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Although it has been demonstrated that p21WAF1/Cip1 could be induced by transforming growth factor-beta1 (TGF-beta1) in a Smad-dependent manner, the cross-talk of Smad signaling pathway with other signaling pathways still remains poorly understood. In this study, we investigated a possible role of hydrogen peroxide (H2O2)-ERK pathway in TGF-beta1 induction of p21WAF1/Cip1 in human keratinocytes HaCaT cells. Using pharmacological inhibitors specific for MAP kinase family members, we found that ERK, but not JNK or p38, is required for TGF-beta1 induction of p21WAF1/Cip1. ERK activation by TGF-beta1 was significantly attenuated by treatment with N-acetyl-l-cysteine or catalase, indicating that reactive oxygen species (ROS) generated by TGF-beta1, mainly H2O2, stimulates ERK signaling pathway to induce the p21WAF1/Cip1 expression. In support of this, TGF-beta1 stimulation caused an increase in intracellular ROS level, which was completely abolished by pretreatment with catalase. ERK activation does not appear to be associated with nuclear translocation of Smad-3, because ERK inhibition did not affect nuclear translocation of Smads by TGF-beta1, and H2O2 treatment alone did not cause nuclear translocation of Smad-3. On the other hand, ERK inhibition ablated the phosphorylation of Sp1 by TGF-beta1, which was accompanied with the disruption of interaction between Smad-3 and Sp1 as well as of the recruitment of Sp1 to the p21WAF1/Cip1 promoter induced by TGF-beta1, indicating that ERK signaling pathway might be necessary for their interaction. Taken together, these results suggest that activation of H2O2-mediated ERK signaling pathway is required for p21WAF1/Cip1 expression by TGF-beta1 and led us to propose a cooperative model whereby TGF-beta1-induced receptor activation stimulates not only a Smad pathway but also a parallel H2O2-mediated ERK pathway that acts as a key determinant for association between Smads and Sp1 transcription factor.

Lysophosphatidic acid (LPA) mediates diverse biological responses, including cell migration, through the activation of G-protein-coupled receptors. Recently, we have shown that LPA stimulates p21-activated kinase (PAK) that is critical for focal adhesion kinase (FAK) phosphorylation and cell motility. Here, we provide the direct evidence that p85 beta-PIX is required for cell motility of NIH-3T3 cells by LPA through FAK and p38 MAP kinase phosphorylations. LPA induced p85 beta-PIX binding to FAK in NIH-3T3 cells that was inhibited by pretreatment of the cells with phosphoinositide 3-kinase inhibitor, LY294002. Furthermore, the similar inhibition of the complex formation was also observed, when the cells were transfected with either p85 beta-PIX mutant that cannot bind GIT or dominant negative mutants of Rac1 (N17Rac1) and PAK (PAK-PID). Transfection of the cells with specific p85 beta-PIX siRNA led to drastic inhibition of LPA-induced FAK phosphorylation, peripheral redistribution of p85 beta-PIX with FAK and GIT1, and cell motility. p85 beta-PIX was also required for p38 MAP kinase phosphorylation induced by LPA. Finally, dominant negative mutant of Rho (N19Rho)-transfected cells did not affect PAK activation, while the cells stably transfected with p85 beta-PIX siRNA or N17Rac1 showed the reduction of LPA-induced PAK activation. Taken together, the present data suggest that p85 beta-PIX, located downstream of Rac1, is a key regulator for the activations of FAK or p38 MAP kinase and plays a pivotal role in focal complex formation and cell motility induced by LPA.

Cells change their gene expression profile dynamically in various conditions. By taking the advantage of ChIP, we examined the transcription profile of Saccharomyces cerevisiae genes in response to DNA damaging agents such as MMS or 4NQO. Gene expression profiles of different groups of genes roughly correlated with that revealed by Northern blot assay or microarray method. Damage-inducible genes showed increased cross-linking signals of RNA polymerase II, TFIIH, and TFIIF, meanwhile damage repressible genes decreased them, which means that gene expression is mainly regulated at the level of transcription. Interestingly, the characteristic occupancy pattern of TFIIH and polymerase with phosphorylated carboxy-terminal domain (CTD) in promoter or in coding regions was not changed by the presence of DNA damaging agents in both non-inducible and inducible genes. ChIP data showed that the extent of phosphorylation of CTD per elongating polymerase complex was still maintained. These findings suggest that overall increase in CTD phosphorylation in response to DNA damage is attributed to the global shift of gene expression profile rather than modification of specific polymerase function.

