STUDIES ON SULFHYDRYL GROUPS DURING CELL DIVISION OF SEA URCHIN EGG : III. —SH Groups of KCl-Soluble Proteins and Their Change during Cleavage

Sea urchin egg proteins extracted with KCl are mostly TCA-soluble and, conversely, those extracted with TCA are KCl-soluble. Both groups are water-insoluble and show fluctuations in—SH content during the division cycle. The fluctuation of the—SH groups of the KCl-soluble protein of the whole egg is due to a —SH—S—S— interchange within the freely reacting groups and not within the sluggish and masked —SH groups of the protein. The —SH content of the KCl-soluble protein of the egg cortex also fluctuates in a similar way.     

THE REACTIONS OF IODINE AND IODOACETAMIDE WITH NATIVE EGG ALBUMIN

The following experimental results have been obtained. 1. Native egg albumin treated with iodine and then denatured no longer gives a nitroprusside test or reduces dilute ferricyanide in neutral Duponol PC solution. 2. More iodine is needed to abolish the ferricyanide reduction if the reaction between native egg albumin and iodine is carried out at pH 6.8 than if the reaction is carried out at pH 3.2. At pH 6.8 iodine reacts with tyrosine as well as with cysteine. 3. Cysteine and tryptophane are the only amino acids with reducing groups which are known to react with dilute iodine at pH 3.2 The reducing power of cysteine is abolished by the reaction with iodine, whereas the reducing power of tryptophane remains intact. Pepsin and chymotrypsinogen which contain tryptophane but not cysteine, do not react at all with dilute iodine at pH 3.2. 4. Native egg albumin treated with iodoacetamide at pH 9.0 and then denatured by Duponol PC reduces only 60 per cent as much dilute ferricyanide as egg albumin which has not been treated with iodoacetamide. 5. The SH group is the only protein reducing group which is known to react with iodoacetamide. The simplest explanation of the new observation that the SH groups of egg albumin can be modified by reactions with the native form of the protein is that the native egg albumin has free and accessible but relatively unreactive SH groups which can react with iodine and iodoacetamide despite the fact that they do not react with ferricyanide, porphyrindin, or nitroprusside. Preliminary experiments suggested by the results with egg albumin indicate that the tobacco mosaic virus is modified by iodine at pH 2.8 without being inactivated and that the tobacco mosaic and rabbit papilloma viruses are not inactivated by iodoacetamide at pH 8.0.     

Dimethylsulfoniopropionate and Methanethiol Are Important Precursors of Methionine and Protein-Sulfur in Marine Bacterioplankton

Organic sulfur compounds are present in all aquatic systems, but their use as sources of sulfur for bacteria is generally not considered important because of the high sulfate concentrations in natural waters. This study investigated whether dimethylsulfoniopropionate (DMSP), an algal osmolyte that is abundant and rapidly cycled in seawater, is used as a source of sulfur by bacterioplankton. Natural populations of bacterioplankton from subtropical and temperate marine waters rapidly incorporated 15 to 40% of the sulfur from tracer-level additions of [³⁵S]DMSP into a macromolecule fraction. Tests with proteinase K and chloramphenicol showed that the sulfur from DMSP was incorporated into proteins, and analysis of protein hydrolysis products by high-pressure liquid chromatography showed that methionine was the major labeled amino acid produced from [³⁵S]DMSP. Bacterial strains isolated from coastal seawater and belonging to the α-subdivision of the division Proteobacteria incorporated DMSP sulfur into protein only if they were capable of degrading DMSP to methanethiol (MeSH), whereas MeSH was rapidly incorporated into macromolecules by all tested strains and by natural bacterioplankton. These findings indicate that the demethylation/demethiolation pathway of DMSP degradation is important for sulfur assimilation and that MeSH is a key intermediate in the pathway leading to protein sulfur. Incorporation of sulfur from DMSP and MeSH by natural populations was inhibited by nanomolar levels of other reduced sulfur compounds including sulfide, methionine, homocysteine, cysteine, and cystathionine. In addition, propargylglycine and vinylglycine were potent inhibitors of incorporation of sulfur from DMSP and MeSH, suggesting involvement of the enzyme cystathionine γ-synthetase in sulfur assimilation by natural populations. Experiments with [methyl-³H]MeSH and [³⁵S]MeSH showed that the entire methiol group of MeSH was efficiently incorporated into methionine, a reaction consistent with activity of cystathionine γ-synthetase. Field data from the Gulf of Mexico indicated that natural turnover of DMSP supplied a major fraction of the sulfur required for bacterial growth in surface waters. Our study highlights a remarkable adaptation by marine bacteria: they exploit nanomolar levels of reduced sulfur in apparent preference to sulfate, which is present at 10⁶- to 10⁷-fold higher concentrations.     

