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At least 235 records · Page 13Linked to original sources
PubMed2001

Preparation of potent cytotoxic ribonucleases by cationization: enhanced cellular uptake and decreased interaction with ribonuclease inhibitor by chemical modification of carboxyl groups.

Carboxyl groups of bovine RNase A were amidated with ethylenediamine (to convert negative charges of carboxylate anions to positive ones), 2-aminoethanol (to eliminate negative charges), and taurine (to keep negative charges), respectively, by a carbodiimide reaction. Human RNase 1 was also modified with ethylenediamine. Surprisingly, the modified RNases were all cytotoxic toward 3T3-SV-40 cells despite their decreased ribonucleolytic activity. However, their enzymatic activity was not completely eliminated by the presence of excess cytosolic RNase inhibitor (RI). As for native RNase A and RNase 1 which were not cytotoxic, they were completely inactivated by RI. More interestingly, within the cytotoxic RNase derivatives, cytotoxicity correlated well with the net positive charge. RNase 1 and RNase A modified with ethylenediamine were more cytotoxic than naturally occurring cytotoxic bovine seminal RNase. An experiment using the fluorescence-labeled RNase derivatives indicated that the more cationic RNases were more efficiently adsorbed to the cells. Thus, it is suggested that the modification of carboxyl groups could change complementarity of RNase to RI and as a result endow RNase cytotoxicity and that cationization enhances the efficiency of cellular uptake of RNase so as to strengthen its cytotoxicity. The finding that an extracellular human enzyme such as RNase 1 could be effectively internalized into the cell by cationization suggests that cationization is a simple strategy for efficient delivery of a protein into cells and may open the way of the development of new therapeutics.

3T3 Cells↗
PubMed1997

From ribonuclease A toward bovine seminal ribonuclease: a step by step thermodynamic analysis.

A proline, a leucine, and two cysteine residues, introduced at positions 19, 28, 31, and 32 of bovine pancreatic RNase A, i.e. the positions occupied by these residues in the subunit of bovine seminal RNase, the only dimeric RNase of the pancreatic-type superfamily, transform monomeric RNase A into a dimeric RNase, endowed with the same ability of BS-RNase of swapping its N-terminal segments. The thermodynamic consequences of the progressive introduction of these four residues into RNase A polypeptide chain have been studied by comparing the temperature- and urea-induced denaturation of three mutants of RNase A with that of a stable monomeric derivative of BS-RNase. The denaturation processes proved reversible for all proteins, and well represented by the two-state N<-->D transition model. The progressive introduction of the four residues into RNase A led to a gradual shift of the protein stability toward that characteristic of monomeric BS-RNase, which, in turn, is markedly less stable than RNase A with respect to both temperature- and urea-induced denaturation. On the other hand, the thermal stability of a dimeric active mutant of RNase A is found to approach that of wild-type seminal RNase.

Animals↗
PubMed1993

Two distinct secretory ribonucleases from human cerebrum: purification, characterization and relationships to other ribonucleases.

Two RNAases from human cerebrum were purified to an electrophoretically homogeneous state and their molecular masses were 22.0 kDa (tentatively called RNAase HB-1) and 19.0 kDa (RNAase HB-2). Analyses of the amino acid compositions, N-terminal amino acid sequences and catalytic properties of these enzymes provided strong evidence that they were strictly related to the secretory (sec) RNAases, such as the pancreatic enzyme, very similar immunologically to urinary sec RNAase, but clearly distinguishable from urinary non-secretory (nonsec) RNAase. There were several differences between HB-1 and HB-2, namely their immunological reactivities with specific antibodies, heat-stabilities, attached carbohydrate moieties and molecular masses. In particular, HB-2 appeared to be nonglycosylated, in view of its lack of affinity for several conjugated lectins, the absence of hexosamine and no change in electrophoretic mobility before and after peptide:N-glycosidase F digestion, whereas HB-1 and human sec RNAases purified from kidney, pancreas and urine all appeared to be glycosylated, as they moved to the same position as HB-2 when electrophoresed after glycosidase digestion. An antibody against urinary sec RNAase inhibited 75% and 20% of the total activity of the crude cerebral extract against RNA at pH 8.0 and 6.0 respectively, whereas an antibody against urinary nonsec RNAase had no such inhibitory effect. These findings suggest that yet another type(s) of cerebral RNAase, which is unable to cross-react immunologically with sec and nonsec RNAases, may exist. Two RNAases corresponding to HB-1 and HB-2 were identified in fresh cerebrospinal fluid.

Amino Acid Sequence↗
PubMed1966

Studies on the function of intracellular ribonucleases. V. Ribonuclease activity in ribosomes and polysomes prepared from rat liver and hepatomas.

The RNase activity and properties of ribosome and polysome preparations from normal rat liver and some hepatomas have been examined. Polysome and ribosome preparations from the Novikoff, McCoy MDAB, and Dunning hepatomas had considerably higher specific RNase activity than corresponding preparations from normal rat liver, Novikoff ascites, or Morris 5123 hepatomas. The optimum pH of the RNase was approximately 8.5 for all samples tested, and the samples showed no evidence of latent RNase activity when treated with 3 M sodium chloride, EDTA, urea, or p-chloromercuribenzenesulfonic acid. The RNase activity appeared to be associated principally with breakdown products and/or subunits smaller than 80S. In the presence of Mg(++) ions, subunits could reaggregate to form monomer ribosomes indistinguishable from the natural products, but some of the reassociated ribosomes could contain RNase activity which had been bound to the smaller particles. Similar results were obtained with spermine. In the hepatomas, evidence was obtained for the preexistence of considerable amounts of the smaller, RNase-containing subunits in the cell. When a small amount of crystalline bovine pancreatic RNase was added to partly dissociated ribosomes, the RNase was found only in association with the smaller subunits, and little or no enzyme was taken up by ribosomes or polysomes. The results have led to the conclusion that RNase is not a normal constituent of the ribosome or polysome, but that RNase may become associated with these particulates if dissociation and reassociation take place. Some implications of these findings for the stability of messenger RNA and for the mechanism of its breakdown are discussed.

Animals↗