Purification and characterization of truncated ribonuclease inhibitor.
Ontology highlight
ABSTRACT:
SUBMITTER: Hofsteenge J
PROVIDER: S-EPMC1150084 | biostudies-other | 1991 Apr
REPOSITORIES: biostudies-other
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Project description:BACKGROUND: Most isolates of Pectobacterium carotovorum subsp. carotovorum (Pcc) produce bacteriocins. In this study, we have determined that Pcc strain F-rif-18 has a chromosomal gene encoding the low-molecular-weight bacteriocin, Carocin S2, and that this bacteriocin inhibits the growth of a closely related strain. Carocin S2 is inducible by ultraviolet radiation but not by mutagenic agents such as mitomycin C. RESULTS: A carocin S2-defective mutant, TF1-2, was obtained by Tn5 insertional mutagenesis using F-rif-18. A 5706-bp DNA fragment was detected by Southern blotting, selected from a genomic DNA library, and cloned to the vector, pMS2KI. Two adjacent complete open reading frames within pMS2KI were sequenced, characterized, and identified as caroS2K and caroS2I, which respectively encode the killing protein and immunity protein. Notably, carocin S2 could be expressed not only in the mutant TF1-2 but also in Escherichia coli DH5? after entry of the plasmid pMS2KI. Furthermore, the C-terminal domain of CaroS2K was homologous to the nuclease domains of colicin D and klebicin D. Moreover, SDS-PAGE analysis showed that the relative mass of CaroS2K was 85 kDa and that of CaroS2I was 10 kDa. CONCLUSION: This study shown that another nuclease type of bacteriocin was found in Pectobacterium carotovorum. This new type of bacteriocin, Carocin S2, has the ribonuclease activity of CaroS2K and the immunity protein activity of CaroS2I.
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Project description:Onconase (ONC) is an amphibian member of the bovine pancreatic ribonuclease (RNase A) superfamily that exhibits innate antitumoral activity. ONC has been granted both orphan-drug and fast-track status by the U.S. Food and Drug Administration for the treatment of malignant mesothelioma, and is poised to become the first chemotherapeutic agent based on a ribonuclease. Investigations into the mechanism of ribonuclease-based cytotoxicity have elucidated several important determinants for cytotoxicity, including efficient deliverance of ribonucleolytic activity to the cytosol and preservation of conformation stability. Nevertheless, the most striking similarity between ONC and bovine seminal ribonuclease, another naturally cytotoxic ribonuclease, is their insensitivity to inhibition by the potent cytosolic ribonuclease inhibitor protein (RI). RI typically binds to its ribonuclease ligands with femtomolar affinity--an extraordinary feat considering the modest sequence identity among the bound ribonucleases. Mammalian ribonucleases such as RNase A or its human homologue, RNase 1, have the potential to be more attractive chemotherapeutic agents than ONC owing to their higher catalytic activity, low potential for immunogenicity, favorable tissue distribution, and high therapeutic index, but are limited by their sensitivity to RI. These non-toxic mammalian ribonucleases can be transformed into potent cytotoxins by engendering them with RI-evasion using protein engineering strategies such as site-directed mutagenesis, multimerization, fusion to a targeting moiety, and chemical modification. In several instances, these engineered ribonucleases exhibit greater cytotoxicity in vitro than does ONC. Herein, we review the biochemical characteristics of RIribonuclease complexes and progress towards the development of mammalian ribonuclease-based chemotherapeutics through the elicitation of RI-evasion.
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Project description:Human angiogenin (ANG) is a homologue of bovine pancreatic ribonuclease (RNase A) that induces neovascularization. ANG is the only human angiogenic factor that possesses ribonucleolytic activity. To stimulate blood vessel growth, ANG must be transported to the nucleus and must retain its catalytic activity. Like other mammalian homologues of RNase A, ANG forms a femtomolar complex with the cytosolic ribonuclease inhibitor protein (RI). To determine whether RI affects ANG-induced angiogenesis, we created G85R/G86R ANG, which possesses 10(6)-fold lower affinity for RI but retains wild-type ribonucleolytic activity. The neovascularization of rabbit corneas by G85R/G86R ANG was more pronounced and more rapid than by wild-type ANG. These findings provide the first direct evidence that RI serves to regulate the biological activity of ANG in vivo.
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Project description: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.
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