Cusativin, a new cytidine-specific ribonuclease accumulated in seeds of Cucumis sativus L.

Abstract

Dry seeds of Cucumis sativus L. were found to contain a heat-sensitive endoribonuclease of a novel type which we have named cusativin. It was purified to apparent electrophoretic homogeneity by chromatography through S-Sepharose Fast Flow, Sephadex G-75, CM-Sepharose, Superdex 75-FPLC (fast protein liquid chromatography) and Mono S-FPLC. It is a single unglycosylated polypeptide chain with an apparent molecular mass (M(r)) of 22900. Polyclonal anti-cusativin antibodies raised in rabbits only reacted with melonin, the translation inhibitor from Cucumis melo L. Functional, Western blot and enzyme-linked immunosorbent assay (ELISA) analyses indicated that cusativin is present in the coat and cotyledons of dry seeds, but not in embryonic axes. Cusativin is accumulated in maturing seeds. By contrast, after seed germination there is degradation of the cusativin present in cotyledons but not that present in the seed coat. The preference of cusativin for polynucleotide cleavage was poly(C) >> poly(A) acids, poly(U) and poly(G) being unaffected by cusativin. Under the denaturing conditions used for RNA sequencing, cusativin acted only on poly(C). Cusativin proved to be useful for RNA sequencing, in particular, complementing the data obtained with RNase CL3. Cusativin represents a new class of plant RNase and, as far as we are aware, is the first plant enzyme that shows cleavage specificity for cytidine under the denaturing conditions of RNA sequencing.

Ebulitins: A new family of type 1 ribosome‐inactivating proteins (rRNA N‐glycosidases) from leaves of Sambucus ebulus L. that coexist with the type 2 ribosome‐inactivating protein ebulin 1

“…All chemicals, biochemicals and radioactive materials were of the highest purity available and were obtained as in previous reports [5,16]. Mature leaves from Sambucus ebulus L. were collected from Cobos de Cerrato (Palencia, Spain) in the summer.…”

“…Rat liver and plant cell-free translation systems (wheat germ, Vicia sativa and Cucumis melo) were prepared as described previously [16,20,21]. The cell-free extracts were filtered through Sephadex G-25 to remove low M~ compounds and stored in small aliquots under liquid N 2 until use.…”

“…The cell-free extracts were filtered through Sephadex G-25 to remove low M~ compounds and stored in small aliquots under liquid N 2 until use. Translation was coded by endogenous messengers and in all cases the reaction mixtures were optimized for concentrations of ions and the 30,000 × g supernatant [16,20,21]. Control reaction mixtures without inhibitor were carried out separately.…”

“…Protein was hydrolysed with 100 pl of 5.7 M HC1 containing 0.05% (v/v) 2-mercaptoethanol in evacuated and sealed tubes at 110°C for 24 h. Analysis was performed as indicated elsewhere [16].…”

“…SDS-polyacrylamide gel electrophoresis of proteins was carried out as has been reported previously [16].…”