Active center and subunit structure of transaldolase from Candida utilis.

Crystalline transaldolase (type III) isolated from Candida utilis is composed of two identical subunits, as shown by the following lines of evidence. 1. Tryptic digestion of the performic acid oxidized enzyme yields the number of ninhydrin- and arginine-positive peptides expected for identical subunits. 2. All attempts to separate both subunits by molecular weight or charge differences have failed. 3. Cyanogen bromide cleavage and sodium dodecyl sulfate gel electrophoresis of S-carboxymethylated transaldolase revealed four distinct peptides designated C₂ to C₅ according to their decreasing molecular weight and one additional peak, C₁, in low yield, presumably an aggregate or partially degraded peptide.By chromatography on Sephadex G-100 the maleylated cyanogen bromide digest from ¹⁴C-labeled β-giyceryl-transaldolase could be separated into four peptide peaks which have been analyzed for their amino acid composition. The largest peptide C₂ with a molecular weight of 16,800 was identified as the active site containing fragment. The four fragments together account for all amino acid residues in the entire protein.From transaldolase (type I) containing four methionine residues three cyanogen bromide peptides could be identified. By addition of the individual peptides a molecular weight of 37,100 ± 3500 could be calculated, which is half the molecular weight of the native enzyme. From experimental data presented so far both isoenzymes of transaldolase can be regarded as “half-of-the-sites” enzymes.     

Deoxyribose-5-P aldolase: subunit structure and composition of active site lysine region

Deoxyribose-5-P aldolase, a Class I aldolase, from Salmonella typhimurium has a molecular weight of 57,000 and is composed of two subunits of 28,500 molecular weight. Evidence from fingerprint analysis of tryptic digests and carboxypeptidase digestion suggests that the two subunits are identical. The COOH-terminal tyrosine residue which is removed by carboxypeptidase digestion appears to be necessary for catalytic activity but not for substrate binding. A tryptic peptide containing the “active site” lysyl residue modified by acetaldehyde has been purified and the following amino acid composition determined: (Ala₃, Gly₃, Thr₃, Asx₂, Ser, Ile, Phe, 1N-Lys)-Lys.     

Preparation and characterization of calcium-binding and other hydrophobic proteins from synaptic membranes

A number of hydrophobic proteins have been separated and purified to varying degrees from synaptic membranes derived from bovine brain. The proteins, which have been obtained using preparative acrylamide gel electrophoresis, have been analyzed for molecular weight, amino acid composition, peptide mapping, N-terminal amino acids, and for their ability to bind calcium and ATP. A number of the proteins bound calcium, the greatest binding being associated with a component having a molecular weight of 1.5 · 10⁴, a binding capacity of 4 calcium/molecule, and a K_m of 1.5 · 10^?5 M. An acidic tryptic peptide derived from this protein was evidently responsible for the calcium-binding. ATP binding appeared to be confined largely to the higher molecular weight proteins. From the peptide mapping there appears to be a similar acidic component in a number of the proteins exhibiting calcium-binding. ATP-binding was associated mainly with the high molecular weight proteins, particularly those which consisted of numerous basic tryptic peptides.     

Arginine metabolism in developing soybean cotyledons : I. Relationship to nitrogen nutrition

The free and protein amino acid composition of Glycine max (L.) Merrill cotyledons was determined for the entire developmental period using high performance liquid chromatography. Arginine constituted 18% of the total protein nitrogen throughout development, and there was a linear arginine nitrogen accumulation rate of 1212 nanomoles per cotyledon per day between 16 and 58 days after anthesis. Arginine and asparagine were major constituents of the free amino acid pool, constituting 14 to 62% and 2 to 41% of the total free amino acid nitrogen, respectively. The urea cycle intermediates, citrulline, ornithine, and argininosuccinate were also detected in the free pool. A comparison of the amino acid composition of cotyledonary protein and of seedcoat exudate suggested that 72% of the cotyledon's arginine requirement is satisfied by in situ biosynthesis, and that 20% of the transformed nitrogen is incorporated into arginine. Also, [1-¹⁴C]glutamate and [U-¹⁴C]glutamine were fed to excised cotyledons. After 4 hours, ¹⁴C was incorporated into protein and released as ¹⁴CO₂, but none was incorporated into the C-1 and C-6 positions of free and protein arginine, determined using arginine-specific enzyme-linked assays. It is not currently known whether arginine biosynthesis in the cotyledon involves glutamate delivered from the mother plant or glutamate derived in situ.     

