Academic literature on the topic 'BS-RNase'

Bovine seminal RNase (BS-RNase) is a dimeric RNase selectively cytotoxic for malignant cells. No information is available on its pathway from the extracellular matrix through the cytosol, where it degrades rRNA. An investigation of this pathway is reported here, carried out by immunofluorescence studies, by assessing the effects on BS-RNase cytotoxicity of drugs that affect specific intracellular compartments and by assaying the behaviour of a protein variant, BS-RNase-KDEL (BS-RNase in which a Lys-Asp-Glu-Leu peptide segment is inserted at the C-terminal ends of the subunit chains), endowed with a consensus sequence that directs proteins to the endoplasmic reticulum. BS-RNase was found to bind both normal and malignant cells and to be internalized by both cell types in endosome vesicles. Non-cytotoxic RNases, such as RNase A and a monomeric derivative of BS-RNase, did not bind to the cell surface and were not internalized. However, an engineered, dimeric and cytotoxic variant of RNase A bound effectively and permeated cells. The results of immunofluorescence studies, the effects of nigericin, monensin and brefeldin A on the cytotoxic action of seminal RNase, and the behaviour of the BS-RNase-KDEL variant, led to the conclusion that the pathway of BS-RNase in malignant cells from the extracellular matrix to the cytosol has two essential intracellular stations: endosomes and the trans-Golgi network. In normal cells, however, the protein does not progress from the endosomal compartment to the Golgi complex.

Bovine seminal ribonuclease (BS-RNase) is a homologue of RNase A with special biological properties, including potent immunosuppressive activity. A mutant BS-RNase was created in which His-119, the active-site residue that acts as a general acid during catalysis, was changed to an aspartic acid. H119D BS-RNase formed a dimer with quaternary structure similar to that of the wild-type enzyme but with values of kcat. and kcat./Km for the cleavage of UpA [uridylyl(3′-->5′)adenosine] that were 4 x 10(3)-fold lower. The mutant protein also demonstrated dramatically decreased immunosuppressive, anti-tumour, aspermatogenic, and embryotoxic activities. The catalytic activity of BS-RNase is therefore necessary for its special biological properties.

Abstract. The effect of bovine seminal ribonuclease (BS RNase) on bone marrow cells in miniature pigs was studied. BS RNase at the concentration 20 and l00 ug/ml preincubated 1 h with bone marrow cells from normal miniature pigs did not influence the formation of CFU-GM and BFU-E colonies cultivated in methylcellulose medium. The bone marrow cells kept in 4 °C survived better in the presence of BS RNase than control cells cocultivated with bovine serum albumin (BSA). There was proved that pig bone marrow cells are able to bind the BS RNase on their surface without any morphological damage. For the experimental bone marrow transplantation in miniature pigs the whole body irradiation of 8G with a cobalt source was used. Two recipients from seven accepted semi-incompatible bone marrow cells preincubated 2 h with BS RNase (10 mg/5 x l08 cells). No recipient from eight transplanted control animals accepted semi-incompatible bone marrow cells preincubated with BSA.

We have studied the inhibition of bovine pancreatic RNAase (RNAase A) and bovine seminal RNAase in its native dimeric form (RNAase BS-1) and in monomeric carboxymethylated form (MCM RNAase BS-1) by human placental RNAase inhibitor (RNAase inhibitor) in order to understand the effect of enzyme structure on its response to the inhibitor. Study of the inhibition as a function of inhibitor concentration revealed that RNAase A and MCM RNAase BS-1 were inhibited fully and the inhibitor-sensitivities of the two were comparable. But under identical inhibitor concentrations RNAase BS-1 was found to be virtually insensitive to the inhibitor; at higher (3-10-fold) inhibitor concentrations marginal inhibition of the native enzyme could be observed. When RNAase BS-1 was pretreated with 5 mM-dithiothreitol (DTT) and assayed, it exhibited greater inhibitor-sensitivity, presumably as a result of its partial monomerization on exposure to DTT. This DTT-mediated change in the response of RNAase BS-1 to the inhibitor did not, however, seem to occur either in the assay conditions (which included DTT) or even when the enzyme was pretreated with DTT in the presence of the substrate, suggesting an effect of the substrate on the enzyme behaviour towards the inhibitor. Independently, gel-filtration runs revealed that, although DTT treatment caused monomerization of RNase BS-1, this change did not take place when DTT treatment was carried out in the presence of the substrate. From our observations, we infer that differential inhibitor-sensitivity of the dimeric and monomeric forms of RNAase BS-1, the relative contents of the two forms and the influence of the substrate on them may be important determinants of the net enzyme activity in the presence of the inhibitor.

