Enrico Bignetti

The half-of-the-sites reactivity of horse liver alcohol dehydrogenase in the presence of alcohol substrates

Abstract We investigated by stopped-flow techniques the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase varying the concentration of the reagents, pH and temperature. The course of the reaction under enzymelimiting conditions is biphasic and the measured amplitude of the initial step corresponds under saturation conditions to half of the total enzyme concentration (half-burst). The fast initial step (with a maximum rate of 20 s −1 at pH 7.0) shows an isotope effect of approximately 2, which indicates that this rate contains a contribution from a hydrogen transfer. It is also shown that this rate differs by at least one order of magnitude with respect to that of the hydrogen transfer during benzaldehyde reduction. The half-of-the-sites reactivity of alcohol dehydrogenase in the initial transient process is obtained independent of reagent concentration, pH and/or temperature. It is obtained also when coenzyme analogues are substituted for NAD, and when different alcohols are substituted for benzyl alcohol. These data are taken to demonstrate unequivocally that the half-of-the-sites reactivity of alcohol dehydrogenase cannot be due to an interplay of rate constants (as proposed by various authors) and must rather be ascribed to a kinetic non-equivalence of the two subunits when active ternary complexes are being formed. When oxidation of benzyl alcohol is carried out in the presence of 0.1 m -isobutyramide (which makes a very tight complex with NADH at the enzyme active site), reaction stops after formation of an amount of NADH product that is equivalent to one half of the enzyme active site concentration . This is considered in the light of the pyrazole experiment designed by McFarland & Bernhard (1972), in which reduction of benzaldehyde is carried out in the presence of pyrazole (which forms a very tight ternary complex with NAD at the enzyme active site). In this case, reaction stops after formation of an amount of NAD-product which is equivalent to the total enzyme active site concentration . It is shown that accommodation of these two seemingly contradictory sets of data poses severe restrictions on the alcohol dehydrogenase mechanism. In particular, it is shown that the only mechanism that adheres to such requirements is one in which the two subunits have distinct and alternating functions in each enzyme cycle. These two functions are the triggering of the chemical transformation and the chemical transformation itself. It is also shown that binding of NAD-substrate to one subunit triggers chemical reactivity in the other NAD-alcohol-containing subunit, whereas on aldehyde reduction, the triggering event is desorption of alcohol product from the first reacted subunit.

Microspectrophotometric measurements on single crystals of coenzyme containing complexes of horse liver alcohol dehydrogenase

In protein crystallography, difference electron density calculations is a wideIy used method to study ligand binding. Interpretation of difference peaks from data to limited resolution gives the position of the ligand on the protein molecule and the rough shape of the bound substance. However, the detailed chemicat nature of the bound molecute often cannot be identified from the difference peaks themselves 6yen at high resolution. For example, structure determinations by difference electron density techniques of dehydrogenase complexes with coenzyme wilt never solve the question in which oxidation state the bound eofactor is present witbJr~ the crystal. The use of electron dense labels on ligands, like Br, C1, and t substituents, frequently has been u~d in ligand Nnding studies in protein crystallography, 8-Br-ADPribose was used in the work with liver alcohol dehydrogenase (LADII) for the identfficatioll of the adenine binding pocket [ I ]. 4-Br-benzaldehyde was introduced as a substrate [2] and the coenzyme analogue 3-I~pyridine adenine dinucteotide was used to demonstrate the binding of an oxidized coenzyme species to the enzyme [3]. Althou~ useful in the X,ray analysis, heavier substituents on ligands might distort the binding mode to the protein. Nonprodue. rive binding can also occur. Obviously, complementary methods of m~alysis of single crystals are necessary when one intends to examine and compare protein structures with naturally occurring cofactors or subst~ates present in different oxidation state. Ill the work with tile flavodoxin semiquinone (radical) structure [4], visible spectra ofsingte cD~tals were recorded [51 which showed that the semiquinone form predominated in the crystals used for X.ray data collection. Structure determinations of LADH complexes have accumulated information about coenzyme analogue binding, inhibitor interactions and conformational changes induced by NADH and substrate [6]. Our goal is to describe in structural terms as many as possible of the individual steps in the process going :from an aldehyde substrate to an alcohot product. In the search for c~stal]ine N,M)Lcontaining complexes the need for additional analytieal methods arose as crystallization experiments on all types of LADH complexes have been performed in an al coholic medium, 4 ;methyl-2,4-pen tanediol (MPD). It seemed especially important to check tbr NADH formation within the single crystals since MPD could act as a substrate during crFstallizatiom Furthermore, in our study of the transient complex between LADH1,4,5,6-tetra.hydronicotinamide adenine dinucleotide (H2NADH) and the chromophoric substrate trans-4-N~Ldimetbylaminocinnamatdehyde (DACA) [71 we needed rNiable tools to measure aldehyde binding within the single c~stal. We report here the use of single crystal microspectrophotometric measurements as a convenient and rapid method to: (I) Detect NADH present ~ the lattice; (2) Follow DACA binding in the active site and O) Test substrate conversion in cD~stals ofcompIexes used for X-ray data collections,

Immunocytochemical localization of pyrazine-binding protein in bovine nasal mucosa

SummaryPolyclonal antibodies have been raised against purified bovine pyrazine-binding protein, a protein that binds the odorant 2-isobutyl-3-methoxypyrazine. These antibodies have been utilized in immunocytochemical experiments to localize the pyrazine-binding protein in bovine nasal mucosa. Tissue fragments, macroscopically identified as olfactory and respiratory mucosa, were fixed in Bouins fluid and embedded in paraffin. Consecutive serial sections were processed for immunofluorescence studies and restained either with haematoxylin-eosin or with periodic acid Schiff-Alcian Blue. In both olfactory and respiratory mucosa, only seromucous tubulo-acinar glands were specifically labelled. These glands are located in the lamina propria underlying typical respiratory epithelium, even in those tissues that are macroscopically defined as olfactory mucosa.