The eukaryotic mRNAs produced by RNA polymerase II (Pol II) are capped with an inverted 7-methyl-guanosine (m7G) linked to the first residue of the mRNA (36). This event occurs by a series of three enzymatic reactions; The 5′ triphosphate end of the nascent RNA Pol II transcript is cleaved by 5′ RNA triphosphatase to produce the diphosphate end. RNA guanylyltransferase forms a covalent enzyme-GMP complex and subsequently caps the RNA substrate by adding a guanosine residue in a 5′-5′ triphosphate linkage. The cap is then methylated by RNA (guanine-7) methyltransferase (23, 39). In higher eukaryotes, a bifunctional monomeric polypeptide carries both the RNA triphosphatase and guanylyltransferase activities, while the capping enzyme from yeast is a complex of RNA triphosphatase and guanylyltransferase subunits (40). The polypeptides are encoded by the CET1 and CEG1 genes, respectively in Saccharomyces cerevisiae, and both are essential for the cell viability. Capping, the first mRNA modification, occurs by the time the transcript is only 25 to 30 nucleotides long in an early transcription phase. Such cotranscriptional capping is mediated by recruitment of capping enzyme machinery to the phosphorylated carboxy-terminal domain (CTD) of the largest subunit of Pol II (7, 15, 22, 51). The CTD of Pol II has an unusual structure with many heptapeptide repeats (YSPTSPS). The capping enzyme binds directly and specifically to the phosphorylated CTD of Pol II via the Ceg1 subunit (yeast) or the guanylyltransferase domain (metazoan). Furthermore, the guanylyltransferase activity of Ceg1 associated with phosphorylated CTD is allosterically regulated by both the Cet1 and phosphorylated CTD to ensure it has a coordinated capping activity (8). The mammalian capping enzyme is also allosterically regulated by an interaction with the phosphorylated CTD (16, 25). The CTD phosphorylated at serine 5 of the heptapeptide repeat appears to be important for the capping reaction because it stimulates the guanylyltransferase activity, although both serine 2 phosphorylation and serine 5 phosphorylation can mediate the protein interaction. An interesting extension to these findings was added by a chromatin immunoprecipitation assay, which provides in vivo evidence that the capping enzyme machinery interacts dynamically with Pol II during a transcription cycle (20, 35). The capping enzyme interacts with Pol II immediately after the serine 5 of CTD is phosphorylated. As serine 5 phosphorylation decreases in an early elongation phase, the capping enzyme dissociates from the transcription complex. Recently, data from several groups have raised the concept of “checkpoints” in transcription, especially in an early phase (summarized in reference 26). As operated during the cell cycle to ensure that each phase of the cycle is complete before the next one begins, checkpoint in early transcription is suggested to play a role in ensuring that only the properly modified RNA at the 5′ end is extended. The Pol II transcription is thus subjected to checkpoint control for the coordinated transcription with mRNA capping (6, 25, 26). Several transcription factors have been reported to play in this window. DSIF, a human homolog of the yeast transcription factor Spt4/Spt5, increases the pausing of Pol II and thus plays a role as a negative factor (42, 46, 49). Within this temporal and spatial interval, while Pol II with the hypophosphorylated CTD is paused at the promoter-proximal region, many factors are intended to target the capping enzymes to increase their recruitment or to enhance their catalytic activities. In addition to TFIIH, which creates a binding epitope for the capping enzyme on CTD by serine 5 phosphorylation, as described above, Spt5 itself interacts with the triphosphatase and guanylyltransferase components of the capping enzyme (21, 27, 44). In the case of human immunodeficiency virus (HIV), DSIF/Spt5-induced transcription arrest allows HIV-encoded Tat to interact with capping enzymes and to stimulate their catalytic activities (5, 6). Phosphorylation of Pol II CTD is critical for the transition to the elongation phase. At this step, the elongation factor P-TEFb, a DRB-sensitive protein kinase, phosphorylates the Pol II CTD and Spt5 (19, 29, 38). HIV Tat also interacts with P-TEFb in this step (29). In S. cerevisae, Ctk1 kinase complex and the Bur1 kinase complex facilitate the transcription elongation (18, 50). The phosphorylation of Pol II CTD is thus meant to lead to the formation of the processive transcription elongation complexes. According to recent reports, the capping enzyme in Schizosaccharomyces pombe interacts with Cdk9/Pch1, a yeast P-TEFb homolog (28). How does the capping enzyme fit into the complicated scheme to delineate it in the order of pausing, capping, and the reversion of pausing in the checkpoint model? If the elongation-competent transition does not occur until the RNA is capped and the capping is the major determinant to shift the Pol II status, it could be indicative that a capping enzyme plays a critical role in regulation of an early transcription in addition to its role in simple cap formation. To study whether the capping enzyme plays a key role in coordinating mRNA processing and transcription elongation, we used a well-characterized yeast system. Because Ceg1, a capping enzyme subunit, contributes to transcription via its typical cap formation activity, this study examined various ceg1 temperature-sensitive alleles to determine if there is any additional role in transcription elongation. Among them ceg1-63 displayed 6-azauracil (6AU) sensitivity and a defect in PUR5 induction by 6AU treatment. This study shows that transcription through the pause sites artificially inserted at the promoter-proximal region was severely inhibited in ceg1-63. We show that such an elongation defect was coupled to the reduced guanylyltransferase activity. However, it happened independently of the turnover of uncapped transcripts. These results indicate that the transcription ternary complexes are held at the promoter as long as their RNAs have not been properly capped. That is, capping enzyme plays a critical role in the promoter-proximal checkpoint window by reinforcing the checkpoint security circuit and probably by reversing the transcription arrest in time. This finding also strongly supports the transcription checkpoint model, in which an early transcription is tightly regulated.

The functional role of protein carboxylmethylation (PCM) has not yet been clearly elucidated in the tissue level. The biochemical feature of PCM in porcine spleen was therefore studied by investigating the methyl accepting capacity (MAC) of natural endogenous substrate proteins for protein carboxyl O-methyltransferase (PCMT) in various conditions. Strong acidic and alkaline-conditioned (at pH 11.0) analyses of the MAC indicated that approximately 65% of total protein methylation seemed to be mediated by spleen PCMT. The hydrolytic kinetics of the PCM products, such as carboxylmethylesters (CMEs), under mild alkaline conditions revealed that there may be three different kinds of CMEs [displaying half-times (T1/2) of 1.1 min (82.7% of total CMEs), 13.9 min (4.6%), and 478.0 min (12.7%)], assuming that the majority of CME is base-labile and may be catalyzed by class I PCMT. In agreement with these results, several natural endogenous substrate proteins (14, 31 and 86 kDa) were identified strikingly by acidic-conditioned electrophoresis, and their MAC was lost upon alkaline conditions. On the other hand, other proteins (23 and 62 kDa) weakly appeared under alkaline conditions, indicating that PCM mediated by class II or III PCMT may be a minor reaction. The MAC of an isolated endogenous substrate protein (23-kDa) was also detected upon acidic-conditioned electrophoresis. Therefore, our data suggest that most spleen PCM may be catalyzed by class I PCMT, which participates in repairing aged proteins.

