Ethionine-induced changes in the activities of S-adenosylmethionine synthetase isozymes from rat liver

Isozyme patterns of $\text{S}$-adenosylmethionine synthetase have been measured with and without dimethylsulfoxide in rat liver incuced by ethionine. The activity of the α-form is increased following administration of ethionine plus adenine for 2 consecutive days, and gradually decreased to control level on the 7th day after treatment, whereas the activity of β-form is relatively unaffected. Methyl-deficient transfer RNA and enhanced levels of transfer RNA-methylating enzymes were found in the livers of female rats after the treatment for 2 days, following which they gradually returned to control level.     

Studies on the activities of some methyltransferases in the livers and tumor cells from tumor-bearing mice

The activities of $\text{S}$-adenosylmethionine synthetase isozymes and some methyltransferases have been measured in liver and tumor cells of tumor-bearing mice. Following intraperitoneal transplantation of Ehrlich ascites tumor cells into mice, the activity of the β-form of the synthetase isozymes markedly increased, whereas that of the α-form did not increase so much, and the activity of tRNA methyltransferases increased gradually, while that of phospholipid, glycine and guanidoacetate methyltransferases did not. It was shown that tumor cells have only the γ-form of the synthetase and that the activity of tRNA methyltransferases in the tumor cells was very high, while that of other methyltransferases was not detectable.     

S-Adenosylmethionine synthetase isozyme patterns from rat hepatoma induced by N-2-fluorenylacetamide

Isozyme patterns of $\text{S}$-adenosylmethionine synthetase have been measured with and without dimethylsulfoxide in hepatoma of rats induced by $\text{N}$-2-fluorenylacetamide. The isozymes of α- and β-types existing in normal rat liver gradually decreased with the progress of hepatocarcinoma, and the kidney type γ-enzyme appears along with disappearance of both α- and β-enzymes. The liver from rat fetus contains a greater part of γ-type enzyme.     

Synthesis of S-adenosylmethionine synthetase isozymes from rat and mouse livers. Immunochemical studies

The alpha- and beta-forms of S-adenosylmethionine synthetase in rat liver were completely fractionated by chromatography on a hydrophobic resin, phenyl-Sepharose. The alpha-form was eluted in low-ionic strength buffer, and the beta-form was eluted with 50% dimethylsulfoxide. The alpha-form is less sensitive to dimethylsulfoxide, whereas the beta-form is strikingly stimulated by dimethylsulfoxide, after removal of the dimethylsulfoxide. The levels of the alpha-form activity in rat liver after treatment with ethionine and adenine for 2 consecutive days, and those of the beta-form activity in mouse liver on the 12th day after transplantation of Ehrlich ascites tumor cells, were increased several fold compared to normal liver. Immunochemical titrations with specific antibody against the beta-form as well as kinetic studies indicated that the observed increase in the levels of each activity from the S-adenosylmethionine synthetase isozymes is due to an increase in the cellular content of the enzyme.     

Effects of nitrous oxide-induced inactivation of cobalamin on methionine and S-asenosylmethionine metabilism in the rat

Inhalation of nitrous oxide oxidises cobalamin and, in turn, inactivates methionine synthetase which forms methionine from homocysteine and which requires cob[I]alamin as a co-factor. This study was planned to determine the effect of virtual cessation of methionine synthesis via a cobalamn-dependeent pathway, on tissue levels of methionine, S-adenosylmethionine and on related enzymes. The level of methionine in liver fell initially after exposure to N₂O but was restored to pre-N₂O levels after 6 days despite continuing N₂O exposure. Brain methionine fell within 12 h of N₂O exposure but the fall was not significant. The restoration of methionine levels is accompanied by an increase in activity of betaine homoysteine methyltransferase in liver but this enzyme was not detected in brain. The activity of methionine synthetase remained very low in both liver and brain as long as N₂O inhalation was continued. There was an initial rise in liver S-adenosyl-methionine levels followed by a steady fall to 40% of its initial level after 11 days of N₂O exposure. However, there was no change in the level of S-adenosylmethionine in brain during this period. The data indicate that either brain meets its requirement by increased methionine uptake from plasma or that there are alternate pathways in brain for methionine synthesis other than those requiring a cobalamin coenzyme.     

