Hepatic metabolism of sulfur amino acids in db/db mice.

To determine the effect of type-2 diabetes and obesity on the hepatic metabolism of sulfur amino acids, hepatic sulfur amino acid metabolism was determined in db/db mice. Hepatic methionine was markedly decreased in db/db mice, although the hepatic activity of betaine homocysteine methyltransferase was increased. The decrease in hepatic methionine was reflected by decreased sulfur-containing methionine metabolites, including S-adenosylmethionine, homocysteine, cysteine, and hypotaurine in liver and plasma. In contrast, S-adenosylhomocysteine, putrescine, and spermidine were increased in db/db mice. The hepatic level and activity of methionine adenosyltransferase I/III, an S-adenosylmethionine synthesizing enzyme, were significantly increased. These results suggest that increased polyamine synthesis, in conjunction with decreased hepatic methionine levels, is partly responsible for the reduction in hepatic S-adenosylmethionine. Decreased homocysteine in liver and plasma may be attributable to the decrease in hepatic methionine and upregulation of hepatic betaine homocysteine methyltransferase. Glutathione in liver and plasma did not change despite decreased γ-glutamylcysteine ligase activity. The decreased hepatic hypotaurine may be attributable to the downregulation of cysteine dioxygenase. The major finding of this study is that db/db mice exhibited decreases in hepatic methionine and its sulfurcontaining metabolites.

Further studies on the hepatoprotective effects of Anoectochilus formosanus.

The purpose of this study was to investigate the hepatoprotective effects of Anoectochilus formosanus effective fraction (AFEF) on chronic liver damage induced by carbon tetrachloride (CCl4) in mice. CCl4 (5%; 0.1 mL/10 g body weight) was given twice a week for 9 weeks, and mice received AFEF throughout the whole experimental period. Plasma GPT, hepatic levels of hydroxyproline and malondialdehyde were significantly lower in mice treated with AFEF compared with those treated with CCl4 only. Liver pathology in the AFEF-treated mice was also improved. RT-PCR analysis showed that AFEF treatment increased the expression of methionine adenosyltransferase 1A and decreased the expression of collagen(alpha1)(I) and transforming growth factor-beta1. These results clearly demonstrated that AFEF reduced the hepatic damage induced by CCl4 in mice.

Visualizing the proteome of Escherichia coli: an efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes.

To investigate the feasibility of conducting a genomic-scale protein labeling and localization study in Escherichia coli, a representative subset of 23 coding DNA sequences (CDSs) was selected for chromosomal tagging with one or more fluorescent protein genes (EGFP, EYFP, mRFP1, DsRed2). We used lambda-Red recombination to precisely and efficiently position PCR-generated DNA targeting cassettes containing a fluorescent protein gene and an antibiotic resistance marker, at the C-termini of the CDSs of interest, creating in-frame fusions under the control of their native promoters. We incorporated cre/loxP and flpe/frt technology to enable multiple rounds of chromosomal tagging events to be performed sequentially with minimal disruption to the target locus, thus allowing sets of proteins to be co-localized within the cell. The visualization of labeled proteins in live E. coli cells using fluorescence microscopy revealed a striking variety of distributions including: membrane and nucleoid association, polar foci and diffuse cytoplasmic localization. Fifty of the fifty-two independent targeting experiments performed were successful, and 21 of the 23 selected CDSs could be fluorescently visualized. Our results show that E. coli has an organized and dynamic proteome, and demonstrate that this approach is applicable for tagging and (co-) localizing CDSs on a genome-wide scale.

Effect of fasting on methionine adenosyltransferase expression and the methionine cycle in the mouse liver.