To define the mechanism of arsenite-induced tumor promotion, we examined the role of reactive oxygen species (ROS) in the signaling pathways of cells exposed to arsenite. Arsenite treatment resulted in the persistent activation of p70(s6k) and extracellular signal-regulated kinase 1/2 (ERK1/2) which was accompanied by an increase in intracellular ROS production. The predominant produced appeared to be H(2)O(2), because the arsenite-induced increase in dichlorofluorescein (DCF) fluorescence was completely abolished by pretreatment with catalase but not with heat-inactivated catalase. Elimination of H(2)O(2) by catalase or N-acetyl-L-cysteine inhibited the arsenite-induced activation of p70(s6k) and ERK1/2, indicating the possible role of H(2)O(2) in the arsenite activation of the p70(s6k) and the ERK1/2 signaling pathways. A specific inhibitor of p70(s6k), rapamycin, and calcium chelators significantly blocked the activation of p70(s6k) induced by arsenite. While the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 completely abrogated arsenite activation of p70(s6k), ERK1/2 activation by arsenite was not affected by these inhibitors, indicating that H(2)O(2) might act as an upstream molecule of PI3K as well as ERK1/2. Consistent with these results, none of the inhibitors impaired H(2)O(2) production by arsenite. DNA binding activity of AP-1, downstream of ERK1/2, was also inhibited by catalase, N-acetyl-L-cysteine, and the MEK inhibitor PD98059, which significantly blocked arsenite activation of ERK1/2. Taken together, these studies provide insight into mechanisms of arsenite-induced tumor promotion and suggest that H(2)O(2) plays a critical role in tumor promotion by arsenite through activation of the ERK1/2 and p70(s6k) signaling pathways.

We previously reported the presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P whose activity was induced by methanol. In the present study, the methanol-induced NOS was purified 900-fold from S. aureus by means of Mono Q ion exchange column, 2',5'-ADP-agarose affinity column, and Superdex 200HR gel permeation column chromatography. The purified bacterial NOS showed two protein bands with 67 and 64 kDa molecular mass on SDS-PAGE. However, the molecular mass of the NOS was 135 kDa on Superdex 200HR gel permeation column chromatography, indicating that the native enzyme exists as a heterodimer. This bacterial NOS had K(m) value of 13.4x10(-6) M for L-arginine and V(max) of 35.3 nmol min(-1) mg(-1) protein. In addition, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, calmodulin and Ca(2+) were required as cofactors in the conversion of L-arginine to L-citrulline, and NOS inhibitors selectively inhibited the activity of the purified NOS.

The antimetabolite 5-fluorouracil (5-FU) is one of the more prominent clinical antitumor agents available for the treatment of stomach and colorectal cancers. In the present study, we characterized the effects of 5-FU on nitric oxide (NO) production by cells from the stomach cancer cell line NCI-N87. A cytokine mixture [interleukin (IL)-1beta/interferon (IFN)-gamma] increased the production of NO by stomach cancer cells in a concentration- and time-dependent manner. Pretreatment with 5-FU inhibited the production of NO that was stimulated by the cytokine mixture and reduced the expression of iNOS. The cytokine mixture activated nuclear factor kappaB (NF-kappaB) in a concentration- and time-dependent manner, which was blocked by 5-FU pretreatment. The pretreatment with 5-FU stabilized IkappaBalpha and inactivated IkappaB kinase. Collectively, these data suggest that the efficacy of 5-FU may include the inactivation of IkappaB kinase and the inhibition of NO production.

Doxorubicin (DOX) is an active and broad spectrum chemotherapeutic agent. Increased inducible nitric oxide synthase (NOS) expression and/or activity have been reported in several human tumors. While the relationship between DOX treatment and the enzymatic activity of endothelial NOS has been well characterized, little is known about the effects of DOX on the expression of iNOS in human cancer cells. In the present study, we characterized the effects of DOX on the nitric oxide (NO) production by colorectal cancer cells, DLD-1. IFN-gamma/IL-1beta (CM) increased the production of NO, whereas pretreatment of DOX inhibited the production of NO in response to CM in a dose dependent manner. The increased expressions of iNOS mRNA and protein by CM were completely blocked by DOX without affecting the iNOS mRNA stability. However, DOX activated nuclear factor-kappaB (NF-kappaB) in response to CM. Furthermore, the expression of inhibitor kappaB alpha was reduced by DOX in a dose dependent manner. Collectively, DOX inhibited the production of NO by DLD-1 cells, which is not linked to well known transcription factor, NF-kappaB. Therefore, further studies on the possible mechanisms of inhibitory effects of NO production by DOX would be worth pursuing.

We have previously reported on the identification of the endogenous transmethylation inhibitor oligosaccharide-linked acyl carrier protein (O-ACP). In this study, the role of the transmethylation reaction on cell cycle progression was evaluated using various transmethylase inhibitors, including O-ACP. O-ACP significantly inhibited the growth of various cancer cell lines, including NIH3T3, ras-transformed NIH3T3, MDA-MB-231, HT-1376, and AGS. In addition, exposure of ras-transformed NIH3T3 to O-ACP caused cell cycle arrest at the G0/G1 phase, which led to a decrease in cells at the S phase, as determined by flow cytometry. In contrast, transmethylase inhibitors did not affect the expression of p21(WAF1/Cip1), a well known inhibitor of cyclin dependent kinase, indicating that the cell cycle arrest by transmethylase inhibitors might be mediated by a p21(WAF1/Cip1)-independent mechanism. Therefore, O-ACP, a novel transmethylase inhibitor, could be a useful tool for elucidating the novel role of methylation in cell proliferation and cell cycle progression.

We previously reported that apicidin, a novel histone deacetylase inhibitor, inhibited the proliferation of tumor cells via induction of p21(WAF1/Cip1). In this study, we determined the molecular mechanisms by which apicidin induced the p21(WAF1/Cip1) gene expression in HeLa cells. Apicidin induced p21(WAF1/Cip1) mRNA independent of the de novo protein synthesis and activated the p21(WAF1/Cip1) promoter through Sp1-3 site located at -82 and -77 relative to the transcription start site. This transcriptional activation appears to be mediated by protein kinase C (PKC), because calphostin C, a PKC inhibitor, significantly attenuated the activation of p21(WAF1/Cip1) promoter via Sp1 sites, which was accompanied by a marked suppression of p21(WAF1/Cip1) mRNA and protein expression induced by apicidin. Consistent with the transcriptional activation of p21(WAF1/Cip1) promoter by apicidin, apicidin treatment led to the translocation of PKCepsilon from cytosolic to particulate fraction, which was reversed by pretreatment with calphostin C, indicating the involvement of PKC in the transcriptional activation of p21(WAF1/Cip1) via Sp1 sites by apicidin. However, the PKC-mediated transcriptional activation of p21(WAF1/Cip1) by apicidin appears to be independent of the histone hyperacetylation, because apicidin-induced histone hyperacetylation was not affected by calphostin C. Furthermore, a PKC activator, phorbol 12,13-dibutyrate, alone induced the transcriptional activation of p21(WAF1/Cip1) promoter, p21(WAF1/Cip1) mRNA, and protein expression without induction of the histone hyperacetylation, suggesting that the transcriptional activation of p21(WAF1/Cip1) by apicidin might have been mediated by a mechanism other than chromatin remodeling through the histone hyperacetylation. Taken together, these results suggest that the PKC signaling pathway plays a pivotal role in the transcriptional activation of the p21(WAF1/Cip1) gene by apicidin.

