Stereoselective oxidation of aromatic sulfides and sulfoxides in the binding domain of bovine serum albumin

The oxidation of aromatic sulfides with achiral oxidizing agents, e.g., sodium metaperiodate (NaIO₄) and hydrogen peroxide (H₂O₂) in the binding domain of bovine serum albumin (BSA), furnished a strong asymmetric bias (max 81%) of the product sulfoxides in fairly high chemical yields. The kinetic resolution of racemic aromatic sulfoxides was also carried out in the chiral binding domain, and the remaining unchanged sulfoxides showed optical purities ranging over 1–33% at ca. 50% completion of oxidation. The combination of the two stereoselective oxidations above mentioned produced several optically active sulfoxides of >90% optical purity in ca. 50% chemical yield. The present method constitutes a successful biomimetic approach to achieving stereoselectivities as high as obtained by sulfur-oxidizing microorganisms.     

Neuroprotective effects of (E)-3,4-diacetoxystyryl sulfone and sulfoxide derivatives in vitro models of Parkinson's disease

(E)-3,4-dihydroxystyryl aralkyl sulfones and sulfoxides have been reported as novel multifunctional neuroprotective agents in previous studies, which as phenolic compounds display antioxidative and antineuroinflammatory properties. To further enhance the neuroprotective effects and study structure-activity relationship of the derivatives, we synthesized their acetylated derivatives, (E)-3,4-diacetoxystyryl sulfones and sulfoxides, and examined their neuroprotective effects in vitro models of Parkinson’s disease. The results indicate that (E)-3,4-diacetoxystyryl sulfones and sulfoxides can significantly inhibit kinds of neuron cell injury induced by toxicities, including 6-OHDA, NO, and H₂O₂. More important, they show higher antineuroinflammatory properties and similar antioxidative properties to corresponding un-acetylated compounds. Thus, we suggest that (E)-3,4-diacetoxystyryl sulfones and sulfoxides may have potential for the treatment of neurodegenerative disorders, especially Parkinson’s disease.     

RuBP carboxylase determination by enzymic estimation of D-3-PGA formed

RuBPcarboxylase activity was measured in extracts of barley (Hordeum Vulgare L., cv. HOP) seedlings both with the standard radiometric method and by measuring D-3-phosphoglyceric acid formed enzymically in a two stage assay. In the different conditions used, characterized by different NaHCO₃ concentrations, different pH and the presence and absence of oxygen, essentially the same ratio of D-3-PGA formed per ¹⁴CO₂ fixed was obtained. This ratio respected the known stoichiometry of two molecules of D-3-PGA formed per CO₂ fixed. It is suggested that measurement of D-3-PGA enzymically in a two stage assay can be routinely used for the determination of RuBP case activity instead of the radiometric method. The advantages and the validity of the method are discussed.     

RuBP carboxylase determination by enzymic estimation of D-3-PGA formed

RuBPcarboxylase activity was measured in extracts of barley (Hordeum Vulgare L., cv. HOP) seedlings both with the standard radiometric method and by measuring D-3-phosphoglyceric acid formed enzymically in a two stage assay. In the different conditions used, characterized by different NaHCO₃ concentrations, different pH and the presence and absence of oxygen, essentially the same ratio of D-3-PGA formed per ¹⁴CO₂ fixed was obtained. This ratio respected the known stoichiometry of two molecules of D-3-PGA formed per CO₂ fixed.It is suggested that measurement of D-3-PGA enzymically in a two stage assay can be routinely used for the determination of RuBP case activity instead of the radiometric method. The advantages and the validity of the method are discussed.Abbreviations Bicine N, N-bis-(2-hydroxyethyl)-glycine - NADH nicotinamide adenine dinucleotide, reduced - PGA phosphoglyceric acid - RuBP ribulose-1-5-bisphosphate     

Escherichia coli formylmethionine tRNA: methylation of specific guanine and adenine residues catalyzed by HeLa cells tRNA methylases and the effect of these methylations on its biological properties

