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     

Cystathionine in rat brain: Catabolism in vivo

The content of cystathionine was measured in 35 rat brains; the range was 10–120 nmol/g wet weight and thus the variability of cystathionine content in rat brain was emphasized. The regional distribution of cystathionine was also determined: the highest level was found in cerebellum; the lowest level was observed in the white and gray matter of the hemispheres. These results are different from those obtained in other species. The radioactive metabolites formed froml-[³⁵S]cystathionine injected intracisternally were measured in brains of rats killed at the following times after injection: 0.25, 1, 2, 4, 6, 9, 16, and 27 hr. The radioactivity was found both in the proteins and in the acid-soluble fraction. In the acid-soluble fraction the radioactivity was found in various ninhydrin-reacting compounds: (cysteic + cysteine sulfinic) acid, taurine, reduced and oxidized glutathione, cystine, cystathionine, and a compound tentatively identified as the mixed disulfide of cysteine and glutathione. The radioactivity of cystathionine decreased exponentially between the 1st and the 27th hour after injection and its half-life was estimated to be about 5 hr. The radioactivity in the other ninhydrin-reacting compounds increased until the 9th hour after injection, then decreased. Half of this radioactivity was present in reduced glutathione, the rest being shared equally between: (cysteic + cysteine sulfinic) acid, taurine, and the mixed disulfide. It is worthwhile to note that the radioactivity in the cystine fraction was always very low.     

Synthesis of taurine in rat liver and brain in vivo

The in vivo formation of taurine and the analysis of labeled taurine precursors was examined in rat brain and liver at different times after an intracisternal injection of [³⁵S]cysteine and an intraperitoneal injection of [³H]cysteine, simultaneously administered. The distribution pattern of radioactivity was similar in liver and brain. Most of the labeling in both organs (85% in brain and 80% in liver) was recovered in glutathione (oxidized and reduced), cysteic acid, cysteine sulfinic acid, hypotaurine, cystathionine, and a mixed disulfide of cysteine and glutathione. The relative rates of labeling of cysteine sulfinic acid and taurine in liver and brain suggest than in vivo, liver possesses a higher capacity for taurine synthesis than brain. A small amount of [³H]taurine was detected in brain after intraperitoneal injection of [³H]cysteine. The time of appearance of this [³H]taurine as well as the fact that it occurs when [³H]cysteine is not detectable in brain or plasma suggests that it was probably not synthesized in brain from labeled precursors but formed elsewhere and transported into the brain through an exchange process.     

Utilization of cystine by dermatophytes on a gelatin medium

All 16 strains of dermatophytes investigated utilized cystine (added to the gelatin medium) as a source of sulfur and also of carbon and nitrogen. Excess sulfur oxidized and excreted to the medium, primarily as inorganic sulfate. Six strains used up all cystine and excreted more than 90% stoichiometric amount of sulfur. Cystine utilization proceeded in parallel with the development of the culture and was terminated during the stationary phase or as late as in the autolytic phase. Other strains did not use up cystine completely and excreted 17-70% sulfur in the oxidized form. In addition to sulfate, sulfite was always produced during the initial growth phases and in poorly growing strains. Free sulfite was only rarely detected; it usually reacted with the residual cystine yielding S-sulfocysteine that was also used up later. Specific features of cystine metabolism (known from Microsporum gypseum) are generally valid in dermatophytes.     

Utilization of cystine by dermatophytes on glucose-peptone media

All the 16 strains of dermatophytes tested here metabolized cystine (3 mmol/L) in two glucose-peptone media with a different C: N ratio. Cystine was utilized as a sulfur source and, in addition, as a carbon and nitrogen source, in parallel with growth. Excess sulfur was excreted to the medium after its oxidation as inorganic sulfate and sulfite. In a physiologically alkaline medium the growth was fast and was accompanied by a pH increase and cystine was utilized intensively. Eleven species used up all cystine available. Sulfate was the main oxidation product, sulfite was produced at a low concentration, at the beginning of growth in particular. Only traces of thiol compounds (cysteine) were present in the medium. In a physiologically acid medium growth was soon limited by a decreased pH (below 5.0) but cystine continued to be utilized at an identical rate. All cystine was used up by 5 species. The tendency to produce sulfite in addition to sulfate further increased and sulfite was often the predominant product. Concentrations of thiol compounds were also substantially higher. Thus, dermatophytes can utilize cystine even under conditions that do not support good growth and increase the sulfite production.     

