Semisynthetic studies on bovine pancreatic ribonuclease

The S-peptide of the enzyme bovine pancreatic ribonuclease has been used as a model for covalent semisynthesis. Methods for side-chain protection, enzymatic cleavage of the peptide chain at the level of the single arginine-10 and for selective deprotection of the alpha-carboxyl function of this residue, have been examined. The partially protected [1-10] sequence has been coupled to a solid-phase generated [11-15] sequence attached to the polymer. After deblocking from the solid-support, the [1-15] semisynthetic peptide was complexed with native S-protein to give a complex with high biological activity.     

Kinetic studies on turtle pancreatic ribonuclease: a comparative study of the base specificities of the B2 and P0 sites of bovine pancreatic ribonuclease A and turtle pancreatic ribonuclease

Kinetic constants for the transesterification of eight dinucleoside phosphates CpX and UpX by bovine and turtle pancreatic ribonuclease were determined. Both ribonucleases have a preference for purine nucleotides at the position X. However, bovine ribonuclease, like other mammalian ribonucleases, prefers 6-amino bases at this site, while turtle ribonuclease prefers 6-keto bases. This difference in specificity at the B2 site may be explained by the substitution of glutamic acid at position 111 by valine in turtle ribonuclease. These results have been confirmed by inhibition studies with the four nucleoside triphosphates. Inhibition studies with pT and pTp showed that a cationic binding group (P0) for the 5'-phosphate of the pyrimidine nucleotides bound at the primary B1 site is present in turtle ribonuclease, although lysine at position 66 in bovine ribonuclease is absent in turtle ribonuclease. However, the side chain of lysine 122 in turtle ribonuclease is probably located in the correct position to take over the role as cationic P0 site.     

Modification of amino acids and bovine pancreatic ribonuclease A by kethoxal.

Kethoxal (3-ethoxy-2-ketobutanal) reacts with the guanidino group of Nalpha-acetylarginine to produce four derivatives, reactive to periodate, stable at pH 7, with 15% reverting to arginine on acid hydrolysis. Other amino acids with blocked alpha-amino groups do not react, except the epsilon-amino of lysine (slowly). The pK of the mixed Kethoxal-Nalpha-acetylarginine derivatives is 5.8-6.1. Kethoxal reacts at neutral pH with arginyl residues of bovine pancreatic ribonuclease A. In the presence of an active-site ligand, arginine-39 and arginine-85 react at about equal rates. The loss of enzymic activity at pH 7 is proportional to the combined loss of these residues. The enzymic activity toward RNA is 20-25% of that of native RNAase at pH 7, and 90-100% at pH 5. In the absence of an active site ligand, arginine-10 is also modified with the loss of almost all enzymic activity, although arginine-10 is not an active-site residue. Arginine-33 is unreactive. Kethoxal-modified RNAase undergoes cross-linking in solution at pH 7 or in the freeze-dried state, Incubation at pH 9 in the presence of homoarginine results in partial regeneration of arginyl residues and activity at pH 7. Kethoxal modification of arginines-39 and -85 appears to raise the pK of lysine-41 by about 1 unit, as indicated ty the pH dependence of arylation by 2-carboxy-4,6-dinitrochlorobenzene. The claims of Patthy and Smith (J. Biol, Chem. (1975) 250, 565-569), and of Takahashi (J. Biol. Chem. (1968) 243, 6171-6179) that arginine-39 is a more important functional residue than is arginine-85 are questioned.     

1H-NMR studies on the binding subsites of bovine pancreatic ribonuclease A

The titration curves of the C-2 histidine protons of an RNAase derivative (a covalent derivative obtained by reaction of bovine pancreatic RNAase A (EC 3.1.27.5) with 6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate) were studied by means of 1H-NMR spectroscopy at 270 MHz. The interaction of natural (5'AMP, 5'GMP, 5'IMP) and halogenated purine mononucleotides (cl6RMP, br8AMP) with RNAase A was also monitored by using the same technique. The slight change observed in the pK values of the active centre histidine residues of the RNAase derivative, with respect to those in the native enzyme, can be considered as evidence that the phosphate of the label does not interact directly either with His-12 or 119 in the p1 site, but the p2 site as proposed previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571--579). Lys-7 and/or Arg-10 are proposed as part of the p2 phosphate-binding subsite. The pK values of His-12 and 119 and the shift of an aromatic resonance of the native enzyme found on interaction with some purine nucleotides, can be interpreted by postulating that the interaction of 5'AMP, 5'GMP and 5'IMP takes place not only in the so-called purine-binding site B2R2p1 but also in the primary pyrimidine-binding site B1R1 and p0 of RNAase A.     

