Marcos Mares-Guia

CHILLING-INDUCED CHANGES IN MEMBRANE FLUIDITY AND ANTIOXIDANT ENZYME ACTIVITIES IN COFFEA ARABICA L. ROOTS

Exposure of coffee to low temperatures caused growth inhibition, changes in metabolic rates, and membrane alterations. Root tissue exposed to 10 °C evolved significantly lower rates of metabolic heat compared with controls grown at 25 °C, and the values were closely associated with the observed root growth inhibition. Electron paramagnetic resonance spectra of intact tissues showed that the spin probe 5-doxylstearic acid was capable to intercalate within the cellular membrane lipids. Indeed, at the depth of the 5th carbon atoms of the alkyl chains, the nitroxide radical detected more rigid membranes in seedlings exposed to 10 °C compared with 25 °C treated samples. Ascorbate peroxidase and catalase activities did not show appreciable changes under chilling conditions, while guaiacol peroxidase activity increased 55 % compared to the control. On the other hand, glutathione reductase activity decreased, in parallel to a significant decline in the capacity to reduce triphenyl-tetrazolium. Our results showed a marked correlation between lipid peroxidation and root tissue damage, which seemed to be associated with increased membrane rigidity.

Release of lipopolysaccharide (LPS) from cell surface of Trypanosoma cruzi by EDTA

Abstract LPS release from the cell surface of culture forms (epimastigotes) of Y and CL stocks of Trypanosoma cruzi was achieved by EDTA (85 mM final concentration). Following the EDTA treatment (5 min at 28°C) the parasites remained active, had normal morphology and displayed full growth when inoculated onto blood agar slants. The presence of LPS in the preparations was detected both colorimetric ally and by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The heterogeneity of the surface LPS was demonstrated by using Procion Red-dyed LPS subjected to SDS-PAGE. Our kinetic data on 14 C-arginine transport suggest that the observed alterations in cell permeability may be due to an effect of EDTA, associated or not wilh LPS removal from the cell surface.

Hydrophobic interactions in the trypsin active center. The sensitivity of the hydrophobic binding site to side chain modifications in competitive inhibitors of the amidinium type

Abstract An investigation was carried out with model compounds designed to allow the study of the effects of small substituents (methyl groups), uncharged isosteres, methyl substitution on the benzamidinium nucleus, and extension of the aromatic side chain, on the binding properties of the hydrophobic binding site of the trypsin active center. Formamidine-HCl, guanidine-HCl, methylguanidine sulfate, meta - and para -toluamidines, as well as beta-naphthamidine-HCl were shown to be competitive inhibitors of trypsin. The methyl group contributes about −0.45 Kcal/mole to the free-energy of binding to the active center of the enzyme. If the methyl group is meta - or para - to the aromatic ring of benzamidine, there is a slight decrease in binding as compared to benzamidine itself, probably due to steric hindrance. In the amidine compounds, exchanging the phenyl ring of benzamidine for the naphthyl ring of beta-naphthamidine results in no alteration in the free-energy of binding. The results are discussed in terms of their usefulness in the design of compounds suitable for the labeling of the hydrophobic binding site of the trypsin active center.

Studies on the mechanism of rat urinary kallikrein catalysis, and its relation to catalysis by trypsin☆

Abstract The pH-activity curves for rat urinary kallikrein catalysis of benzoyl- l -arginine ethyl ester and benzoyl- dl -arginine p-nitroanilide hydrolysis suggest the participation of a histidyl residue in the catalytic process. A specific active center reagent of trypsin, 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), did not inactivate the kallikrein at pH 7.2, 8.0, or 8.92. This was interpreted as the result of a fitting between TLCK and kallikrein, different from that between TLCK and trypsin, such that renders alkylation of a histidyl residue impossible. Phenylguanidine sulfate, cyclohexylguanidine sulfate, benzamidine-HCl, and β-naphthamidine-HCl were shown to competitively inhibit the kallikrein. The Ki values found demonstrate differences between binding of the inhibitors to kallikrein and to trypsin, but assure the proposition of hydrophobic and anionic binding sites in the kallikrein active center. The data prove that trypsin and the trypsin-like kallikreins can be distinguished on the basis of the lack of reactivity of the latter toward TLCK.

Thrombin-like enzyme from Lachesis muta muta venom: isolation and topographical analysis of its active site structure by means of the binding of amidines and guanidines as competitive inhibitors