The effect of fasting on mouse liver methionine adenosyltransferase (MAT I/ III) expression and the regulation of methionine metabolism were investigated. The mRNA level, protein level, and activity of MAT I/III were increased by fasting for 10 or 16 h. In spite of the increase of MAT I/III activity, S-adenosylmethionine, the product of methionine due to MAT I/III, decreased. S-Adenosylhomocysteine, which is made from S-adenosylmethionine by its coupling to methyltransferase, increased as a result of fasting for 16 h. These results suggest that the total methylation reactions using S-adenosylmethionine are stimulated in the fasting mouse liver. However, the DNA methylation level was not changed by fasting for 16 h. Glutathione, which is made by the transsulfuration pathway from homocysteine, decreased due to fasting. Regulation of supplementation of S-adenosylmethionine may occur in the fasting mouse because MAT I/III activity increases and the flow to glutathione is decreased.

High-performance liquid chromatography determination of methionine adenosyltransferase activity using catechol-O-methyltransferase-coupled fluorometric detection.

A nonradioactive, sensitive, rapid, and specific method for the determination of methionine adenosyltransferase activity has been established. In this method, the methyl group of S-adenosyl-L-methionine was enzymatically transferred to esculetin with the aid of catechol-O-methyltransferase and then the resulting scopoletin was extracted with n-hexane:ethyl acetate (7:3, v/v) and measured by high-performance liquid chromatography with Si 60 column and fluorometric detection with excitation and emission wavelengths at 347 and 415 nm, respectively. The detection limit for scopoletin was about 100 fmol. Using this method to determine MAT activity in HL-60 cells required only about 2.5 microg of protein and the incubation time needed for enzymatic reaction is less than 30 min. The HPLC analysis procedure took only 5 min per sample. The kinetic study showed that MAT in HL-60 cells exhibited negative cooperativity with a Hill coefficient of 0.5. The values of K(m) and V(max) were 6.1+/-0.3 microM and 135.4+/-1.5 nmol AdoMet formed/mg protein/h, respectively.

Characterization of sams genes of Amoeba proteus and the endosymbiotic X-bacteria.

As a result of harboring obligatory bacterial endosymbionts, the xD strain of Amoeba proteus no longer produces its own S-adenosylmethionine synthetase (SAMS). When symbiont-free D amoebae are infected with symbionts (X-bacteria), the amount of amoeba SAMS decreases to a negligible level within four weeks, but about 47% of the SAMS activity, which apparently comes from another source, is still detected. Complete nucleotide sequences of sams genes of D and xD amoebae are presented and show that there are no differences between the two. Long-established xD amoebae contain an intact sams gene and thus the loss of xD amoeba's SAMS is not due to the loss of the gene itself. The open reading frame of the amoeba's sams gene has 1,281 nucleotides, encoding SAMS of 426 amino acids with a mass of 48 kDa and pI of 6.5. The amino acid sequence of amoeba SAMS is longer than the SAMS of other organisms by having an extra internal stretch of 28 amino acids. The 5'-flanking region of amoeba sams contains consensus-binding sites for several transcription factors that are related to the regulation of sams genes in E. coli and yeast. The complete nucleotide sequence of the symbiont's sams gene is also presented. The open reading frame of X-bacteria sams is 1,146 nucleotides long, encoding SAMS of 381 amino acids with a mass of 41 kDa and pI of 6.0. The X-bacteria SAMS has 45% sequence identity with that of A. proteus.

Correlation between the expression of methionine adenosyltransferase and the stages of human colorectal carcinoma.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet) from ATP and L-methionine. AdoMet is the major methyl donor in most transmethylation reactions in vivo, and it is also the propylamino donor in the biosynthesis of polyamines. In the present study, we assessed MAT activity in human colons with colorectal carcinoma and the values were compared with those of morphologically normal adjacent mucosa. Higher levels of MAT activity were observed in the colorectal carcinoma than in the normal colon. The ratio of MAT activity in tumor tissue versus normal tissue seemed to be correlated well will the stage of the colorectal tumor. Furthermore, immunoblot analysis showed that the high levels of MAT activity observed in colorectal carcinoma were due to the increased amounts of MAT protein. Immunohistochemical analysis revealed that MAT was most abundant in goblet cells, particularly in granules in the supranuclear area of these cells. In the colorectal carcinoma tissues, MAT was strongly stained in the cancerous cells and localized in granules in the supranuclear region. The results of this preliminary study suggest that determination of the relative ratio of MAT activity in both normal and tumor regions in human colorectal carcinoma could be a clinically useful tool for determining the stage of malignancy of colorectal carcinomas.