Incorporation of cadmium into proteins in a cadmium tolerant fungi

Aspergillus carbonarius and a strain of Penicillium, a cadmium tolerant fungi, are able to metabolize cadmium chloride up to 2% (w/v). Their amino acids analysis on cadmium free and cadmium chloride containing media indicated certain disorders in their metabolic activities. Cystathionine was only detected in both fungi in the presence of cadmium chloride. However, cadmium was incorporated into several types of low and high molecular weight proteins. The amino acids hydrolyzates of a cadmium containing protein are characterized by the presence of high levels of sulfur amino acids; cysteine and methionine. Ethylasparagine was detected in the hydrolyzate of that cadmium containing protein as well.     

The periodate oxidation of amino acids with reference to studies on glycoproteins

1. All α-amino acids are oxidized by periodate, but at different rates. 2. The rates of oxidation of individual α-amino acids vary with pH. In general, oxidation proceeds more rapidly at alkaline pH. 3. Serine, threonine, cysteine, cystine, methionine, proline, hydroxyproline, tryptophan, tyrosine and histidine are rapidly and extensively oxidized by periodate. 4. Cysteine, cystine, methionine, tryptophan, tyrosine and histidine are oxidized by periodate when they are substituted in the carboxyl and amino groups, as in a polypeptide chain.     

Enzyme Regulation in C(4) Photosynthesis : PURIFICATION AND PROPERTIES OF THIOREDOXIN-LINKED NADP-MALATE DEHYDROGENASE FROM CORN LEAVES

NADP-malate dehydrogenase, a light-modulated enzyme of C₄ photosynthesis, was purified to homogeneity from leaves of corn. The pure enzyme was activated by thioredoxin m that was reduced either photochemically (with ferredoxin and ferredoxin-thioredoxin reductase) or chemically (with dithiothreitol). Unactivated corn leaf NADP-malate dehydrogenase had a molecular weight of 50,000 to 60,000 and was chromophorefree. The enzyme appeared to have a high content of serine and glycine and to contain both S—S and SH groups. Consequently, NADP-malate dehydrogenase seems to be capable of undergoing reversible oxidation/reduction during its photoregulation.     

Meat meal in the diet of the early-weaned pig IV. The supplementation of diets with tryptophane,lysine and methionine

Two experiments were conducted with 72 pigs between 28 and 56 days of age to study the effect of tryptophane supplementation on their performance when fed on diets containing wheat and meat meal.In the first experiment, pigs were fed on a basal diet (Diet 1) or on the same diet supplemented with calcium dihydrogen phosphate (Diet 2), bone meal (Diet 3) or bone meal plus tryptophane (Diet 4), all to 3.1% calcium. The weight gains of the pigs (315 g day^?1) fed on Diet 3 were significantly lower than that of the pigs fed on the other three diets (363 g day^?1). The feed conversion ratios showed a similar trend. Diet 3 contained 0.16% tryptophane while the other diets contained 0.18–0.19% tryptophane. The crude protein, lysine and methionine contents of all diets were similar.In the second experiment, a basal diet containing meat meal and bone meal was supplemented with tryptophane, lysine plus methionine or all three amino acids. Feed intake was increased by all amino acid supplements. Weight gains were improved significantly (57%) by the addition of all three amino acids to the diets, but the improvements due to tryptophane alone (28%) or methionine plus lysine (35%) were not significant. Tryptophane supplementation alone or with lysine plus methionine increased the nitrogen retention of the pigs.It was concluded that the requirement for tryptophane of pigs between 28 and 56 days of age was greater than 0.16% of diets containing wheat and meat meal.     