Occurrence of hydroxyproline in a toxin from the marine snail Conus geographus

A 22-residue peptide toxin from the venomous marine snail Conus geographus (L.) was found to have a most unusual amino acid composition: Lys₄, Arg₃, $\text{1}\text{2}\text{Cys}{}_6$, Asx₂, Glx₂, Thr, Ala, plus three residues of trans-4-hydroxyproline. Absence of Gly and Pro indicates that the hydroxyproline must be in sequences different from those in which hydroxyproline occurs in collagen and other proteins.     

Comparative specificity of microbial acid proteinases for synthetic peptides. II. Effect of secondary interaction

To investigate the effect of “secondary interaction” on hydrolysis by various acid proteinases from molds and yeasts, synthetic peptides

amino acid residues) were used as substrates. Pepsin was used for the comparative study. These peptides were split at the peptide bonds indicated by the arrows, permitting examination of the effect of residue X distant by two or three amino acid residues from the hydrolytic site in the peptides. According to the system of Schechter and Berger (Biochem. Biophys. Res. Commun. 27; 157, 1967), the amino acid residues in peptide substrates were numbered P₁, P₂, etc. toward the N-terminal direction from the site of hydrolysis, and P₁′, P₂′, etc. toward the C-terminal direction. The results indicated that hydrolysis by these microbial enzymes is affected by at least six amino acid residues (P₁-P₃ and P₁′-P₃′) in peptide substrates, as is seen with pepsin. Elongation of the peptide chain with suitable amino acid residues from P₁ to P₂ or P₃ and from P₁′ to P₂′ or P₃′ in peptide substrates resulted in much or less increase of hydrolysis depending upon the species of the enzyme producers.     

Studies on Bacterial Protease

Some physicochemical properties and amino acid composition of the alkaline protease of B. amylosacchariticus were determined. The molecular weight and sedimentation coefficient were estimated to be 22,700 and 2.89 s, respectively, and the amino terminal amino acid was identified to be alanine. The enzyme contained 15.9% of nitrogen and was composed of 220 residues of amino acid: lys₆, his₅, arg₃, asp₂₀, thr₁₄, ser₃₇, glu₁₂, pro₁₀, gly₂₅ ala₂₇ val₂₀, met₃, isoleu₁₂, leu₁₂, tyr₉, phe₂, try₃ and amide ammonia₁₆ The results indicate that protein nature and chemical properties of the alkaline protease presented here are distinct from those of alkaline proteases obtained from the other strains of B. subtilis, such as subtilopeptidase A, B and BPN’     

Antioxidant Properties of the Peptides Isolated From Ganoderma lucidum Fruiting Body

Ganoderma lucidum is widely used as traditional medicine for centuries particularly in China, Japan and Korea. Many bioactive metabolites isolated from G. lucidum were therapeutically active against various diseases. The peptide isolated from water extract of G. lucidum was purified by employing Sephadex G-25, Sephadex G-50 and reverse phase HPLC column chromatography. The antioxidant property of the peptide fractions was determined by various in vitro methods. All fractions obtained from Sephadex G-25 and fraction G from Sephadex G-50 are effective antioxidants and comparably fraction C has the highest antioxidant activity. The molecular weight of purified peptide determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration chromatography and Matrix-assisted laser desorption ionization time-of-flight-mass spectrometer was found to be 2.8, 3.34 and 3.35?kDa respectively. The amino acid composition of the peptide was rich in phenylalanine, aspartic acid, proline, histidine and isoleucine. Peptide isolated in the present investigation suggests that has beneficial antioxidant properties may be due to its low molecular weight and specific amino acid composition.     

