Comparison of denaturation of tryptophan synthase α-subunits from Escherichia coli,Salmonella typhimurium, and an interspecies hybrid

Guanidine hydrochloride-induced denaturation and thermal denaturation of three kinds of tryptophan synthase α subunit have been compared by circular dichroism measurements. The three α subunits are from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid in which the C-terminal domain comes from E. coli (α-2 domain) and the N-terminal domain comes from S. typhimurium (α-1 domain). Analysis of denaturation by guanidine hydrochloride at 25 °C showed that the α-2 domain of S. typhimurium was more stable than the α-2 domain of E. coli, but the α-1 domain of S. typhimurium was less stable than the α-1 domain of the E. coli protein; overall, the hybrid protein was slightly less stable than the two original proteins. It is concluded that the stability to guanidine hydrochloride denaturation of each of the domains of the interspecies hybrid is similar to the stability of the domain of the species from which it originated. The E. coli protein was more stable to thermal denaturation than the other proteins near the denaturation temperature, but the order of their thermal stability was reversed at 25 °C and coincided with that obtained from guanidine hydrochloride-induced denaturation.     

Structure—activity relationships in the mutagenicity of quinone methides of 7-hydroxyflavylium salts for Salmonella typhimurium

Several synthetic 7-hydroxyflavylium salts related to apigeninidin, a natural 3-deoxyanthocyanidin, have been studied in the Ames mutagenicity test using strain TA1537 of Salmonella typhimurium. Under the neutral pH conditions of the test, these flavylium salts are deprotonated through ionization of the C7-OH (pK_a′ = 4.2–4.4) to form quinone methides. Only the quinone methides of 4-methyl-7-hydroxyflavylium chloride and 4′-methoxy-4-methyl-7-hydroxy-flavylium chloride showed mutagenicity. Responses of 4–8 times the background were observed at the higher doses (1000 μg/plate), both with and without metabolic activation. It was concluded that the induction of frameshift mutagenicity by this group of compounds is caused by those quinone methides that have non-ionic, stable polycyclic structures at neutral pH.     

Na+-dependent methyl β-thiogalactoside transport in Salmonella typhimurium

We have studied the role of sodium ions in methyl β-thiogalactoside (TMG) transport via the melibiose permease (TMG II) in SalmonellaTMG uptake via TMG Il in anaerobic, starved and metabolically poisoned cells is dependent on an inward-directed Na⁺ gradient.Cells which have been partially depleted of endogenous substrates show H⁺ extrusion upon sodium-stimulated TMG influx.Measurements of the electrochemical H⁺ gradient in cells, starved in different ways for endogenous substrates, suggest that this proton extrusion is probably not linked to the actual translocation mechanism but is the result of metabolism induced by TMG plus Na⁺ uptake.     

Specificity and stereospecificity of α-chymotrypsin

1. The optically pure p-nitrophenyl esters of the d and l enantiomers of N-acetyl-tryptophan, N-acetylphenylalanine and N-acetyl-leucine, and the p-nitrophenyl ester of N-acetylglycine, have been prepared. 2. These materials are all substrates of α-chymotrypsin, and the rates of deacylation of the corresponding acyl-α-chymotrypsins have been determined. 3. As the size of the amino acid side chain increases, the l series deacylate progressively faster than the N-acetylglycyl-enzyme, and the d series progressively more slowly. 4. The results are interpreted in terms of a three-locus model of the enzyme's active site, which accounts for the interrelationship between substrate specificity and stereospecificity observed. 5. The concepts of negative specificity and of specificity saturation are introduced.     

