Jesús M. Fominaya

Oxidation of sulfhydryl groups of ribonuclease inhibitor in epithelial cells is sufficient for its intracellular degradation.

Ribonuclease inhibitor (RI) is a cytoplasmic protein (50 kDa) that inhibits a variety of pancreatic type RNases. The porcine inhibitor contains 30 cysteine residues, all of which occur in the reduced state. It is well known that in vitro modification of the thiol groups inactivates the protein and greatly increases its susceptibility to proteolysis. Here we show that oxidation of thiol groups in RI can also occur within the cell. Induction of an oxidative insult in cultured LLC-PK1 cells, either with a general oxidant, H2O2, or with a thiol-specific oxidant, diamide, led to the loss of RI activity. By using specific antibodies it was demonstrated that the decrease correlated with a decline in the amount of RI protein in the cells. Furthermore, analysis of RI mRNA levels and half-life of the protein excluded inhibition of the synthesis of RI as the cause of its depletion. The results indicate that oxidation of thiol groups in RI is sufficient to cause its rapid inactivation and disappearance from the cell. Most likely this results from intracellular degradation of the protein.

Alkaline ribonuclease from the insect Ceratitis capitata

Abstract An alkaline ribonuclease has been purified from Ceratitis capitata larvae. The enzyme preparation gives a single band on SDS-polyacrylamide gel electrophoresis using both silver-stain and ribonucleolytic activity detection. This enzyme has been characterized as a cyclizing endonuclease with optimum pH value at 8.0–8.3. The enzyme preparation does not exhibit phosphatase activity. Poly(C) is the only homopolyribonucleotide degraded under standard assay conditions. Neither native nor denatured DNA is hydrolyzed by this enzyme. The protein is a single polypeptide chain of about 18 000 molecular weight; its secondary structure is composed of 19% α-helix, 10% β-structure, 71% aperiodic conformation with an average number of residues per helical segment of 9. The amino acid composition of this alkaline RNAase is also reported. Latent alkaline ribonuclease has been observed in crude insect homogenates. The insect enzyme is inhibited by the RNAase inhibitor from human placenta.

Ribonuclease-RNAase inhibitor complex from rat testis.: Purification of the RNAase inhibitor

The RNAase inhibitor from rat testis has been purified to homogeneity. The purified protein appeared as a single spot after two-dimensional electrophoresis. The calculated Mr value is 48,000 which coincides with that obtained for the native protein on gel filtration chromatography, thus indicating a single polypeptide chain. The amino acid composition and the characteristics of the inhibitor activity are reported and compared to those of other RNAase inhibitors from mammalian tissues. The naturally occurring ribonuclease-RNAase inhibitor complex from rat testis has also been studied and compared with the rat testis inhibitor-RNAase A as model complex. The ribonuclease released from the natural rat testis complex showed heterogeneity of size. The significance of the rat testis ribonuclease/RNAase inhibitor system is discussed in terms of the important functionality of this organ.

Polyadenylated-RNA during the development of Ceratitis capitata

Abstract Polyadenylated-RNA has been isolated at different stages of the life cycle of the insect Ceratitis capitata using chromatography on oligo-dT-cellulose. The molecular characterization of this RNA fraction has been carried out by melting, sedimentation, electrophoretic and CD studies. The distribution of this polynucleotide fraction during the development has been related to physiological changes of this holometabolous insect. The results obtained are discussed in terms of the presence of RNA-degrading activities previously reported for this insect.

Residues 287-301 of human ribonuclease inhibitor do not affect ribonuclease activity and inhibitor binding ; a reply

Residues 287-301 of human placental ribonuclease inhibitor have been reported to inhibit pancreatic ribonuclease A in a similar way as the entire inhibitor (Crevel-Thieffry, I., Cotterill, S. and Schuller, E. (1992) Biochim. Biophys. Acta 1122, 107-112). Using three different assays, we were unable to observe inhibition by the synthetic peptide. Moreover, the peptide did not compete with the entire inhibitor for binding to RNAase A.

Characterization of the nucleolytic activity from the digestive juice of Ceratitis capitata larvae

1. 1. The ability of the digestive juice of Ceratitis capitata larvae to degrade polynucleotides has been studied: optimum pH, cationic dependence and kinetic studies of the hydrolytic reaction. 2. 2. The relative catalytic efficiency of the digestive juice against different polynucleotides is reported. 3. 3. The existence of analogous nucleolytic activities in other insects is discussed as a general feature in the diet digestion of these organisms.