Characterization of ribonucleases and ribonuclease inhibitor in subcellular fractions from rat adrenals

1. The presence of two RNA-degrading enzymes, one with optimum activity at pH5.6 (acid ribonuclease) and the other with optimum activity at pH7.8 (alkaline ribonuclease), in rat adrenals has been demonstrated. The acid ribonuclease was localized in the mitochondrial fraction whereas the alkaline ribonuclease was present in mitochondria as well as in the supernatant fraction. Freezing and thawing of mitochondria and treatment with Triton X-100 gave a three- to four-fold increase in acid-ribonuclease activity, whereas the mitochondrial alkaline-ribonuclease activity was practically unaffected. 2. The amount of free ribonuclease in the adrenal supernatant was small. Treatment of the supernatant fraction with N-ethylmaleimide resulted in release of large amounts of ribonuclease activity, indicating the presence of a ribonuclease inhibitor having reactive thiol groups. 3. Considerable amounts of free ribonuclease inhibitor in excess over the bound alkaline ribonuclease are present in the rat-adrenal supernatant fraction. The inhibitor is heat-labile and non-diffusible. A 400-500-fold purification of the ribonuclease inhibitor was achieved by ammonium sulphate fractionation, treatment with calcium phosphate gel and DEAE-cellulose chromatography. It is concluded that the adrenal inhibitor is protein in nature, similar to the inhibitor present in rat liver.     

Interaction of human placental ribonuclease with placental ribonuclease inhibitor

The interactions of human placental ribonuclease inhibitor (PRI) with bovine pancreatic ribonuclease (RNase) A and human angiogenin, a plasma protein that induces blood vessel formation, have been characterized in detail in earlier studies. However, studies on the interaction of PRI with the RNase(s) indigenous to placenta have not been performed previously, nor have any placental RNases been identified. In the present work, the major human placental RNase (PR) was purified to homogeneity by a five-step procedure and was obtained in a yield of 110 micrograms/kg of tissue. The placental content of angiogenin was also examined and was found to be at least 10-fold lower than that of PR. On the basis of its amino acid composition, amino-terminal sequence, and catalytic properties, PR appears to be identical with an RNase previously isolated from eosinophils (eosinophil-derived neurotoxin), liver, and urine. The apparent second-order rate constant of association for the PR.PRI complex, measured by examining the competition between PR and angiogenin for PRI, is 1.9 X 10(8) M-1 s-1. The rate constant for dissociation of the complex, determined by HPLC measurement of the rate of release of PR from its complex with PRI in the presence of a scavenger for free PRI, is 1.8 X 10(-7) s-1. Thus the Ki value for the PR.PRI complex is 9 X 10(-16) M, similar to that obtained with angiogenin, and 40-fold lower than that measured with RNase A. Complex formation causes a small red shift in the protein fluorescence emission spectrum, with no significant change in overall intensity. The fluorescence quantum yield of PR and the Stern-Volmer constant for fluorescence quenching by acrylamide are both high, possibly due to the presence of an unusual posttranslationally modified tryptophan residue at position 7 in the primary sequence.     

The antioxidant effects of ribonuclease inhibitor

Ribonuclease inhibitor (RI) is an acidic cytosolic glycoprotein with molecular weight of about 50 kDa, which contains 32 cysteine residues. It is possibly that RI may have antioxidant effect by thiol-disulfide exchange reaction. We studied the effects of RI over-expression on the rat glial cell line C6 injured with H2O2. The transfected C6 cells with RI cDNA (C6') had higher viability, less LDH leakage and MDA contents, but more GSH contents compare that in the control C6 cells. In transfected C6 cells, the activities of CAT and GST were higher than that in the control C6 cells. Without H202 stress, the activities of CAT and GST in the C6' cells were 1.73 and 3.62 times that in the control C6 cells, respectively; With 1.00 mmol/L H2O2 stress, the activities of CATand GSTin the C6' cells were 3.38 and 2.11 times that in the C6 cells, respectively. These results suggest that the over-expression RI has antioxidant activity and it is able to protect cells from per-oxidative injuries. Moreover, we investigated whether RI has a protective role against mouse hepatic damage in vivo. The mice pretreated with different doses of human RI were injected by CC14. The results show that the SOD activities of therapy groups were significantly higher than that of the control group (p < 0.01), while the contents of MOD and activities of ALT and AST in blood were remarkably lower than that of the control group (p < 0.01). Pathological examination shows that the degree of damage was alleviated with RI therapy. These results suggest that RI has the protective role against mouse hepatic damage induced by CC14. The anti-oxidative effects of RI may play an important role in cell protection from per-oxidative injuries.     