The pyrazine-binding protein and olfaction

1. The present results provide circumstantial evidence, but not a proof, that the Pyrazine-binding Protein is an odorant carrier molecule of fundamental importance. 2. At first sight a role for a secretory protein in olfaction is not obvious. 3. Odorants freely diffuse in air, in water and in lipids, and the use of carrier proteins, would seem superfluous unless a very special combination with the odorant occurs [Gaupp E. (1902) In Anatomie des Frosches, 2nd Edn, pp. 673. Vieweg-Verlag, Braunschweig]. 4. The possibility should be considered that the Pyrazine-binding Protein and the urinary proteins belong to a large family of species-specific secretory molecules which, with the odorant bound, directly stimulate the receptor cell.

Light and gtp effects on the turbidity of frog visual membrane suspensions

SummaryThe time course of turbidity changes of frog visual membranes, dependent on osmotic shocks, on light and on nucleotide substrates or effectors of enzyme activities, were measured as absorption changes in a rapid mixing stopped-flow spectrophotometer.As a result of studies on different preparations, it is concluded that light can cause both rapid (within 50 msec) and slow (within 90 sec) changes in the turbidity of visual membranes, not associated with permeability changes, and that they are affected by GTP or its analog guanyl-5′-yl imidodiphosphate; however, the light and GTP effects are lost when a water soluble fraction containing the light-sensitive enzyme cGMP-phosphodiesterase, is removed from the rod outer segments membranes.It is suggested that the fast light and GTP-sensitive response is related to the activation of cGMP-phosphodiesterase.

Occurrence and role of umami molecules in foods

Glutamate is a multifunctional amino acid. It plays a key role in central neurotransmission, in intermediate metabolism of carbohydrate as well as in taste, representing the major ligand having the umami taste. Glutamate is one of the main constituents of dietary proteins and is also consumed in many prepared foods as a flavour enhancer in the form of glutamate salts. Umami perception is based on multiple receptor systems distributed in the oral cavity and in the gastrointestinal tract which activates a number of regions of the brain involved in different functions, from food identification to the formation of an affective value related to a particular food, which may influence appetitive behaviour. Future research on umami taste and umami compounds will be fundamental in gaining a better understanding of their physiological significance and to promote their status in a healthy and pleasant diet.

Preparation of an affinity resin for odorants by coupling odorant binding protein from bovine nasal mucosa to Sepharose 4B

Abstract The soluble Odorant Binding protein (OBP) of bovine nasal mucosa is a dimer of non-covalently bound subunits that specifically recognizes several odorants of different chemical classes. Monomerization and binding activity impairment are concomitant reversible processes that occur below pH 6.0. In order to avoid this inconvenience, a mild pH condition has been chosen to couple native OBP to CNBr-activated Sepharose 4B resin. Above pH 6.0, the OBP-Sepharose matrix shows high specificity and affinity for odorants, thus opening new perspectives in odorant-sensor technology.

Hake fish bone as a calcium source for efficient bone mineralization.

Abstract Calcium is recognized as an essential nutritional factor for bone health. An adequate intake is important to achieve or maintain optimal bone mass in particular during growth and old age. The aim of the present study was to evaluate the efficiency of hake fish bone (HBF) as a calcium source for bone mineralization: in vitro on osteosarcoma SaOS-2 cells, cultured in Ca-free osteogenic medium (OM) and in vivo on young growing rats fed a low-calcium diet. Lithotame (L), a Ca supplement derived from Lithothamnium calcareum, was used as control. In vitro experiments showed that HBF supplementation provided bone mineralization similar to standard OM, whereas L supplementation showed lower activity. In vivo low-Ca HBF-added and L-added diet similarly affected bone deposition. Physico-chemical parameters concerning bone mineralization, such as femur breaking force, tibia density and calcium/phosphorus mineral content, had beneficial effects from both Ca supplementations, in the absence of any evident adverse effect. We conclude HBF derived from by-product from the fish industry is a good calcium supplier with comparable efficacy to L.

Evaluation of cathepsin B levels in fresh thighs selected for cured raw ham production.

Excessive meat tenderization in cured raw Parma ham has recently been correlated with abnormal levels of cathepsin B in freshly slaughtered thigh meat. We have developed a visual assay employing the substrate Z-Arg-Arg-NNapOMe for the quantitative detection of active cathepsin B levels in pork thigh muscle homogenates. The work was based on a kinetic characterization, in steady state condition, of pig muscle cathepsin B with several peptidyl chromophoric substrate analogs. This assay can easily and safely be performed by non-specialized personnel directly in the slaughterhouse or in the factory, for an early quality evaluation of thighs selected for Parma ham production. Our characterization has further indicated that the catalytic properties of porcine muscle cathepsin B and those of isoforms from other animal and plant species are practically identical. This is particularly evident in the commercially available bovine spleen isoform, which was employed as a model enzyme in most of the experiments.

A Psychophysical Approach to Test: “The Bignetti Model”

1Department of Veterinary Sciences, University of Parma, Via del Taglio 10, Parma 43126, Italy 2Department of Food Science, University of Parma, Via del Taglio 10, Parma 43126, Italy 3Department of Neurosciences, University of Parma, Via del Taglio 10, Parma 43126, Italy *Corresponding author Enrico Bignetti, MD Professor of Clinical Biochemistry and Molecular Biology Department of Veterinary Sciences University of Parma Via del Taglio 10 Parma 43126, Italy Tel. +39 0521 032710 E-mail: Enrico.bignetti@unipr.it; biriko@icloud.com