Differential expression of S-adenosylmethionine synthetase isozymes in different cell types of rat liver.

Mammalian S-adenosylmethionine (AdoMet) synthetase exists as two isozymes, liver-type and nonhepatic-type enzymes, which are the products of two different genes. It is known that the liver-type isozyme is only expressed in adult liver. Whereas, the nonhepatic-type isozyme is widely distributed in various tissues. In addition to the liver-type isozyme, a minor amount of the nonhepatic-type isozyme is also detected in adult liver. To investigate the distribution of these two isozymes in the liver in detail, the localization of these two isozymes was examined in each cell type of liver using a combination of cell fractionation technique and Western blot analysis. In the parenchymal cells, the liver-type isozyme protein was predominantly expressed, and a small amount of the nonhepatic-type isozyme protein was also detected. On the other hand, in the stellate cells the nonhepatic-type isozyme protein was exclusively or only expressed. Interestingly, a large amount of both isozymes were present in endothelial and Kupffer cell fraction. Using both antibodies to anti-rat nonhepatic-type and liver-type isozymes, respectively, immunohistochemical analysis clearly confirmed these results. In addition, in cultured hepatocellular carcinoma cells (FAA-HTC1), the nonhepatic-type isozyme protein only was detected, and the liver-type isozyme protein completely disappeared. This result indicates that the changes in the isozyme expression is regulated within the parenchymal cells. Administration of hepatotoxic drug carbon tetrachloride (CCl4) to rats resulted in about 40% to 50% reduction of enzyme activity in parenchymal cells and stellate cells compared with those of control rats. However, enzyme activity in endothelial and Kupffer cell fraction was not changed.

Abnormal metabolism of S-adenosyl-L-methionine in hypoxic rat liver. Similarities to its abnormal metabolism in alcoholic cirrhosis.

S-Adenosylmethionine (AdoMet) is an important biologic methylating agent for nucleic acids, phospholipids, biologic amines, and proteins. Previous studies indicated that hepatic AdoMet synthetase and hepatic levels of AdoMet are subnormal in patients with alcoholic cirrhosis. This abnormality limits the patients' capacity to convert phosphatidylethanolamine to phosphatidylcholine by way of phosphatidylethanolamine-N-methyltransferase (PEMT). Because alcoholic consumption appears to be associated with hepatic hypoxia, we previously measured AdoMet concentration in liver cells under acute hypoxia and found the level to be decreased substantially. In the present study, we determined whether a similar metabolic abnormality was also observed in rats maintained under physiologic hypoxia for 9 days and administered standard rat chow. The study showed that AdoMet levels in hypoxic rat (ave +/- SD) were significantly lower than those in the control (36.8 +/- 11.6 vs. 60.4 +/- 2.3 nmol/g liver; P < 0.05). Also significantly lower in the hypoxic group were the activities of AdoMet synthetase (0.60. +/- 0.07 vs. 0.97 +/- 0.20 U; P < 0.05) and PEMT (26.2 +/- 4.2 vs. 35.6 +/- 5.8 U; P < 0.02). The mRNA levels of AdoMet synthetase also declined in hypoxia indicating that hypoxia may modulate the gene expression of hepatic AdoMet synthetase. Thus, in vivo hypoxia may have an important effect on 1-carbon metabolism.

The use of a spectrophotometric assay to study the interaction of S-adenosylmethionine synthetase with methionine analogues.

We have developed a continuous spectrophotometric assay for S-adenosylmethionine synthetase and, using this assay, have examined the interaction of five potential inhibitors with the E. coli enzyme. S-Vinylhomocysteine and S-allylhomocysteine were found to be substrates, while S-(methanethio)cysteine and S-(methanethio)homocysteine were found to be competitive inhibitors. S-Cyanohomocysteine is neither a substrate nor an inhibitor.

