Effects of cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid (CLA) isomers on immune function in healthy men.

OBJECTIVES: To study the effects of two different mixtures of the main conjugated linoleic acid (CLA) isomers cis-9, trans-11 CLA and trans-10, cis-12 CLA on human immune function. DESIGN: Double-blind, randomized, parallel, reference-controlled intervention study. SUBJECTS AND INTERVENTION: Seventy-one healthy males aged 31-69 y received one of the following treatments: (1). mixture of 50% c9,t11 CLA and 50% t10,c12 CLA isomers (CLA 50:50); (2). mixture of 80% c9,t11 CLA and 20% t10,c12 CLA isomers (CLA 80:20); and (3). sunflower oil fatty acids (reference). The treatments were given as supplements in softgel capsules providing a total of 1.7 g (c9,t11+t10,c12) CLA fatty acids (50:50) or 1.6 g (c9,t11+t10,c12) CLA glycerides (80:20) per day in treatment groups for 12 weeks. RESULTS: Almost twice as many subjects reached protective antibody levels to hepatitis B when consuming CLA 50:50 fatty acids (15/24, 62%) compared with subjects consuming the reference substance (7/21, 33%, P=0.075). In subjects consuming CLA 80:20 glycerides this was 8/22 (36%). Other aspects of immune function, ie DTH responses, NK cell activity, lymphocyte proliferation and production of TNF-alpha, IL1-beta, IL6, IFN-gamma, IL2, IL4, and PGE(2), were not affected. CONCLUSION: This is the first study that suggests that CLA may beneficially affect the initiation of a specific response to a hepatitis B vaccination. This was seen in the CLA 50:50, but not in the CLA 80:20 group.

Analysis of conjugated linoleic acid-enriched triacylglycerol mixtures by isocratic silver-ion high-performance liquid chromatography.

Silver-ion HPLC (Ag-HPLC) was applied to the fractionation of a triacylglycerol (TAG) sample enriched (>80%) with conjugated linoleic acid (CLA). After conversion of the TAGs to fatty acid methyl esters using sodium methoxide in methanol, Ag-HPLC (dual-column; isocratic solvent system of 0.1% acetonitrile in hexane; UV detection at 233 nm) was used to determine the CLA isomer distribution (50:50 mixture of 9c 11t- and 10t,12c-18:2). Three or four Ag-HPLC columns connected in series (0.6-1.0% acetonitrile in hexane as solvent; UV detection at 206 nm) were used to analyze the sample in TAG form. Elution times for CLA-enriched TAGs averaged 30 min or less. Isocratic solvent conditions were used to eliminate the solvent equilibration times (often 30 min or more) required between sample injections when solvent programming is used. The ratio of TAGs containing three vs. only two CLA molecules was found to be approximately 3 to 1. Ag-HPLC has thus been shown to be a useful method for rapidly analyzing not only CLA isomers as esters, but also in the TAG form.

Oxygen exchange with water in heme-oxo intermediates during H2O2-driven oxygen incorporation in aromatic hydrocarbons catalyzed by microperoxidase-8.

The present paper describes the oxygen incorporation into naphthalene and anthracene by H2O2-driven microperoxidase-8, forming alpha-naphthol and anthraquinone, respectively. Microperoxidase-8 is a minienzyme containing a histidinyl-coordinated Fe3+-protoporphyrin IX cofactor covalently attached to an eight-amino-acid peptide. Additional experiments were performed to investigate whether the reaction mechanism involved is like that of peroxidase and/or cytochrome P-450. A reaction pathway like that of cytochrome P-450 implies oxygen transfer to the substrate from the as yet uncharacterized iron-oxo species formed in the reaction of the heine cofactor with H2O2. In contrast, a peroxidase-type reaction chemistry involves reaction pathways proceeding by initial one-electron oxidation of, or H-abstraction from, the substrate, followed by incorporation of oxygen from sources other than the iron-oxo species, i.e. from other than H2O2. The results of the present study exclude Fenton-type chemistry and prove that the minicatalyst is able to catalyze the oxygen incorporation by both peroxidase and cytochrome P-450 types of reaction pathways, while exchange occurs between the high-valency iron-oxo species and H2O. The mechanistic implications of this exchange for cytochrome P-450 are discussed.

