Methionine metabolism in apple tissue in relation to ethylene biosynthesis

A comparison of the rate of ethylene production by apple fruit to the methionine content of the tissue suggests that the sulfur of methionine has to be recycled during its continuous synthesis of ethylene. The metabolism of the sulfur of methionine in apple tissue in relation to ethylene biosynthesis was investigated. The results showed that in the conversion of methionine to ethylene the CH₃S-group of methionine is first incorporated as a unit into S-methylcysteine. By demethylation, S-methylcysteine is metabolized to cysteine. Cysteine then donates its sulfur to form methionine, presumably through cystathionine and homocysteine. This view is consistent with the observation that cysteine, homoserine and homocysteine were all converted to methionine, in an order of efficiency from least to greatest. For the conversion to ethylene, methionine was the most efficient precursor, followed by homocysteine and homoserine. Based on these results, a methionine-sulfur cycle in relation to ethylene biosynthesis is presented.     

Stimulation of ethylene production in apple tissue slices by methionine

Methionine can induce more than a 100% increase in ethylene production by apple tissue slices. The increased amount of ethylene derives from carbons 3 and 4 of methionine. Only post-climacteric fruit tissues are stimulated by methionine, and stimulation is optimum after 8 months' storage. Copper chelators such as sodium diethyl dithiocarbamate and cuprizone very markedly inhibit ethylene production by tissue slices. Carbon monoxide does not effect ethylene production by the slices. These data suggest that the mechanism for the conversion of methionine to ethylene, in apple tissues, is similar to the previously described model system for producing ethylene from methionine and reduced copper. Therefore, it is suggested that one of the ethylene-forming systems in tissues derives from methionine and proceeds to ethylene via a copper enzyme system which may be a peroxidase.     

Methionine metabolism and ethylene biosynthesis in senescent flower tissue of morning-glory

In immature rib segments prepared from morning-glory (Ipomoea tricolor) flower buds, the major soluble metabolite formed from tracer amounts of l-methionine-U-(14)C was S-methylmethionine (SMM). In segments of senescing ribs, (14)C was progressively lost from SMM and appeared in free methionine. Immature segments contained about 4 nmoles of free methionine and about 16 nmoles of SMM per 30 segments. As the segments senesced, the methionine content increased about 10-fold while the SMM content remained unchanged; during this time about 0.8 nmole of ethylene was produced per 30 segments. Tracer experiments with l-methionine-U-(14)C, l-methionine-methyl-(3)H, and l-homocysteine thiolactone-(35)S indicated that SMM was capable of acting as a methyl donor, and that in senescent segments the methyl group was utilized for methionine production with homocysteine serving as methyl acceptor. Of the 2 molecules of methionine produced in this reaction, 1 was re-methylated to SMM, and the other contributed to the observed rise in the content of free methionine.Internal pools of methionine and SMM were prelabeled (but not significantly expanded) by overnight incubation on 10 mum l-methionine-U-(14)C. The specific radioactivity of the ethylene subsequently evolved during the senescence of the segments closely paralleled the specific radioactivity of carbon atoms 3 plus 4 of free methionine extracted from the tissue, demonstrating that methionine was the major precursor of ethylene in this system. The specific radioactivity of carbon atoms 3 plus 4 of extracted SMM was about twice that of the free methionine.Based on these results, a scheme for methionine biosynthesis in senescent rib tissue is presented. The operation of this pathway in the control of ethylene production is discussed.     

Methionine metabolism in mammals. Adaptation to methionine excess

We conducted a systematic evaluation of the effects of increasing levels of dietary methionine on the metabolites and enzymes of methionine metabolism in rat liver. Significant decreases in hepatic concentrations of betaine and serine occurred when the dietary methionine was raised from 0.3 to 1.0%. We observed increased concentrations of S-adenosylhomocysteine in livers of rats fed 1.5% methionine and of S-adenosylmethionine and methionine only when the diet contained 3.0% methionine. Methionine supplementation resulted in decreased hepatic levels of methyltetrahydrofolate-homocysteine methyltransferase and increased levels of methionine adenosyltransferase, betaine-homocysteine methyltransferase, and cystathionine synthase. We used these data to simulate the regulatory locus formed by the enzymes which metabolize homocysteine in livers of rats fed 0.3% methionine, 1.5% methionine, and 3.0% methionine. In comparison to the model for the 0.3% methionine diet group, the model for the 3.0% methionine animals demonstrates a 12-fold increase in the synthesis of cystathionine, a 150% increase in flow through the betaine reaction, and a 550% increase in total metabolism of homocysteine. The concentrations of substrates and other metabolites are significant determinants of this apparent adaptation.     

