M.A. Rojo

Isolation and partial characterization of a new ribosome-inactivating protein from Petrocoptis glaucifolia (Lag.) Boiss.

Petrocoptis glaucifolia, a paleoendemic member of the Caryophyllaceae from the North of Spain, was found to contain at least five proteins that inhibit protein synthesis in a rabbit reticulocyte lysate. One of them, for which the name petroglaucin is proposed, was purified to apparent electrophoretic homogeneity by chromatography through S-Sepharose Fast Flow, Sephadex G-75 and CM-Sepharose Fast Flow. The apparent Mr of the preparation was 27500. This protein does not contain appreciable glycan chains and displays 45.8% of NH2-terminal amino-acid sequence homology with some ribosome-inactivating proteins from Saponaria officinalis, another member of the Caryophyllaceae. Petroglaucin shows the following functional properties: (i) it strongly inhibits the rabbit-reticulocyte-lysate system and Vicia sativa cell-free extracts, both coded by endogenous messengers, and also inhibits poly(U)-directed polyphenylalanine synthesis by Vicia sativa cell-free extracts and purified rat-liver ribosomes; (ii) it shows much less inhibitory capacity in wheat-germ, Cucumis sativus and rat-liver cell-free systems coded by endogenous messengers; (iii) the inhibitory effects on purified rat-liver ribosomes were irreversible; (vi) it promotes the release of adenine from purified rat-liver ribosomes. The total activity of this translational inhibitor has been found to increase up to 11-fold during its purification, indicating that some regulatory factor that normally blocks the translational inhibitory activity of the ribosome-inactivating protein in crude extracts of the plant is removed during purification.

Isolation and characterization of two new N-glycosidase type-1 ribosome-inactivating proteins, unrelated in amino-acid sequence, from Petrocoptis species

Two new N-glycosidase type-1 ribosome-inactivating proteins (RIPs), denoted petroglaucin 1 and petrograndin, respectively, were isolated from the plantsPetrocoptis glaucifolia (Lag.) Boiss sp.viscosa (Rothm.) Laínz andPetrocoptis grandiflora Rothm. These new RIPs do not share H2N-terminal amino-acid sequence homology with petroglaucin (now denoted as petroglaucin 2), the only other type-1 RIP to be isolated fromP. glaucifolia (Arias et al. (1992) Planta186, 532–540). Petroglaucin 1 shares amino-acid sequence homology with RIPs from Cucurbitaceae while petroglaucin 2 and petrograndin do so with saporins and dianthin 30 (Caryophyllaceae). The new RIPs strongly inhibited protein synthesis at subnanomolar concentrations in rabbit reticulocyte lysates and other eukaryotic cell-free systems, but they were inactive on bacterial ribosomes.

Development of a cell-free translation system from Cucumis melo: preparation, optimization and evaluation of sensitivity to some translational inhibitors

Abstract A very active cell-free translation system was prepared from 4–5-day-old embryonic axes of melon (Cucumis melo L.), a species whose dry seeds contain a powerful translational inhibitor. The system was optimized for Mg2+, K+, NH4+, high speed supernatant, total wheat germ tRNA, time and temperature. Using a 30 000 × g supernatant, the system translates endogenous messengers and polyuridylic acid very efficiently. Melon ribosomes were inhibited in vitro by several well-known eukaryotic inhibitors including melonin, the protein inhibitor present in the dry seeds of C. melo. Our results suggest that the protein inhibitor does not affect the activity of melon ribosomes neither in vivo nor during their isolation.

Protein phosphorylation in a cell-free translation system from Vicia sativa

Abstract The phosphorylation of Vicia sativa cell-free extracts able to carry out translation efficiently has been studied. Several bands were highly phosphorylated. The inhibitor of translation, fusidic acid, strongly reduces the level of phosphorylation of some proteins but not others.

Changes in sensitivity of in vitro rat brain protein synthesis to the acute action of ethanol and isopropanol as a consequence of the long-term ingestion of isopropanol

Long-term treatment of rats with isopropanol in the drinking water results in a change or process of adaptation occurring in in vitro brain protein synthesis which increases the resistance of the ribosomal machinery to the acute effect of either ethanol or isopropanol. Such an increase was observed both in the system coded by endogenous messenger and in the system coded by polyuridylic acid. In both translation systems, the adaptation seems to affect the ribosomal step of polypeptide chain elongation. The increase in resistance to the alkanols apparently did not affect the inhibitory action of puromycin, fusidic acid and cycloheximide on the ribosome.