δ-Aminolevulinate dehydratase from Raphanus sativus cotyledons

Phytochrome induces δ-aminolevulinate dehydratase (ALAD) activity in radish seedling cotyledons under continuous far red light. Analysis of the enzymatic activity in etioplasts vs total activity shows a constant ALAD level in these organelles (10 %) in etiolated seedlings. In far red irradiated seedlings, the percentage of enzyme detected into etioplasts increases up to 45 % of the total. Comparative kinetic studies of ALAD activity detected in the cytoplasm and the etioplasts indicate an increase in both compartments with a maximum value reached respectively at 96 and 120 hr from sowing. Treatment with cycloheximide shows a very fast abolition of cytoplasmic ALAD activity which is always correlated to an etioplast decrease with a time shift of ca 24 hr. Erythromycin acts only on the cytoplasmic level of ALAD, and only for far red irradiated seedlings, with an increase of activity twice the level detected in untreated ones. This unexpected effect is discussed.     

Corrinoids from Methanosarcina barkeri: Structure of the α-ligand

5-Hydroxybenzimidazole is the only base detected in cobamide compounds from methanol-grown Methanosarcina barkeri. 5-Hydroxybenzimidazolylcobamide accounted for about 83 and 90% of the total corrinoids of whole cells and cell-free extracts, respectively. Probably, the rest of the corrinoids are base-less.     

The stereospecificity of biosynthesis of squalene and β-amyrin in Pisum sativum

The distribution of deuterium in squalene and β-amyrin, biosynthesized from mevalonic acid-6,6,6-d₃ in Pisum sativum, has been examined b     

Purification and properties of erythro-β-hydroxyasparate dehydratase from Micrococcus denitrificans

1. The novel enzyme, erythro-beta-hydroxyaspartate dehydratase, a key enzyme of the beta-hydroxyaspartate pathway (Kornberg & Morris, 1963, 1965), has been purified 30-fold from extracts of glycollate-grown Micrococcus denitrificans. The purified preparation was devoid of erythro-beta-hydroxyaspartate-aldolase activity, and free from enzymes that act on oxaloacetate. 2. Properties of the purified dehydratase were studied by direct assay of the enzymic formation of oxaloacetate and ammonia from added erythro-beta-hydroxyaspartate. 3. The enzyme was highly substrate-specific, utilizing only the l-isomer of erythro-beta-hydroxyaspartate (K(m), 0.43mm, and V(max.), 99mumoles of oxaloacetate formed/min./mg. of protein at pH9.15 and 30 degrees ). Of many compounds tested, only maleate was a competitive inhibitor (K(i), 32mm at pH7.6). 4. The optimum pH for activity was about 9.5. The K(m) varied with pH, showing a marked optimum at pH7.8. The V(max.) also varied with pH in a manner suggesting the presence in the enzyme-substrate complex of a dissociable group of pK'(a) about 8.5. 5. Carbonyl reagents were inhibitory, but of three thiol reagents tested only p-chloromercuribenzoate was inhibitory. 6. A partially resolved preparation of the enzyme was activated four-fold by the addition of pyridoxal phosphate and thereby restored to half activity. 7. EDTA (0.1mm) was almost completely inhibitory, activity being restored by bivalent cations (Mg(2+), Ca(2+) and Mn(2+)); no activation by univalent cations was observed. 8. The findings are discussed in the light of reported properties of related hydroxyamino acid dehydratases.     

Metabolites of Cercospora. Taiwapyrone,an α-pyrone of unusual structure from Cercospora taiwanensis

(+)-Mellein (1), cis-3S,4S-4-hydroxymellein (3), and taiwapyrone (4), a new α-pyrone, have been isolated from the mycelium of Cercospora taiwanensis, grown on potato-agar. The structure and absolute configuration of (3) and (4) have been elucidated.     