Variations in Sulfhydryl, Disulfide, and Protein Content during Synchronous and Asynchronous Growth of HeLa Cells

The cellular contents of protein-bound and nonprotein sulfhydry (—SH) and disulfide (—SS—) groups were measured in both asynchronous and synchronous HeLa S3 cultures. About 90% of these groups are associated with proteins, the majority in the —SH form. The content of protein-bound groups, and hence the total content of —SH and —SS— groups (28 × 10^-15 moles/cell, or 1.1 × 10^-6 moles/g protein on average), changes in parallel with the protein content (which varies between 2 and 4 × 10^-10 g/cell) as asynchronous populations pass from the lag through the exponential to the stationary phase of growth. The concentration of nonprotein —SH groups, in contrast, increases 10-fold during lag phase and decreases in stationary phase; it follows the protein concentration closely during the exponential phase, at a level of about 2.8 × 10^-15 moles/cell. In synchronous cultures the protein content doubles during the cell cycle, possibly in an exponential fashion. The total —SH and —SS— content also doubles, but the rate of increase appears to fluctuate. The concentrations of the protein-bound groups show 2- to 3-fold fluctuations per unit protein: protein-bound —SH groups and mixed —SS— linkages rise to maxima while protein-bound —SS— groups fall to a minimum at the G₁/S transition, and fluctuations in these groups occur again during G₂. In addition, the protein-bound —SH concentration falls continuously during the S phase. The nonprotein —SH concentration undergoes the largest (relative) fluctuations, dropping from 4 × 10^-15moles/cell in early G₁ to about 0.4 × 10^-15 moles/cell (of standard protein content) at the end of G₁, and then rising to 30 times this value by the end of S.     

Rhipicephalus sanguineus: amino acid composition of egg shell

Acid hydrolysis of the protein fraction of a batch of egg shells of Rhipicephalus sanguineus was followed by determination of the amino acids in the hydrolysate. Using thin layer chromatography, the amino acids—lysine, arginine, aspartic acid, serine, glycine, glutamic acid, alanine, threonine, valine, tyrosine, isoleucine, and leucine were identified. Alkaline hydrolysis of the fraction followed by TLC revealed the presence of tryptophane. Chitin was revealed utilizing a chitosan test.     

Amino-acid synthesis in adult Dacus oleae (Gmelin) (Diptera Tephritidae) determined with [U-14C] glucose

The ability of adult Dacus oleae for amino-acid synthesis from [U-14C] glucose was investigated. The relatively high specific activity radiometric measurements indicated that both sexes were able to synthesize the amino acids: alanine, aspartic acid, cystine, glutamic acid, glycine, hydroxyproline, proline and tyrosine; therefore, these amino acids are considered as nutritionally dispensable for D. oleae. On the other hand, the amino acids: arginine, histidine, leucine, isoleucine, lysine, methionine, phenylalanine, serine, threonine, and valine, showed a very low specific activity and therefore are considered as nutritionally indispensable. It was not possible to conclude about tryptophane, since the acid hydrolysis destroyed this amino acid.     

Cysteine supplementation reverses methionine restriction effects on rat adiposity: significance of stearoyl-coenzyme A desaturase