We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) by ergolide, sesquiterpene lactone from Inula britannica. iNOS activity in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS. Ergolide markedly decreased the production of prostaglandin E(2) (PGE(2)) in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner, without alteration of the catalytic activity of COX-2 itself. Ergolide decreased the level of iNOS and COX-2 protein, and iNOS mRNA caused by stimulation of LPS/IFN-gamma in a concentration-dependent manner, as measured by Western blot and Northern blot analysis, respectively. Ergolide inhibited nuclear factor-kappaB (NF-kappaB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-gamma. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF-kappaB as well as IkappaB-alpha degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF-kappaB itself or an upstream molecule of NF-kappaB. Ergolide also directly inhibited the DNA-binding activity of active NF-kappaB in LPS/IFN-gamma-pretreated RAW 264.7 macrophages. These results demonstrate that the suppression of NF-kappaB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF-kappaB resulted from blockade of the degradation of IkappaB and the direct modification of active NF-kappaB, leading to the suppression of the expression of iNOS and COX-2, which play important roles in inflammatory signalling pathway.

Excessive nitric oxide (NO) produced by inducible NO synthase (iNOS) acts as a causative regulator in various inflammatory disease states. Carpesium divaricatum has been used in Korean traditional herbal medicine for its antipyretic, analgesic, vermifugic, and anti-inflammatory properties. We investigated the molecular mechanism for the suppression of lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-induced NO production in RAW 264.7 macrophages by the sesquiterpene lactone 2beta,5-epoxy-5,10-dihydroxy-6alpha-angeloyloxy-9beta-isobutyloxy-germacran-8alpha,12-olide (C-1), which has been identified recently as a new compound from C. divaricatum. C-1 decreased NO production in LPS/IFN-gamma-stimulated RAW 264.7 cells in a concentration-dependent manner, with an IC50 of approximately 2.16 microM; however, it had no direct effect on the iNOS activity of fully LPS/IFN-gamma-stimulated RAW 264.7 cells. Furthermore, treatment with C-1 led to a decrease in iNOS protein and mRNA. These effects appear to be due to inhibition of nuclear factor-kappaB (NF-kappaB) activation through a mechanism involving stabilization of the NF-kappaB/inhibitor of the kappaB (I-kappaB) complex, since inhibition of NF-kappaB DNA binding activity by C-1 was accompanied by a parallel reduction of nuclear translocation of subunit p65 of NF-kappaB and I-kappaBalpha degradation. Taken together, the results suggest that the ability of C-1 to inhibit iNOS gene expression may be responsible, in part, for its anti-inflammatory effects.

The presence of the nitric oxide synthase (NOS) enzyme from Salmonella 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 degrees C and 43 degrees C. The activity was inhibited by NOS inhibitors such as aminoguanidine and N(G),N(G)-dimethyl-L-arginine. Taken together, partially purified NOS in S. typhimurium is assumed to be a different isoform of mammalian NOSs.

The level of expression and cellular localization of isoenzymes of nitric oxide synthase (NOS) was detected in human stomach tumor tissues. Tumor tissues showed 70% higher activity of NOS than that of normal tissues (P0.01). Poorly differentiated adenocarcinoma tend to have higher activity (P0.05) than well differentiated and moderately differentiated tumor tissues. Aminoguanidine (AG), 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), NG-monomethyl-L-arginine (L-NMMA), and Nomega-nitro-L-arginine (L-NNA) inhibited NOS activity in tumor tissues by 18, 14, 11 and 13%, respectively. The TNF-alpha mRNA expression was correlated with the inducible NOS (iNOS) level, which was high in adenocarcinomas and low in normal tissues. Tumor tissues showed higher expression of iNOS in gland epithelial cells but the level of eNOS was significantly decreased with an exception of concentrated localization in the proliferating capillary endothelium. These results revealed that isoforms of NOS might contribute differentially to growth and progression of human stomach tumor.

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

Enzymatic methylation of endogenous proteins in several cancer cell lines was investigated to understand a possible relationship between protein-arginine methylation and cellular proliferation. Cytosolic extracts prepared from several cancer cells (HeLa, HCT-48, A549, and HepG2) and incubated with S-adenosyl-L-[methyl-3H]methionine revealed an intensely [methyl-3H]-labeled 20-kDa polypeptide. On the other hand, cytosolic extracts prepared from normal colon cells did not show any methylation of the 20-kDa protein under identical conditions. To identify nature of the 20-kDa polypeptide, purified histones were methylated with HCT-48 cytosolic extracts and analyzed by SDS-PAGE. However, none of the histones comigrated with the methylated 20-kDa polypeptide, indicating that it is unlikely to be any of the histone subclasses. The [methyl-3H]group in the 20-kDa polypeptide was stable at pH 10-11 (37 degrees C for 30 min) and methylation was not stimulated by GTPgammaS (4 mM), thus the reaction is neither carboxyl methylesterification on isoaspartyl residues, nor on C-terminal farnesylated cysteine. The present study together with the previous identification of N(G)-methylated arginine residues in the HCT-48 cytosol fraction suggests that this novel endogenous 20-kDa arginine-methylation is a cellular proliferation-related posttranslational modification reaction.

