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R Bru

Publications and source records attributed to R Bru.

At least 19 recordsLinked to original sources
PubMed1997

Kinetic characteristics of the enzymatic conversion in presence of cyclodextrins: study of the oxidation of polyunsaturated fatty acids by lipoxygenase.

The capability of cyclodextrins (CDs) to greatly enhance the solubility in water of poorly water-soluble substances makes them an ideal alternative system for studying the expression of enzyme activity with such substrates in aqueous solution. In order to evaluate the behaviour of the enzymes in presence of CDs a study of the lipoxygenase (LOX)-catalyzed oxidation of polyunsaturated fatty acids (PUFA) as model reaction has been carried out. This was done by using LOX from two different sources (soybean and potato tuber), at two pH values (6.3 and 9.0), with two substrates (linoleic acid and arachidonic acid) and three types of CD (beta-CD, methyl-beta-CD and monoglucosyl-beta-CD). PUFA have been shown to form inclusion complexes of 1:2 stoichiometry which are in equilibrium with free PUFA and free CD, thus complexation is governed by two equilibrium constants, K1 and K2 (J.M. López-Nicolás et al., Biochem. J. 308 (1995) 151-154; R. Bru et al., Colloids Surf. 97 (1995) 263-269). For the oxidation of PUFA by LOX in the presence of beta-CD we propose a model in which free PUFA is the only effective substrate, thus the oxidation of the complexed substrate requires the previous dissociation of the complex. The equilibrium constants of complex formation are determined by both a physico-chemical and an enzymatic method. In spite of giving quite similar results, the second was proven to be more accurate so it was employed in further studies. CD was shown to slow down the reaction rate of LOX, specifically due to the increase of Km, Vmax remaining unchanged. That apparent inhibition is due to removal of effective (free) substrate in the form of inclusion complexes. This 'sequestered' substrate can, however, be converted since it is in equilibrium with the free. The feasibility of realizing a CD-mediated accurate control over the conversion rate is demonstrated in the experiment called 'cyclodextrin assay' in which the concentration of the free substrate is calculated by using the equilibrium constants of complex formation and setting the initial concentrations of total substrate and total CD. From the observation of the reaction progress curves in the conditions of the CD assay, we have studied some characteristic parameters of the oxidation of PUFA by LOX in this new medium, such as enzymatic activity, duration of linear product accumulation and the lag phase.

Arachidonic Acid↗
PubMed1997

Cell-linked and extracellular cholesterol oxidase activities from Rhodococcus erythropolis. Isolation and physiological characterization.

Rhodococcus erythropolis cells growing in a cholesterol-free glycerol-containing mineral medium displayed very low levels of a cell-wall-bound cholesterol oxidase activity. Addition of cholesterol induced a marked increase in the synthesis of this enzyme, which reached a maximum within 6 days and was subsequently followed by the appearance of extracellular cholesterol oxidase in the culture broth. Significant levels of induction were only achieved when cholesterol emulsified with Tween 80. The presence of chloramphenicol at the time of induction completely prevented the emergence of both enzymatic forms, suggesting the requirement of de novo protein synthesis. Upon transfer of cholesterol-growing cultures to fresh medium lacking cholesterol, the extracellular cholesterol oxidase was quickly erased, while the activity of the particulate enzyme decreased sharply. The electrophoretic pattern on native Western blotting as well as on sodium dodecyl sulphate/polyacrylamide gels, together with kinetic data, strongly support the idea that the particulate and extracellular cholesterol oxidases are two different forms of the same enzyme with an estimated molecular mass of 55 kDa.

Chloramphenicol↗
PubMed1996

Triton X-114-aided purification of latent tyrosinase.

Mushroom tyrosinase was partially purified using an aqueous two-phase system with Triton X-114. The purification achieved was 5.5-fold from a crude extract of mushroom pileus, with a high recovery of 84%. The phenols were reduced to 8% of the original content, avoiding pre- and post-purification tanning of the enzyme. The enzyme obtained was latent and was activated 3-fold by trypsin, 2.7-fold by changes in the pH and to different extents by cationic and anionic detergents, the latter being the more effective. There was also a synergistic effect between trypsin and detergent, at low detergent concentrations. When kinetically characterized, latent enzyme showed both monophenolase and diphenolase activities, the latter activity displaying an unexpected lag period before reaching the steady-state rate. This behaviour is characteristic of a hysteretic enzyme, and has not been previously described for this enzyme. In addition, inhibition studies with substrate analogues were carried out, tropolone being found to be the most effective inhibitor.