Comparative specificity of microbial acid proteinases for synthetic peptides. 3. Relationship with their trypsinogen activating ability

The specificities of acid proteinases from Aspergillus niger, Aspergillus saitoi, Rhizopus chinensis, Mucor miehei, Rhodotorula glutinis, and Cladosporium sp., and that of swine pepsin, were determined and compared with ability of the enzymes to activate trypsinogen. Various oligopeptides containing l-lysine, Z-Lys-X-Ala, Z-Lys-(Ala)_m, Z-Lys-Leu-(Ala)₂, and Z-(Ala)_n-Lys-(Ala)₃ (X = various amino acid residues, m = 1–4, n = 1–2) were used as substrates. Of the enzymes which are able to activate trypsinogen, most split these peptides at the peptide bond formed by the carbonyl group of l-lysine. For the peptides to be susceptible to the enzymes it was essential that the chain extended for two or three amino acid residues on the C-terminal side of the catalytic point, and that a bulky or hydrophobic amino acid residue formed the imino-side of the splitting point. The rate of hydrolysis was markedly accelerated by elongation of the peptide chain with l-alanine on the N-terminal side of the catalytic point. Thus, of the substrates used, Z-(Ala)₂-Lys-(Ala)₃ was the most susceptible to the microbial acid proteinases possessing trypsinogen activating ability. On the other hand, M. miehei enzyme and pepsin, which do not activate trypsinogen, showed very little peptidase activity on the peptides.     

Partial structure and properties of the ferredoxin from Rhodymenia palmata

A ferredoxin of MW 11 000 was isolated from the marine alga Rhodymenia palmata (Palmaria palmata). In its oxidised form the ferredoxin had absorption maxima at 276, sh 281, 328, 423 and 465 nm, and contained a single [2Fe-2S] cluster. The midpoint potential of the ferredoxin was ?400 mV and it effectively mediated electron transport in NADP⁺-photoreduction by higher plant chloroplasts, and pyruvate decarboxylation by the phosphoroclastic system of an anacrobic bacterium. The amino acid composition was Lys₃, His₁, Arg₁, Asx₁₂, Thr₉, Ser₈, Glx₁₃, Pro₄, Gly₈, Ala₇, Cys₅, Val₈, Ile₄, Leu₉, Tyr₄, Phe₂; tryptophan and methionine were absent from the molecule. The N-terminal amino acid region consisting of ca half the total amino acid sequence was determined using an automatic sequencer.     

Molecular cloning and biochemical characterization of the first Na-channel α-type toxin peptide (Acra4) from Androctonus crassicauda scorpion venom

Due to the medical importance played in Turkey by stings of the scorpion Androctonus crassicauda, its venom has been studied with more attention. In this communication we report a new toxic peptide, named Acra4, because it is the fourth peptide completely characterized from venom of this scorpion. The peptide contains 64 amino acid residues stabilized by four disulfide bridges, with a molecular weight of 6937 Da. Purification of the lethal peptide was performed by three steps of high performance liquid chromatography (HPLC) separations, and the molecular weight was determined by mass spectrometry analysis and the full amino acid sequence was obtained by direct Edman degradation in conjunction with gene cloning. The LD₅₀ of Acra4 was 50.5 ng/20 g mouse body weight (95% confidence intervals from 48.8 to 52.2 ng/20 g mouse body weight). Additionally, from a sample of cDNA of A. crassicauda four genes were cloned displaying sequence similarities to known scorpion toxins, and are reported here as potentially toxic peptides, named Acra5 to Acra8. Electrophysiological studies of Acra4 were performed using Na⁺-channels expressed in F11 cell culture, by patch-clamp recordings. This is the first time that such peptide from A. crassicauda having a specific Na⁺-channel α-type effect is reported. Its affinity toward Na⁺-channels in F11 cell line is in the order of 1 μM concentration.     