Nitric oxide synthesized by inducible nitric oxide synthase (iNOS) has been implicated as a mediator of inflammation in rheumatic and autoimmune diseases. The effects of higenamine, a tetrahydroisoquinoline compound, on induction of NOS by bacterial lipopolysaccharide (LPS) were examined in murine peritoneal macrophages. LPS-induced nitrite/nitrate production was markedly inhibited by higenamine which at 0.01 mM, decreased nitrite/nitrate levels by 48.7+/-4.4%. This was comparable to the inhibition of LPS-induced nitrite/nitrate production by tetrandrin (49.51+/-2.02%) at the same concentration. Northern and Western blot analysis of iNOS expression demonstrated that iNOS expression was significantly attenuated following co-incubation of peritoneal macrophages with LPS (10 microg/ml; 18 hrs) and higenamine (0.001, 0.01 mM; 18 hrs). These results suggest that higenamine can inhibit LPS-induced expression of iNOS mRNA in murine peritoneal macrophages. The clinical implications of these findings remain to be established.

Recombinant TIS21 protein was overexpressed in Escherichia coli harboring the expression vector plasmid pQE-30 carrying the TIS21 cDNA coding sequence containing an extra 120 nucleotides upstream. Employing this protein consisting of 158 amino acid residues of the main chain plus 40 residues of the fusion peptide. It was found that one of the protein methylase I group [S-adenosylmethionine:nuclear protein/histone-arginine N-methyltransferase; BC 2.1.1.23; J. Biol. Chem., 269, 1075 (1994)] methylated this protein. The methylation products were identified as guanidino-N-methylated arginines. Some of the kinetics of the reaction are described.

The presence of L-arginine methylester (AME), L-arginine ethylester (AEE), or N-nitro-L-arginine methylester (NAME) in the growth media of Staphylococcus aureus increased the nitric oxide synthase (NOS) activity approximately 5- to 14-fold. The increase of NOS activity was confirmed by two assay methods, namely assaying the formation of L-[3H] citrulline from L-[3H] arginine and NO formation. The increase of NOS activity was most likely due to increased de novo synthesis, demonstrated by Western immunoblot analysis. The addition of methanol to the culture medium also increased the NOS activity as much as that found with the above three compounds. Evidence is presented to show that AME, AEE, or NAME gave rise to the formation of methanol in vivo by the action of intracellular esterase(s) and that methanol is subsequently involved in the induction of NOS in this bacterial system.

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 apparentK m 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.

Nitric oxide (NO) is thought to be a second messenger involved in secretion. Upon stimulating pancreatic acinar cells with cholecystokinin- pancreozymin (CCK-PZ), NO formation has been shown to be associated with increased levels of cGMP (Seoet al., 1995). To elucidate the signaling pathway of VIP-induced enzyme secretion, we investigated the NO and cGMP synthesis steps as potential steps where two signal pathways triggered by CCK-PZ and VIP interact. The results obtained in this work provide evidence that increase in pancreatic enzyme secretion by treatment with VIP has no relationship with NOS activity and cGMP level. This conclusion was derived from the following findings that VIP treatment of rat pancreatic tissue increased amylase release as well as protein output in a dose- and time-dependent manner, whereas NOS activity and cGMP synthesis were not affected by VIP treatment as monitored by NOS activity assay and determining cGMP level, which was further confirmed by a NOS-inhibitor study. Consequently, CCK-PZ or VIP increases enzyme secretion in rat pancreatic tissue, but the two hormones are different in their mode of action. Together the results suggest that signaling pathway of VIP-induced enzyme secretion might either bypass the NO and cGMP synthesis steps or lie on a distinct pathway from CCK-PZ-induced pathway.

In pancreatic cells, NO formation is associated with increased levels of cGMP and endocrine/ exocrine secretion. In the present study, the role of NO in the regulation of exocrine secretion was investigated in rat pancreatic tissues. Treatment of rat pancreatic tissue with cholecystokinin-pancreozymin (CCK-PZ) resulted in an significant increase in arginine conversion to citrulline, the amount of nitrite/nitrate, the release of amylase, and the level of cGMP. Furthermore, CCK-PZ-stimulated increase of amylase release and conversion of arginine to citrulline transformation were counteracted by the inhibitor of NO synthase, NG-nitro-L-arginine methyl ester. The results on the time course of CCK-PZ-induced citrulline formation and rise of cGMP level indicate that NO synthase and guanylate cyclase are activated within the first seconds of stimulation. The kinetics of citrulline accumulation correlate well with those of cGMP rise, which further confirms the conclusion that NO mediates the response to CCK-PZ by cGMP.

The activity of S-famesylcysteine O-methyltransferase was assayed by incubating the enzyme with a syntheticin vitro substrate, [N-acetyl-S-trans, trans-famesyl-L-cysteine (AFC)], together with S-adenosyl-L-[methyl-14C]methionine. The resulting methylesterification product, [N-acetyl-S-trans, trans-famesyl-L-cysteine (methyl-14C) ester (AFCME)], was then analyzed either directly on HPLC or by converting the AFC[methyl-14C]ME to [methyl-14C] alcohol by basehydrolysis. Employing these two analytical methods, it was established that a peptide purified from rat liver cytosol fraction [Int. J. Biochem., 25, 1157 (1993)] strongly inhibited the above enzyme activity with IC50 of 7.1X10−8 M. Also, the S-famesylcysteine O-methyltransferase from several human colon cancer cells was also equally inhibited by the peptide.

1. 1. A proteinaceous inhibitor for S-adenosyl-l-methionine (AdoMet)-dependent transmethylation reactions has been purified to apparent homogeneity from rat liver cytosolic fraction. 2. 2. The peptide was made up of 29 amino acid residues with a molecular weight of 2,584. Glycine accounted for 52% of the total amino acids. 3. 3. Employing AdoMet: protein-carboxyl O-methyltransferase (Protein methylase II) and bovine serum γ-globulin as in vitro substrate, the mode of inhibition was found to be non-competitive with Ki value of 1.9 × 10−8 M. 4. 4. When the inhibitor was present in the reaction mixture together with S-adenosyl-l-homocysteine (AdoHcy), which is a competitive inhibitor for AdoMet, the extent of inhibition exceeded that exerted by each individual inhibitor alone, suggesting that the sites of the inhibitors on the enzyme molecule are different. 5. 5. Almost a stoichiometric relationship exists between the enzyme and the inhibitor molecule, the ratio being approx one.