Stearoyl-CoA desaturase-1 (SCD1) is a key enzyme in fatty acid and energy metabolism, but little is known about its nutritional regulation. Dietary methionine restriction in rats decreases hepatic Scd1 mRNA and protein, increases energy expenditure, and decreases fat-pad mass/body-weight% (FM/BW%). In humans, plasma concentrations of the methionine product, cysteine, are associated with obesity. To determine which consequences of methionine-restriction are mediated by decreased cysteine availability, we monitored obesity-related variables in 4 dietary groups for 12 weeks: control-fed (CF), methionine-restricted (MR), MR supplemented with 0.5% l-cysteine (MR+Cys) and CF+Cys rats. MR lowered weight gain and FM/BW% despite higher food intake/weight than CF, and lowered serum cysteine. Hepatic Scd1 expression was decreased, with decreased serum SCD1 activity indices (calculated from serum fatty acid profile), decreased serum insulin, leptin and triglycerides, and higher adiponectin. Cysteine supplementation (MR+Cys) essentially reversed all these phenotypes and raised serum cysteine but not methionine to CF levels. Adding extra cysteine to control diet (CF+Cys) increased serum taurine but did not affect serum cysteine, lipids, proteins, or total weight gain. FM/BW% and serum leptin were modestly decreased. Our results indicate that anti-obesity effects of MR are caused by low cysteine and that dietary sulfur amino acid composition contributes to SCD1 regulation.     

Generation of a free alpha-amino group by Raney nickel after 2-nitro-5-thiocyanobenzoic acid cleavage at cysteine residues: application to automated sequencing.

The selective reaction of SH containing proteins and peptides with NTCB (2-nitro-5-thiocyanobenzoic acid) has been reported (Degani, Y., & Patchornick, A. (1974) Biochemistry 13, 1; Jacobson, G.A., Schaffer, M.H., Stark, G.R., & Vanaman, T.C. (1973) J. Biol. Chem. 248, 6583). With this reagent, cysteinyl peptide bonds are selectively cyanylated and subsequently cleaved under alkaline conditions. In the present study we have successfully cleaved the beta-chains of guinea pig hemoglobin at the single cysteine and the peptides thus obtained were separated. However, the C-terminal peptide was blocked at its N terminal by a thiazolidine ring and hence could not be used for Edman degradation sequence analysis. Deblocking of this peptide was successfully done by Raney nickel in the buffer medium of pH 7.0, and also in water, at 50 degrees C for 6 to 10 h. The Raney nickel reagent is used in large excess by weight (at least ten times the weight of sulfur compound) over the compound to be desulfurized. Under these conditions, control experiments on cysteine, methionine, and some other amino acids showed that only the sulfur containing amino acids are degraded by Ni(H). Cysteine and methionine were rapidly converted to alanine and beta-aminobutyric acid, respectively. Gel electrophoresis of the iminothiazolidine peptide after exposure to Ni(H) showed no breakage of the chain.     

THE REDUCING GROUPS OF PROTEINS

1. Intact, unhydrolyzed proteins possess in addition to SH groups other reducing groups which can be oxidized by ferricyanide. 2. The activity of these reducing groups, like that of SH groups, is enhanced by denaturation of the protein and by increase of pH and temperature. 3. These groups differ from SH groups in the manner in which their activity is dependent on concentration of ferricyanide and time of contact with ferricyanide. 4. The activity of these groups is increased if protein SH groups are present. 5. The number and activity of these groups varies from protein to protein. 6. These groups are probably contained in the tyrosine and tryptophane components of proteins. 7. The significance of these reducing groups for an understanding of protein denaturation and the reducing properties of tissues is indicated.     

Probing weakly polar interactions in cytochrome c.

Theoretical, statistical, and model studies suggest that proteins are stabilized by weakly polar attractions between sulfur atoms and properly oriented aromatic rings. The two sulfur-containing amino acids, methionine and cysteine, occur frequently among functional alleles in random mutant libraries of Saccharomyces cerevisiae iso-1-cytochrome c genes at positions that form a weakly polar aromatic-aromatic interaction, the wild-type protein. To determine if a weakly polar sulfur-aromatic interaction replaced the aromatic-aromatic interaction, the structure and stability of two variants were examined. Phenylalanine 10, which interacts with tyrosine 97, was replaced by methionine and cysteine. The cysteine was modified to form the methionine and cysteine analog, S-methyl cysteine (CysSMe). Proton NMR studies indicate that changing Phe 10 to Met or CysSMe affects only local structure and that the structures of sulfur-containing variants are nearly identical. Analysis of chemical shifts and nuclear Overhauser effect data indicates that both sulfur-containing side chains are in position to form a weakly polar interaction with Tyr 97. The F10M and F10CSMe variants are 2-3 kcal mol-1 less stable than iso-1-cytochrome c at 300 K. Comparison of the stabilities of the F10M and F10CSMe variants allows evaluation of the potential weakly polar interaction between the additional sulfur atom of F10CSMe and the aromatic moiety of Tyr 97. The F10CSMe;C102T variant is 0.7 +/- 0.3 kcal mol-1 more stable than the F10M;C102T protein. The increased stability is explained by the difference in hydrophobicity of the sulfur-containing side chains. We conclude that any weakly polar interaction between the additional sulfur and the aromatic ring is too weak to detect or is masked by destabilizing contributions to the free energy of denaturation.     

