Fate of ($ ) S-methyl-L-cysteine sulfoxide in Chinese cabbage, Brassica pekinensis RUPR.

($) S-methyl-L-cysteine sulfoxide (MCS) was scarcely found inseeds of Chinese cabbage, but was present in relatively largeamounts in all plant parts after germination. Changes in MCScontent paralleled those for soluble sulfur content of tissue. When Na₂³⁵SO₄ was fed to plants, the ³⁵S was predominantly incorporatedinto MCS in the free amino acid fraction in both sulfur-sufficientand deficient plants, but it was incorporated to a greater extentin the former. ³⁵S-MCS was metabolized more readily in deficientthan in sufficient plants, and its sulfur was found not onlyin various soluble compounds but in the insoluble fractionsfrom plants as well. These results indicate that MCS is a conspicuousconstituent in the free amino acid pool of Chinese cabbage andmay play an important role in sulfur metabolism by acting asa soluble pool for organic sulfur. (Received June 22, 1970; )     

Biosynthesis of s-methylcysteine in radish leaves

Investigation on the biosynthesis of S-methyl-L-cysteine in radish leaves has shown that it is formed by the methylation of cysteine. This conclusion is based on: A) the relatively high recovery of radioactivity in methylcysteine sulfoxide after the administration of cysteine or methyl-labeled methionine to radish leaves; B) the nearly complete recovery of label from methyl-labeled methionine in the methyl group of methylcysteine sulfoxide; and C) the similarity in the ratio of tritium to ¹⁴C in methylcysteine sulfoxide and in its methyl group to this ratio in the methyl group of methionine given to radish leaves. Direct evidence for the synthesis of methylcysteine in radishes was obtained for the first time.     

Incorporation of methylsulfonylmethane sulfur into guinea pig serum proteins

Methionine, an essential amino acid, and cysteine are the major sulfur-containing amino acids in the body and both are thought to be synthesized predominantly in plants and micro-organisms. Methylsulfonylmethane (MSM) is a natural constituent of the environment in which it is found in plants, in milk and urine of both bovines and humans, is a normal oxidation product of dimethyl sulfoxide (DMSO) also in the natural environment and may be part of the natural global sulfur cycle. To determine whether sulfur from methylsulfonylmethane (MSM) is incorporated into sulfur amino acids, I fed 35S-MSM to guinea pigs. 35S was incorporated into peptidyl methionine and cysteine of guinea pig serum proteins. The specific activity of 35S-methionine was 30% greater than for 35S-cysteine, suggesting a precursor-product relationship. Total specific activity of serum proteins was increased by only 30% with a 100% increase of administered 35S-MSM, suggesting a limiting step in synthesis. Approximately 1% of the radioactivity was recovered in serum proteins, none in the feces and most was excreted in the urine. Microorganisms of intestinal lumen may be responsible for the incorporation of the 35S of MSM into sulfur amino acids. MSM may provide a source of sulfur for essential animal methionine by mechanisms not yet elucidated in either animals or micro-organisms.     

ORIGIN OF AMINO ACIDS CONSTITUTING CELLULAR PROTEIN IN GERMINATING CONIDIOSPORES OF ASPERGILLUS NIGER

Formation of pool amino acids in germinating spores of Aspergillusniger strain 1617 was investigated. The pool amino acids comprisedmainly glutamic acid and alanine. Small amounts of pyruvateand -ketoglutarate were found to increase almost in parallelwith the course of increase in the amount of free amino acidsup to the stage of onset of active protein synthesis. Asparticglutamictransaminase activity was exhibited even in dormant spores andit developed in response to the increase in cellular protein.Alanine-glutamic transaminase activity, on the other hand, waslacking in dormant spores and appeared at the stage of accumulationof amino acids preceding protein synthesis. It was revealed from the experiments with ³⁵S-labeled sporesthat the dormant spores of this fungus contain two unidentifiedsulfur substances, and the sulfur of these substances is incorporatedinto the sulfur amino acids of the protein synthesized in germinatingspores. ¹Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo (Received September 11, 1959; )     

Reduced sulphur allocation from three-year-old needles of Norway spruce (Picea abies [Karst] L.)