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.

Protein carboxylmethylation methylates the free carboxyl groups in various substrate proteins by protein carboxyl O-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 gamma-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 antibody could be carboxylmethylated via spleen PCMT to a level similar to gamma-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.

We show that a histone deacetylase (HDAC) inhibitor apicidin increases the transcriptional activity of cyclin E gene, which results in accumulation of cyclin E mRNA and protein in a time- and dose-dependent manner. Interestingly, apicidin induction of cyclin E gene is found to be mediated by Sp1- rather than E2F-binding sites in the cyclin E promoter, as evidenced by the fact that specific inhibition of Sp1 leads to a decrease in apicidin activation of cyclin E promoter activity and protein expression, but mutation of E2F-binding sites of cyclin E promoter region fails to inhibit the ability of apicidin to activate cyclin E transcription. In addition, this transcriptional activation of cyclin E by apicidin is associated with histone hyperacetylation of cyclin E promoter region containing Sp1-binding sites. Our results demonstrate that regulation of histone modification by an HDAC inhibitor apicidin contributes to induction of cyclin E expression and this effect is Sp1-dependent.

Treatment with ergolide, a sesquiterpene lactone from Inula britannica var chinensis, caused the induction of apoptosis in Jurkat T cells, which was confirmed by DNA fragmentation, caspase-3 activation and cleavage of poly(ADP-ribose) polymerase in response to ergolide. Furthermore, mitochondrial dysfunction appeared to be associated with ergolide-induced apoptosis, because Bax translocation and cytochrome c release were stimulated by ergolide. In parallel, the nuclear factor-κB (NF-κB) signaling pathway was significantly inhibited by ergolide, which was accompanied by down-regulation of cell survival molecules, such as X-chromosome-linked inhibitor of apoptosis and Bcl-2. In addition, the JNK signaling pathway was involved in ergolide-induced apoptosis. Collectively, our results identified a new mechanism for the anti-cancer property of ergolide, attributable to the induction of apoptosis through down-regulation of cell survival signal molecules resulting from inhibition of the NF-κB signaling pathway.

The molecular mechanism underlying the suppression of lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-induced nitric oxide (NO) and prostaglandin (PG) E(2) production was investigated in RAW 264.7 macrophages treated with sesquiterpene lactones, zaluzanin-C and estafiatone, isolated from Ainsliaea. Zaluzanin-C and estafiatone decreased NO production in LPS/IFN-gamma-stimulated RAW 264.7 macrophages with an IC50 of about 6.61 microM and 3.80 microM, respectively. In addition, these compounds inhibited the synthesis of PGE(2) in LPS/IFN-gamma-treated RAW 264.7 macrophages. Furthermore, treatment with zaluzanin-C and estafiatone resulted in a decrease in inducible No Synthase (iNOS) and Cyclooxygenase-2 (COX-2) protein and mRNA expression levels. Zaluzanin-C and estafiatone inhibited nuclear factor-kappaB (NF-kappaB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-gamma. This effect was accompanied by parallel reduction of phosphorylation and degradation of inhibitor of kappaB (IkB). In addition, these effects were completely blocked by treatment with cysteine, indicating that the inhibitory effect of zaluzanin-C and estafiatone might be mediated by alkylation of either NF-kappaB itself or an upstream molecule of NF-kappaB. These results demonstrate that the suppression of NF-kappaB activation by zaluzanin-C and estafiatone might be attributed to inhibition of nuclear translocation of NF-kappaB resulting from blockade of the degradation of IkappaB, leading to suppression of the expression of iNOS and COX-2, which play important roles in inflammatory signaling pathways.

Lysophosphatidic acid (LPA), one of the naturally occurring phospholipids, stimulates cell motility through the activation of Rho family members, but the signaling mechanisms remain to be elucidated. In the present study, we investigated the roles of p21-activated kinase 1 (PAK1) on LPA-induced focal adhesion kinase (FAK) phosphorylation and cell motility. Treatment of human melanoma cells A2058 with LPA increased phosphorylation and activation of PAK1, which was blocked by treatment with pertussis toxin and by inhibition of phosphoinositide 3-kinase (PI3K) with an inhibitor LY294002 or by overexpression of catalytically inactive mutant of PI3Kgamma, indicating that LPA-induced PAK1 activation was mediated via a Gi protein and the PI3Kgamma signaling pathway. In addition, we demonstrated that Rac1/Cdc42 signals acted as upstream effector molecules of LPA-induced PAK activation. However, Rho-associated kinase, MAP kinase kinase 1/2 or phospholipase C might not be involved in LPA-induced PAK1 activation or cell motility stimulation. Furthermore, PAK1 was necessary for FAK phosphorylation by LPA, which might cause cell migration, as transfection of the kinase deficient mutant of PAK1 or PAK auto-inhibitory domain significantly abrogated LPA-induced FAK phosphorylation. Taken together, these findings strongly indicated that PAK1 activation was necessary for LPA-induced cell motility and FAK phosphorylation that might be mediated by sequential activation of Gi protein, PI3Kgamma and Rac1/Cdc42.

We previously reported that hydrogen peroxide (H2O2) mediates mitogen activation of ribosomal protein S6 kinase 1 (S6K1) which plays an important role in cell proliferation and growth. In this study, we investigated a possible role of H2O2 as a molecular linker in Rac1 activation of S6K1. Overexpression of recombinant catalase in NIH-3T3 cells led to the drastic inhibition of H2O2 production by PDGF, which was accompanied by a decrease in S6K1 activity. Similarly, PDGF activation of S6K1 was significantly inhibited by transient transfection or stable transfection of the cells with a dominant-negative Rac1 (Rac1N17), while overexpression of constitutively active Rac1 (Rac1V12) in the cells led to an increase in basal activity of S6K1. In addition, stable transfection of Rat2 cells with Rac1N17 dramatically attenuated the H2O2 production by PDGF as compared with that in the control cells. In contrast, Rat2 cells stably transfected with Rac1V12 produced high level of H2O2 in the absence of PDGF, comparable to that in the control cells stimulated with PDGF. More importantly, elimination of H2O2 produced in Rat2 cells overexpressing Rac1V12 inhibited the Rac1V12 activation of S6K1, indicating the possible role of H2O2 as a mediator in the activation of S6K1 by Rac1. However, H2O2 could be also produced via other pathway, which is independent of Rac1 or PI3K, because in Rat2 cells stably transfected with Rac1N17, H2O2 could be produced by arsenite, which has been shown to be a stimulator of H2O2 production. Taken together, these results suggest that H2O2 plays a pivotal role as a mediator in Rac1 activation of S6K1.