Changes in subcellular distribution of S-adenosylmethionine decarboxylase in regenerating and in developing rat liver

Distribution of the activity of S-adenosylmethionine decarboxylase in homogenates of rat liver generating after partial hepatectomy and during development are reported. In the stages of rapid growth of liver remaining after partial hepatectomy, and increased activity of S-adenosylmethionine decarboxylase in the supernatant fractions is accompanied by a decreased activity in the crude nuclear fractions. Prior to birth, in the liver of the developing rat, all activity of S-adenosylmethionine decarboxylase is in the supernatant fraction. After birth, activity in the crude nuclear fraction increases rapidly, reaching adult values by the end of weaning.     

Analysis of the 5′ non-coding region of rat liver S-adenosylmethionine synthetase mRNA and comparison of the Mr deduced from the cDNA sequence and the purified enzyme

A 3 kb cDNA coding for rat liver S-adenosylmethionine (AdoMet) synthetase has been isolated. The M_r of the protein has been unequivocally determined by cDNA sequencing and enzyme purification on a thiopropyl-Sepharose column. The length of the mRNA 5′ non-coding region has been defined by primer-extension analysis. The rat liver cloned cDNA has been also used to detect S-adenosylmethionine synthetase mRNA in human liver.     

Subellular distribution of S-adenosylamenthionine decarboxylase in rat liver. Evidence of decarboxylation of S-adenosylmenthionine separate from synthesis of spermidine

The acitivity of S-adenosylmethionine decarboxylase in rat liver homogenates is localized chiefly in the crude nuclear fraction, probably associated with membrane fragments, with the remainder in the supernatant fraction. This distribution is not paralleled by the activity of the cytoplasmic enzyme, lactate dehydrogenase. The spermidine-synthesizing activity of whole homogenate is recovered entirely in the supernatant fraction. Measurement of various kinetic parameters in crude fractions provided no positive evidence for isozymes of S-adenosylmethionine decarboxylase. Some species do not possess a sedimentable fraction of S-adenosylmethionine decarboxylase activity in liver. In those species all activity present in the whole homogenate of liver is released into the supernatant fraction.     

Purification,properties and regulation of the level of bovine S-adenosylmethionine decarboxylase during lymphocyte mitogenesis

S-Adenosylmethionine decarboxylase was purified from the livers of calves treated with methylglyoxal bis (guanylhydrazone) to elevate the level of the enzyme. Purified bovine S-adenosylmethionine decarboxylase was similar in specific activity and subunit molecular weight (32 000) to the enzymes previously isolated from rat and mouse. The bovine liver enzyme immunologically crossreacted with S-adenosylmethionine decarboxylase from resting and mitogenically activated bovine lymphocytes. The rate of enzyme synthesis in activated lymphocytes was determined by labeling the cells with [³H]leucine and isolating the radioactive decarboxylase by affinity chromatography and sodium dodecyl sulfate gel electrophoresis. The rate of enzyme syntheis was increased 10-fold by 9 h after mitogen treatment, which accounts for the initial increase in cellular enzymatic. There was no further incraese in the rate of S-adenosylmethionine decarboxylase synthesis that correlated with a second elevation of activity occuring at approx. 24 h after mitogenic activation. It was concluded that the second increase in enzyme activity was due to lengthening the intracellular half-life of the enzyme by 2-fold.     

