Effect of oral contraceptives on lacrimal fluid peroxidase activity in women.

Lacrimal fluid peroxidase has been supposed to be involved in the protection against oxidative damage to the ocular surface. Our recent findings showed the existence of significant cyclic variations in lacrimal fluid peroxidase activity that were positively correlated with those of 17beta-estradiol plasma levels throughout the menstrual cycle of fertile women. In the present study lacrimal fluid peroxidase activity of 8 healthy normocyclic women using low-dose oral contraceptives during the monthly cycle was determined. Data showed that low-dose oral contraceptives caused a decrease in lacrimal fluid peroxidase activity and a lack of its cyclic pattern with respect to the enzyme activity of 8 untreated age-matched women. Moreover, this result suggests that lacrimal fluid peroxidase activity could be regulated by estrogen.

Variations of lacrimal fluid peroxidase activity in female and male rats.

Previous research from this laboratory showed that human lacrimal fluid peroxidase has cyclic variations during the menstrual cycle, correlated with plasma levels of 17beta-oestradiol. In the present investigation, variations of enzyme activity and total protein content during the oestrous cycle of young adult female rats are analysed. Effects from circadian rhythm and a gender-related influence are also examined. In female rats, as in women, lacrimal fluid peroxidase activity shows cyclic variations; in fact, it significantly (p < 0.05) changes during the different phases of the oestrous cycle. In contrast, in males such variations do not occur. Thus, we suggest that gender seems to exert a significant influence on the secretion of this specific tear protein, probably by a direct effect of oestrogens.

Nuclear damage induced by liposomes containing fitc-labelled saporin on human melanoma cells in vitro.

Ribosome-inactivating proteins are enzymes of plant origin which de-adenilate the major ribosomal RNA, making it unable to bind the elongation factor and thus arresting protein synthesis. Recently the N-glycosidase activity of these enzymes has been extended also to deoxyribonucleotides substrates. In the present study we report the successful entrapment of the type 1 ribosome-inactivating protein saporin, covalently labelled with fluorescein isothiocyanate (FITC) into L-alpha lecitin/cholesterol liposomes and describe its delivery to human melanoma cells in vitro. The fluorescein reacted toxin maintained its enzymatic activity, although to a reduced extent; its interaction with liposomes resulted in the entry of the protein through the lipid bilayers. The resulting vesicles are carriers that can deliver the toxin inside cells; as a consequence the cytotoxic effects of the encapsulated enzyme were evident at a concentration two order of magnitude lower than that of the native one. In particular the nuclear damage, as revealed by micronuclei formation, was evident within 44 hr. The intracellular dynamics of the enzyme, as analyzed by confocal microscopy, point to an endocytic pathway of vesicles entry.

Antiproliferative effect and apoptotic response in vitro of human melanoma cells to liposomes containing the ribosome-inactivating protein luffin.

The present study describes the liposome-mediated delivery of the type 1 ribosome-inactivating protein luffin to human melanoma cells in vitro. Luffin from Luffa cylindrica seeds has been successfully incorporated into lecithin/cholesterol and lecithin/cholesterol/dicetylphosphate negatively charged liposomes. The exposure of melanoma cells to the two types of liposomes resulted in the inhibition of protein synthesis and cell growth; apoptotic cell death was verified by means of TUNEL reaction and quantitation of cytosolic oligonucleosome-bound DNA. The toxicity of encapsulated luffin varied with the lipid composition of the vesicles; the strongest effect was observed with lecithin/cholesterol liposomes. These results identify liposome-incorporated luffin as a possible alternative to immunotoxins for the treatment of human melanoma in situ.

Effects of surfactants on the stabilization of the bovine lactoperoxidase activity.