We previously reported that apicidin arrested human cancer cell growth through selective induction of p21(WAF1/Cip1). In this study, the apoptotic potential of apicidin and its mechanism in HL60 cells was investigated. Treatment of HL60 cells with apicidin caused a decrease in viable cell number in a dose-dependent manner and an increase in DNA fragmentation, nuclear morphological change, and apoptotic body formation, concomitant with progressive accumulation of hyperacetylated histone H4. In addition, apicidin converted the procaspase-3 form to catalytically active effector protease, resulting in subsequent cleavages of poly(ADP-ribose) polymerase and p21(WAF1/Cip1). Incubation of HL60 cells with z-DEVD-fmk, a caspase-3 inhibitor, almost completely abrogated apicidin-induced activation of caspase-3, DNA fragmentation, and cleavages of poly(ADP-ribose) polymerase and p21(WAF1/Cip1). Moreover, these effects were preceded by an increase in translocation of Bax into the mitochondria, resulting in the release of cytochrome c and cleavage of procaspase-9. The addition of cycloheximide greatly inhibited activation of caspase-3 by apicidin by interfering with cleavage of procaspase-3 and DNA fragmentation, suggesting that apicidin-induced apoptosis was dependent on de novo protein synthesis. Consistent with these results, apicidin transiently increased the expressions of both Fas and Fas ligand. Preincubation with NOK-1 monoclonal antibody, which prevents the Fas-Fas ligand interaction and is inhibitory to Fas signaling, interfered with apicidin-induced translocation of Bax, cytochrome c release, cleavage of procaspase-3, and DNA fragmentation. Taken together, the results suggest that apicidin might induce apoptosis through selective induction of Fas/Fas ligand, resulting in the release of cytochrome c from the mitochondria to the cytosol and subsequent activation of caspase-9 and caspase-3.

Autotaxin (ATX) is a recently described member of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family of proteins with potent tumor cell motility-stimulating activity. Like other NPPs, ATX is a glycoprotein with peptide sequences homologous to the catalytic site of bovine intestinal alkaline phosphodiesterase (PDE) and the loop region of an EF-hand motif. The PDE active site of ATX has been associated with the motility-stimulating activity of ATX. In this study, we examined the roles of the EF-hand loop region and of divalent cations on the enzymatic activities of ATX. Ca 2+ or Mg 2+ was each demonstrated to increase the PDE activity of ATX in a concentration-dependent manner, whereas incubation of ATX with chelating agents abolished this activity, indicating a requirement for divalent cations. Non-linear regression analysis of enzyme kinetic data indicated that addition of these divalent cations increases reaction velocity predominantly through an effect on V max. Three mutant proteins, Ala 740 -, Ala 742 -, and Ala 751 -ATX, in the EF-hand loop region of ATX had enzymatic activity comparable to that of the wild-type protein. A deletion mutation of the entire loop region resulted in slightly reduced PDE activity but normal motility-stimulating activity. However, the PDE activity of this same deletion mutant remained sensitive to augmentation by cations, strongly implying that cations exert their effect by interactions outside of the EF-hand loop region.

We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) by two beta-carboline alkaloids isolated from Melia azedarach, 4,8-dimethoxy-1-vinyl-beta-carboline (compound 1, C-1) and 4-methoxy-1-vinyl-beta-carboline (compound 2, C-2). iNOS activity in a cell-free extract of lipopolysaccharide/interferon-gamma-stimulated RAW 264.7 cells was found to be markedly increased, and this increase was prevented by C-1 and C-2, accompanied by the parallel reduction in nitrite accumulation in culture medium. However, C-1 and C-2 had no further effect on the iNOS activity prepared from fully lipopolysaccharide/interferon-gamma-stimulated RAW 264.7 cells. Treatment with C-1 or C-2 decreased the levels of iNOS protein and mRNA in a concentration-dependent manner. In addition, prostaglandin E(2) production, cyclooxygenase-2 protein and DNA binding of nuclear factor-kappaB (NF-kappaB) in lipopolysaccharide-stimulated RAW 264.7 cells were reduced by these compounds. These results indicate that C-1 and C-2 primarily inhibit iNOS and cyclooxygenase-2 activities via the suppression of de novo synthesis of these two enzymes, and that the inhibition of iNOS expression may be associated with the inhibition of NF-kappaB activation. Taken together, the results suggest that suppression of iNOS and cyclooxygenase-2 induction by lipopolysaccharide is responsible for the anti-inflammatory activity of these alkaloids through selective inhibition of the expression of genes, which play important roles in inflammatory signaling pathways.

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.

The enzyme responsible for the synthesis of nitric oxide (NO) from L-arginine in mammalian tissues is known as nitric oxide synthase (NOS) (EC.1.14.13.39). In the present study, the role of NO in the regulation of exocrine secretion was investigated in rat pancreatic acinar cells. Treatment of rat pancreatic acinar cells with cholecystokinin-octapeptide (CCK-OP) resulted in an increase in the arginine conversion to citrulline, the amount of NOx, the release of amylase, and the level of cGMP. Especially, CCK-OP-stimulated increase of arginine to citrulline transformation, the amount of NOx and cGMP level were completely counteracted by the inhibitor of NOS, NG-monomethyl-L-arginine (MMA), by contrast, that of amylase release was partially reduced. Furthermore, MMA-induced decrease of NOS activity and amylase release showed dose-dependent pattern. The data on the time course of CCK-OP-induced citrulline formation and cGMP rise indicate that NOS and guanylate cyclase were activated by treatment of CCK-OP. However, the mechanism of agonist-stimulated guanylate cyclase activation in acinar cells remains unknown. Therefore, activation of NOS is one of the early events in receptor-mediated cascade of reactions in pancreatic acinar cells and NO, not completely, but partially mediate pancreatic enzyme exocrine secretion.

The presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P was established by western blot analysis. The identity of citrulline formed from L-arginine by the NOS was confirmed by both TLC and HPLC and the other product, NO, by directly measuring the production of nitrogen oxides (NOx) in the reaction mixture. The activity was inhibited by typical NOS inhibitors such as N-nitro-L-arginine methylester and NG,NG-dimethyl-L-arginine with the IC50 of 4.9 x 10(-4) and 2.5 x 10(-4) M, respectively. The NOS activity was maximum at pH 6.5 and at 47.5 degrees C. These results indicate that the NOS of S. aureus ATCC6538P is an isoform distinct from mammalian NOS.

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.

The wide distribution of the inhibitor among various subcellular fractions and rat organs8) and its inhibitory activity on many AdoMet-dependent methyltransferases strongly suggest the importance ofin vivo methylation. Since 120 micrograms inhibitor in 0.050 m/incubation mixture inhibits the protein methylase II activity by 50% (Fig. 4) and the molecular mass of the inhibitor is assumed to be 2,482 Da, the final concentration of the inhibitor needed to inhibit the enzyme activity by 50% is calculated to be 0.97 μM. This value is extremely close to the reported K i values of S-adenosyl-L-homocysteine towards various methyltransferases (1–10 μM)11). Furthermore, since the present inhibitor is primarily membrane-bound, thein situ concentration of the inhibitor in the membrane might even be higher. To what extent the present inhibitor participates in modulation of biological methyltransferation remains to be explored.

The activities of three S-adenosylmethionine:protein methyltransferases (protein methylase I, II, and III) were measured throughout the mitotic cycle of synchronized HeLa S-3 cells in an attempt to determine the biological function of these enzymes. 1. The activities of protein methylase I and II, which catalyze the methylation of protein-arginine residues and carboxyl groups, increase more or less continuously until they have doubled by the end of G2 phase. These increases were not obviously correlated with major cell cycle events such as DNA replication or histone synthesis and modification. 2. The activity of protein methylase III (S-adenosylmethionine:protein-lysine methyltransferase) remains relatively constant through G1 and early S phase of the cell cycle, then begins increasing until it reaches over three to four times its initial level by the end of G2. The increase in protein methylase III activity is temporally correlated with the increased mono- and dimethylation of the e-amino groups of histone lysine residues. 3. By inhibiting protein and RNA synthesis it was determined that this increase in protein methylase III activity in late S and G2 of the HeLa cell cycle was probably due to the de novo synthesis of enzyme molecules of those times. 4. The same 3- to 4-fold increase in the activity of protein methylase III occurs at the usual time after mitosis even if DNA and histone synthesis were prevented by treatment of the cells with cytosine arabinoside. In such treated cells, the enzyme apparently "overmethylated" the already mono- and dimethylated lysine residues of previously synthesized histone molecules to the di- and trimethyllysine state. These data suggest that protein methylase III synthesis is not triggered by the appearance of its normal substrate but rather is coupled with some other unknown cell cycle event.

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.

1. When purified calf thymus histones were incubated with [1-14C]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-14C]histone and [acetyl-14C]polylysine revealed that practically all of the incorporated radioactivity was as e-N-acetyllysine. The presence of e-N-[acetyl-14C]acetyllysine was also confirmed by paper chromatography.

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.

Autotaxin (ATX), an exo-nucleotide pyrophosphatase and phosphodiesterase, stimulates tumor cell motility at sub-nanomolar levels and augments invasiveness and angiogenesis. We investigated the role of G protein-coupled phosphoinositide 3-kinase gamma (PI3Kgamma) in ATX-mediated tumor cell motility stimulation. Pretreatment of human melanoma cell line A2058 with wortmannin or LY294002 inhibited ATX-induced motility. ATX increased the PI3K activity in p110gamma, but not p85, immunoprecipitates. This effect was abrogated by PI3K inhibitors or inhibited by pertussis toxin. Furthermore, stimulation of tumor cell motility by ATX was inhibited by catalytically inactive form of PI3Kgamma, strongly indicating the crucial role of PI3Kgamma for ATX-mediated motility in human melanoma cells

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.

Protein methylase I (S-adenosylmethionine: protein-arginine methyltransferase, EC 2.1.1.23) has been purified from calf brain approximately 120-fold with a 14% yield. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate protein. The enzyme has an optimum pH of 7.2 and pI value of 5.1. The Km values for S-adenosyl-L-methionine, histone H4, and an ancephalitogenic basic protein are 7.6 X 10(-6), 2.5 X 10(-5), and 7.1 X 10(-5) M, respectively, and the Ki value for S-adenosyl-L-homocysteine is 2.62 X 10(-6) M. The enzyme is highly specific for the arginine residues of protein, and the end products after hydrolysis of the methylated protein are NG,NG-di(asymmetric), NG,N'G-di(symmetric), and NG-monomethylarginine. The ratio of [14C]methyl incorporation into these derivatives by enzyme preparation at varying stages of purification remains unchanged at 40:5:55, strongly indicating that a single enzyme is involved in the synthesis of the three arginine derivatives. The kinetic mechanism of the protein methylase I reaction was studied with the purified enzyme. Initial velocity patterns converging at a point on the extended axis of abscissas were obtained with either histone H4 or S-adenosyl-L-methionine as the varied substrate. Product inhibition by S-adenosyl-L-homocysteine with S-adenosyl-L-methionine as the varied substrate was competitive regardless of whether or not the enzyme was saturated with histone H4. On the other hand, when histone H4 is the variable substrate, noncompetitive inhibition was obtained with S-adenosyl-L-homocysteine under conditions where the enzyme is not saturated with the other substrate, S-adenosyl-L-methionine. These results suggest that the mechanism of the protein methylase I reaction is a Sequential Ordered Bi Bi mechanism with S-adenosyl-L-methionine as the first substrate, histone H4 as the second substrate, methylated histone H4 as the first product, and S-adenosyl-L-homocysteine as the second product released.

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.