ABSTRACT A 320-nucleotide RNA with several characteristic features was expressed in Bacillus subtilis to study RNA processing. The RNA consisted of a 5′-proximal sequence from bacteriophage SP82 containing strong secondary structure, a Bs-RNase III cleavage site, and the 3′-proximal end of the ermC transcriptional unit. Comparison of RNA processing in a wild-type strain and a strain in which the pnpA gene, coding for polynucleotide phosphorylase (PNPase), was deleted, as well as in vitro assays of phosphate-dependent degradation, showed that PNPase activity could be stalled in vivo and in vitro. Analysis of mutations in the SP82 moiety mapped the block to PNPase processivity to a particular stem-loop structure. This structure did not provide a block to processivity in the pnpA strain, suggesting that it was specific for PNPase. An abundant RNA with a 3′ end located in the ermCcoding sequence was detected in the pnpA strain but not in the wild type, indicating that this block is specific for a different 3′-to-5′ exonuclease. The finding of impediments to 3′-to-5′ degradation, with specificities for different exonucleases, suggests the existence of discrete intermediates in the mRNA decay pathway.

"Zero-length" dimers of ribonuclease A, a novel type of dimers formed by two RNase A molecules bound to each other through a zero-length amide bond [Simons, B.L. et al. (2007) Proteins 66, 183-195], were analyzed, and tested for their possible in vitro cytotoxic activity. Results: (i) Besides dimers, also trimers and higher oligomers can be identified among the products of the covalently linking reaction. (ii) The "zero-length" dimers prepared by us appear not to be a unique species, as was instead reported by Simons et al. The product is heterogeneous, as shown by the involvement in the amide bond of amino and carboxyl groups others than only those belonging to Lys66 and Glu9. This is demonstrated by results obtained with two RNase A mutants, E9A and K66A. (iii) The "zero-length" dimers degrade poly(A).poly(U) (dsRNA) and yeast RNA (ssRNA): while the activity against poly(A).poly(U) increases with the increase of the oligomer's basicity, the activity towards yeast RNA decreases with the increase of oligomers' basicity, in agreement with many previous data, but in contrast with the results reported by Simons et al. (iv) No cytotoxicity against various tumor cells lines could be evidenced in RNase A "zero-length" dimers. (v) They instead become cytotoxic if cationized by conjugation with polyethylenimine [Futami, J. et al. (2005) J. Biosci. Bioengin. 99, 95-103]. However, polyethylenimine derivatives of RNase A "zero-length" dimers and native, monomeric RNase A are equally cytotoxic. In other words, protein "dimericity" does not play any role in this case. Moreover, (vi) cytotoxicity seems not to be specific for tumor cells: polyethylenimine-cationized native RNase A is also cytotoxic towards human monocytes.
"Zero-length" dimers of ribonuclease A, a novel type of dimers formed by two RNase A molecules bound to each other through a zero-length amide bond [Simons, B.L. et al. (2007) Proteins 66, 183-195], were analyzed, and tested for their possible in vitro cytotoxic activity. Results: (i) Besides dimers, also trimers and higher oligomers can be identified among the products of the covalently linking reaction. (ii) The "zero-length" dimers prepared by us appear not to be a unique species, as was instead reported by Simons et al. The product is heterogeneous, as shown by the involvement in the amide bond of amino and carboxyl groups others than only those belonging to Lys66 and Glu9. This is demonstrated by results obtained with two RNase A mutants, E9A and K66A. (iii) The "zero-length" dimers degrade poly(A).poly(U) (dsRNA) and yeast RNA (ssRNA): while the activity against poly(A).poly(U) increases with the increase of the oligomer's basicity, the activity towards yeast RNA decreases with the increase of oligomers' basicity, in agreement with many previous data, but in contrast with the results reported by Simons et al. (iv) No cytotoxicity against various tumor cells lines could be evidenced in RNase A "zero-length" dimers. (v) They instead become cytotoxic if cationized by conjugation with polyethylenimine [Futami, J. et al. (2005) J. Biosci. Bioengin. 99, 95-103]. However, polyethylenimine derivatives of RNase A "zero-length" dimers and native, monomeric RNase A are equally cytotoxic. In other words, protein "dimericity" does not play any role in this case. Moreover, (vi) cytotoxicity seems not to be specific for tumor cells: polyethylenimine-cationized native RNase A is also cytotoxic towards human monocytes.