocytochrome c had a more profound effect on the pI of the protein. The result also indicated that conjugation of heme to apocytochrome c increased its pI value, resulting in the more "compact" and basic structure of the protein. The observed magnitude of the pI change subsequent to the methylation of apocytochrome c (decrease of 0.90 unit) seemed to be contradictory to the predicted increase in the value, since the positive charge is fixed on the quaternary amino group of trimethyllysine and there is no proton to titrate. Trimethylation of epsilon-NH2 group of Res-72 lysine of apocytochrome c could disrupt any possible hydrogen bond formed by the nitrogen atom of Res-72 lysine residues, as visualized by a space-filling model. The model and observed shift in the "effective charge" of the protein strongly suggest that conformational change in the apoprotein takes place upon methylation. This presumably altered conformation along with the decrease in pI caused by methylation may play a role in enhancement of apocytochrome c import into mitochondria.

Chromatography, Gel↗
PubMed1988

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.

Amino Acids↗