Purified HeLa cell tRNA methylases have been used for site-specific methylations of Escherichia coli formylmethionine transfer ribonucleic acid (tRNA^fMet). Guanine-N²-methylase catalyzed the methylation of a specific guanine residue (G₂₇) and adenine-1-methylase that of a specific adenine residue (A₅₉). The combined action of both of these enzymes leads to a total incorporation of two methyl groups and results in the methylation of both G₂₇ and A₅₉.The effect of introducing additional methyl groups on the function of tRNA has been studied by a comparison in vitro of the biological properties of tRNA^fMet and enzymically methylated tRNA^fMet. It was found that none of the following properties of E. coli tRNA^fMet are altered to any significant extent by methylation: (a) rate, extent, and specificity of aminoacylation, (b) ability of methionyl-tRNA to be enzymically formylated, and (c) ability of formylmethionyl-tRNA to initiate protein synthesis in cell-free extracts of E. coli in the presence of f2 RNA as messenger. Also, the temperature versus absorbance profile of the doubly methylated tRNA^fmet was virtually identical to that of the E. coli tRNA^fMet, and enzymically methylated tRNA^fmet resembled tRNA^fMet in that both were resistant to deacylation by E. coli, N-acylaminoacyl-tRNA hydrolase.     

The formation of unhydrated propionaldehyde by dioldehydrase.

When dl-1,2-propanediol is converted to propionaldehyde by dioldehydrase, an enzyme which requires B₁₂-coenzyme, the product is unhydrated propionaldehyde. Its formation was demonstrated by measuring the rate of reduction of the enzymically formed aldehyde by NADH, catalyzed by yeast alcohol dehydrogenase.     

Mode of action of sulfoxides in preventing loss of activity of 11β-hydroxylase in cultured bovine adrenocortical cells

Previously, loss of 11β-hydroxylase activity when adrenocortical cells are incubated with the pseudosubstrate cortisol was found to be reduced when the concentration of oxygen was lowered, or when butylated hydroxyanisole (BHA) or dimethyl sulfoxide (Me₂SO) were included in the medium. In the present experiments, we tested the hypothesis that Me₂SO protects 11β-hydroxylase by scavenging OH^? radicals. Substances known to react with OH^? at high rates and non-toxic enough to be used at concentrations of 10–100 mM, including several alcohols, benzoate and radioprotectant thiols, did not prevent loss of activity of 11β-hydroxylase in the presence of 50 μM cortisol. Two of the alcohols, ethanol and glycerol, as well as Me₂SO, were radioprotective in cultured bovine adrenocortical cells. Therefore free OH^? radicals do not appear to be involved in loss of 11β-hydroxylase activity. When sulfoxides other than dimethyl sulfoxide were tested for their ability to protect 11β-hydroxylase in the presence of cortisol, several aryl sulfoxides, particularly dibenzyl sulfoxide, as well as dipropyl sulfoxide, were active at concentrations to $\text{1}\text{200}$ of that required for Me₂SO. Previously, we have demonstrated that 11β-hydroxylase inhibitors, particularly metyrapone, effectively protect against loss of 11β-hydroxylase activity in the presence of pseudosubstrates and therefore we examined whether sulfoxides may act by directly inhibiting 11β-hydroxylase. Me₂SO showed an ED₅₀ for inhibition of 11β-hydroxylase activity of > 1 M, in contrast to its ED₅₀ for protection of 34 mM. For metyrapone, however, the ED₅₀ for inhibition of the enzyme (250 nM) was close to that for protection of activity (270 nM). The other sulfoxides showed ED₅₀-values for inhibition of 11β-hydroxylase that were substantially higher than the ED₅₀-values for protection. Sulfoxides may have a mixed mode of action in protection of 11β-hydroxylase activity, as previously shown for phenols; they may protect by radical scavenging, but may also need to bind close to the active site of the enzyme where destructive radicals may be formed.     

Hydrogenation as an approach to study of reactions of oxidizing polyphenols with plant proteins

Coupling of oxidation products of o-diphenols with -NH₂ groups of plant proteins can damage nutritional availability of lysine residues. Relevant model coupling products (before or after reductive acetylation or permethylation) are unstable to acid hydrolysis. Hydrogenation over Rh/Al₂O₃, at room temperature and atmospheric pressure, gave cyclohexane derivatives stable to hydrolysis and retaining, with only partial hydrogenolysis, all groups originally attached to the aromatic nucleus. Plant bulk proteins were hydrogenated with substantial conversion of their aromatic amino acids; their S-containing amino acids were desulphurized. The technique is therefore promising for study of the fate of lysine residues in “enzymically browned” proteins.     

A rapid method for the assay of mitochondrial ATPase activity.

