Alessandra Cambi

Changes of lysosomal enzyme activities in sea bass (Dicentrarchus labrax) eggs and developing embryos

The sea bass Dicentrarchus labrax is a pelagic egg spawner; sinking eggs are unable to develop into embryos, and this is a limitation in the controlled reproduction of this species. The eggs were divided into good and poor quality, by virtue of their ability to float or sink in seawater. High levels of cathepsins B, D, and L were detected in the eggs, whereas no cathepsin A, C, and E activity was detected. Cathepsin D was found at significantly higher levels in sinking eggs, whereas cathepsin L was more abundant in floating eggs. Since degradation of yolk proteins is essential for the early development of the embryo, the levels of cathepsins A, B, C, D, E, and L were tested in different stages of embryo development. Cathepsin A activity was detectable from the morula stage at which time cathepsin B activity reached its maximal level. Cathepsins A and L reached maximal activity during segmentation, and this corresponded with major changes in the electrophoretic pattern of yolk proteins during embryogenesis suggesting their involvement in yolk protein mobilization at this time. Cathepsin D reached its maximal activity during hatching.

HUMAN PLACENTA CYTIDINE DEAMINASE: PROTON-LINKED ENZYME ACTIVITY AND SUBSTRATE BINDING.

In this work we focused our attention on some catalytic site requirements for human placenta cytidine deaminase (CDA). The effect of pH on substrate binding and catalysis was studied between pH 3.0 and pH 11.0. The results could be discussed postulating the presence of two classes of ionizable groups in the active site of CDA. The kinetic parameters pH‐dependence has been discussed considering the presence of four zinc atoms per each enzyme tetramer. Furthermore fluorescence studies on the enzyme and on enzyme‐inhibitor complexes, examined by using polar and non polar quenchers, allowed to define the substrate(inhibitor)‐dependent accessibility of the tryptophan‐containing pocket on each CDA monomer.

Studies on cysteine residues involved in the active site of human cytidine deaminase

The three-dimensional structure of homodimeric E. coli cytidine deaminase (CDA)1 showed a zinc atom coordinated to histidine (H 102) and two cysteine residues (C 129 and C 132) in the active site. The role of zinc in catalysis is to bind and activate a water molecule for attack on the pyrimidine ring with consequent elimination of ammonia. The human CDA cDNA has been cloned and the protein expressed in E. coli is a tetramer composed of identical 16.2 KDa subunits each containing a firmly bound zinc atom in the active site.2 Comparison of the deduced amino acid sequence of the human recombinant CDA with that of E. coli enzyme1 identified the putative zinc coordinating residues as C99, C 102 and C65, the latter replacing H102 of the E. coli enzyme. To confirm the proposed role of the three indicated cysteines in catalysis of human CDA, we have studied the effect of specific thiolic reagents, and we have constructed and characterized three mutant proteins by site directed mutagenesis in which each of the cysteines, C99, C102 and C65 were replaced by an alanine residue.

Human placenta cytidine deaminase: a zinc metalloprotein

Cytidine deaminase, a tetrameric enzyme purified from human placenta, was shown to contain a single atom of tightly bound zinc per subunit by Inductively Coupled Plasma Optical Emission Spectrometry analysis. The metal appears to be involved in catalysis, as suggested by the inhibition exerted by 1,10‐phenanthroline and dipicolinic acid. This hypothesis is further supported by the finding that the presence of substrate protects the enzymatic activity from dipicolinic acid inhibition. Furthermore the total cysteine residues per subunit were investigated by sulphydryl groups titrating agents.