Agaricus↗
PubMed1995

Use of 'soluble lipids' for biochemical processes: linoleic acid-cyclodextrin inclusion complexes in aqueous solutions.

The equilibria of linoleic acid (LA)-cyclodextrin (CD) complexes were studied to investigate the behaviour of 'soluble lipids' in solution as a function of factors that typically affect biochemical processes, such as pH, temperature and CD structure. The above complexes are formed with a stoicheiometry of 1:2 in solution. The first CD molecule interacts with LA through hydrogen bonds when the pH is below the fatty acid pK; hydrophobic interactions may also play an important role at high pH. The second CD molecule makes only hydrophobic contact with the LA hydrocarbon chain. The formation of hydrogen bonds is dependent on the inner diameter of the CD whereas the strength of the hydrophobic interactions between CD and LA can be related to the presence of hydrophobic groups in the CD. The first CD molecule interacts more strongly with LA at increased temperatures. The quantitative description of the LA-CD interaction allows absolute control of the effects produced by the lipid on biochemical processes.

Cyclodextrins↗
PubMed1994

An octaethylene glycol monododecyl ether-based mixed micellar assay for lipoxygenase acting at neutral pH.

Using the detergent octaethylene glycol monododecyl ether (C12E8), a spectrophotometric mixed micellar assay for lipoxygenas (LOX) activity was designed. Potato LOX was able to use the linoleic acid (LA) solubilized in C12E8 micelles, displaying the characteristic induction period of LOX-catalyzed LA peroxidation. In the mixed micellar system, LOX responds to the LA surface concentration expressed as mol% (=[lipid]*100/([detergent]-cmc)) and not to the molarity of the LA. For both potato and soybean LOX, Vmax was independent of the mixed micelle concentration, while Km was independent as well when expressed as mol% but was dependent on C12E8 concentration when expressed in molar. In mixed micelles, H2O2 shortened the induction period, while 13-hydroperoxylinoleic acid and t-butyl hydroperoxide completely removed it. C12E8/LA proved to be a reliable system for assaying LOX activity at pH values around neutrality. Like Tween 20, this system avoided the turbidity problems arising from the protonated fatty acid and did not interfere with the uv-absorption band of the hydroperoxide product. However, this system is superior to the commonly used Tween 20 because it permits investigation of the lipid requirements of LOX since the concentration-independent Km can be determined both in mol% and as the absolute number of lipids per micelle. In addition, the detergent did not affect the enzyme through any side effects.

Detergents↗
PubMed1994

Phase separation of biomolecules in polyoxyethylene glycol nonionic detergents.

The advantage of aqueous two-phase systems based on polyoxyethylene detergents over other liquid-liquid two-phase systems lies in their capacity to fractionate membrane proteins simply by heating the solution over a biocompatible range of temperatures (20 to 37 degrees C). This permits the peripheral membrane proteins to be effectively separated from the integral membrane proteins, which remain in the detergent-rich phase due to the interaction of their hydrophobic domains with detergent micelles. Since the first reports of this special characteristic of polyoxyethylene glycol detergents in 1981, numerous reports have consolidated this procedure as a fundamental technique in membrane biochemistry and molecular biology. As examples of their use in these two fields, this review summarizes the studies carried out on the topology, diversity, and anomalous behavior of transmembrane proteins on the distribution of glycosyl-phosphatidylinositol-anchored membrane proteins, and on a mechanism to describe the pH-induced translocation of viruses, bacterial endotoxins, and soluble cytoplasmic proteins related to membrane fusion. In addition, the phase separation capacity of these polyoxyethylene glycol detergents has been used to develop quick fractionation methods with high recoveries, on both a micro- and macroscale, and to speed up or increase the efficiency of bioanalytical assays.

Animals↗
PubMed1993

sn-1,2-diacylglycerol cholinephosphotransferase from pig liver: mixed micellar assay and kinetic analysis of the partially pure enzyme.