Role of Methionine in Cephalosporin Synthesis

Methionine has an almost unique stimulatory effect on biosynthesis of cephalosporins (by Cephalosporium acremonium). No other sulfur-containing compound tested, except dl-methionine-dl-sulfoxide, replaced methionine. dl-Methionine stimulated the synthesis of cephalosporins when added after the growth phase. The utilization of inorganic sulfate was repressed by methionine. Experiments with l-methionine-S(35) showed that essentially all the sulfur in the cephalosporins was derived from methionine. Sulfur-labeled compounds found in the soluble pool from cells grown with methionine-S(35) were methionine, homocysteine, taurine, cystathionine, cysteic acid, glutathionine, and cysteine. dl-Serine-3-C(14) was incorporated into the antibiotics, and its utilization was stimulated by methionine. l-Cysteine had a sparing effect on the incorporation of methionine-S(35) and serine-C(14) into the antibiotics. The data are consistent with the hypothesis that a cystathionine-mediated pathway is operative in the transfer of sulfur between methionine and cysteine.     

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.     

Ultrasound-Induced Formation of S-Nitrosoglutathione and S-Nitrosocysteine in Aerobic Aqueous Solutions of Glutathione and Cysteine

S-Nitrosocompounds are formed when aqueous solutions of cysteine or glutathione are exposed to ultrasound (880 kHz) in air. The yield of the S-nitrosocompounds was as high as 10% for glutathione and 4% for cysteine of the initial thiol concentrations (from 0.1 to 10 mM) in the aqueous solutions. In addition to the formation of S-nitrosocompounds, thiol oxidation to disulfide forms was observed. After the oxidation of over 70% of the sulfhydryl groups, formation of peroxide compounds as well as cysteic acid derivatives was recorded. The formation of the peroxide compounds and peroxide radicals in the ultrasound field reduced the yield of S-nitrosocompounds. S-Nitrosocompounds were not formed when exposing low-molecular-weight thiols to ultrasound in atmospheres of N₂ or CO. In neutral solutions, ultrasound-exposed cysteine or glutathione released NO due to spontaneous degradation of the S-nitrosocompounds. N₂O₃, produced due to the spontaneous degradation of the S-nitrosocompounds in air, nitrosylated sulfhydryl groups of glutathione manifested in the appearance of new absorption bands at 330 and 540 nm. The nitrogen compounds formed in an ultrasound field modified the sulfhydryl groups of apohemoglobin and serum albumin. The main target for ultrasound-generated oxygen free radicals were cystine residues oxidized to cysteic acid residues.     

Sulfur requirement of Gonium (Volvocaceae)

The sulfur requirement of six strains of three species of Goniumhas been investigated. These strains can grow well with sulfide,sulfite, bisulfite, thiosulfate or sulfate in light and darkness.They are the first algae shown to utilize sulfide as a sulfursource. However, organic sulfur sources (methionine, cystine,cysteine, homocysteine, homocystine and taurine) were ineffectivefor growth of Gonium. (Received December 6, 1975; )     