Engineering of cysteine and methionine biosynthesis in potato

Summary. Methionine and cysteine, two amino acids containing reduced sulfur, are not only an important substrate of protein biosynthesis but are also precursors of various other metabolites such as glutathione, phytochelatines, S-adenosylmethionine, ethylene, polyamines, biotin, and are involved as methyl group donor in numerous cellular processes. While methionine is an essential amino acid due to an inability of monogastric animals and human beings to synthesise this metabolite, animals are still able to convert methionine consumed with their diet into cysteine. Thus, a balanced diet containing both amino acids is necessary to provide a nutritionally favourable food or feed source. Because the concentrations of methionine and cysteine are often low in edible plant sources, e.g. potato, considerable efforts in plant breeding and research have been and are still performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their synthesis, transport, and accumulation in plants. During the last decade molecular tools have enabled the isolation of most of the genes involved in cysteine and methionine biosynthesis, and the efficient plant transformation technology has allowed the creation of transgenic plants that are altered in the activity of individual genes. The physiological analysis of these transgenic plants has contributed considerably to our current understanding of how amino acids are synthesised. We focused our analysis on potato (Solanum tuberosum cv. Désirée) as this plant provides a clear separation of source and sink tissues and, for applied purposes, already constitutes a crop plant. From the data presented here and in previous work we conclude that threonine synthase and not cystathionine gamma-synthase as expected from studies of Arabidopsis constitutes the main regulatory control point of methionine synthesis in potato. This article aims to cover the current knowledge in the area of molecular genetics of sulfur-containing amino acid biosynthesis and will provide new data for methionine biosynthesis in solanaceous plants such as potato. Received December 19, 2001 Accepted January 7, 2002     

SULFHYDRYL AND DISULFIDE GROUPS OF PROTEINS : I. METHODS OF ESTIMATION

1. Methods have been described for reducing protein S-S groups, for oxidizing protein SH groups, and for estimating protein S-S and SH groups. 2. It has been found necessary in estimating the cystine content of proteins by the Folin-Marenzi method to take into account any cysteine that may be present. 3. A method for estimating the cysteine content of proteins has been described. 4. With these methods, estimations have been made of the S-S and SH groups and of the cystine and cysteine contents of a number of proteins. 5. In a denatured, but unhydrolyzed protein, the number of S-S and SH groups is equivalent to the quantity of cystine and cysteine found in the protein after hydrolysis.     

Amino acid composition and chemical functional group modification of protease (TI-Ajl) inhibitor from Actinomyces janthinus 118]

The amino acid composition of the protease (TI-Ajl) inhibitor from Actinomyces janthinus 118 has been determined. It was shown that the TI-Ajl, S-SI and plasminostreptin inhibitors of trypsin and subtilisin have a number of common features: number of double bonds, tryptophane and tyrosine residues, prevalence of the acidic amino acids over the basic ones, ets. The effect of chemical modification of amino groups, arginine, tyrosine and methionine residues on the inhibitory activity of TI-Ajl was studied. The data obtained are indicative of the presence of two active centers of the inhibitor. The antitrypsin center contains a lysine residue.     