A method is described which permits the rapid simultaneous assay of numerous samples for ATPase activity. The sample is incubated with ATP and PEP, and the pyruvate liberated is enzymically coupled with NADH oxidation. The reaction is terminated and cleared after a suitable time interval by addition of sodium dodecyl sulphate (SDS) and A₃₄₀ read directly.     

Sensitive method for determination of peroxidase activity in tissue by means of coupled oxidation reaction

A simple colorimetric one-step method for determination of peroxidase activity in tissue is described. The method utilizes enzymically released H₂O₂ from glucose oxidation and o-dianisidine as hydrogen donor. The sensitivity of the method is at least ten times greater than existing methods. The influence of H₂O₂ concentration, buffer composition, and catalase interference is studied and discussed. An alternative fluorometric modification is briefly described.     

Changes in Metabolite Levels in Kalanchoë daigremontiana and the Regulation of Malic Acid Accumulation in Crassulacean Acid Metabolism

Changes in glucose-6-P, fructose-6-P, fructose-1,6-diP, 6-phospho-gluconate, phosphoenolpyruvate, 3-phosphoglycerate, and pyruvate levels in the leaves of the Crassulacean plant Kalanchoë daigremontiana Hammet et Perrier were measured enzymically during transitions from CO₂-free air to air, air to CO₂-free air, and throughout the course of acid accumulation in darkness. The data are discussed in terms of the involvement of phosphoenolpyruvate carboxylase in malic acid synthesis and in terms of the regulation of the commencement of malic acid synthesis and accumulation through the effects of CO₂ on storage carbohydrate mobilization and its termination through the effects of malic acid on phosphoenolpyruvate carboxylase activity.     

Absorption of Iron by Enzymically Isolated Leaf Cells

Iron uptake was studied using cells enzymically isolated from green tobacco leaves. Absorption was increased both by light and succinate as probable energy sources. Bicarbonate in the incubation mixture was inhibitory, and citrate also reduced absorption presumably by chelation with the metal. Absorption of iron was temperature sensitive and optimal at 25°C. Temperature coefficients and activation energies suggested that absorption was energy mediated. NaN₃ and DNP inhibited uptake at concentrations of 10-³M and 10^?4M, respectively. The inhibition caused by DNP was not negated by an external supply of ATP. The results suggest that iron absorption is an active metabolic process in cells enzymically isolated from green tobacco leaves. Cells from Fe-chlorotic leaves of PI 54619–5–1 soybean absorbed less iron than those derived from healthy leaves of the same variety, while leaf cells from the variety Hawkeye showed no such differences.     

Synthesis of chiral fluorine-tagged reference standards for the 19F NMR-based stereochemical analysis of sulfoxides at trace analytical levels

Chiral fluorine-tagged sulfoxides of known absolute configuration have been synthesized. These compounds are required as reference standards to validate a ¹⁹F NMR-based micromethod for the stereochemical analysis of biosynthetic fatty acyl sulfoxides.     

Penetration of iron and some organic substances through isolated cuticular membranes

Cuticular membranes were isolated enzymically from tomato fruits and from the dorsal and ventral surfaces of the leaves of Euonymus japonicus. Penetration of Fe from FeSO₄ and FeEDDHA (ferric ethylenediamine di(o-hydroxyphenylacetate) in the absence and presence of urea through the isolated cuticular membranes was studied. Fe from FeSO₄ penetrated more rapidly through the cuticles than Fe from FeEDDHA. Urea reduced the penetration of Fe from FeSO₄ and FeEDDHA. Binding of Fe on the inner surfaces of tomato fruit cuticles was also reduced by EDDHA.     

Kinetic properties of fractions of extracellular NAD+ nucleosidase from Streptococcus pyogenes as an example of host selection by a pathogen: possible role of serum albumin in the organism

Preparative isoelectric focusing was used to separate free bacterial NAD⁺ nucleosidase from its complex with a bound host component. Both fractions were characterized by optimum temperature and activation energy of denaturation. The bacterial product is enzymically inactive. The enzymically active structure is formed upon binding to the host component. Only the host organism can provide the suitable, activating structure. The host component in the present system is added to the cultivation medium with a beef heart extract but it can be replaced by serum albumin. The possible role of albumin as a carrier structure for flexible and enzymically inactive peptides is discussed. Different peptides bound to albumin can provide different enzyme activities. The term binary enzyme is coined, referring to a situation where the two enzyme components are coded at genetically distant loci. The pathogen makes use of the carrier structure of albumin type and produces another polypeptide invested with an enzyme activity convenient for the pathogen.     