Comparison of the structure of turtle pancreatic ribonuclease with those of mammalian ribonucleases

There are 33 invariant amino acid positions out of 132 positions in 42 investigated sequences of ribonucleases from a number of mammalian species and a reptile (snapping turtle, Chelydra serpentina). These invariant residues are unequally distributed over 3 different parts of the molecule. The lobe of the S-protein part of the molecule, which lacks one disulfide bridge and has two shortened loops in turtle ribonuclease, has the lowest percentage of invariant residues, although the active-site residue His 119 is located in this part.     

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.     

Analysis of the interactions of ribonuclease inhibitor with kanamycin

The interaction of ribonuclease inhibitor (RI) with kanamycin was studied by molecular modeling. The preliminary binding model was constructed using the Affinity module of the Insight II molecular modeling program and the key residues involved in the combination of RI binding to kanamycin were determined. Meanwhile, we determined relevant surface characteristics determining the interaction behavior. The modeling results indicated that electrostatic interactions and H-bond forces may work as major factors for the molecular interaction between kanamycin and RI. The above results are useful for elucidating the molecular principles upon which the selectivity of a kanamycin is based. The quartz-crystal microbalance (QCM) is a new method usually used to monitor the binding function of macromolecules with samples online in a flow-injection analysis (FIA) system. The experimental results demonstrate that kanamycin has an extraordinary affinity to the basic protein RI, and our result is consistent with the molecular modeling results. These principles can in turn be used to study the molecular recognition mechanism and design a mimic of kanamycin for the development of new RI binders.Figure Proposed binding model of kanamycin to RI obtained by the computer-aided docking method and optimized with molecular mechanics with the CVFF forcefield.     

Interaction of onconase with the human ribonuclease inhibitor protein

One of the tightest known protein-protein interactions in biology is that between members of the ribonuclease A superfamily and the ribonuclease inhibitor protein (RI). Some members of this superfamily are able to kill cancer cells, and the ability to evade RI is a major determinant of whether a ribonuclease will be cytotoxic. The archetypal cytotoxic ribonuclease, onconase (ONC), is in late-stage clinical trials for the treatment of malignant mesothelioma. We present here the first measurement of the inhibition of the ribonucleolytic activity of ONC by RI. This inhibition occurs with K_i = 0.15 μM in a solution of low salt concentration.     

Revisiting the action of bovine ribonuclease A and pancreatic-type ribonucleases on double-stranded RNA

Single-strand-preferring ribonucleases of the pancreatic type, structurally and/or catalytically similar to bovine RNase A but endowed with a higher protein basicity, are able to degrade double-stranded RNA (dsRNA) or DNA: RNA hybrids under standard assay conditions (0.15 M NaCl, 0.015 M sodium citrate, pH 7), where RNase A is inactive. This enzyme too, however, becomes quite active if assay conditions are slightly modified or its basicity is increased (polyspermine-RNase). In the attempt to review these facts, we have analyzed and discussed the role that in the process have the secondary structure of dsRNA as well as other variables whose influence has come to light in addition to that of the basicity of the enzyme protein, i.e., the ionic strength, the presence of carbohydrates on the RNase molecule, and the structure (monomeric or dimeric) of the enzyme. A possible mechanism by which dsRNAs are attacked by pancreatic-type RNases has been proposed.Abbreviations RNase Ribonuclease - dsRNA Double-stranded RNA - ssRNA Single-stranded RNA - poly(A) poly(U), poly(I) : poly(C) Double-stranded Homopolymers formed between Polyadenylate and Polyurydilate, and Polyinosinate and Polycytidylate, respectively - poly(dA): poly (rU) Double-stranded complex formed between Polydeoxyriboadenylate and Polyribouridylate - poly(A), poly(C) Polyadenylate and Polycytidylate, respectively - poly[d(A-T)] Double-stranded Homopolymers formed between Polydeoxyriboadenilate and Polydeoxyribothymidylate - poly(dA-dT) : poly (dA-dT) Double-stranded alternating copolymers - SSC 0.15 M Sodium Chloride, 0.015 M Sodium Citrate pH 7     

Interaction of human pancreatic ribonuclease with human ribonuclease inhibitor. Generation of inhibitor-resistant cytotoxic variants