Bovine lactoperoxidase (LPO) is taken as a model protein of mammalian peroxidases to investigate the activity and the stability of the enzyme in the presence of different surfactants. The cationic benzalkonium chloride (Bz) has proved efficient in preserving the enzymatic activity for over 10 days, while the native enzyme completely lost its activity within 3-4 days. The presence of Bz allows the enzyme to preserve its secondary structure for a long time, as shown in CD spectra, and creates a more hydrophobic environment for the enzyme, as indicated in fluorescence studies. Moreover, this surfactant at a concentration of 0.01% (0.3 mM) increases the lactoperoxidase activity in the first 2 h of incubation at 37 degrees C. Both hydrophobic and electrostatic interactions of the cationic surfactant seem to be responsible for the enzyme activation and stabilization, and this is a promising result in view of industrial applications of enzymes.

Type B monoamine oxidase activity in human brain malignant tumors.

An increase of monoamine oxidase (MAO) activity was observed in Central Nervous System (CNS) malignant tumors, but the isoform responsible was not identify (Marcozzi et al., 1985). In the present work we report additional data in order to ascertain whether the type A or B MAO isoform is increased in some malignant human tumors of CNS. In the homogenated tissues the amine oxidase activity was determined by the chemiluminescent method, using different and specific substrates or inhibitors of MAO A and B and copper-dependent enzymes. 19 samples from 4 different types of tumors and relative peritumoral tissues were analysed. The highest activity of was imputable to type B MAO.

A simple method for the purification of type 1 RIPs.

We describe a straightforward and simple method for obtaining pure and active preparations of type 1 ribosome inactivating proteins (RIPs). The very high isoelectric point values, characteristic of these proteins, allow this purification in a single chromatographic step.

A rapid procedure for the purification of human salivary peroxidase.

An improvement to the purification method for human salivary peroxidase is presented. The enzyme is obtained at a higher degree of purity in two chromatographic steps instead of four, and avoiding lyophilation treatment. Differences in electrophoretic pattern confirm the genetic polymorphism of the peroxidase of human saliva.

Protein extraction by reverse micelles: a study of the factors affecting the forward and backward transfer of alpha-chymotrypsin and its activity.

alpha-chymotrypsin is taken as a model protein to investigate three aspects of the protein extraction by reverse micelles: (1) the comparison between the two forward transfer techniques, i.e., the liquid-liquid and the solid state-liquid transfer; (2)the back-transfer, i.e., the capability of the protein to be recovered from the micellar solution; and (3) the maintainance of the enzyme activity at the end of the extraction cycle. Concerning the forward transfer from the liquid phase, we study first the effect of salt initially present in the aqueous phase on the equilibrium concentration of the extracted species; further, we study the forward protein extraction from the solid state, and the effect of pH, salt, and protein concentration on the transfer efficiency. Concerning the back transfer, we find the somewhat surprising result, that the percentage of protein back-extraction depends on the type and concentration of salt used for the forward transfer. Preliminary data concerning an alternative method for the back-transfer using silica gel to liberate the protein from the micellar environment, are presented. Finally, it is found that the enzyme activity depends again on the type and concentration of salt used for the forward transfer.

Inhibition of copper-dependent amine oxidases by some hydrazides of pyrrol-1-ylbenzoic and pyrrol-1-ylphenylacetic acids.

Some hydrazides of pyrrol-1-ylbenzoic and pyrrol-1-ylphenylacetic acids were prepared, and their effect on copper-dependent amine oxidases (Cu-AOs) and FAD monoamine oxidases (MAOs) activities was tested. The compounds were not substrates for Cu-AO enzymes but acted as noncompetitive inhibitors. Hydrazides of pyrrol-1-ylphenylacetic acids were highly specific for plasma amine oxidase (Ki = 0.5-1 microM). In contrast, all the hydrazides were weak inhibitors of MAO activity. Incubation with the hydrazide derivatives led to irreversible inactivation of Cu-AOs. Therefore, the inhibition implied two distinct steps. The first one consisted of the rapid formation of the enzyme-inhibitor complex and was reversed by dialysis. In the second step, the complex was irreversibly transformed, probably by the formation of a Schiff base between the hydrazide and the prosthetic carbonyl group of the enzyme.