sn-1,2-Diacylglycerol cholinephosphotransferase from pig liver microsomes was partially purified through a procedure involving solubilization with sodium cholate and chromatography on Sepharose 6B. The resulting preparation was 19-fold enriched with respect to microsomes and was shown to be very sensitive to different detergents. Sodium cholate gave the best yields in activity. In a mixed micellar assay with Triton X-100 a strong dependence of the enzyme activity on the concentration of mixed micelles was observed, due to Triton X-100 acting as an inactivator. Soja phosphatidylcholine added exogenously protected the enzyme against detergent inactivation and stimulated the enzyme activity. Dioleoyl-phosphatidylcholine had a similar stimulatory effect, whereas didecanoyl- or dioctanoyl-phosphatidylcholine did not; thus long-chain phosphatidylcholines seem to be essential in the activation of cholinephosphotransferase. In a mixed micellar assay with sodium cholate no inactivation of the enzyme could be detected and it was found that soja phosphatidylcholine stimulates the activity in a greater extent than in Triton X-100 mixed micelles. The phospholipid activates the enzyme in a noncompetitive way with an activation constant of 176 mol%. Km was estimated as 1.54 mol% with a Vmax = 30 nmol/min/mg protein. Those results support an activation mechanism by phosphatidylcholine interacting at sites different from the active center. The high activation constant led to the conclusion that cholinephosphotransferase requires a lipidic boundary for full activation. No activation by substrate was observed. Short-chain diacylglycerides such as dihexanoyl-, dioctanoyl-, or didecanoylglycerol can be used as substrates although the enzyme in this case has only 5 to 10% of the activity it has for dioleoylglycerol or egg diglycerides.

Animals↗
PubMed1993

Catalytic activity of elastase in reverse micelles.

The activity of porcine pancreatic elastase has been studied in reverse micelles formed by AOT (sodium bis(2-ethylhexyl) sulfosuccinate) in isooctane. For the two substrates succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide and succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, the catalytic constant, kcat, in reverse micelles increases with increasing wo until, at high wo, the value of kcat measured in bulk buffer solution is approached (wo = [H2O/]AOT]). In analogy to alpha-chymotrypsin--and in apparent contrast to many other enzymes--elastase does not show a maximum in the kcat-wo profile. Within the wo range of 8 to 35, for both substrates, the Michaelis constant Km (as expressed relative to the total volume of the solution, Km,overall) increases with increasing wo.

Amino Acid Sequence↗
PubMed1991

Product inhibition of alpha-chymotrypsin in reverse micelles.

The alpha-chymotrypsin-catalyzed hydrolysis of succinyl-L-alanyl-L-alanyl- L-prolyl-L-phenylalanyl p-nitroanilide has been studied in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. It has been found that alpha-chymotrypsin is strongly inhibited competitively by the acidic peptide product which is formed during the course of the reaction. It has also been shown that the application of the integrated form of the Michaelis-Menten equation can be useful to detect possible inhibition effects and abnormal kinetic behavior of enzymes in reverse micelles. Furthermore, it has been shown that the turnover number (kcat) at low water content is lower than in water and increases as the water content in the system is lower than in water and increases as the water content in the system (wo = [H2O]/[AOT]) increases, kcat reaching the value in water at high wo. If however, initial velocity data, as obtained under conditions where the enzyme is not saturated with substrate, are plotted against wo, the curves are bell-shaped, with a maximum around wo = 15.

Amino Acid Sequence↗
PubMed1991

Trypsin-SBTI interaction in reverse micelles. A slow intermicellar exchange-dependent binding.

Solubilisate exchange between reverse micelles must take place before any reaction inside reverse micelles occurs if the reactants are confined to the aqueous micellar core. When the interacting species are 2 small molecules or one small molecule and one macromolecule, it has been shown that the exchange is faster than the typical turnover of an enzymatic reaction. The study of the interaction between 2 macromolecules (trypsin and soybean trypsin inhibitor) in reverse micelles carried out in this work reveals that the exchange between these macromolecule-containing reverse micelles slows down by a thousand times and the limiting-step in the exchange, the fusion, by 10(6) times. Both reverse micellar size (omega 0 = [water]/[surfactant]) and temperature affected the rate of the fusion process. A hypothesis for the proposed active role of macromolecules in the exchange process is also given.

Arginine↗
PubMed1990

The effect of substrate partitioning on the kinetics of enzymes acting in reverse micelles.

A theoretical model for the expression of enzymic activity in reverse micelles previously developed [Bru. Sánchez-Ferrer & García-Carmona (1989) Biochem. J. 259, 355-361] was extended in the present work. The substrate concentration in each reverse-micelle phase (free water, bound water and surfactant apolar tails) and the organic solvent was expressed as a function of the total substrate concentration, taking into account its partition coefficients, that is, partitioning of the substrate in a multiphasic system. In each phase the enzyme expresses a catalytic constant and a Km. Thus the whole reaction rate is the addition of the particular rates expressed in each domain. This model was compared with that developed for a biphasic system [Levashov, Klyachko, Pantin, Khmelnitski & Martinek (1980) Bioorg. Khim. 6, 929-943] by fitting the experimental results obtained with mushroom tyrosinase (working on both 4-t-butylcatechol and 4-methylcatechol) to the two models. The parameters which characterize reverse micelles, omega 0 (water/surfactant molar ratio) and theta (fraction of water) were investigated. The omega 0 profile was shown to be hyperbolic for both substrates. Activity towards 4-t-butylcatechol decreases as theta increases, this observation being attributable to a dilution of the substrate. A Km of 7.8 M for 4-t-butylcatechol could be calculated on the basis of the biphasic model, whereas it was 13.5 mM when calculating on the basis of our model. A new parameter, rho (= [substrate]/theta), was defined to characterize those substrates that mainly solubilize in the reverse micelle ('micellar substrates').

