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.     

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.     

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.     

Modification of bovine pancreatic ribonuclease A with 6-chloropurine riboside

The chemical modification of bovine pancreatic ribonuclease A by 6-chloropurine riboside was studied to obtain information about the role of the purine nucleoside moiety of the ribonucleic acid in the enzyme-substrate interaction. The residues involved in the reaction were identified, after performic acid oxidation and trypsin digestion, by reverse-phase HPLC peptide mapping. The labeled peptides were detected by following the absorbance at 254 nm, and amino acid analyses of these peptides showed that the reaction had taken place with the amino groups of Lys-1, -37, -41, and -91. The specificity of the reaction was unaffected by changing the ligand:protein molar ratio. Partial separation of the reaction products was accomplished by means of chromatography on CM-Sepharose: four labeled fractions corresponding to mono- and bisubstituted derivatives were found. One of the monosubstituted fractions (fraction E) contained a homogeneous protein with the nucleoside bound to the alpha-amino group of Lys-1 whereas the other (fraction D) was a mixture of derivatives labeled in the epsilon-amino group of Lys-1, -37, -41, and -91. Kinetic studies of these two monosubstituted fractions were performed with cytidine 2',3'-phosphate and ribonucleic acid as substrates. These derivatives showed a noncompetitive inhibition-like behavior with respect to RNase A. Results support the existence of several RNase A regions with affinity for purine nucleosides.     

A semisynthetic bovine pancreatic ribonuclease containing a unique nitrotyrosine residue

A fully active semisynthetic ribonuclease, RNase 1-118:111-124, may be prepared by enzymatically removing six residues from the COOH terminus of the protein (positions 119-124) and then complementing the inactive RNase 1-118 with a chemically synthesized peptide containing the COOH-terminal 14 residues of the molecule (RNase 111-124) [M. C. Lin, B. Gutte, S. Moore, and R. B. Merrifield (1970) J. Biol. Chem. 245, 5169-5170]. Nitration of tyrosine-115 in the peptide followed by complex formation with RNase 1-118 affords a fully active enzyme containing a unique nitrotyrosine residue in a position which is known and which is very likely to be completely exterior to the active site region. The binding constant between the tetradecapeptide and RNase 1-118 (5 X 10(6) M-1 at pH 6.0) is not changed by the nitration. Crystals of the nitrated complex are isomorphous with those of RNase 1-118:111-124, for which a refined 1.8-A structure has recently been obtained.     

Isolation and characterization of monodeamidated derivatives of bovine pancreatic ribonuclease A

The isolation and characterization of the initial intermediates formed during the irreversible acid denaturation of enzyme Ribonuclease A are described. The products obtained when RNase A is maintained in 0.5 M HCl at 30 degrees for periods up to 20 h have been analyzed by ion-exchange chromatography on Amberlite XE-64. Four distinct components were found to elute earlier to RNase A; these have been designated RNase Aa2, Aa1c, Aa1b, and Aa1a in order of their elution. With the exception of RNase Aa2, the other components are nearly as active as RNase A. Polyacrylamide gel electrophoresis at near-neutral pH indicated that RNase Aa1a, Aa1b, and Aa1c are monodeamidated derivatives of RNase A; RNase Aa1c contains, in addition, a small amount of a dideamidated component. RNase Aa2, which has 75% enzymic activity as compared to RNase A, consists of dideamidated and higher deamidated derivatives of RNase A. Except for differences in the proteolytic susceptibilities at an elevated temperature or acidic pH, the monodeamidated derivatives were found to have very nearly the same enzymic activity and the compact folded structure as the native enzyme. Fingerprint analyses of the tryptic peptides of monodeamidated derivatives have shown that the deamidations are restricted to an amide cluster in the region 67-74 of the polypeptide chain. The initial acid-catalyzed deamidation occurs in and around the 65-72 disulfide loop giving rise to at least three distinct monodeamidated derivatives of RNase A without an appreciable change in the catalytic activity and conformation of the ribonuclease molecule. Significance of this specific deamidation occurring in highly acidic conditions, and the biological implications of the physiological deamidation reactions of proteins are discussed.     

Activity change during unfolding of bovine pancreatic ribonuclease A in guanidine

Bovine pancreatic ribonuclease A loses almost completely its activity in 2-3 M guanidine, whereas only very slight conformational changes can be detected when following its unfolding by changes in its intrinsic fluorescence at 305 nm and ultraviolet absorbance at 287 nm. Reactivation on diluting out the denaturant is a time-dependent process, indicating that the inactivation is not due to inhibition by a reversible association of the enzyme with guanidine. The kinetic method of following the substrate reaction, in the presence of the denaturant previously proposed for use in the study of rapid inactivation reactions (Tian, W.X. and Tsou, C.-L. (1982) Biochemistry 21, 1028-1032), is applied to examine the inactivation rates of this enzyme during guanidine denaturation, and these have been compared with the unfolding rates as followed by fluorescence and absorbance changes. It is shown that during the unfolding of this enzyme in guanidine, the inactivation of the enzyme occurs within the dead time of mixing in a stopped-flow apparatus and is at least several orders of magnitude faster than the unfolding reaction as detected by the optical parameters. It appears that, as in the case of creatine kinase reported previously, the active site of a small enzyme stabilized by multiple disulfide linkages, such as ribonuclease A, is also situated in a region which is much more liable to being perturbed by denaturants than is the molecule as a whole.