Hepatic sulfur amino acid metabolism in rats with chronic renal failure.

We recently demonstrated elevated plasma amino acid concentrations and abnormal responses to amino acid supplementation (e.g., elevated methionine and phenylalanine) in children with chronic renal failure (CRF). We also recently developed an improved model of CRF in which animals manifest abnormal tissue amino acid levels, marked anorexia and growth failure. The objective of the current study was to determine the etiology of elevations of sulfur amino acids in animals with chronic renal failure. Chronic renal failure, defined as creatinine clearance less than 30% of control values, was induced in male rats in a two-stage surgical procedure. Four groups were studied over 2, 4 and 6 wk: control (non-operated) control (sham-operated), pair-fed (sham-operated and pair-fed with uremics) and CRF. Animals with CRF were anorexic and growth-retarded. Although plasma sulfur amino acid levels tended to be lower in the uremic animals than in controls, hepatic tissue concentrations were higher. Methionine adenosyltransferase was higher, but cystathione synthase and cystathionase activities were not significantly different in rats with CRF compared to pair-fed controls. We conclude that uremia, not malnutrition, affected sulfur amino acid metabolism and that with CRF, a normal adaptive response to elevated methionine levels was occurring, sufficient to normalize sulfur amino acid pool size. Alternative causes of elevated sulfur amino acids must be sought.

Effects of selenomethionine on cell growth and on S-adenosylmethionine metabolism in cultured malignant cells.

The effects of selenomethionine (SeMet) on the growth of 17 cultured cell lines were studied. SeMet in the culture medium of three hepatoma cell lines promoted cell growth at subcytotoxic levels (1-20 microM), but the growth of malignant lymphoid and myeloid cells was not stimulated. L-SeMet was cytotoxic to all 17 cell lines when assayed after culture for 3-10 days. A 50% growth inhibition was observed by 30-160 microM-SeMet in a culture medium containing 100 microM-methionine. SeMet cytotoxicity to normal (fibroblasts) and malignant cells was rather similar, excluding specific antineoplastic cytotoxicity. Cytotoxicity was increased by decreasing concentrations of methionine. The DL form of SeMet was less cytotoxic than the L form. L-SeMet was metabolized to a selenium analogue of S-adenosylmethionine approximately as effectively as the natural sulphur analogue methionine in malignant R1.1 lymphoblasts. Concomitantly, S-adenosylmethionine pools were decreased. This occurred early and at cytotoxic SeMet levels. Methionine adenosyltransferase activity was not altered by SeMet treatment. ATP pools were not affected early, and decreases in the synthesis of DNA and protein took place late and were apparently related to cell death. RNA synthesis was slightly stimulated at low cytotoxic SeMet levels by 24 h, but was markedly inhibited after 48 h. The SeMet analogue of S-adenosylmethionine could be effectively utilized in a specific enzymic transmethylation. Neither S-adenosylhomocysteine nor its selenium analogue accumulated in the treated cells. These findings together suggest a direct or indirect involvement of S-adenosylmethionine metabolism in SeMet cytotoxicity, but exclude a gross blockage of transmethylations.

Decreased activities of S-adenosylmethionine synthetase isozymes in hereditary hepatitis in Long-Evans rats.

Isozyme patterns of S-adenosylmethionine synthetase have been measured with or without dimethylsulfoxide in liver of LEC rat hereditary hepatitis. The activities of the alpha- and beta-forms are decreased with age after birth, and decreased to a half level of 36 weeks after birth. Concentration of S-adenosylmethionine in the liver is almost a half level of control rat. However, the activity of glycine- and tRNA-methyltransferases in the liver shows no significant change.

Formation of S-adenosylethionine in liver of rats chronically fed with DL-ethionine.