Nitrogen Nutrition and Xylem Sap Composition of Peanut (Arachis hypogaea L. cv Virginia Bunch)

The principal forms of amino nitrogen transported in xylem were studied in nodulated and non-nodulated peanut (Arachis hypogaea L.). In symbiotic plants, asparagine and the nonprotein amino acid, 4-methyleneglutamine, were identified as the major components of xylem exudate collected from root systems decapitated below the lowest nodule or above the nodulated zone. Sap bleeding from detached nodules carried 80% of its nitrogen as asparagine and less than 1% as 4-methyleneglutamine. Pulse-feeding nodulated roots with ¹⁵N₂ gas showed asparagine to be the principal nitrogen product exported from N₂-fixing nodules. Maintaining root systems in an N₂-deficient (argon:oxygen, 80:20, v/v) atmosphere for 3 days greatly depleted asparagine levels in nodules. 4-Methyleneglutamine represented 73% of the total amino nitrogen in the xylem sap of non-nodulated plants grown on nitrogen-free nutrients, but relative levels of this compound decreased and asparagine increased when nitrate was supplied. The presence of 4-methyleneglutamine in xylem exudate did not appear to be associated with either N₂ fixation or nitrate assimilation, and an origin from cotyledon nitrogen was suggested from study of changes in amount of the compound in tissue amino acid pools and in root bleeding xylem sap following germination. Changes in xylem sap composition were studied in nodulated plants receiving a range of levels of ¹⁵N-nitrate, and a ¹⁵N dilution technique was used to determine the proportions of accumulated plant nitrogen derived from N₂ or fed nitrate. The abundance of asparagine in xylem sap and the ratio of asparagine:nitrate fell, while the ratio of nitrate:total amino acid rose as plants derived less of their organic nitrogen from N₂. Assays based on xylem sap composition are suggested as a means of determining the relative extents to which N₂ and nitrate are being used in peanuts.     

Enzymatic peptide synthesis by the recombinant proline-specific endopeptidase from Flavobacterium meningosepticum and its mutationally altered Cys-556 variant

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) was investigated for its potential as a catalyst in peptide synthesis. Using an activated peptide ester or a peptide amide as the acyl component, the enzyme catalyzed kinetically controlled aminolysis and transpeptidation respectively, with various amino acid amides as acyl acceptors. To a certain extent the nucleophile preference reflected the amino acid preference in the S₁-position of the enzyme in peptide hydrolysis: the highest fractions of aminolysis were obtained using amino acid amides with hydrophobic side-chains (e.g. Leu-NH₂, Phe-NH₂). PSE also catalyzed the thermodynamically controlled condensation of short peptides with a free carboxyterminus and various amino acid amides. This enabled us to examine the acceptance of different acyl components in the substrate-binding site of the enzyme with regard to their amino acid composition: In the S₁ position proline was clearly favored, but alanine was also accepted, whereas the S₂ subsite accepted various amino acids rather unspecifically. Since PSE was shown to be extremely sensitive against water-miscible organic solvents, an alternative approach was used to increase yields in enzymatic peptide synthesis: a derivative of PSE in which the catalytic Ser-556 is converted to a Cys was constructed by protein engineering. This mutant (PSE_cys) exhibited a dramatically increased peptide ligase activity in aqueous solution.     

Purification and properties of enolase from swine kidney

The first, enolase (2-phospho-d-glycerate hydrolyase, EC 4.2.1.11) to be isolated from a gluconeogenic tissue, swine kidney, was purified more than 600-fold to near homogeneity, as estimated from sedimentation equilibrium and velocity measurements and from disc electrophoresis patterns. The physical properties of the enzyme were examined. Purified kidney enolase has a s^0.87%_20,w = 5.87 S, M_r = 90,000 ± 4,500, e^0.1%_280,1cm = 1.07/mg/ml, a Stokes radius of 37.0 Å, and an apparent subunit molecular weight of 52,000.The amino acid composition was determined and compared with those of mammalian muscle enolases. The partial specific volume calculated from the amino acid composition was found to be 0.728 cc/g. Swine kidney enolase had 12 cysteines per mole; in the native enzyme, two reacted with DTNB.The enzyme was stabilized by magnesium, sucrose, or glycerol; activity lost, on prolonged storage could be completely recovered by treatment with mercaptoethanol and EDTA at 37 °C. Some evidence was obtained for the existence of active monomers of this enzyme. This form of swine kidney enolase was quite unstable, however. The pH optimum was at 6.8. The Michaelis constants for 2-phospho-d-glycerate and phosphoenolpyruvate were 5.10^?5m and 10^?4m; that for magnesium was 4.10^?4m. Substrate inhibition was found for 2-phosphoglycerate but not for phosphoenolpyruvate. No inhibition is seen under comparable conditions with mammalian enolases from glycolytic tissues. This finding is discussed.     