Effect of bovine seminal ribonuclease and bovine pancreatic ribonuclease A on bovine oocyte maturation

Bovine seminal ribonuclease (BS-RNase) contains the MxM (noncovalent dimer) and M=M (free monomer) in constant ratio. The aim of this work was to evaluate the effect of BS-RNase, its monomer and dimer forms, and also various mutants of this enzyme on meiotic completion in cattle oocytes. It was found that BS-RNase has irreversible effects on the meiotic maturation of bovine oocytes in vitro, particularly on the completion of meiosis. The effect of BS-RNase is dose-dependent. In medium supplemented with 1 microg/ml, the results were comparable with those of the control (70% MII oocytes after 24 hr of culture). Whereas 5 microg/ml reduced the number of MII oocytes to 50%, 10 and 25 microg/ml arrested this process completely. The MxM form and RNase A at 5 microg/ml inhibited the maturation rate by 71 and 48%, respectively, but a less significant effect was observed for the M=M form, or the carboxymethylated monomers MCM31 and MCM32 (21%, 16%, and 42% MII oocytes, respectively, in comparison with control). These data demonstrate that bovine ribonucleases can have variable detrimental effects on the maturation of bovine oocyte. J. Exp. Zool. 287:394-399, 2000.     

Structural studies of a folding intermediate of bovine pancreatic ribonuclease A by continuous recycled flow

A new technique, continuous recycled flow (CRF) spectroscopy, has been developed for observing intermediates of any thermally induced, reversible reaction with a half-life of 10 s or longer. The structure can be probed by any spectroscopic method which does not perturb the system. Prolonged signal acquisitions of 8 h for ribonuclease A are possible. CRF was used to investigate the structure of the slow-folding intermediates of chemically intact ribonuclease A (RNase A) during thermal unfolding/folding under acidic conditions. The following conclusions were reached on the basis of the proton nuclear magnetic resonance and far-ultraviolet circular dichroism spectra of a folding intermediate(s): (A) The conformation of the detected folding intermediate(s) is similar to that of the heat-denatured protein. There is only limited formation of new structures. (B) The N-terminal alpha-helix is partially stable under these conditions and is in rapid (less than 10 ms) equilibrium with the denatured conformation. (C) There are long-range interactions between the hydrophobic residues of the N-terminal alpha-helix and the rest of the protein. These interactions persist well above the melting point. (D) An aliphatic methyl group reports on the formation of a new structure(s) that lie(s) outside of the N-terminal region. (E) The structures detected in chemically modified, nonfolding forms of the RNase A are also present in the folding intermediate(s). There are, however, additional interactions that are unique to chemically intact RNase A.     

Kinetic studies on the depolymerization of polyadenylic acid by ribonuclease A.

Studies were conducted on the depolymerization of polyadenylic acid (poly (A)) by RNAse A (EC 3.1.4.22) depending on the pH (pH 5-8). The results showed that depending on the pH, the ratio Vmax/Km was analogous to that described earlier for nucleoside-2', 3'-cyclophosphates and dinucleoside phosphates. This indicates that depolymerization of poly (A), transesterification and hydrolysis of specific substrates is achieved by the same ionizing groups of the enzyme with pKa 5.4 and pKb 6.4. The rate of degradation of poly (A) is also influenced by the state of adenine ionization, the protonation of which leads to the formation of a double helical poly (A), and does not serve as a substrate for RNAse A. The low rate for the depolymerization of poly (A) in the presence of RNAse A is related to a decrease in the turnover number of the enzyme, and an increase in the molecular weight of the enzyme (RNAse dimer), leads to a decrease in the Km, and does not effect Vmax. This indicates that the rate of depolymerization of polynucleotides is determined by orientation of factors. On the basis of the comparison of the resultant kinetic data, and the structure of the enzyme inhibitory complexes of RNAse S, which were studied by the method of x-ray structural analysis, a conclusion was reached on the kinetic characteristics of RNAse A specificity with respect to polymeric substrates, which is determined by the orinetation of the ribose phosphate relative to the catalytic groups of the active site.     

Interferon-gamma activates the cleavage of double-stranded RNA by bovine seminal ribonuclease

Bovine seminal ribonuclease (BS-RNase), a dimeric homologue of RNase A, cleaves both single- and double-stranded RNA and inhibits the growth of tumor cells. Its catalytic activity against double-stranded RNA, either homopolymeric ([3H]polyA/polyU) or mixed sequence, is enhanced by bovine or human recombinant interferon-gamma (IFN-gamma). Activation is seen with as little as 4-10 interferon units per assay. Enhancing the degradation of double-stranded RNA, an intermediate in the growth cycle of many viruses, could contribute to IFN-gamma's ability to control cell growth and induce an antiviral state.     

Expression of bovine pancreatic ribonuclease A in Escherichia coli

A synthetic gene for bovine pancreatic ribonuclease A (RNase A) has been expressed in Escherichia coli as a fusion protein with beta-galactosidase linked by the tetrapeptide Ile-Glu-Gly-Arg. RNase A was cleaved from the fusion using factor Xa, and the resulting product purified and reconstituted. The isolated RNase A was chromatographically, catalytically, and immunologically identical with authentic RNase A. This work argues that the method suggested by Nagai and Thogersen [Nagai, K. & Thogersen, H. C. (1984) Nature (Lond.) 309, 810-812] for releasing fusion proteins is quite general, even when applied to particularly complicated expression problem. The procedure here makes RNase A available for the first time as a model for studying structure-function relationships in proteins using site-directed mutagenesis.