Evidence for an intracellular sulfur cycle in cucumber leaves

H₂S emission from cucumber (Cucumis sativus L.) leaf discs supplied with L-cysteine in the dark is inhibited 80–90% by aminooxyacetic acid (AOA), an inhibitor of pyridoxal-phosphate dependent enzymes. Exposure to L-cysteine in the light enhanced the emission of H₂S in response to this sulfur source. Turning off the light reduced the emission of H₂S to the rate observed in continuous dark; turning on the light enhanced the emission of H₂S to the rate observed in continuous light. Therefore, in the light H₂S emission in response to L-cysteine becomes a partially light-dependent process. Treatment with cyanazine, an inhibitor of photosynthetic electron transport, reduced H₂S emission in the light to the rate observed in continuous dark, but did not affect H₂S emission in the dark. In leaf discs pre-exposed to L-cysteine in the light, treatment with cyanazine+ AOA inhibited the emission of H₂S in response to L-cysteine completely. Therefore, only part of the H₂S emitted in response to this sulfur source is derived from a light-independent, but pyridoxal-phosphate-dependent process; the balance of the H₂S emitted is derived from a light-dependent process that can be inhibited by cyanazine. When cucumber leaf discs were supplied with a pulse of L-[^35S]cysteine, radioactively labeled H₂S was emitted in two waves, one during the first hour of exposure to L-cysteine, and a second after 3–4 h; unlabeled H₂S, however, was emitted continuously. The second wave of emission of labeled H₂S was not observed in pulse-chase experiments in which sulfate or cyanazine were added to the treatment solution after 3 h of exposure to L-cysteine, or when the lights was turned off. The labeling pattern of sulfur compounds inside cucumber cells supplied with a pulse of L-[³⁵S]cysteine showed that the labeled H₂S released from L-cysteine partially enters first the sulfite, then the sulfate pool of the cells. The radioactively labeled sulfate, however, is not incorporated into L-cysteine, but enters the H₂S pool of the cells again. These observations are consistent with the idea of an intracellular sulfur cycle in plant cells. The L-cysteine taken up by the leaf discs seems to be desulfhydrated in a light-independent, but pyridoxal-phosphate-dependent process. The H₂S synthesized this way may be partially released into the atmosphere; the other part of the H₂S produced in response to L-cysteine may be oxidized to sulfite, then to sulfate, which is subsequently reduced via the light-depent sulfate assimilation pathway. In the presence of excess L-cysteine, synthesis of additional cysteine may be inhibited, and the sulfide moiety may be split off carrier bound sulfide to enter the H₂S pool of the cells again. It is suggested that the function of this sulfur cycle may be regulation of the free cysteine pool.Abbreviation AOA aminooxyacetic acid     

Organic sulphur sources for the growth of the dermatophyte microsporum gypseum

The suitability of 30 organic compounds (of them 19 sulphur-containing amino acids) at a concentration of 1mm as sulphur sources for the growth of the dermatophyteMicrosporum gypseum was investigated. The dry mass of the mycelium after an 11-d growth served as a measure of utilizability. Of sulphur amino acids cystine, cysteine, reduced and oxidized glutathione, cysteic and cysteinesulphinic acids, S-sulphocysteine, lanthionine, taurine and serine sulphate were the best sources. Methionine and methionine-sulphone were utilized slightly less effectively. Other compounds were medium to poor sources and only S-carboxymethylcysteine was not utilized at all. All organic compounds that are not of amino acid type were poor sulphur sources or were utilized at all. Sodium dodecyl sulphate inhibited germination and growth completely.     

Adenosine 5'-Phosphosulf ate Sulfotransferase from Euglena: Enzyme-Bound Intermediates

Adenosine 5'-phosphosulfate sulfotransferase (APSST) purifiedfrom Euglena gracilis Klebs var. bacillaris mutant W₁₀BSmL byammonium sulfate precipitation, Sephadex G-100 gel filtration,reactive blue agarose, reactive dye agarose and DEAE-cellulosecan be labeled by incubation with AP³⁵S and separated from smallradioactive compounds on Sephadex G-50. Most of the label isnot exchangeable with nonradioactive APS and therefore is notassociated with bound substrate. On non-inactivating SDS-PAGE,a radioactive band at the position of native APSST tetramershows APSST activity (measured as acid-volatile radioactivity).Labeled protein hydrolyzed with Pronase yields radioactive S-sulfocysteine,indicating that at least one cysteine residue of APSST acceptsa sulfo group from APS to form E-S-SO₃^–. A labeled lowmolecular weight compound can be separated from the proteinby paper electrophoresis or by treatment with acidic proteindenaturing reagents such as trifluoroacetic acid (TFA) or trichloroaceticacid (TCA). This labeled compound (perhaps the sulfo-carrier)behaves as a strong acid on paper electrophoresis and is stabilizedby iodoacetamide or acidic conditions but degrades to thiosulfate,sulfate and other compounds as the pH is raised. The radioactivityin APSST is exchangeable with sulfite or thiosulfate. AMP inhibitsAPSST in the formation of acid-volatile radioactivity by competingwith APS, but APA inhibits APSST activity uncompetitively. AK_m of 0.1 µM for APS and K_i of 0.1 mM for AMP and 0.6mM for APA are obtained when a saturating amount of dithiothreitol(DTT) is used as the thiol. A mechanism is proposed for theinitial reaction(s) catalyzed by APSST. ¹Present address: Boyce Thompson Institute for Plant Research,Tower Road, Ithaca, NY 14853, U.S.A.     