The ³⁵S-labelled sulphur compounds glutathione, cysteine, and-glutamylcysteine were fed to 6-year-old spruce trees via thecut surface of a single 3-year-old needle. After 1–3 hexposure, uptake of the radiotracer into the fed needle, exportinto other parts of the plant and distribution between needles,bark and wood along the transport path were analysed. Uptakeof cysteine into the exposed needle was one order of magnitudeand uptake of -glutamylcysteine two orders of magnitude lowerthan that of glutathione. Independent of the thiol applied,the current year's sprouts were the preferential sinks of exported³⁵S. Transport towards basipetal parts of the twig amountedto less than 10% of total ³⁵S export in all cases. After feeding³⁵S-cysteine and ³⁵S--glutamylcysteine, ³⁵S-glutathione wasfound along the transport path, in particular in distant partsof the twig. This was also observed when ³⁵S-GSH was fed. Thisresult confirms the significance of glutathione as the majorlong-distance transport form of reduced sulphur in spruce twigs.In xylem sap of trunk sections of spruce, cysteine rather thanglutathione was the main thiol. Cysteine concentrations in thexylem sap of the trunk amounted to 260–500 nmol l^–1.Glutathione concentrations were 2–5 times and -glutamylcysteineconcentrations 4–16 times lower than those of cysteine. Key words: Glutathione, -glutamylcysteine, cysteine, long-distance transport     

ROLE OF SULFUR IN THE CELL DIVISION OF CHLORELLA, WITH SPECIAL REFERENCE TO THE SULFUR COMPOUNDS APPEARING DURING THE PROCESS OF CELL DIVISION. I.

1. The role of sulfur in the cell division of Chlorella was studiedby following the fate of the sulfur supplied to the sulfur-deficientcells using ³⁵S as a tracer.
2. The sulfur-deficient cells whichwere unable to perform celldivision were made capable of divisionby the provision of ³⁶S-labeledsulfate under non-photosynthesizingconditions. Soon after theprovision of sulfate the labeledsulfur went rapidly into thecold perchloric acid (PCA)-solublefraction of algal cells,almost entirely in the form of sulfateand/or some other inorganicsulfur substance (s). With the lapseof time, more or less remarkablechanges occurred in the patternof ³⁵S-distribution in differentfractions of cell material.It was noticed that, at the onsetof cell division, a sulfur-containingpeptide-nucleotide compound(s)(SPN), which has been reportedearlier, appeared in a largequantity in the cold PCA-solublefraction, and that its quantitydecreased gradually during thesubsequent process of cell division,suggesting that the compoundwas transformed into some othersubstance (s), presumably withits nucleotide moiety going intonucleic acids and the peptidemoiety going into some essentialproteins.
3. Another noteworthyphenomenon observed during the process ofcell division wasthe incorporation of ³⁶S in a group of hotPCA-soluble substances.These sulfur substances were revealedto be sulfur-containingnucleotidic compounds, which might possiblybe some essentialcomponents of, or substances in close relationto, deoxypentosenucleic acid (DNA).

(Received March 1, 1960; )     

Studies on the metabolism of a sulfur-oxidizing bacterium VI1. Fractionation and reconstitution of the elementary sulfur-oxidizing system of Thiobacillus thiooxidans

The sulfur-oxidizing system of a strain of Thiobacillus thiooxidanswas obtained in cell-free state. The system is resolved intothree fractions and can be reconstituted from these fractions.Both the soluble and particulate fractions are required forthe oxidation of elementary sulfur. The soluble fraction wasfurther separated into two fractions, the collodion membrane-permeable(S-P)and the impermeable(S-IP). S-P contains a low molecular weight,relatively heat stable substance(s) which is indispensable forthe reconstitution of the sulfur-oxidizing system and was identifiedas a pyridine nucleotide. The function of S-P can be replacedby NAD or NADP, but not by cysteine nor GSH. Oxidation of NADH₂ and NADPH₂ is catalyzed by the particulatefraction. Oxidation of the latter is much more rapid than thatof the former. Oxidation of NADPH₂ as well as sulfur oxidationis inhibited by cyanide, pCMB and CO, the CO-inhibition beingphoto-irreversible. However, strong inhibitors of sulfur oxidationsuch as DDC, 8-hydroxyquinoline and salicylaldoxime have noeffect on the oxidation of NADPH₂. The optimum pH values for sulfur and sulfite oxidations by thecell-free extract are shifted to the neutral side in comparisonwith pH values by intact cells. ¹V = References(I). ²Partly supported by a grant from the Ministry of Education. (Received April 3, 1969; )     

Ablation of the mammalian methionine sulfoxide reductase A affects the expression level of cysteine deoxygenase

Methionine sulfoxide reductases (Msrs) are able to reduce methionine sulfoxide to methionine both in proteins and free amino acids. By their action it is possible to regulate the function of specific proteins and the cellular antioxidant defense against oxidative damage. Similarly, cysteine deoxygenase (CDO) may be involved in the regulation of protein function and antioxidant defense mechanisms by its ability to oxidized cysteine residues. The two enzymes' involvement in sulfur amino-acids metabolism seems to be connected. Lack of methionine sulfoxide reductase A (MsrA) in liver of MsrA-/- led to a significant drop in the cellular level of thiol groups and lowered the CDO level of expression. Moreover, following selenium deficient diet (applied to decrease the expression levels of selenoproteins like MsrB), the latter effect was maintained while the basal levels of thiol decreased in both mouse strains. We suggest that both enzymes are working in coordination to balance cellular antioxidant defense.     