Basidiomycota↗
PubMed1990

Partial purification of a thylakoid-bound enzyme using temperature-induced phase partitioning.

Triton X-114 was used to partially purify broad bean polyphenol oxidase, a thylakoid membrane-bound enzyme, in latent form, free of phenolic compounds and chlorophylls, with a high recovery rate. The activation of the latent enzyme by detergents or trypsin was 10 times higher than that obtained when the enzyme was purified by other methods used in plant biochemistry, such as acetone powders and ammonium sulfate fractionation. The kinetic parameters of the latent and activated enzyme are also given.

Catechol Oxidase↗
PubMed1989

A theoretical study on the expression of enzymic activity in reverse micelles.

The present work deals with a theoretical model of catalysis by enzymes entrapped in reverse micelles. Three aspects of the enzyme-reverse-micelle system have been considered: structure, dynamics and enzyme distribution and catalysis in reverse micelles. A proposed structural model of reverse micelles [El Seoud (1984) in Reverse Micelles (Luisi, P. L. & Straub, B. E., eds.), p. 81, Plenum Press, New York] consists of three domains: surfactant apolar tails, bound water and free water. Dynamics are based on a dynamic equilibrium of association-dissociation that lead one to consider the dispersed polar phase as a pseudo-continuous phase [Luisi, Giomini, Pileni & Robinson (1988) Biochim. Biophys. Acta 947, 207-246]. Enzyme is distributed among the reverse-micelle domains and it expresses a catalytic constant for each one of them. The overall activity is calculated taking into account the volume in which enzyme is solubilized, and expressed as a function of the whole volume (V). The characteristic parameters of reverse micelles, omega 0 (= [H2O]/[surfactant]) and theta (= % water, v/v), were investigated as modulators of enzymic activity. Three basic patterns of modulation by omega 0 were found depending on which domain the enzyme expressed the highest catalytic constant. Combinations of those basic patterns lead to other modulation types that can be found experimentally, such as superactivation. Other combinations predict behaviour patterns not described to date, such as superinhibition. Dependence of catalytic activity on theta was only stated at omega 0 values around a critical value, which coincides with the appearance of free water.

Catalysis↗
PubMed1989

Novel procedure for extraction of a latent grape polyphenoloxidase using temperature-induced phase separation in triton x-114.

Polyphenoloxidase from grape berries is extracted only by nonionic detergents with a hydrophilic-lipophilic balance between 12.4 and 13.5. The enzyme was partially purified in latent form, free of phenolics and chlorophylls, by using temperature phase partitioning in a solution of Triton X-114. This method permits the purification of the enzyme with the same fold purification as the commonly used method, but with a yield three times higher and a 90% reduction in time needed. The latent enzyme can be activated by different treatments, including trypsin and cationic and anionic detergents. Cetyltrimethylamonium bromide was found to be the most effective detergent activator, followed by sodium dodecyl sulfate. Polyphenoloxidase in grape berries, in spite of being an integral membrane protein, had an anomalous interaction with Triton X-114, remaining in the detergent-poor phase after phase separation. This could be explained by its having a short hydrophobic tail that anchors it to the membrane.

Journal Article↗
PubMed1988

Chemical and enzymic oxidation by tyrosinase of 3,4-dihydroxymandelate.

Tyrosinase usually catalyses the conversion of monophenols into o-diphenols and the oxidation of diphenols to the corresponding o-quinones. Sugumaran [(1986) Biochemistry 25, 4489-4492] has previously proposed an unusual oxidative decarboxylation of 3,4-dihydroxymandelate catalysed by tyrosinase. Our determination of the intermediates involved in the reaction demonstrated that 3,4-dihydroxybenzaldehyde is not the first intermediate appearing in the medium during the enzymic reaction. Re-examination of this new activity of tyrosinase has demonstrated that the product of the enzyme action is the o-quinone, which, owing to its instability, evolves to the final product, 3,4-dihydroxybenzaldehyde, by a chemical reaction of oxidative decarboxylation.

Ascorbic Acid↗