Comparative aspects of utilization of sulfonate and other sulfur sources by Escherichia coli K12

Selected biochemical features of sulfonate assimilation in Escherichia coli K-12 were studied in detail. Competition between sulfonate-sulfur and sulfur sources with different oxidation states, such as cysteine, sulfite and sulfate, was examined. The ability of the enzyme sulfite reductase to attack the C-S linkage of sulfonates was directly examined. Intact cells formed sulfite from sulfonate-sulfur. In cysteine-grown cells, when cysteine was present with either cysteate or sulfate, assimilation of both of the more oxidized sulfur sources was substantially inhibited. In contrast, none of three sulfonates had a competitive effect on sulfate assimilation. In studies of competition between different sulfonates, the presence of taurine resulted in a decrease in cysteate uptake by one-half, while in the presence of isethionate, cysteate uptake was almost completely inhibited. In sulfite-grown cells, sulfonates had no competitive effect on sulfite utilization. An E. coli mutant lacking sulfite reductase and unable to utilize isethionate as the sole source of sulfur formed significant amounts of sulfite from isethionate. In cell extracts, sulfite reductase itself did not utilize sulfonate-sulfur as an electron acceptor. These findings indicate that sulfonate utilization may share some intermediates (e.g. sulfite) and regulatory features (repression by cysteine) of the assimilatory sulfate reductive pathway, but sulfonates do not exert regulatory effects on sulfate utilization. Other results suggest that unrecognized aspects of sulfonate metabolism, such as specific transport mechanisms for sulfonates and different regulatory features, may exist.     

Increase in tissue cysteine level and excretion of sulfate and taurine after intragastric administration ofl-2-oxothiazolidine-4-carboxylate in rats

Summary Five mmol ofl-2-oxothiazolidine-4-carboxylate (OTC)/kg of body weight was administered into the stomach of rats, and cysteine levels in tissues and sulfate and taurine excreted in the urine were determined. The cysteine (plus cystine expressed as cysteine) concentration in the liver increased to 170–200% of the original level at 30 min and that in the blood to 160% at 60 min after the OTC administration. These high levels were maintained until 8 h after the administration and decreased gradually thereafter. Excretion of sulfate and taurine increased after the OTC administration and the increase corresponded to 26% and 15%, respectively, of the OTC administered. These findings suggest that at least about 40% of the OTC administered into the stomach was taken up and converted to cysteine, which was metabolized to sulfate and taurine.     

The role of the Golgi complex in sulfate metabolism

This investigation was designed to determine if sulfate metabolism is the function of a particular cell organelle, or whether the site of sulfation varies, depending upon the type of cell and the class of sulfated compound. Rats and mice were injected intravenously with inorganic sulfate labeled with ³⁵S (H₂ ³⁵SO₄), and were then killed by vascular perfusion of fixative 5–30 min later. Several tissues were prepared for electron microscope autoradiography. 14 different types of specialized cells which incorporated the labeled sulfate were analyzed. In every case, the sulfate was initially detected in the smooth membranes and vesicles of the Golgi complex. Available evidence indicates that these cells were engaged in the synthesis of several different sulfated compounds, including mucopolysaccharides, glycoproteins, lipids, and steroids. These results lead to the generalization that the enzymes required for the transfer of inorganic sulfate to a variety of acceptor molecules are located in the Golgi complex.     

Observations on the fate of cystathionine in rat brain.

Direct intracerebral administration of ³⁵S-cystathionine has been shown to be capable of labelling the normal pools of cystathionine in rat brain tissue and thus capable of yielding useful information about the metabolic fate of the amino acid. Cystathionine was shown to be metabolised slowly to yield inorganic SO₄^2?, taurine and cystine. Sub-cellular fractionation studies concerned with the distribution of ³⁵S-cystathionine coupled with similar investigations involving enzymes of the transsulphuration pathway indicate that cystathionine fulfill some of the requirements of a neurotransmitter substance.     