Dissociation equilibrium of human recombinant interferon gamma.

The biologically active form of interferon gamma is a dimer composed of two noncovalently bound identical polypeptide chains of 17 kDa each. In this study, it was found that dissociation of the dimer into monomers significantly reduced the fluorescence quantum yield and the efficiency of the intermolecular Tyr to Trp radiationless energy transfer. The same process caused significant changes in the fluorescence decay and in the fluorescence anisotropy decay. The kinetic and thermodynamic parameters of the dimer-monomer equilibrium were determined by fluorescence measurements at different temperatures and by a theoretical mathematical model. Dissociation of the dimers into monomers was an endothermic process and was favored by concentrations of the protein lower than 1 microM and by increasing the temperature. It was accompanied by formation of aggregates, a slow and partially reversible process leading to inactivation of the interferon. It is suggested that certain monomeric conformers are competent for aggregation.

A 1H nuclear magnetic resonance method for investigating the phospholipase D-catalyzed hydrolysis of phosphatidylcholine in liposomes.

Liposomes with mean diameters between 45 and 73 nm have been prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at pH 8.0; and a new methodology is described which allows one to quantitatively follow the phospholipase D-catalyzed transformation of POPC to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid and free choline. The method does not require a special sample preparation; it takes advantage of the fact that the chemical shift of the protons of the three methyl groups in free choline differs from the chemical shift of the choline methyl protons in POPC. Measurements have been carried out under different experimental configurations and they have been paralleled by electron and light microscopy studies, partially using a fluorescently labeled phospholipid. It has been found that for a fixed concentration of the Ca2+-independent phospholipase D from Streptomyces sp. AA 586 the initial velocity and the reaction yields depend on the size of the vesicles. The smaller the vesicles, the higher the yields and the lower the initial rates. Furthermore, the size of the liposomes does not change during hydrolysis of the external POPC layer.

A comparative picosecond-resolved fluorescence study of tryptophan residues in iron-sulfur proteins.

The fluorescence intensity and anisotropy decays of the intrinsic tryptophan emission from six Fe/S proteins (ranging from the very simplest ones to enzyme complexes containing one, two or more Trp residues) were measured. All proteins were examined in the reduced and the oxidized state. In either redox state each protein exhibits ultrarapid tryptophan fluorescence decay on the picosecond timescale contributing up to 93% of the total decay. Correlation times in the range of 1 ns or less were found for all six iron-sulfur proteins reflecting internal Trp motion. In addition, some proteins exhibit longer correlation times reflecting segmental motion and overall protein tumbling. The ultrarapid fluorescence decay in iron-sulfur proteins indicates efficient radiationless energy transfer between distant tryptophan residues and iron-sulfur clusters. Such an energy transfer mechanism can be accounted for by referring to the three-dimensional structures of rubredoxin and ferredoxin in calculating the transfer efficiency of the single tryptophan-iron-sulfur couple.

Reverse micelles as a water-property-control system to investigate the hydration/activity relationship of alpha-chymotrypsin.