Optimization of antibiotic production by the cyanobacteriumScytonema sp. TISTR 8208 immobilized on polyurethane foam

The cyanobacteriumScytonema sp. TISTR 8208 was cultivated under illumination with a polyurethane foam sheet as the biomass-supporting material. The cyanobacterium produced an antibiotic with a broad spectrum in the post-exponential phase of growth. Modification of the composition of the BGA medium by adding 1.5 g 1^?1 NaNO₃, increasing the Fe₂(SO₄)₃.6H₂O concentration to 0.025 g 1^?1 not adding NaCl, using an initial pH of 7.0, incubated at 35 °C, and at the light intensity of 90 µmol photon m^?2 s^?1, enabled a 28-fold increase in antibiotic production. The antibiotic was stable when treated at 70 °C for 2 h and unstable at 100 °C for 75 min. Since the activity was completely lost with protease treatment and the positive result was shown toward ninhydrin test, and the UV and IR spectrum were characterized at 210 nm and 1547 cm^?1, respectively, this antibiotic appears to be a peptide with aromatic amino acid. Analysis by double-focusing mass spectrometry showed the molecular weight of the antibiotic to be 1490.     

Comparative study of various serine alkaline proteinases from microorganisms. Esterase activity against N-acylated peptide ester substrates

Hydrolyses of N-acylated peptide ester substrates by various serine alkaline proteinases from bacterial and mold origin were compared using Ac- or Z-(Ala)_m-X-OMe (m = 0-2 or 0-3; X = phenylalanine, alanine, and lysine) as esterase substrates. The results indicated that the esterase activities of these enzymes were markedly promoted by elongating the peptide chain from P₁ to P₂ or P₃ with alanine, irrespective of the kind of the amino acid residue at the P₁-position (amino acid residues in peptide substrates are numbered according to the system of Schechter and Berger (1)). The effect of the kind of amino acid residue at the P₂-position was further determined using Z-X-Lys-OMe (X = glycine, alanine, leucine, or phenylalanine) as esterase substrates. Alanine was the most efficient residue as X with subtilisins and Streptomyces fradiae Ib enzyme, while leucine or phenylalanine were most efficient with the enzymes from Streptomyces fradiae II, Aspergillus sojae, and Aspergillus melleus. All the serine alkaline proteinases tested in this study were sensitive to Z-Ala-Gly-PheCH₂Cl, the dependence of inhibition on the inhibitor concentration differed among the enzymes.     

Trichotoxin A-40, a new membrane-exciting peptide. Part A. Isolation,characterization and conformation

The new polypeptide antibiotic trichotoxin A-40 is isolated by chloroform/methanol extraction from the dry mycelium of Trichoderma viride NRRL 5242. The lipophilic peptide is purified by chromatography on Kieselgel H-60 and reverse-phase chromatography on Lichrosorb RP-8. The new antibiotic differs in amino acid composition and various chemical and physicochemical properties from similar peptides such as trichotoxin A, the suzukacillins or alamethicins. The amino acid composition is (Pro)₁ (Gly)₁ (Ala)₂ (Leu)₂ (Aib)₁₀ (Glx)₂. (Aib, α-aminoisobutyric acid.) The antibiotic has a carboxyl group which can be esterified by diazomethane, which results in slightly enhanced membrane-modifying activities.The peptide exhibits a right-handed α-helical conformation increasing about two-fold from aqueous to lipophilic media as shown by solvent-dependent circular dichroism measurements. Most of the ¹³C-NMR resonances can be assigned unequivocally and amino acids situated in the α-helical part show characteristic shift differences from those in the non-helical regions. No β-phenylalaninol residue could be identified by ¹³C-NMR and ultraviolet spectroscopy, as can be for alamethicins and suzukacillins. A pronounced hemolytic action is found on human erythrocytes, which develops at micromolar concentrations. Trichotoxin A-40 induces a voltage-dependent ionic conductance in bilayer lipid membranes and it can serve as a new pore-forming model system for structure/activity studies in membrane excitation by peptides.     