Formation of putrescine from ornithine and arginine in tobacco plants

Putrescine was formed when both DL-ornithine-2-¹⁴C and L-arginine-U-¹⁴Cwere injected into tobacco plants. The incorporation rate ofthese amino acids into putrescine was higher in potassium orsulfur deficient plants than in normal plants. Degradation ofputrescine-1,4-¹⁴C injected into tobacco plants was inhibitedby a potassium or sulfur deficiency. Increased accumulationof putrescine in potassium or sulfur deficient plants may bepartly dependent upon this inhibition in the degradation ofputrescine. Detached leaves and roots were also able to formputrescine from DL-ornithine-2-¹⁴C and L-arginine-U-¹⁴C. (Received December 13, 1968; )     

Studies on the metabolism of a sulfur-oxidizing bacterium IV.1 Growth and oxidation of sulfur compounds in Thiobacillus thiooxidans

By immersing a few small cellophane bags containing BaCO³ powderin STARKEY's medium, the duration of lag phase in the growthof Thiobacillus thiooxidans is minimized and the yield of cellsis increased ten times that of the previous method. The activitiesof oxidation for sulfur and sulfite change with growth. Sulfiteis oxidized at a comparable rate to that of sulfur oxidationat pH values between 6.0 and 6.5. In the presence of cysteineor glutathione, thiosulfate can be oxidized at a pH above 5.0.At pH values below 4.5, apparent oxidation of thiosulfate andtetrathionate to sulfate is observed. This result is accountedfor by the facts that thiosulfate is decomposed to sulfur andsulfite under the acidic condition at pH values below 4.5, andthat tetrathionate is reduced to thiosulfate enzymatically.In the oxidation of tetrathionate, oxygen uptake begins aftera lag phase, the duration of which depends on the concentrationsof cells and of tetrathionate. Cysteine is oxidized to cystine.The oxidation is strongly inhibited by metal-chelating agents.The cysteine oxidizing activity is, however, quite stable andis not lost by treating cells with organic solvents, sonic oscillation,by heating or lyophilization. ¹III=References (11). ²Partly supported by a grant from the Ministry of Education.     

ROLE OF SULFUR IN THE CELL DIVISION OF CHLORELLA, WITH SPECIAL REFERENCE TO THE SULFUR COMPOUNDS APPEARING DURING THE PROCESS OF CELL DIVISION II.

1. Chlorella cells, which had been grown synchronously undersulfur-deficient conditions and thus rendered unable to performcell division, were made capable of nuclear and cellular divisionby being supplied with ³⁵S-labeled sulfate and nitrate underphotosynthesizing conditions, and the fate of sulfur duringthese recovery processes was followed. 2. When the S-starved cells were provided with sulfate aloneunder photosynthesizing conditions, cells grew appreciably inmass performing nuclear division but remaining incapable ofcellular division. During these processes most of the ³⁵S wasfound to be incorporated into the protein fraction of algalcells. 3. When the cells which had been stalemated at the above-mentionedstage were supplied with nitrate, they grew further in massand eventually performed cellular division. During this periodthe ³⁵S was found to be distributed not only in the proteinfraction, but also in an appreciable amount in the cold andhot acid-soluble fractions. 4. By paper-electrophoretic experiments it was found that thenature of the sulfur substances appearing in the hot acid-solublefraction changed strikingly during the process of cellular division.Zone electrophoresis and an anion-exchange chromatography ofthese substance isolated from the cells at the completion ofcellular division, disclosed that they were most probably deoxypentosepolynucleotides containing sulfur in some form yet unidentified. 5. It was demonstrated that there exist some antagonistic relationsbetween the protein synthesis and the formation of these sulfur-containingdeoxypentose polynucleotides, and that the former predominatesunder photosynthesizing conditions while the latter outweighsunder nonphotosynthesizing conditions. (Received August 9, 1960; )     

The level of sulfane sulfur in the fungus <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> wild type and mutant strains