Daminozide inhibits ethylene production in apple fruit by blocking the conversion of methionine to aminocyclopropane-1-carboxylic acid (ACC)

At harvest, fruit from apple trees sprayed with daminozide (+daminozide) had lower levels of aminocyclopropane-1-carboxylic acid (ACC) and produced significantly lower amounts of ethylene than untreated (–daminozide) fruit. Flesh discs from the fruit of +daminozide and –daminozide trees were fed precursors of ethylene to determine how daminozide inhibits ethylene production. ACC was metabolized to ethylene regardless of treatment. Methionine (MET), however, was only converted to ethylene by –daminozide fruit, and only after the fruit had been maintained at 4 °C for 5 months. +Daminozide fruit failed to convert MET to ethylene at harvest, as well as after cold storage. When daminozide was added to the incubation media of flesh discs it did not inhibit ethylene production or the conversion of ACC to ethylene. The addition of daminozide did, however, inhibit the metabolism of exogenous MET to ethylene. Aminooxyacetate acid (AOA) blocked both the endogenous production of ethylene and that from MET feeds. Daminozide inhibits ethylene production by preventing the conversion of MET to ACC, but it does not appear to act as a simple competitive inhibitor of ACC synthase activity.Abbreviations ACC aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - AOA aminooxyacetic acid - CH cycloheximide - MET methionine - PUT putrescine Author for correspondence     

Methionine metabolism and ethylene biosynthesis in senescing petals of Tradescantia

The endogenous content of methionine in isolated petals of Tradescantia was found to increase during petal senescence while the levels of S-methylmethionine and protein were found to decline. The increase in free methionine was, at least in part, the result of protein degradation. Methionine and homocysteine were shown to be intermediates in ethylene biosynthesis while S-methylmethionine was not involved. Application of 1-aminocyclopropane-1-carboxylic acid (ACC) to all floral tissues resulted in large stimulations of ethylene production. ACC was shown to be an endogenous amino acid the internal levels of which correlated positively with the rate of ethylene production. Application of l-methionine-[U-¹⁴C] led to a rapid appearance of radioactivity in both ethylene and ACC. The specific radioactivity of C-2 and C-3 of ACC and that of ethylene were found to be nearly identical which indicated that ACC was the immediate precursor of ethylene in senescing petals of Tradescantia.     

Biosynthesis of ethylene. 4-Methylmercapto-2-oxobutyric acid, an intermediate in the formation from methionine

The enzyme responsible for the conversion of methionine into a precursor of ethylene in cauliflower florets is a transaminase. The formation of 4-methyl-mercapto-2-oxobutyric acid by this enzyme has been shown. The oxo acid stimulates the synthesis of ethylene when added to floret tissue, and tracer experiments have shown that (14)C is incorporated into ethylene from the labelled oxo acid. The evidence is consistent with the view that the oxo acid is an intermediate in the formation of ethylene from methionine.     

Ethylene biosynthesis: Methionine as an in-vivo precursor of ethylene in auxin-treated mungbean hypocotyl segments

Summary Ethylene production was induced in excised hypocotyl segments of etiolated mungbean seedlings in response to exogenous auxin. 1,2,3,4-¹⁴C-Methionine was efficiently incorporated into C₂H₄, although cold methionine added at substrate level did not enhance C₂H₄ production. Incorporation of labeled glucose into C₂H₄ was reduced when hypocotyl segments were incubated with cold methionine and homoserine, but the rate of labeling of CO₂ was not affected. Feeding of labeled glucose to segments resulted in production of labeled methionine both in the presence and the absence of auxin, and auxin did not affect rate of methionine synthesis from glucose. The decrease in the amount of endogenous methionine during auxin treatment approximated the amount of C₂H₄ produced. The timecourse pattern of incorporation of radioactivity from labeled methionine into C₂H₄ during removal or re-addition of auxin was very similar to that of C₂H₄ production. The role of endogenous methionine as a C₂H₄ precursor is discussed.     