STUDIES ON THE PATHWAY OF SULFIDE PRODUCTION IN A COPPER-ADAPTED YEAST

Metabolism of some sulfur-containing substances was studiedin a copper-resistant strain of yeast (R), its parent strain(P) and respiratory-deficient(RD) mutants from them. The resultsobtained are as follows:

1. Using sulfate, sulfite and thiosulfate as sulfur sources, Rproducedmore H₂S than P, and both of these had the activityhigher than their RD mutants. All of them produced a large amountof H₂S from cysteine, but only little from methionine, cysteinesulfinic acid and S-sulfocysteine.
2. From sulfite and thiosulfate,P and R produced more H₂S inaerobicthan in anaerobic condition.With sulfate and cysteine, however,H₂S production did not differunder those conditions.
3. In both P and R, the sulfate-to-sulfiteand sulfite-to-sulfidereactions were remarkably lowered byiron and zinc deficiencies.But the cysteine-to-sulfide reactionwas not affected by themetal-deficiencies.
4. H₂S productionfrom sulfate was remarkably depressed by highconcentrationsof pantothenate.
5. Rates of reaction steps on a plausible pathway from sulfatetosulfide and to organic sulfur compounds areestimated forthe strainsused. R is characterized by its largecapacity ofthe reaction step from sulfate to sulfite, and excessivesulfitethus formed is liberatedas sulfide not by the way ofcysteine.

¹Present address: Research Reactor Institute, Kyoto University,Kumatori-cho, Sennan-gun, Osaka     

Tissue and regional distribution of cysteic acid decarboxylase

A sensitive and rapid assay method for cysteic acid decarboxylase was developed which combined the selectivity of ion exchange resin (a complete retention of the substrate, cysteic acid, and exclusion of the product, taurine) with the speed of a vacuum filtration. The synthesis and purification of³⁵S-labeled cysteic acid were described. The validity of the assay was established by the identification of the reaction product as taurine. With this new method, the decarboxylase activity was measured in discrete regions of bovine brain. Putamen had the highest activity, 172 pmol taurine formed/min/mg protein (100%), followed by caudate nucleus, 90%; cerebral cortex, 82%; hypothalamus, 81%; cerebellar cortex, 79%; cerebellar peduncle, 59%; thalamus, 42%; brain stem, 25%; pons, 10%; and corpus callosum, 3%. The decarboxylase activity in various mouse tissues was also determined as follows: liver, 403; brain, 145; kidney, 143; spinal cord, 59; lung, 21; and spleen, 10 pmol taurine formed/min/mg. No activity could be detected in skeleton muscle and heart, suggesting a different biosynthetic pathway for taurine synthesis in these tissues. The advantages and disadvantages of the new assay method are also discussed.This work was supported in part by grant NS 13224 from the National Institutes of Health, U.S.A., and grant from Huntington's Chorea Foundation in memory of Mrs. Ruth Berman.This work was presented at the Eighth Annual Meeting of American Society for Neurochemistry in Denver, Colorado, March 1977.     

Multiple Forms of Inorganic Pyrophosphatase from the Leaf of Amarantus blitum L.

The existence of a carrier-bound pathway for inorganic sulfate assimilation has been proposed in Chlorella and Escherichia coli. The possibility that the sulfonyl group of active sulfate is transferred to a specific organic acceptor to form thiosulfate ester was examined with Salmonella typhimurium LT-2. Some 11% of the radioactive products from [³⁵S]-3′-phosphoadenosine 5′-phosphosulfate were transferred to high molecular weight compounds, and the remainder of the product is identified as free inorganic sulfite. Apparent thiosulfonate reductase activity was detected in the reaction mixtures containing S-sulfoglutathione and NADPH as conceivable substrates, but not with partially purified sulfite reductase. The former activity was attributable to the nonenzymatic reaction, sulfitolysis. Through these in vitro experiments the existence of the carrier-bound pathway was disproved.