Cysteine restriction‐specific effects of sulfur amino acid restriction on lipid metabolism

Decreasing the dietary intake of methionine exerts robust anti‐adiposity effects in rodents but modest effects in humans. Since cysteine can be synthesized from methionine, animal diets are formulated by decreasing methionine and eliminating cysteine. Such diets exert both methionine restriction (MR) and cysteine restriction (CR), that is, sulfur amino acid restriction (SAAR). Contrarily, SAAR diets formulated for human consumption included cysteine, and thus might have exerted only MR. Epidemiological studies positively correlate body adiposity with plasma cysteine but not methionine, suggesting that CR, but not MR, is responsible for the anti‐adiposity effects of SAAR. Whether this is true, and, if so, the underlying mechanisms are unknown. Using methionine‐ and cysteine‐titrated diets, we demonstrate that the anti‐adiposity effects of SAAR are due to CR. Data indicate that CR increases serinogenesis (serine biosynthesis from non‐glucose substrates) by diverting substrates from glyceroneogenesis, which is essential for fatty acid reesterification and triglyceride synthesis. Molecular data suggest that CR depletes hepatic glutathione and induces Nrf2 and its downstream targets Phgdh (the serine biosynthetic enzyme) and Pepck‐M. In mice, the magnitude of SAAR‐induced changes in molecular markers depended on dietary fat concentration (60% fat >10% fat), sex (males > females), and age‐at‐onset (young > adult). Our findings are translationally relevant as we found negative and positive correlations of plasma serine and cysteine, respectively, with triglycerides and metabolic syndrome criteria in a cross‐sectional epidemiological study. Controlled feeding of low‐SAA, high‐polyunsaturated fatty acid diets increased plasma serine in humans. Serinogenesis might be a target for treating hypertriglyceridemia.     

Studies of Sulfate Utilization by Algae. 7. In vivo Metabolism of Thiosulfate by Chlorella

Chlorella pyrenoidosa Chick (Emerson strain 3) utilizes thiosulfate for growth as effectively as sulfate, and more effectively than a variety of organic sulfur compounds containing sulfur in various oxidation states. Thiosulfates, differentially labeled with ³⁵S in either the SH— or SO₃ — sulfur moieties, were used to follow the incorporation of thiosulfate-sulfur into constituents of the insoluble fraction and of the soluble pools. Labeled sulfate was also used for purposes of comparison. Label from both sulfur atoms of thiosulfate and from sulfate is incorporated into the cysteine, homocysteine, and glutathione of the soluble pools, and into the methionine and cystine of protein in the insoluble fraction. Label from SO₃-sulfur of thiosulfate is incorporated more slowly into protein methionine and cystine than label from the SH-sulfur. Moreover, the SO₃-sulfur of thiosulfate is recovered largely as sulfate in both the soluble pools and the insoluble fraction, while only a trace of SH-sulfur is recovered as sulfate in either case. Consistent with this, the metabolism of the SO₃-sulfur of thiosulfate more closely resembles the metabolism of sulfate. Thus it would appear that exogenous thiosulfate undergoes early dismutation in which the SO₃-sulfur is preferentially oxidized, and the SH-sulfur is preferentially incorporated in a reduced state. These results are discussed in relation to the conversion of sulfate to thiosulfate by cell-free extracts of Chlorella previously described.     

Spectrophotometric measurement of the liberation of the alpha-amino group of cysteine residues in polypeptides

1. The reaction between β-bromopyruvic acid and SH groups of cysteine residues in reduced ribonuclease and in some other polypeptides was investigated. 2. One molecule of the acid was found to be necessary to block one SH group in reduced ribonuclease. The stoicheiometry of the interaction and the spectral characteristics of the compound formed suggested that the product is and S-oxalomethyl (R·S·CH₂·CO·CO₂H) derivative of reduced ribonuclease. 3. Digestion of reduced S-oxalomethylated ribonuclease by trypsin or chymotrypsin induced changes in the spectrum that could be attributed to the liberation of the α-amino group of S-oxalomethylated cysteine residues from peptide bonds. The spectral changes that accompanied the hydrolysis of specific peptide bonds in reduced S-oxalomethylated ribonuclease and S-oxalomethylated co-poly(l-Lys,l-CySH) allowed the kinetics of the digestion to be followed. 4. Possible applications of the spectrophotometric method in the study of protein structure are discussed.