Defective transport in S-adenosylmethionine synthetase mutants of Escherichia coli

The transport of several metabolites is decreased in mutant strains of Escherichia coli (Met K, E4 and E40), which contain decreased levels of S-adenosylmethionine synthetase. The rates and extents of uptake for lysine, leucine, methionine, and α-methylglucoside in both whole cells and membrane vesicles isolated from these mutants are 2- to 10-fold lower than in corresponding preparations from wild-type cells, although proline uptake is normal. The addition of S-adenosylmethionine to cultures of strain E40 can partially restore the rate and extent of lysine uptake. Lysine transport is lower in mutant vesicles in the presence of either d-lactate, succinate, α-hydroxylbutyrate, or NADH even though these substrates are oxidized at rates comparable to those in wild-type vesicles. This suggests that the defect is not related to the ability of vesicles to oxidize electron donors, but is very likely related to the ability of mutant vesicles to couple respiration to lysine transport. In addition, temperature-induced efflux of α-methylglucoside phosphate and dinitrophenol-induced efflux of lysine are similar in both the mutant and wild-type membranes, indicating that the barrier properties of the membrane and the activity of the lysine carrier are normal.     

Effect of adenosine metabolites on methyltransferase reactions in isolated rat livers

Adenosine is rapidly metabolized by isolated rat livers. The major products found in the perfusate were inosine and uric acid while hypoxanthine could also be detected. S-Adenosylhomocysteine was also excreted when the liver was perfused with both adenosine and L-homocysteine. A considerable portion of the added adenosine was salvaged via the adenosine kinase reaction. The specific radioactivity of the resultant AMP reached 75–80% of the added [8-¹⁴C]adenosine within 90 min. When the liver was perfused with adenosine alone, hydrolysis of S-adenosyllhomosysteine, via S-adenosylhomocysteine hydrolase, appeared to be blocked resulting in the accumulation of this compound. As the intracellular level of S-adenosylhomocysteine increased, the rates of various methyltransferase reactions were reduced, resulting in elevated levels of intracellular S-adenosylmethionine. When the liver was perfused with normal plasma levels of methionine the S-adenosylmethionine : S-adenosylhomocysteine ratio was 5.3 and the half-life of the methyl groups was 32 min. Upon further addition of adenosien the S-adenosylmethionine : S-adenosylhomocysteine ratio shifted to 1.7 and the half-life of the methyl groups to 103 min. In the presence of adenosine and L-homocysteine such inordinate amounts of S-adenosylhomocysteine accumulated in the cell that methylation reactions were completely inhibited. Although adenine has been found to be a product of the S-adenosylhomocysteine hydrolase only trace quantities of this compound were detectable in the tissue after perfusing the liver with high concentrations of adenosine for 90 min.     

Selenite toxicity,depletion of liver S-adenosylmethionine,and inactivation of methionine adenosyltransferase

The possibility that dimethyl selenide production depletes liver S-adenosylmethionine was explored as a biochemical basis for selenite toxicity. Toxic doses of selenite (25 nmol/ g body weight) were found to rapidly decrease mouse liver S-adenosylmethionine and increase S-adenosylhomocysteine, indicative of an increased rate of transmethylation. However, S-adenosylmethionine levels remained depressed beyond the time when dimethyl selenide synthesis ceased, suggesting that selenite inactivated methionine adenosyltransferase. This was found to be the case in vivo by measuring the effect of graded doses of selenite on the conversion of the methionine analog, ethionine, to S-adenosylethionine. In vitro studies also indicated inactivation of this enzyme by selenite. Liver homogenates from mice injected with 25 nmol of selenite/g, as above, were found to have less than 50% of the methionine adenosyltransferase activity of saline-injected controls.     

Significance of alterations in the metabolomics of sulfur-containing amino acids during liver regeneration