alpha-Chymotrypsin, solubilized in hydrated reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in n-octane, was used as a model system for studying the involvement of different water structures (strongly bound water, disordered water, water clusters and bulk water) in the development of the catalytically active conformation of the enzyme. Results presented in this study indicate a characteristic dependence of the stability/activity profile on the water content of the reverse-micellar system for values of wo of approximately 5 (wo is defined as [H2O]/[AOT]). The results are consistent with heat-capacity measurements for proteins. At very low wo values, the conformation of alpha-chymotrypsin changes to a very rigid structure in comparison to the structure observed in water. This is demonstrated by the overall center of gravity of the tryptophan fluorescence spectrum of the enzyme at wo = 0.65, which is blue shifted in comparison to the spectrum in bulk water indicating that the enzyme is in an apolar environment. In the absence of a hydration shell, the protein is to a great extent frozen and inactive. A small increase in the level of enzyme hydration (up to wo = 2.3) causes an increase in the amount of strongly bound water associated with the enzyme and the enzyme displays a high catalytic activity. Upon further addition of water, a new unstable water structure with unfavourable enthalpy is developed and the enzyme activity declines, reaching a minimum at wo = 5.1. A new increase of water content within a relatively small range, wo = 5-8, causes a dramatic increase in enzymic activity, reminiscent of a cooperative hydration dependence. In the range wo = 10-29, the effect of hydration on the activity is complete which shows that the enzyme activity depends on the amount of water in contact with the enzyme and not on the total amount of bulk water in the system. The experimental results on enzyme incubation at different wo values followed by dilution to constant high wo, are indicative of inactive conformational substates of alpha-chymotrypsin. It is demonstrated that highly active enzyme conformations at very low, wo values occur via an induced fit mechanism of substrate binding.

Comparison of the dynamic structure of alpha-chymotrypsin in aqueous solution and in reversed micelles by fluorescent active-site probing.

A highly fluorescent anthraniloyl (Ant) group was covalently attached to the active site of alpha-chymotrypsin (CT), probably at Ser195. Ant-CT is stable at neutral pH for months, allowing a detailed fluorescence study of Ant-CT as a model protein to investigate its physical properties in 0.1 M Tris/HCl, pH 8.2, and in reversed micelles of n-octane, 0.1 M Tris/HCl, pH 8.2, and sodium bis(2-ethylhexyl)sulfosuccinate (AOT). Steady-state fluorescence measurements of the progressive red-shift of the center of gravity of the emission band as function of degree of hydration, wo, defined as [H2O]/[AOT], indicate that the average polarity in the vicinity of the probe is approaching that of bulk water at wo > 12. Time-resolved fluorescence measurements of Ant-CT in water and in reversed micelles showed that the active site has different properties in reversed micelles compared to those in water. Some specific changes at very low water content (0.6 < wo < 5) can be observed, which correlate with enzyme activity measurements in the same wo region (unpublished results). These effects are, for instance, significant changes in the average fluorescence lifetime and the internal flexibility of the probe. The overall rotational-correlation time of the enzyme in AOT reversed micelles seems to be independent on wo (5 < wo < 29), which suggests that the enzyme creates its own micelle.

The specificity in the elementary steps of alpha-chymotrypsin catalysis. A temperature study with a series of N-acetyl-L-amino acid methyl esters.

The method of 'added nucleophile' (methanol) was employed to study the steady-state kinetics of the alpha-chymotryptic hydrolysis of methyl esters of N-acetyl-L-amino acids of the RCH(NHCOCH3)-C(O)OCH3 type, which are derivatives of alpha-aminobutyric acid, norvaline, norleucine, phenylalanine, alpha-aminoheptanoic acid and alpha-aminooctanoic acid. In the 5 to 30 degrees C temperature range at pH 8.0 the elementary rate and binding constants for both hydrolysis (Ks, k2, k3) and methanolysis (k-2) of the intermediate acylenzyme have been determined. The following results are discussed: 1. The enthalpy-entropy compensation phenomenon, which has previously been established to take place in alpha-chymotryptic catalysis, is characterized by two values of isokinetic temperatures (Tc), i.e., about 200 K at the step of binding the substrate and 430--460 K at the chemical steps of the enzymatic reaction. 2. It is demonstrated how the free energy and enthalpy, as well as entropy, change along all the reaction coordinate. To explain the results obtained, a mechanism for enzyme-substrate interaction is suggested according to which the sorption of substrate group R on the enzyme is different in the metastable intermediates and in the transition states of the reaction: (i) in the enzyme-substrate complex and in the acylenzyme, substrate group R merely emerges from water to be buried in the protein. Two facts point to the hydrophobic nature of the E-R interaction. First, the change in the free energy is exactly delta GRextr, that is the free energy of the transfer (extraction) of substrate group R from water to the organic solvent. Second, the motive force of the E-R interaction is the change in the entropy only. (ii) In the transition state the situation is different, i.e., the E-R interaction becomes thermodynamically still more favourable (the free energy change is 2 delta GRtrans) and involves thermodynamically favourable changes not only in the entropy (as is the case in the intermediates), but also in the enthalpy. Hence the E-R interaction in the transition states is accompanied by conformational and solvational changes of the enzyme-substrate system and, first and foremost, of the entire protein.