Characteristics of α-glucan phosphorylase from Chlorella vulgaris

α-Glucan phosphorylase from Chlorella vulgaris has been partially purified. In the direction of glucan phosphorolysis the apparent K_m for Pi was ca 2.4 mM at pH 7.1. In the direction of glucan synthesis the K_m for G1P was ca 0.12 mM at pH 6.2. The enzymic activity was inhibited by physiological concentrations of ADP, ATP, ADPG and UDPG. In the direction of starch degradation in the presence of 2.4 mM Pi the I_0.5 values for ADP and ATP were ca 1.6 and 2.9 mM, respectively, while in the direction of synthesis in the presence of 0.12 mM G1P the values were ca 0.23 and 1.4 mM, respectively. The Hill plots for starch degradation showed n values of 2.2 for ADP and 2.2 for ATP and values of 1.5 and 1.2, respectively, for starch synthesis. Both ADPG and UDPG were linear competitive inhibitors either with respect to Pi or with respect to GIP. The K_i values for ADPG and UDPG in the direction of phosphorolysis were shown to be ca 0.11 and 0.51 mM, respectively, and those in the direction of synthesis 0.033 and 0.15 mM, respectively.     

The structure and stereochemistry of lantanilic acid,the β,β-dimethylacryloyl ester of lantaninilic acid,isolated from Lantana camara

The structure of lantanilic acid, a new triterpene isolated from the leaves of Lantana camara, has been determined as the β,β-dimethylacryloyl ester of lantaninilic acid.     

Synthesis of α- and β-N-oxalyl-l-α,β-diaminopropionic acids and their isolation from seeds of Lathyrus sativus

The α- and β-N-oxalyl derivatives of l-α,β-diaminopropionic acid have been chemically synthesized and also isolated from seed extracts of Lathyrus sativus. Chemical and physical properties of the natural and synthetic isomers were in good agreement. The toxicity of the α-isomer to chicks was evaluated and compared with that of the β-isomer.     

Extracellular expression and biochemical characterization of α-cyclodextrin glycosyltransferase from Paenibacillus macerans

The cgt gene encoding α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01 was expressed in Escherichia coli as a C-terminal His-tagged protein. After 90 h of induction, the activity of α-CGTase in the culture medium reached 22.5 U/mL, which was approximately 42-fold higher than that from the parent strain. The recombinant α-CGTase was purified to homogeneity through either nickel affinity chromatography or a combination of ion-exchange and hydrophobic interaction chromatography. Then, the purified enzyme was characterized in detail with respect to its cyclization activity. It is a monomer in solution. Its optimum reaction temperature is 45 °C, and half-lives are approximately 8 h at 40 °C, 1.25 h at 45 °C and 0.5 h at 50 °C. The recombinant α-CGTase has an optimum pH of 5.5 with broad pH stability between pH 6 and 9.5. It is activated by Ca²⁺, Ba²⁺, and Zn²⁺ in a concentration-dependent manner, while it is dramatically inhibited by Hg²⁺. The kinetics of the α-CGTase-catalyzed cyclization reaction could be fairly well described by the Hill equation.     

Synthesis and initial evaluation of novel,non-peptidic antagonists of the αv-integrins αvβ3 and αvβ5

The discovery, synthesis and preliminary SAR of a novel class of non-peptidic antagonists of the α_v-integrins α_vβ₃ and α_vβ₅ is described. High-throughput screening of an extensive series of ECLiPS? compound libraries led to the identification of compound 1 as a dual inhibitor of the α_v-integrins α_vβ₃ and α_vβ₅. Optimization of compound 1 involving, in part, introduction of two novel constraints led to the discovery of compounds 15a and 15b with reduced PSA and much improved potency for both the α_vβ₃ and α_vβ₅ integrins. Compounds 15a and 15b were shown to have promising activity in functional cellular assays and compound 15a also exhibited a promising Caco-2 permeability profile.     

(−)-3β-Acetoxydrimenin from the leaves of Drimys winteri

A new natural product, 3β-acetoxydrimenin was isolated from the petrol extract of the leaves of Drimys winteri which also contains the known compounds safrol, drimenol and polygodial. The structure of the new compound was determined by chemical and spectroscopic methods.     