It has been known that liver regeneration is accompanied with a profound change in the metabolomics of sulfur-containing substances in liver. However, its physiological significance in the liver regenerative process is still unclear. Our previous work showed that buthioninesulfoximine and phorone, both widely used to deplete intracellular glutathione (GSH) in biological experiments, induced contrasting changes in the sulfur-containing amino acid metabolism in liver. In this study we employed these GSH-depleting agents to evaluate the role of sulfur-containing substances in the early phase of liver regeneration. Male rats treated with buthioninesulfoximine or phorone were subjected to two-thirds partial hepatectomy (PHx). At the doses used, the magnitude of GSH depletion after PHx was comparable, but buthioninesulfoximine administration inhibited the progression of liver regeneration as determined by liver weight increase, elevation of serum alanine aminotransferase activity, and cyclin D1 and proliferating cell nuclear antigen (PCNA) protein expressions, whereas liver recovery was significantly accelerated in the phorone-treated rats, suggesting that the role of GSH in this process is minimal. Hepatic concentrations of methionine, S-adenosylmethionine, cysteine, taurine and GSH were all elevated by PHx. Methionine adenosyltransferase activity was also induced in the remnant liver. Buthioninesulfoximine administration depressed the elevation of S-adenosylmethionine, but increased the catabolism of cysteine to taurine. In contrast, S-adenosylmethionine elevation was augmented whereas cysteine, hypotaurine and taurine were decreased in the phorone-treated rats. PHx elevated hepatic putrescine and spermidine, but lowered spermine concentrations. Buthioninesulfoximine administration increased putrescine further, but decreased spermidine and spermine concentrations. On the contrary, both spermidine and spermine concentrations were elevated in the rats treated with phorone. The results suggest that the availability of S-adenosylmethionine plays a critical role in the progression of liver regeneration via enhancement of polyamine synthesis. These findings raise the possibility that regulating hepatic transsulfuration reactions may be capable of modifying the recovery process after liver injury.     

Effect of urethan on polyamine and DNA synthesis in the regenerating rat liver

When a single dose of urethan was injected into the peritoneal cavity of rats immediately after partial hepatectomy, DNA synthesis was delayed by 12 h. The induction of ornithine decarboxylase which was induced biphasically following partial hepatectomy was also reduced and delayed by 14–15 h by the administration of urethan. S-Adenosylmethionine decarboxylase activity in urethan-treated rat liver at 20 h and 29 h after operation was significantly lower than that of untreated animals. This enzyme activity was shown to increase thereafter, reaching a higher level than in untreated rats at 37–42 h. Hepatic spermidine content changed biphasically in a manner similar to DNA synthesis. These results suggest that the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase may correlate with DNA synthesis and that an increase of spermidine concentration is necessary to DNA synthesis.     

Methionine analogs inhibit production of β-subunit of soybean 7S protein

We have previously reported that exogenous methionine inhibits production of the β-subunit of the 7S storage protein in cultured soybean cotyledons, and that this inhibition involves lack of functional mRNA for the β-subunit. Analogs of methionine were used to study this inhibition. Cycloleucine, norleucine, norvaline and S-ethylcysteine treatments prevented accumulation of the β-subunit. The effects of cycloleucine and norleucine on β-subunit synthesis might have been indirect, since these compounds inhibited growth and caused a 2- to 3-fold increase in free methionine concentration. Norvaline did not affect free methionine concentration, but it did inhibit growth. Treatment with a combination of S-ethylcysteine and aminoethoxyvinylglycine prevented appearance of the β-subunit without inhibiting growth or raising the S-adenosylmethionine concentration. Thus, accumulation of S-adenosylmethionine does not appear to mediate the effect of exogenous methionine on β-subunit production. Treatment with S-ethylcysteine raised free methionine concentration only 34%, so S-ethylcysteine was probably acting directly to inhibit β-subunit production. Measurements of free methionine concentrations in seeds of different sizes, taken from intact plants, suggested that the relatively late appearance of the β-subunit in normal soybean seed development may be due to the presence of high levels of free methionine in very young seeds.     

S-adenosylmethionine synthetase in cultured normal and oncogenically-transformed human and rat cells

We have investigated the enzymatic formation of S-adenosylmethionine in extracts of a variety of normal and oncogenically-transformed human and rat cell lines which differ in their ability to grow in medium in which methionine is replaced by its immediate precursor homocysteine. We have localized the bulk of the S-adenosylmethionine synthetase activity to the post-mitochondrial supernatant. We show that in all cell lines a single kinetic species exists in a dialyzed extract with a K_m for methionine of about 3–12 μM. In selected lines we have demonstrated a requirement for Mg²⁺ in addition to that needed to form the Mg·ATP complex for enzyme activity and have shown that the enzyme can be regulated by product feedback inhibition. Because we detect no differences in the enzymatic ability of these cell extracts to utilize methionine for S-adenosylmethionine formation in vitro, we suggest that the failure of oncogenically-transformed cell lines to grow in homocysteine medium may result from the decreased methionine pools in these cells or from the loss of ability of these cells to properly metabolize homocysteine, adenosine, or their cellular product S-adenosylhomocysteine.     