Biosynthesis of ethylene from methionine in aminoethoxyvinylglycine-resistant avocado tissue

This study was conducted to determine if aminoethoxyvinylglycine (AVG) insensitivity in avocado (Persea americana Mill., Lula, Haas, and Bacon) tissue was due to an alternate pathway of ethylene biosynthesis from methionine. AVG, at 0.1 millimolar, had little or no inhibitory effect on either total ethylene production or [(14)C] ethylene production from [(14)C]methionine in avocado tissue at various stages of ripening. However, aminoxyacetic acid (AOA), which inhibits 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (the AVG-sensitive enzyme of ethylene biosynthesis), inhibited ethylene production in avocado tissue. Total ethylene production was stimulated, and [(14)C]ethylene production from [(14)C]methionine was lowered by treating avocado tissue with 1 millimolar ACC. An inhibitor of methionine adenosyltransferase (EC 2.5.1.6), l-2-amino-4-hexynoic acid (AHA), at 1.5 millimolar, effectively inhibited [(14)C]ethylene production from [(14)C]methionine in avocado tissue but had no effect on total ethylene production during a 2-hour incubation. Rates of [(14)C]AVG uptake by avocado and apple (Malus domestica Borkh., Golden Delicious) tissues were similar, and [(14)C]AVG was the only radioactive compound in alcohol-soluble fractions of the tissues. Hence, AVG-insensitivity in avocado tissue does not appear to be due to lack of uptake or to metabolism of AVG by avocado tissue. ACC synthase activity in extracts of avocado tissue was strongly inhibited (about 60%) by 10 micromolar AVG. Insensitivity of ethylene production in avocado tissue to AVG may be due to inaccessibility of ACC synthase to AVG. AVG-resistance in the avocado system is, therefore, different from that of early climacteric apple tissue, in which AVG-insensitivity of total ethylene production appears to be due to a high level of endogenous ACC relative to its rate of conversion to ethylene. However, the sensitivity of the avocado system to AOA and AHA, dilution of labeled ethylene production by ACC, and stimulation of total ethylene production by ACC provide evidence for the methionine --> SAM --> ACC --> ethylene pathway in avocado and do not suggest the operation of an alternate pathway.     

An evaluation of 4-s-methyl-2-keto-butyric Acid as an intermediate in the biosynthesis of ethylene

Stimulation of ethylene production by cauliflower (Brassica oleracea var. botrytis L.) tissue in buffer solution containing 4-S-methyl-2-keto-butyric acid is not due to activation of the natural in vivo system. Increased ethylene production derives from an extra-cellular ethylene-forming system, catalyzed by peroxidase and other factors, which leak from the cauliflower tissue and cause the degradation of 4-S-methyl-2-keto-butyric acid. This exogenous ethylene-forming system is similar to the ethylene-forming horseradish peroxidase system which utilizes methional or 4-S-methyl-2-keto-butyric acid as substrate. We conclude that 4-S-methyl-2-keto-butyric acid is probably not an intermediate in the biosynthetic pathway between methionine and ethylene.     

Impact of methionine oxidation as an initial event on the pathway of human prion protein conversion

Prion diseases comprise a group of fatal neurodegenerative disorders characterized by the autocatalytic conversion of the cellular prion protein PrP^C into the infectious misfolded isoform PrP^Sc. Increasing evidence supports a specific role of oxidative stress in the onset of pathogenesis. Although the associated molecular mechanisms remain to be elucidated in detail, several studies currently suggest that methionine oxidation already detected in misfolded PrP^Sc destabilizes the native PrP fold as an early event in the conversion pathway. To obtain more insights about the specific impact of surface-exposed methionine residues on the oxidative-induced conversion of human PrP we designed, produced, and comparatively investigated two new pseudosulfoxidation mutants of human PrP 121–231 that comprises the well-folded C-terminal domain. Applying circular dichroism spectroscopy and dynamic light scattering techniques we showed that pseudosulfoxidation of all surface exposed Met residues formed a monomeric molten globule-like species with striking similarities to misfolding intermediates recently reported by other groups. However, individual pseudosulfoxidation at the polymorphic M129 site did not significantly contribute to the structural destabilization. Further metal-induced oxidation of the partly unfolded pseudosulfoxidation mutant resulted in the formation of an oligomeric state that shares a comparable size and stability with PrP oligomers detected after the application of different other triggers for structural conversion, indicating a generic misfolding pathway of PrP. The obtained results highlight the specific importance of methionine oxidation at surface exposed residues for PrP misfolding, strongly supporting the hypothesis that increased oxidative stress could be one causative event for sporadic prion diseases and other neurodegenerative disorders.