A serine protease enzyme was purified from Lachesis muta muta venom, with 40% yield, by gel filtration on Sephadex G-100 and affinity chromatography on Sepharose-agmatin. Homogeneity of the enzyme preparation was demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the enzyme had a relative mol. wt of 45,000. The molar extinction coefficient at 280 nm was 62,127 (M x cm)-1. The enzyme hydrolysed Bz-Arg-Nan with Ks = 0.233 +/- 0.08 mM and kcat = 2.80 +/- 0.07 sec-1. All the amidines and guanidines tested for their inhibitory effect on thrombin-like enzyme behaved as competitive inhibitors of the enzyme with Ki values in the range 6.2 microM to 42.3 mM for amidines and 0.19 mM to 9.31 mM for guanidines. Dissociation constant values were analyzed in terms of the binding of the inhibitors with the subsite S1, the specificity pocket of the enzyme, Ki values were discussed in accordance with those for trypsin inhibition. beta-Naphthamidine was the strongest inhibitor, while guanidine was the weakest. The differences among the Ki values were interpreted in terms of the shape of the enzyme active site. For meta- and para-substituted benzamidinium ions a good correlation was found between log l/Ki and sigma Hammett values of the substituents. The substituent effects in the pi-electrons of the benzamidine ring were considered in the frame of Hückel molecular orbital theory. A model for the binding of p-benzamidine derivatives with the primary specificity S1 subsite of the enzyme active site was proposed.

Microcalorimetric evaluation of metabolic heat rates in coffee (Coffea arabica L.) roots of seedlings subjected to chilling stress

In numerous tropical and sub-tropical plant species, such as coffee, exposure to low temperatures can cause extensive tissue damage, the seedlings being particularly sensitive to chilling stress. This condition usually induces changes in the metabolic rates, and there are indications that this process can be evaluated by monitoring heat evolution by microcalorimetry. We studied the responses of coffee seedlings to chilling stress by measuring root growth and metabolic heat rates in apical root segments of coffee seedlings exposed for 6 days to temperatures ranging from 5 to 25°C. The metabolic heat rates were measured in a heat conduction calorimeter. Root growth was progressively hindered as the seedlings were exposed to temperatures below 15°C; low temperature-induced growth inhibition was closely correlated with the lowering of metabolic heat rates. An Arrhenius plot of metabolic heat rate revealed a break in the line at 15°C, suggesting the occurrence of a metabolic transition at this temperature. The microcalorimetric technique provides a sensitive, non-invasive method for evaluating plant growth responses to chilling stress.

Reaction of the main proteolytic fraction of Schistosoma mansoni cercarial enzymes with synthetic substrates and inhibitors of proteolytic enzymes

Abstract The purified proteolytic fraction of Schistosoma mansoni cercarial enzymes (PCF) was inhibited by Diisopropylphosphofluoridate (DFP). Its esterolytic activity was not affected by either of two specific active center reagents of proteolytic enzymes: (1) 1-chloro-3-tosylamido-7-ammo-2-heptanone (trypsin) and (2) l -1-tosylamido-2-phenylethyl chloromethyl ketone (chymotrypsin). Furthermore, PCF did not destroy the biological activity of bradykinin on the isolated guinea pig ileum, nor did it release bradykinin from purified dog plasma bradykininogen. The pentapeptide Cbz-Gly-Pro-Leu-Gly-Pro, of which the bond Leu-Gly is split by collagenase, is hydrolyzed rather slowly by PCF. The fingerprint of the products of hydrolysis showed several spots instead of two spots to be expected if only a true collagenase were present. The possibility of including PCF in the group of serine enzymes is discussed.

Microcalorimetric determination of glucose utilization by leishmania

Abstract Heat conduction calorimetry of Leishmania amazonensis parasites was carried out using 1 to 6 × 10 8 cells (promastigotes), dependent on glucose concentrations in the calorimeter and dependent on glucose used as carbon source in cell culture. Leishmania produce aerobic heat from endogenous sources even when given no exogenous carbon. However, depending on how cells were grown (adaptation) they respond to glucose addition with increased heat production up to a limit roughly around 25 μ M glucose, plateauing thereafter. Heat production ranges from 8 to 135 × 10 −4 millical/(sec)(10 8 cells) when dissolved oxygen is adequate.

Kinetic parameters for the activation of α- and β-trypsins by the methyl ester of tosyl-l-arginine (Tos-l-Arg-OMe)

Abstract The kinetic parameters describing the activation of α- and β-trypsins by the methyl ester of tosyl- l -arginine were determined at 37 °C, pH 8.0, according to the model proposed by C. G. Trowbridge, A. Krehbiel, and M. Laskowski, Jr. (1963 , Biochemistry2, 843-50). The parameters, calculated through a computer program called ATIV3, were: Km(2) = 19.2 mM and k′cat = 653 s−1, for α-trypsin; Km(2) = 50.3 mPm and k′cat = 1192 s−1 for β-trypsin. Thus, α-trypsin has a higher affinity for the activator, but β-trypsin has a higher value of the enhanced catalytic constant, k′cat. The ratio k′ cat K m(2) indicates that α-trypsin is more susceptible to activation than the β-form.

Partial Purification of Rat Urinary Kininogenase and its Reactions with Active Center Reagents of Trypsin

The kininogenase from rat urine has the properties of a trypsin-like enzyme, as determined with the help of synthetic substrates or competitive inhibitors (Mares-Guia and Diniz, 1967). The work done previously was carried out with a partially purified preparation of high specific activity. The purification procedure will now be described, as well as the behavior of the enzyme in the presence of specific reagents for the trypsin active center.