The early changes in the metabolism of L-ethionine were examined in rats preexposed to chronic administration of DL-ethionine. The capacity of liver to accumulate S-adenosylethionine after a single injection of L-ethionine decreases rapidly from the onset of the carcinogenic regimen. This drop is caused by diminished S-adenosylethionine synthesis, a consequence of lower activity of the ATP-L-methionine adenosyltransferase. This change is accompanied by the rapid increase of the concentration of free ethionine and ethionine sulfoxide. The concentration of hepatic ATP depends in the control animals on the L-ethionine dose and is inversely related to the S-adenosylethionine concentration, but in DL-ethionine-pretreated rats it becomes gradually independent of the L-ethionine dose. The alterations in L-ethionine metabolism observed are not attributed to the change in the ratio of hepatocytes to oval cells but rather to the functional alterations of hepatocytes.

A study of the mechanism of selenite-induced hypomethylated DNA and differentiation of Friend erythroleukemic cells.

Sodium selenite is a good inducer of hemoglobin production in Friend erythroleukemic cells (FELC). At a concentration of 20 microM 70-80% of the cells produce hemoglobin and the DNA is hypomethylated. What is the mechanism for sodium selenite alteration of the DNA methylation pattern? Experiments with methionine adenosyltransferase (the enzyme that synthesizes adenosylmethionine) showed little effect of selenite on the activity of this enzyme in vitro or in vivo. Therefore, FELC are able to synthesize S-adenosylmethionine in the presence of sodium selenite. When sodium selenite was added to an in vitro assay for DNA methylase, the enzyme was non-competitively inhibited by 80% at 20 microM selenite with a Ki of 6 microM. DNA methylase isolated from control and selenite-treated FELC was purified through a DEAE-Sephacel column and no difference in activity was found. In the presence of selenite, DNA methylase is very sensitive to selenite inhibition, but removal of the selenite restores activity. However, DNA synthesized by FELC grown in the presence of selenite (no DNA methylase activity) was found to be hypomethylated. These results suggest that DNA methylase activity is inhibited in FELC grown in the presence of sodium selenite.

Resistance to multiple adenine nucleoside and methionine analogues in mutant murine lymphoma cells with enlarged S-adenosylmethionine pools.

Adenosine and many adenosine analogues exert toxicity to mammalian cells at the nucleoside level. The mechanism of action of these agents is controversial. Previous experiments suggested that adenosine toxicity could be mediated by the accumulation of S-adenosylhomocysteine (AdoHcy), a potent inhibitor of S-adenosylmethionine (AdoMet) dependent methylation reactions. To analyze this question genetically, adenosine resistant, adenosine kinase deficient mutant clones of a murine T-lymphoma cell line (R1.1) have been selected and analyzed. Compared to parental lymphoma cells, the adenosine resistant mutants had severalfold elevated levels of AdoMet and an increased AdoMet:AdoHcy ratio. The activity of methionine adenosyltransferase was also raised in the mutants. The mutant cells were cross-resistant to agents postulated to cause accretion of AdoHcy, formation of AdoHcy analogues, impairment of AdoMet synthesis, or direct interference with AdoMet dependent reactions. These included 3-deazaadenosine, carbocyclic adenosine, carbocyclic 3-deazaadenosine, formycin A, 8-azaadenosine, 5'-deoxy-5'-methylthiotubercidin, 5'-deoxy-5'-methylthioadenosine, 5'-deoxy-5'-S-isobutylthioadenosine, adenine, cycloleucine, L-ethionine, seleno-DL-ethionine, and (+/-)-2-aminobicyclo[2.1.1]hexane-2-carboxylic acid. These results suggest that diverse purine nucleoside and methionine analogues may block the growth of adenosine kinase deficient cells by interference with AdoMet synthesis and degradation. An increase in AdoMet pools can render mammalian cells cross-resistant to multiple drugs affecting this essential metabolic pathway.

Altered methylation complex isozymes as selective targets for cancer chemotherapy.