Determination of amine oxidases in tissues by peroxidation-induced chemiluminescence of phthalazines.

A method is proposed for the determination of the activity of amine oxidases in purified samples and tissue homogenates. The method is based on the chemiluminescence of luminol and other cyclic hydrazides elicited by the horseradish peroxidase-catalyzed peroxidation using H2O2 produced in the amine oxidase reaction. Several aspects of the chemiluminescence method for determining enzymatic activity in crude tissue extracts are discussed.

Pulse-radiolysis studies on the interaction of one-electron reduced species with blue oxidases. Reduction of type-2-copper-depleted ascorbate oxidase.

The interaction of one-electron reduced metronidazole (ArNO2.-) with native and Type-2-copper-depleted ascorbate oxidase were studied in buffered aqueous solution at pH 6.0 and 7.4 by using the technique of pulse radiolysis. With ArNO2.-, reduction of Type 1 copper of the native enzyme and of the Type-2-copper-depleted ascorbate oxidase occurs via a bimolecular step and at the same rate. Whereas the native protein accepts, in the absence of O2, 6-7 reducing equivalents, Type-2-copper-depleted ascorbate oxidase accepts only 3 reducing equivalents with stoichiometric reduction of Type 1 copper. On reaction of O2.- with ascorbate oxidase under conditions of [O2.-] much greater than [ascorbate oxidase], removal of Type 2 copper results in reduction of all the Type 1 copper atoms, in contrast with reduction of the equivalent of only one Type 1 copper atom in the holoprotein. From observations at 610 nm, the rate of reduction of ascorbate oxidase by O2.- is not dependent on the presence of Type 2 copper. For the holoprotein, no significant optical-absorption changes were observed at 330 nm. It is proposed that electrons enter the protein via Type 1 copper in a rate-determining step followed by a fast intramolecular transfer of electrons within the protein. For the Type-2-copper-depleted protein, intramolecular transfer within the protein, however, is slow or does not occur. In the presence of O2, it is also suggested that re-oxidation of the partially reduced holoprotein occurs at steady state, as inferred from the observations at 330 nm and 610 nm. The role of Type 2 copper in ascorbate oxidase is discussed in terms of its involvement in redistribution of electrons within the protein or structural considerations.

A new crystalline modification of the copper enzyme ascorbate oxidase.

The copper enzyme ascorbate oxidase, purified from green zucchini squash, has been crystallized at pH 5.4 employing the organic solvent 2-methyl-2,4-pentanediol. The crystals obtained are larger than one millimetre and belong to the space group P2(1)2(1)2, with unit cell parameters; a = 106.7 A, b = 105.1 A, c = 113.5 A. The crystallographic asymmetric unit contains two subunits of the enzyme (Mr = 140,000) and the solvent content of the crystals is 46% (v/v). The diffraction pattern extends to 2.5 A resolution; this crystal form is suitable for a X-ray structural investigation.

A reinvestigation on the quaternary structure of ascorbate oxidase from Cucurbita pepo medullosa.

The optical properties, copper content, catalytic activity and quaternary structure of many preparations of ascorbate oxidase purified with two different methods were examined. Fresh samples appeared identical and were characterized by optical ratios A280/A610 = 25 +/- 1 and A330/A610 = 0.8 +/- 0.05, by specific activity toward ascorbate of 3.48 +/- 0.05 mol g-1 min-1 and by a copper content of 8 +/- 0.3 mol/145 000 Mr. The enzyme is composed of two non-covalently linked subunits of slightly different molecular mass (75 000 and 72 000 respectively). These subunits cannot be further resolved by reduction of disulfide bonds. Proteolytic cleavage of the protein chains was observed during purification and storage in the absence of the protease inhibitor 6-amino caproic acid. Ascorbate oxidase exists as a monomer at neutral pH and undergoes reversible association into higher molecular weight species at slightly acid pH values. Association is not accompanied by spectroscopic or catalytic changes.