Polyamines in testosterone-induced hypertrophic and antifolate-induced hyperplastic mouse kidney. Differential effect of α-difluoromethylornithine

In the testosterone-induced hypertrophic and antifolate (N¹⁰-propargyl,5,6-dideazafolic acid, CB 3717)-induced hyperplastic mouse kidney models, a marked increase of two diamine levels — putrescine and cadaverine — occurred which paralled induced ornithine decarboxylase (ODC) activity. Under these conditions the augmentation of spermidine levels was much smaller, while spermine levels were affected differentially — increased by testosterone and decreased by CB 3717; this resulted in an increase of spermidine/spermine ratio in hyperplastic, but not hypertrophic kidney. α-Difluoromethylornithine (DFMO) prevented testosterone- or CB 3717-induced increment of both diamine levels. Spermidine and spermine depletion in response to DFMO was significant in hyperplastic kidney only. DFMO also significantly affected the other biochemical markers of hyperplasia, namely lowered CB 3717-induced cell proliferation rate and increased S-adenosylmethionie decarboxylase (AdoMetDC) activity. In contrast, testosterone-induced hypertrophy was not influenced by DFMO, as judged by the lack of its effect on S-adenosylmethionine synthetase and cystathionine synthase activity. These results indicate that the increase of putrescine levels does not mediate testosterone-induced renal hypertrophy and possibly also antifolate-induced hyperplasia. The involvement of spermidine in mediation of renal hyperplasia is highly possible, while that of spermine is excluded.     

A procedure for the quantitative analysis of the sulphur amino acids of rat tissues

1. A method is described for the quantitative separation of the sulphur compounds in a single sample of tissue by passing an extract through a serial assembly of ion-exchange resins in the order: Dowex 2 (Cl⁻ form), Dowex 1 (CO₃²⁻ form), Amberlite CG-50 (H⁺ form) and Zeo-Karb 225 (H⁺ form). 2. Groups of sulphur amino acids were eluted separately from each column; the recovery of sulphur compounds after their labelling with ³⁵S in vivo by injection of l-[³⁵S]-methionine was 91–106%. Individual sulphur compounds were further resolved by one-dimensional or two-dimensional paper chromatography. 3. Evidence is presented on the occurrence of S-adenosylmethionine and S-adenosylhomocysteine in rat liver and brain. Rat liver and brain contained 83·6 and 31·4mμ-moles/g. respectively of S-adenosylmethionine.     

Effect of methylglyoxal bis (guanylhydrazone) (MGBG) in vivo on the decarboxylation of s-adenosylmethionine and synthesis of spermidine in the rat and guinea pig.

The rates of decarboxylation of $\text{S}$-adenosylmethionine and synthesis of spermidine have been measured in extracts of liver, kidney and brain of the rat and guinea pig after intraperitoneal injection of MGBG, both before and after dialysis. The rate of decarboxylation of $\text{S}$-adenosylmethionine paralleled that of spermidine synthesis in all of the tissues investigated, even when spermidine synthesis was measured using preformed decarboxylated $\text{S}$-adenosylmethionine as substrate instead of $\text{S}$-adenosylmethionine itself. MGBG inhibited CO₂ production and spermidine synthesis to a similar extent in extracts of liver and kidney of both the rat and the guinea pig. After dialysis, a similar increase in both CO₂ production and spermidine synthesis was noted in these extracts. No effects on CO₂ production or spermidine synthesis were noted on extracts of brain of the rat or guinea pig, either before or after dialysis. When MGBG was injected intracisternally, CO₂ production and spermidine synthesis by extracts of brain were inhibited to the same extent, and after dialysis a similar increase in CO₂ production and spermidine synthesis was observed. These results indicate that the effects of MGBG are essentially the same in brain as they are in liver and kidney, and the MGBG injected intraperitoneally does not pass into the brain.