MC2 is a ternary enzyme complex consisting of MAT, methyltransferase and SAHH. Three isozymes of SAHH have been identified from rat and mouse livers based on different kinetic properties. The Km values are 0.35 +/- 0.05 microM, 1.63 +/- 0.38 microM and 0.37 +/- 0.07 mM for SAHH-L, SAHH-I, and SAHH-H respectively. The corresponding low Km isozymes of MAT and SAHH form MCs-L which include RNA MCs, the intermediate Km isozymes form MC-I, and the high Km isozymes form MC-H which is glycine MC. MCs-L are common to all tissues whereas MC-I and MC-H are organ specific enzyme complexes. Low levels of MC-H in the liver of C3H/HeN mouse are correlated with the slow maturation of hepatocytes and the genetic predisposition to develop spontaneous PHC. Rat Novikoff ascites hepatoma and mouse spontaneous PHC have been shown to contain a SAHH isozyme displaying kinetic properties different from the corresponding normal SAHH-L. The abnormal kinetic properties of tumour SAHH are analogous to those of tumour MAT previously shown by the authors to be uniquely associated with malignant tissues. The tumour isozyme, which is named SAHH-LT, has a Km (AR) value of 2.18 +/- 0.22 microM. The altered tumour MC isozymes appear to play an important role in perpetuating malignant growth, because once the tumour growth was inhibited by poly (I) (C), the abnormal kinetic properties were no longer detectable. Thus abnormal tumour MCs may be exploited as selective targets for cancer chemotherapy. Evidence is presented to indicate that antineoplaston is a potent inhibitory effector of tumour rRNA MCs and an effective antitumor agent.

Biochemical RBC abnormalities in drug-free and lithium-treated manic patients.

RBCs from two lithium-free manic patients displayed lower choline transport and higher choline concentrations and methionine S-adenosyltransferase activity than those of controls. Lithium therapy decreased RBC methionine S-adenosyltransferase activity to normal, decreased choline transport further, and increased choline concentrations further.

Effect of chronic phenobarbital treatment on folates and one-carbon enzymes in the rat.

Chronic oral phenobarbital treatment (50 mg/kg every 12 hr for 8 weeks), which was nontoxic and continuously protective against seizures in rats, significantly decreased folate concentration in liver (29%) but not in brain or plasma. The apparent activity of 5,10-methylenetetrahydrofolate reductase (MTR) in liver decreased with initiation of treatment but then increased with a significant correlation to the length of treatment. Phenobarbital also stimulated the activity of this enzyme slightly in vitro. Methionine adenosyltransferase (MAT) activity was inhibited by high concentrations of phenobarbital in vitro but was not affected in vivo. No significant effects of phenobarbital on the activities of serine hydroxymethyltransferase (SHMT) or 5-methyltetrahydrofolate:homocysteine methyltransferase (MHMT) were observed either in vivo or in vitro.

Biochemical studies on the enzymatic deficiencies in hereditary tyrosinemia.

Experiments are described on the effects of succinylacetone and fumarylacetoacetate on delta-aminolevulinic acid dehydratase, methionine adenosyltransferase and p-OH-phenylpyruvate dioxygenase. delta-Aminolevulinic acid dehydratase from human erythrocytes is inhibited non-competitively by succinylacetone (Ki 0.03 mumol/l) and by fumarylacetoacetate (Ki 0.06 mumol/l). The inhibition by succinylacetone is not prevented by dithiothreitol, but the inhibition by fumarylacetoacetate is not observed if dithiothreitol is present. Methionine adenosyltransferase, partially purified from rabbit liver, is not inhibited by succinylacetone but is inhibited by fumarylacetoacetate: 69% inhibition is observed at 1 mmol/l. Human liver p-OH-phenylpyruvate dioxygenase is not inhibited by succinylacetone or fumarylacetoacetate. It is concluded that secondary enzyme deficiencies observed in hereditary tyrosinemia (delta-aminolevulinic acid dehydratase, methionine adenosyl transferase) are the result of inhibition by succinylacetone and fumarylacetoacetate, accumulating as a result of a primary deficiency of fumarylacetoacetase.