The specificity of induction of α-galactosidase from Saccharomyces carlsbergenensis

A number of sugars and derivatives have been tested for their ability to induce the synthesis of α-galactosidase from Saccharomyces carlbergensis. Besides galactose and the substrates of the enzyme melibiose, raffinose and stachyose, D-galacturonic acid, L-arabinose, D-tagatose, methyl-α-D-galactoside, lactose and isopropyl-β-D-thiogalactoside were able to act as inducers. Of these, metyl-α-D-galactoside, lactose, isopropyl-β-D-thiogalactoside and L-arabinose have been shown to be gratuitous inducers with which kinetic studies of induction have been carried out. Lactose was the most efficient inducer, giving a maximal differential rate of synthesis of the enzyme of 110 mU/10⁷ cells at a concentration of 190 mM, followed by L-arabinose (60 mU/10⁷ cells at a concentration of 180 mM, followed by L-arabinose (60 mU/10⁷ cells at 40 mM), isopropyl-β-D-thiogalactoside (43 mU/10⁷ cells at 60 mM) and metyl-α-D-galactoside (25 mU/10⁷ cells at 150 mM). The concentration of inducer required to obtain half-maximal induction was similar for lactose, L-arabinose and isopropyl-β-D-thiogalactoside and about 5-fold higher for methyl-α-D-galactoside. The property of the compounds to act as inducers was compared to their ability to interact with the enzyme and the results discussed in terms of the molecular structures which are recognized by the enzyme and by the induction machinery.     

Phospholipase C and sphingomyelinase activities of the Clostridium perfringens α-toxin

α-Toxin is a major pathogenic determinant of Clostridium perfringens, the causative agent of gas gangrene. α-Toxin has been known for long to be a phospholipase C, but up to now its hydrolytic properties have been studied only through indirect methods, e.g. release of cell contents, or under non-physiological conditions, e.g., in micelles, or with soluble substrates. In this report we characterize the phospholipase C and sphingomyelinase activities of α-toxin using a direct assay method (water-soluble phosphorous assay) with phospholipids in bilayer form (large unilamellar vesicles) in the absence of detergents. The simplest bilayer compositions allowing measurable activities under these conditions were DOPC:Chol (2:1 mol ratio) and SM:PE:Chol (2:1:1 mol ratio) for the PLC and SMase activities respectively. PLC activity was five times higher than SMase activity. Both activities gave rise to vesicle aggregation, after a lag time during which ca. 10% of the substrate was hydrolyzed. Vesicle aggregation, measured as an increase in light scattering, was a convenient semi-quantitative method for estimating the enzyme activities. The optimum pH for the combined PLC and SMase activities was in the 5-7 range, in agreement with the proposed role of α-toxin in aiding the bacterium to escape the fagosome and survive within the cytosol.     

Activation studies with amines and amino acids of the α-carbonic anhydrase from the pathogenic protozoan Trypanosoma cruzi

The activation of a α-class carbonic anhydrase (CAs, EC 4.2.1.1) from Trypanosoma cruzi (TcCA) was investigated with the best known classes of activators, the amino acids and aromatic/heterocyclic amines. The best TcCA activators were l-/d-DOPA and 4-amino-l-phenylalanine, which had activation constants in the range of 0.38–0.83?µM. Low micromolar activators were also l-/d-Trp, l-/d-Tyr, l-Gln, histamine and serotonin (K_As of 1.79–4.92?µM), whereas l-/d-His, l-/d-Phe and l-Asp were less effective activators (K_As of 6.39–18.7?µM). Amines such as dopamine, pyridyl-alkylamines, aminoethyl-piperazine or l-adrenaline, were devoid of activating effects on TcCA. Since the role of autacoids as many of these compounds investigated here is not known for the life cycle of T. cruzi, our work provides new tools for further investigations of factors connected with this protozoan pathogen infection.     

Occurrence of α-ecdysone in the developing embryos of the silkworm, Bombyx mori

From an acetone-ethanol extract of the developing embryos of the silkworm, Bombyx mori, the presence of α-ecdysone, but not of β-ecdysone, was shown by high pressure liquid chromatography and bioassay. The amount of α-ecdysone was estimated to be 0.74 μg per gram of eggs. The absence of a hydroxylating system at C-20 in the embryos is suggested.