The interdependence of the sulfane sulfur metabolism and sulfur amino acid metabolism was studied in the fungus Aspergillus nidulans wild type strain and in mutants impaired in genes encoding enzymes involved in the synthesis of cysteine (a precursor of sulfane sulfur) or in regulatory genes of the sulfur metabolite repression system. It was found that a low concentration of cellular cysteine leads to elevation of two sulfane sulfurtransferases, rhodanase and cystathionine γ-lyase, while the level of 3-mercaptopyruvate sulfurtransferase remains largely unaffected. In spite of drastic differences in the levels of biosynthetic enzymes and of sulfur amino acids due to mutations or sulfur supplementation of cultures, the level of total sulfane sulfur is fairly stable. This stability confirms the crucial role of sulfane sulfur as a fine-tuning regulator of cellular metabolism.     

TASTE OF ACETYLATED AMINO ACIDS

The gustatory properties of amino acids and their acetylatedcounterparts were compared. Application of multidimensionalscaling procedures to semantic differential ratings and similarityjudgments revealed that five unpleasant tasting amino acidsbecame tasteless when acetylated. Also, two extremely unpalatableamino acids became sour. All the acetylated amino acids fallwithin Henning's tetrahedral prism; however, one amino acid,cysteine (crystallized from acid solution) fell outside thetraditional sweet, sour, salty and bitter categories. ^* Assistant Professor of Medical Psychology.     

Phloem transport of sulfur in Ricinus

Mature leaves of Ricinus communis fed with ³⁵SO ₄ ^2- in the light export labeled sulfate and reduced sulfur compounds by phloem transport. Only 1–2% of the absorbed radiosulfur is exported to the stem within 2–3 h, roughly 12% of ³⁵S recovered was in reduced form. The composition of phloem translocate moving down the stem toward the root was determined from phloem exudate: 20–40% of the ³⁵S moved in the form of organic sulfur compounds, however, the bulk of sulfur was transported as inorganic sulfate. The most important organic sulfur compound translocated was glutathione, carrying about 70% of the label present in the organic fraction. In addition, methionine and cysteine were involved in phloem sulfur transport and accounted for roughly 10%. Primarily, the reduced forms of both, glutathione and cysteine are prsent in the siever tubes.Abbreviations CySH cysteine - GSH glutathione - GSSG glutathione disulfide - NEM N-ethylmaleimide - CyS-SCy cystine     

Metabolism of Sulfur Compounds in Bacillus subtilis during Sporulation

Metabolism of various sulfur compounds in Bacillus subtilis during growth and sporulation was investigated by use of tracer techniques, in an attempt to clarify the mechanism involved in the formation of cystine rich protein of the spore coat.

Methionine, homocysteine, cystathionine, cysteine and some inorganic sulfur compounds (sulfate, sulfite and thiosulfate) were utilized by this organism as sulfur sources for its growth and sporulation. Biosynthesis of methionine from sulfate during growth was more or less inhibited by the addition of cysteine, homocysteine or cystathionine to the culture.

It is suggested from these results that in Bacillus subtilis methionine is synthesized from sulfate through cysteine, cystathionine and homocysteine as is the case in Salmonella or Neurospora. The results also suggest that the metabolism of sulfur-containing amino acids in Bacillus subtilis is strongly regulated by methionine and homocysteine.     

Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1

KAAT1 is a neutral amino acid transporter activated by K⁺ or by Na⁺ (9). The protein shows significant homology with members of the Na⁺/Cl^–-dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20–30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of V_max and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na⁺ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1. amino acid transport; structure/function; amino acid modifiers; Manduca sexta     

Studies on the metabolism of a sulfur-oxidizing bacterium V. Comparative studies on sulfur and sulfite oxidizing systems of Thiobacillus thiooxidans

Properties of the oxidation systems of sulfur and sulfite ofa sulfur oxidizing bacterium, Thiobacillus thiooxidans, arecompared by using various inhibitors. Oxidation of sulfur isinhibited by a low concentration of monoiodoacetic acid, NEMand pCMB. Inhibition by pCMB is diminished by the addition ofan equivalent amount of cysteine to that of added pCMB. Althoughinhibition by pCMB is also observed in the oxidation of sulfite,it is not diminished by the addition of excess cysteine andthe extent of inhibition is lower than that in the oxidationof sulfur. Metal chelating agents, such as DDC, 8-hydroxyquinoline, salicylaldoximeand neocuproine have inhibitory effects on the oxidation ofsulfur but do not affect the oxidation of sulfite. Carbon monoxide inhibits the oxidation of sulfur photo-irreversiblyand the oxidation of sulfite photo-reversibly. Alcohols and organic acids, inhibit the oxidation of both sulfurand sulfite. The cell-free extract prepared by sonic disruptionof cells can oxidize sulfite, but not sulfur. The sulfur oxidizingextract can be, however, prepared by disruption under a nitrogenatmosphere. Both the soluble and participate fractions are requiredfor the oxidation of sulfur, while sulfite oxidation is catalyzedby the participate fraction alone. ¹Partly supported by a grant from the Ministry of Education.     