Inhibitory effect of metal ions on alkaline mesentericopeptidase.

The effect of AG+, Cu2+, Cd2+, Co2+ and Ni2+ on the activity of alkaline mesentericopeptidase (EC 3.4.21.-) has been studied. Ag+, Cu2+ and Cd2+ were found to be reversible non-competitive inhibitors of the enzyme. The pH-dependence of Ki for Ag+-inhibition is sigmoidal with a pKa near 6. The Kilim values, calculated for the pH-independent region of the metal-enzyme inhibition, are close to the corresponding dissociation constants of metal-imidazole complexes, thus implying that the inhibitory effect of metal ions on enzyme activity is due to complex formation with the imidazole group of the active site histidine. The method of the two-component inhibition showed that Cu2+ and Ag+ bind to the same ligand of the enzyme molecule. The addition of Cu2+ decreases the rate of deacylation of the hydrolysis of p-nitrophenyl valerate, catalyzed by alkaline mesentericopeptidase in contrast to alpha-chymotrypsin where the acylation step is affected.

Determination of the individual rate and association constants of the hydrolysis catalysed by serine proteinases by means of added nucleophiles in Eisenthal and Cornish-Bowden coordinates.

It is shown that in the three-step enzyme-catalysed hydrolysis the addition of nucleophile shifts the common intersection point in Eisenthal and Cornish-Bowden coordinates when registering the second product P2. The different points obtained at different nucleophile concentrations are situated on a straight line with intercepts Ks on the Km axis and k3 [E]o on the V axis. Since the Eisenthal and Cornish-Bowden method is considered as the best graphic method for determination of the kinetic parameters Km and V of enzyme reactions, the graphic procedure proposed here for determination of k2, k3, k4 and Ks by the method of added nucleophile to be preferred. This procedure was applied for determination of individual constants on the hydrolysis of N-acetylated L-amino acid methyl esters catalysed by alkaline mesentericopeptidase.

A study of the alkaline mesentericopeptidase active site by means of peptide chloromethyl ketones.

The kinetics of inactivation of alkaline mesentericopeptidase was studied using chloromethyl ketone derivatives of amino acids and peptides. It was shown that Tos-LysCH2Cl and Tos-PheCH2Cl did not influence the enzyme activity, while the inhibitory effect of Cbz-Ala-Gly-PheCH2Cl was 35 times that of Cbz-Ala-PheCH2Cl. The dependence of the pseudo-first order rate constant of the enzyme inactivation by Cbz-Ala-Gly-PheCH2Cl on pH and temperature indicated that a group with a pK of 6.59 and deltaHi of 7.7 kcal/mol was the site of the inhibitor's attack. Amino acid analysis of the modified totally inactive enzyme revealed a definite loss of histidine and after performic acid oxidation a recovery of 3-carboxymethyl histidine. The whole set of experimental data is convincing evidence on behalf of a selective alkylation of the N-3 of the active site histidine after treatment with Cbz-Ala-PheCH2Cl and Cbz-Ala-Gly-PheCH2Cl. Alkaline mesenteriocopeptidase possesses an extended active site and only a peptide chloromethyl ketone, covering a determined sequence of theenzyme molecule (S3, S2, S1, S'1, S'2, S'3 ...) is able to provide effective inhibition. The values of the inactivation constant (kinact) for Cbz-Ala-PheCH2Cl and Cbz-Ala-Gly-PheCH2Cl are close to the corresponding values reported for subtilisin Amylosacchariticus.