Carbonic anhydrase and urease: an investigation in vitro on the possibility of a synergic action.

The indirect interactions between the carbonic anhydrase (CA) and urease (UR) are investigated in the present work using rate determinations detected by combined potentiometric measurements. It is shown that, in accord with the mass-action law for the two enzyme catalyzed reactions, the two enzymes assume a synergic pattern: the increase in the rate of removal of CO2 from the solution facilitated by CA increases the rate of production of NH3 consequent from urea dissociation. The experimental system which has been set up to monitor these interactions consists of a potentiometric apparatus to follow the gaseous exchanges of CO2 and NH3 which take place from a buffered solution containing both CA and UR. The results of the present work are consistent with, and add a further support to the finding of Dodgson and Forster, who first demonstrated in vivo the existence of an indirect linkage between urea production and CA catalytic activity.

Synthesis and inhibitory activity on carbonic anhydrase of some new sulpiride analogues studied by means of a new method.

The pharmacological activity of several new sulpiride analogues was studied by means of a new approach, based on a potentiometric technique with a pCO2 sensor, capable of detecting carbonic anhydrase inhibition at equilibrium conditions. This procedure gives results stated as percent of inhibition of enzymatic activity (IP, inhibitory power). To prove the reliability of the proposed approach and to study structure-activity relationships, several new molecules were synthesized and tested in comparison with the two sulpiride enantiomers. A possible inhibition mechanism is discussed in terms of experimental evidence obtained from the interactions between the molecular structures of the new synthesized compounds and carbonic anhydrase.

Potentiometric determination of carbonic anhydrase activity in rabbit carotid bodies: comparison among normoxic, hyperoxic and hypoxic animals.

The catalytic activity of carbonic anhydrase (CA) contained in the glomus cells of mammalian carotid bodies has been determined in vitro by a potentiometric method. Experiments performed on whole rabbit carotid bodies have shown a very low variability, in terms of the overall CA activity, among organs belonging to different animals maintained in normoxic conditions. Repeated assays performed on each carotid body have shown a marked decrease of the overall CA activity after the first assay, thus suggesting the presence of at least two different forms of enzyme. Experiments performed on carotid bodies belonging to rabbits maintained in normal, hyperoxic and hypoxic conditions have shown that the overall CA activity follows the sequence: hypoxic > normoxic > hyperoxic, matching with the corresponding physiological activity of the carotid body.

Nuclear magnetic resonance investigations of calcium antagonist drugs. II: Conformational and dynamic features of verapamil in [2H6]DMSO.

Conformational features of verapamil in [2H6]DMSO have been delineated by measuring 13C and 1H NMR parameters. Spin-lattice relaxation rates were interpreted within the frame of an extended alkane chain with segmental motions hampered by the aromatic substituents at both ends. The effective correlation times were all evaluated in the range 0.16-0.22 ns at 293 K. The aromatic rings were found to lie somewhat out-of-plane with the alkane chain. The NMR data were used to construct a Dreiding model of the most probable spatial arrangement and a molecular modeling system was utilized to represent the "preferred" conformation of verapamil in solution.

Atmospheric pollution originating from the interaction of different gaseous effluents.

During the last few years, several episodes of atmospheric pollution have been reported in a limited area near Guidonia, Rome. The area contains a disposal plant, Inviolata, for the collection of municipal solid waste (MSW) and a famous thermal water resort, the Acque Albule spring, which is a source of water rich in H2S. We conducted a multiparametric study in the areas surrounding the solid waste disposal plant and the Acque Albule spring. The concentration of main gaseous effluent was continuously monitored over a period of 4 months and the data relating to the meteorologic conditions in the area during the last few decades were examined. Our results suggest that most of the atmospheric pollution is due to the interaction of different gaseous effluents. Specifically, the presence of relatively high levels of hydrogen sulfide in the atmosphere, constantly released in large amounts by the Acque Albule springs, and of biogases (mainly hydrocarbons) from the organic matter present in the solid waste continuously unloaded and stored at the disposal plant, lead to mixing and photochemical interactions between these chemical compounds, which in turn are responsible for most of the polluting effects. Such interactions are promoted by the strong solar irradiation in the area that is enhanced by the peculiar local meteorological features that do not allow the pollutants to disperse.

Determination of L-glutamate and L-glutamine in pharmaceutical formulations by amperometric L-glutamate oxidase based enzyme sensors.

An amperometric biosensor for the direct determination of L-glutamate was developed by chemical bonding of L-glutamate oxidase (GAO) on a carboxylic Nylon membrane with polyazetidine prepolymer (PAP), and using a hydrogen peroxide electrode as indicating sensor. The biosensor is specific for L-glutamate and the peculiar analytical properties (linearity range, reproducibility, accuracy) were experimentally determined. Furthermore, the same basic biosensor was also modified to be used and characterized for the direct determination of L-glutamine. This L-glutamine biosensor was obtained by coimmobilizing, on two separate membranes, glutamic acid oxidase and glutaminase (GMN) on the same biosensor. The two sensors were then used for the determination of glutamate and L-glutamine contained in pharmaceutical formulations and the results were compared with those obtained by other analytical methods.

Toxicity testing in environmental monitoring: the role of enzymatic biosensors.

Biological toxicity testing is a rapidly expanding field involving numerous bioanalytical techniques. The enzymatic biosensors are valuable screening tools to identify pollutants and/or toxic agents in the environment and/or in food matrices, thus representing a valid alternative to animal testing in analytical toxicology. Inhibition based biosensors here presented have been proved to represent alternative assays for the toxicity evaluation of warfare agents and endocrine disrupting chemicals as well as algal toxins (phycotoxins) in the contamined sea foods (mainly clams and other mollusks). Results obtained by inhibition studies performed by means of several enzymatic biosensors indicate the reliability of the proposed method and the possibility to extend such an experimental approach to other toxicants as a simple, rapid and cheap biotest, to be used easily also "on the spot".

Determination of carbonic anhydrase activity by a pCO2 sensor.

A potentiometric method capable of determining carbonic anhydrase (CA) activity in vitro and based on the use of a pCO2 sensor is presented. By means of the procedure described here it is possible to follow the rate of CO2 diffusion that takes place in a buffered solution of NaHCO3 in either the presence or the absence of CA. All experimental parameters that affect the speed of HCO3- dehydration, as well as the speed of CO2 diffusion, can be fixed and kept constant for the duration of every assay. The advantage of this method is that the overall dehydration plus diffusion process can be followed as it actually takes place in open thermodynamic systems far from equilibrium. The results obtained strongly confirm the hypothesis of a facilitating role of CA toward the rate of CO2 diffusion.

Interactions between calcium entry blocking drugs and carbonic anhydrase.

The interactions between some of the most common calcium entry blocker drugs (CEB) and the enzyme carbonic anhydrase (CA) are studied in the present work by an electroanalytical approach. The study comprises drugs belonging to the classes of phenylalkylamines, dihydropyridines, benzothiazepines and piperazines. The evaluation of the potential inhibitory power towards CA was performed either by measuring the speed of CO2 diffusion taking place from a buffered solution of NaHCO3, or by monitoring the metabolic activity of yeast cells. The results obtained according to both of these procedures have shown that verapamil and gallopamil are endowed with a relevant inhibitory power on CA catalytic activity, whereas all the other compounds, tested in the same experimental conditions, did not show any effect on CA activity.