Studies on the Mechanism of the Oxidation of Tea Leaf Catechins

In order to understand the mechanism of the enzymic oxidation of epigallocatechin, ethyl gallate was taken up as a model substrate on account of its close structural similarity to the w-trihydroxyphenyl group, which indeed is supposed to be attacked by oxidase, when epigallocatechin is oxidized enzymically. By the action of tea or apple oxidase at pH 5.5, it gave a characteristic polyphenolic substance, which was obtained in prisms, C₁₈H₁₈O₁₀·2H₂O, and proved to be diethyl 4,4′,5,5′,6,6′-hexahydroxydiphenate, as the product was converted into ellagic acid by hydrolysis of ester linkage and then lactone formation.     

Structure of the water-insoluble α-d-glucan of streptococcus salivarius HHT

Water-insoluble, non-adherent α-d-glucans have been obtained from Streptococcus salivarius HHT under two sets of conditions: from a growing culture, or synthesized enzymically by using a glucosyltransferase. In the former case, the glucan ([α]_d + 197°) was shown by methylation analysis to have a slightly branched structure containing a relatively high proportion (80 %) of (1→3)-α-d-glucosidic linkages, together with small proportions of (1→6)- and (1→4)-α-d-glucosidic linkages. The enzymically synthesized glucan had a much less-branched structure, containing 88 % of (1→3)-α-d-glucosidic linkages. Both glucans, on Smith degradation (sequential periodate oxidation, borohydride reduction, and mild acid hydrolysis), gave linear, (1→3)-α-d-glucosidic polysaccharides (yields, 82-90%) that constitute the backbone chains. The presence of small proportions of glycerol, erythritol, 1-O-α-d-glucosyl-d-glycerol, and also 2-O-α-d-glucosyl-d-erythritol in the products of Smith degradation suggests that the short side-chains are attached to the backbone chain by (1→4)-, (1→6)-, and (1→3)-α-d-glucosidic linkages     

Peroxidase activity in human red cell: a biological model for excited state molecules generation.

The presence of enzymically generated triplet acetone in red cells and energy transfer to eosin, rose bengal and 9,10-dibromoanthracene-2-sulfonate was indicate by: (1) product distribution; (2) K_ET τ_o, similar to the 2-methylpropanal/peroxidase/O₂ system; (3) correlation between hemolysis, oxygen uptake and photon emission; (4) membrane protection by energy acceptors, and (5) by comparison of the 2-methylpropanal/peroxidase/O₂ system with 2-methylpropanal/red cells/membranes/O₂ and 2-methylpropanal/acid extractable protein from red cells membrane/O₂ systems, which have a high peroxidase activity.This is the first report of a biological system producing a photohemolysis effect in the dark.     

Steroid 17,20-lyase: the effect of detergents on enzymic activity and microsomal composition

The activity of the steroid 17, 20-lyase from rat testis microsomes was determined following exposure of the microsomes to detergents. Only Triton N-101 and X-100 produced enzymically active supernatants. The supernatant from Triton N-101 treatment consisted of submicrosomal particles enriched in cytochrome P450, flavin, and phospholipid; depleted in RNA and NADPH oxidase; and unchanged in the concentration of cytochrome b₅ and non-heme iron. The activities of the NADPH and NADH cytochrome C reductases were also intact. Lubrol produced an inactive supernatant which contained all the components thought to be necessary for microsomal electron transport with the exception of cytochrome b₅. An assay, specific for cleavage and more expeditious than the chromatographic separation of reactants, is also described.     

An oxygen-18 tracer investigation of the mechanism of myo-inositol oxygenase

In the conversion of $\text{myo}$-inositol to D-glucuronic acid catalyzed by $\text{myo}$-inositol oxygenase only one atom of ¹⁸O from ¹⁸O₂ is incorporated into the product, and it is found exclusively in the carboxyl group. Control experiments indicate that under the reaction conditions no exchange of solvent oxygens with D-glucuronate occurs. To avoid exchange during isolation and analysis the oxygenase product was enzymically reduced to L-gulonate and isolated in that form. The results eliminate one possible mechanism for the oxygenase reaction, but are consistent with two others which seem chemically reasonable.