Indentification of two somatomedin A active polypeptides and in vivo effects of a somatomedin A concentrate

The first chemical characterization of two polypeptides from human serum which stimulate the $\text{in vitro}$ incorporation of ³⁵S sulfate into chick cartilage is described. These two polypeptides, designated Somatomedin A₁ and A₂ have a molecular weight of approximately 7000. Although each peptide contains 1 cysteine residue and has asparagine as amino terminal residue, there are apparent differences in the amino acid composition. Administration of a Somatomedin A concentrate to hypophysectomized rats gave an increase in tibial width similar to that obtained with 20 μg human growth hormone.     

Light,temperature and nitrogen starvation effects on the total lipid and fatty acid content and composition ofSpirulina platensis UTEX 1928

The total lipid and fatty acid content ofSpirulina platensis UTEX 1928 was 7.2 and 2.2% respectively of cellular dry weight under controlled conditions supporting high growth rates. With increases in irradiance from 170 to 870 μmol photon m^?2 s^?1, growth rate increased, total lipid decreased, and fatty acid composition was unaffected. At 1411 μmol photon m^?2 s^?1, total lipid increased slightly and percent composition of the fatty acid gamma linolenic acid increased. Growth and total lipid content ofS. platensis were affected by changes in growth temperature from 25 to 38 °C. With increased growth rate, total lipid content increased. This suggests that the storage of carbon increases at temperatures supporting high growth rates. The degree of saturation increased with temperature. Although the percent composition of gamma linolenic acid was higher at lower growth temperature, production was still primarily a function of growth rate. The effect of temperature on fatty acid content and degree of saturation was of secondary importance. Nitrogen starvation increased total lipid content but decreased fatty acid content as a percentage of dry weight; composition of the fatty acids was unaffected. N-starvation appeared to suspend synthesis of long chain fatty acids inS. platensis, suggesting that some other compound stores fixed carbon when nitrogen is limiting. It was concluded that fatty acid production inS. platensis is maximized by optimizing culture conditions for growth.     

Nonprotein amino acid composition of flatpea (Lathyrus sylvestris L.) as affected by ethephon seed treatments and seedling fertilization

Use of flatpea (Lathyrus sylvestris L.) as a forage is limited because of nonuniform seed germination and the potentially toxic effects of 2,4-diaminobutyric acid (A₂bu), a nonprotein amino acid found in seeds and vegetative tissues. The effects of ethephon (2-chloroethyl phosphonic acid) on seed germination, amino acid leachates of seeds, and amino acid composition (particularly A₂bu) of seedlings were investigated. Germination of flatpea seeds, imbibed for 16 h in 0, 100, 200, 400, 800, and 1600 mg/L ethephon, did not differ, but amino acid leachates tended to increase up to 200 mg/L ethephon and then decline at higher concentrations. The major amino acid constituents in leachates were A₂bu, 4-aminobutyric acid (Abu), and homoserine (Hse). Dry matter accumulation of seedlings grown from ethephon-treated seeds was reduced for second cuttings grown from ethephon-treated seeds and high nitrogen grown plants. During regrowth, free amino acid accumulation was most pronounced in leaves of plants supplied with high nitrogen. The most abundant free amino acids in flatpea tissues were the same as those in seed leachates, but concentration and relative abundance varied with nitrogen level, plant part, and ethephon treatment. Results suggest that ethephon seed treatments can have persistent effects on the growth and amino acid composition of flatpea seedlings grown under different nitrogen regimes.