Behavioral responses of the brittle star Ophiura ophiura to amino acids, acetylcholine and related low-molecular-weight compounds

Feeding behavior of the brittle star Ophiura ophiura includesorienting posture, orienting movements, arm ‘walking’,changing the direction of ‘walking’ arm coilingand ingestion. All sequential behavior patterns were releasedor enhanced by single low-molecular-weight compounds. Stimuliwhich released ‘walking’ behavior at high concentrations(10^–4 M) in all the test animals are listed in decreasingorder of sensitivity: sarcosine, glycine, urea, L-valine, L-leucine,L-methionine, L-homocysteine, L-norvaline, L-norleucine, L-threonine,L-serine, S-methyl-L-cysteine, L-proline. Threshold values forsingle amino acids were as much as 100 times different in differentindividuals and ranged from 3 x 10^–9 to 3 x 10^–7M for the most effective stimulus, sarcosine, and from 10^–6to 10^–4 M for proline. Above 10^–5 M, only L-prolineregularly released a second behavior pattern, the arm coilingresponse, which temporarily inhibited the ‘walking’behavior. Behavioral thresholds for the ‘walking’behavior for L(+)-lactate and L-alanine were higher than thosefor the orienting movements. Thyoglycolic acid and ß-alaninereleased tube feet walking, which is not part of the feedingbehavior. Structure—activity comparisons were studied at estimated10^–5 M concentrations. Gycline, sarcosine, L-valine, L-norvaline,L-leucine, L-isoleucine, DL-norleucine and DL-homocysteine releasedarm ‘walking’ behavior in more than 75% of all thetests. With the exceptions of S-methyl-and S-ethyl-cysteine,and glycine methylester, derivatives of amino acids were noteffective behavioral stimuli in Ophiura ophiura. L-Isomers ofvaline and leucine regularly stimulated the ‘walking’behavior while their D-isomers were effective in some testsand ineffective in others. Acetylcholine iodide, acetyl-ß-methylcholine chloride and choline phosphate chloride regularly released‘walking’ behavior at concentrations above 10^–5M.     

Properties of Recombinant Staphylococcus haemolyticus Cystathionine β‐Lyase (metC) and Its Potential Role in the Generation of Volatile Thiols in Axillary Malodor

Enzymes implicated in cysteine and methionine metabolism such as cystathionine β‐lyase (CBL; EC 4.4.1.8), a pyridoxal‐5′‐phosphate (PLP)‐dependent carbon–sulfur lyase, have been shown to play a central role in the generation of sulfur compounds. This work describes the unprecedented cloning and characterization of the metC‐cystathionine β‐lyase from the axillary‐isolated strain Staphylococcus haemolyticus AX3, in order to determine its activity and its involvement in amino acid biosynthesis, and in the generation of sulfur compounds in human sweat. The gene contains a cysteine/methionine metabolism enzyme pattern, and also a sequence capable to effect β‐elimination. The recombinant enzyme was shown to cleave cystathionine into homocysteine and to convert methionine into methanethiol at low levels. No odor was generated after incubation of the recombinant enzyme with sterile human axillary secretions; sweat components were found to have an inhibitory effect. These results suggest that the generation of sulfur compounds by Staphylococci and the β‐lyase activity in human sweat are mediated by enzymes other than the metC gene or by the concerted activities of more than one enzyme.     

The origin of the sulfur atom in thiamine

The mode of biosynthesis of the thiazole moiety of thiamine, 4-methyl-5β-hydroxyethyl thiazole (MHET) was studied using Salmonella typhimurium as test organism. It was shown by isotope incorporation experiments, that the sulfur atom, but not carbon-3, of cysteine is incorporated into MHET, indicating a separation of the sulfur atom of cysteine from the carbon chain during incorporation. Isotope competition experiments revealed that the incorporation of [³⁵S]cysteine is not significantly diluted by the presence of methionine, homocysteine, and glutathione. No incorporation of label from [¹⁴C]glutamate and [¹⁴C]formate was observed, leaving the origin of the five-carbon unit still in doubt.