Mammalian ribonucleases interact very strongly with the intracellular ribonuclease inhibitor (RI). Eukaryotic cells exposed to mammalian ribonucleases are protected from their cytotoxic action by the intracellular inhibition of ribonucleases by RI. Human pancreatic ribonuclease (HPR) is structurally and functionally very similar to bovine RNase A and interacts with human RI with a high affinity. In the current study, we have investigated the involvement of Lys-7, Gln-11, Asn-71, Asn-88, Gly-89, Ser-90, and Glu-111 in HPR in its interaction with human ribonuclease inhibitor. These contact residues were mutated either individually or in combination to generate mutants K7A, Q11A, N71A, E111A, N88R, G89R, S90R, K7A/E111A, Q11A/E111A, N71A/E111A, K7A/N71A/E111A, Q11A/N71A/E111A, and K7A/Q11A/N71A/E111A. Out of these, eight mutants, K7A, Q11A, N71A, S90R, E111A, Q11A/E111A, N71A/E111A, and K7A/N71A/E111A, showed an ability to evade RI more than the wild type HPR, with the triple mutant K7A/N71A/E111A having the maximum RI resistance. As a result, these variants exhibited higher cytotoxic activity than wild type HPR. The mutation of Gly-89 in HPR produced no change in the sensitivity of HPR for RI, whereas it has been reported that mutating the equivalent residue Gly-88 in RNase A yielded a variant with increased RI resistance and cytotoxicity. Hence, despite its considerable homology with RNase A, HPR shows differences in its interaction with RI. We demonstrate that interaction between human pancreatic ribonuclease and RI can be disrupted by mutating residues that are involved in HPR-RI binding. The inhibitor-resistant cytotoxic HPR mutants should be useful in developing therapeutic molecules.     

The antiviral action of a modified bacterial ribonuclease]

The antiviral activity of a bacterial ribonuclease conjugate with chitosane of Kamchatka crab (in a form of water soluble chito-oligosaccharides) has been studied. The conjugate inhibitory activity for A and B viruses as well as to Sindbis arbovirus in tissue cultures is shown. The preparation efficiency at intramuscular and intranasal administration was observed at experimental influenza infection of white mice.     

Purification of rat liver particulate neutral ribonuclease and comparison of properties with pancreas and serum ribonucleases.

Rat liver particulate neutral ribonuclease (EC 3.1.4.22) was extensively purified (up to 40000-fold). It is shown to be an endonuclease, specific for pyrimidine bases, hydrolysing 5'-phosphate ester bonds. The enzyme specificity, Km, pH optimum, stability in acid medium and thermal stability at high temperature are the same as those of rat pancreatic and serum ribonucleases. Like pancreatic and serum neutral ribonucleases, the hepatic enzyme is sensitive to the liver natural inhibitor. This inhibitor was purified 8000-fold; its association with ribonuclease follows zero-order kinetics. These identical properties for ribonuclease of rat liver, pancreas and serum support the hypothesis [Bartholeyns, Peeters-Joris & Baudhuin (1975) Eur. J. Biochem. 60, 385-393] of an extrahepatic origin for the liver enzyme, the plasma ribonuclease of pancreatic origin being taken up by endocytosis in the liver. Neutral ribonuclease activity was detected in all rat organs investigated; its distribution among tissues is different from the distribution of the natural ribonuclear inhibitor.     

The cytosolic ribonuclease inhibitor contributes to intracellular redox homeostasis

The hypothesis that the cytosolic RNase inhibitor (cRI) has a role in the protection of the cellular redox homeostasis was investigated testing the effects of oxidants and anti-oxidants on normal, primary endothelial HUVE cells, and malignant HeLa cells, before and after their engineering into cRI-deprived cells. We found that cRI plays an important, possibly a key, physiological role in the protection of cells from redox stress, as demonstrated by decreased GSH levels as well as increased oxidant-induced DNA damage in cRI deprived cells.     

Inhibition of human pancreatic ribonuclease by the human ribonuclease inhibitor protein

The ribonuclease inhibitor protein (RI) binds to members of the bovine pancreatic ribonuclease (RNase A) superfamily with an affinity in the femtomolar range. Here, we report on structural and energetic aspects of the interaction between human RI (hRI) and human pancreatic ribonuclease (RNase 1). The structure of the crystalline hRI x RNase 1 complex was determined at a resolution of 1.95 A, revealing the formation of 19 intermolecular hydrogen bonds involving 13 residues of RNase 1. In contrast, only nine such hydrogen bonds are apparent in the structure of the complex between porcine RI and RNase A. hRI, which is anionic, also appears to use its horseshoe-shaped structure to engender long-range Coulombic interactions with RNase 1, which is cationic. In accordance with the structural data, the hRI.RNase 1 complex was found to be extremely stable (t(1/2)=81 days; K(d)=2.9 x 10(-16) M). Site-directed mutagenesis experiments enabled the identification of two cationic residues in RNase 1, Arg39 and Arg91, that are especially important for both the formation and stability of the complex, and are thus termed "electrostatic targeting residues". Disturbing the electrostatic attraction between hRI and RNase 1 yielded a variant of RNase 1 that maintained ribonucleolytic activity and conformational stability but had a 2.8 x 10(3)-fold lower association rate for complex formation and 5.9 x 10(9)-fold lower affinity for hRI. This variant of RNase 1, which exhibits the largest decrease in RI affinity of any engineered ribonuclease, is also toxic to human erythroleukemia cells. Together, these results provide new insight into an unusual and important protein-protein interaction, and could expedite the development of human ribonucleases as chemotherapeutic agents.