Fernando Soriano

Distribution and properties of major ribosome-inactivating proteins (28 S rRNA N-glycosidases) of the plant Saponaria officinalis L. (Caryophyllaceae).

We have studied the distribution of the protein synthesis inhibitory activity in the tissues of Saponaria officinalis L. (Caryophyllaceae). Seven major saporins, ribosome-inactivating proteins, were purified to apparent homogeneity from leaves, roots and seeds using a new procedure of RIPs isolation including ion-exchange and hydrophobic chromatography. They all catalysed the depurination of rat liver ribosomes, which generate the Endos diagnostic rRNA fragment upon treatment with acid aniline, thus indicating that A4324 from the 28S rRNA has been released (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). The molecular mass of saporins by SDS-PAGE ranged between 30.2 and 31.6 kDa and by gel-filtration between 27.5 and 30.1 kDa. Amino acid composition and amino-terminal amino acid sequence indicate that all saporins may be considered isoforms. Only two saporins present in roots were glycosylated (SO-R1 and SO-R3). All saporins are very active on cell-free translation systems derived from rabbit reticulocyte lysates, rat liver, Triticum aestivum L., Cucumis sativus L. and Vicia sativa L. However, they are poor inhibitors of an Escherichia coli translation system. They inhibit protein synthesis in HeLa, BeWo and NB 100 cells, HeLa cells being the most resistant. The enzymatic activity of at least one saporin isoform was dependent on magnesium concentration in the standard rat liver cell-free system.

Identification of major rye secalins as coeliac immunoreactive proteins.

Six distinct gamma- and omega-type secalins, together with two new low molecular mass glycoproteins, have been identified as the major coeliac immunoreactive proteins from a chloroform/methanol soluble extract from rye endosperm. These components were characterized by a combination of reverse-phase high-performance liquid chromatography, immunoblotting using a coeliac serum and microsequencing analysis. This allowed the identification of a group of secalins with different molecular masses according to their N-terminal amino-acid sequence: one omega-type secalin of 40 kDa (omega 1-40); three gamma-type secalins, one of 70 kDa (gamma-70) and two of 35 kDa (gamma-35); as well as two low molecular mass glycoproteins of 15 and 18 kDa, all exhibiting coeliac serum antigenicity. Moreover, four additional rye components, including two low molecular mass proteins, which did not react with coeliac sera, have also been identified. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of the three main purified coeliac immunogenic secalins, gamma-70, gamma-35 and omega 1-40, indicated molecular masses of 71457, 32240 and 39117 Da, respectively. The omega 1-40 secalin displays a significant absorption in the visible region which could be related to its peculiar low capacity to bind both coeliac sera antibodies and Coomassie brilliant blue dye.

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.

A new family of single chain (type 1) ribosome‐inactivating proteins (RIPs), that we have named ebulitins, have been found in mature leaves of Sambucus ebulus L., a caprifoliaceae plant also known to contain a non‐toxic two chain (type 2) RIP named ebulin 1 in its leaves. Ebulitins are basic proteins of M r 32,000, 29,000 and 29,000 for ebulitins α, β and γ, respectively. The simultaneous presence of different basic type 1 and acidic type 2 RIPs in the same plant and in the same tissue is described here for the first time and opens a new door in research into RIPs.

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

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 (Mr) 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.

Chemical and spectroscopic evidence on the homology of three antitumor proteins: α-sarcin, mitogillin, and restrictocin

Abstractα-Sarcin, restrictocin, and mitogillin, three antitumor proteins, have been compared in terms of chemical composition as well as secondary and tertiary structure. The amino acid composition of the three polypeptides showed that restrictocin and mitogillin are essentially identical, α-Sarcin is also very similar to the other two proteins, although it lacks one methionine in its amino acid composition. Peptide maps of restrictocin and mitogillin coincide, except that one additional peptide is present in the mitogillin fingerprint. Although the α-sarcin map is different from the other two fingerprints, seven tryptic peptides with identical chromatographic and electrophoretic properties as well as amino acid composition were identified. The secondary and tertiary structures of mitogillin and restrictocin are identical from circular dichroism and difference spectroscopy studies. α-Sarcin has slightly different spectroscopic properties than the other two proteins. From these studies, the three proteins could be considered homologous polypeptides.

Characterization of distinct α- and γ-type gliadins and low molecular weight components from wheat endosperm as coeliac immunoreactive proteins

Abstract Distinct α- and γ-type gliadins, as well as a few low molecular weight components have been identified as coeliac immunoreactive proteins from a chloroform/methanol extract from wheat endosperm. Characterization of these components involved the combination of reverse-phase high-performance liquid chromatography, immunoblotting following SDS-PAGE using a coeliac serum and microsequencing analysis. This has allowed the identification of a group of gliadins with different molecular weights, according to their N-terminal amino-acid sequence: five α-type gliadins of 31, 35, 38 and two of 45 kDa, one γ 2 -type gliadin of 40 kDa, two γ 3 -type gliadins of 31, and 50 kDa, and two γ-type gliadins with an atypical gliadin N-terminal of 31, and 40 kDa, as well as a few unidentified low molecular weight components and three N-terminal blocked proteins, all exhibiting similar antigenicity.

Enzymic activity of melonin, a translational inhibitor present in dry seeds of Cucumis melo L.

Abstract Melonin, a protein from the dry seeds of Cucumis melo L. that inhibits protein synthesis in cell-free systems, is a ribonuclease (RNase). Melonin degrades poly(C) but not poly(U) at 55°C and in the presence of 7 M urea, melonin degrades both poly(C) and poly(U). Reflecting this, melonin degrades bonds containing cytidine and, to a lesser extent, uridine in non-synthetic RNA substrates. The NH 2 -terminal amino acid of purified melonin was blocked, but the amino acid sequences of some tryptic peptides indicate that melonin has sequence similarity to cusativin, an RNase isolated from Cucumis sativus L., and to the RNases from Petunia inflata and Nicotiana alata that determine gametophytic self-incompatibility.

Complete automatization of peptide maps by reversed-phase liquid chromatography using o-phthalaldehyde pre-column derivatization☆

Abstract The conventional automatic o-phthalaldehyde (OPA) pre-column labelling procedure used for routine amino acid analyses by high-performance liquid chromatography has been applied as a highly sensitive technique for peptide mapping. Tryptic peptide digests are first derivatized with OPA by using an automated pre-column derivatization system and then the fluorescent peptide mixture is automatically analyzed by reversed-phase HPLC. The complete automatization of peptide mapping is achieved in about 45 min with a high resolution and a routine sensitivity in the range of 10–30 picomoles. Peptide map analyses of dansylated tryptic peptides using the above automatic conditions and with an equivalent range of sensitivity are also described. The effectiveness of this method has been exemplified by using two proteins which are nearly identical in their amino acid sequences.

Spectroscopic characterization by photodiode array detection of human urinary and amniotic protein HC subpopulations fractionated by anion-exchange and size-exclusion high-performance liquid chromatography

A procedure for spectroscopic characterization and partial fractionation of human protein HC populations by high-performance liquid chromatography-photodiode array ultraviolet-visible detection is reported. Human protein HC from urine or amniotic fluid fractionated by anion-exchange HPLC in a protein Pak DEAE 5PW appeared to be heterogeneous as judged by the asymmetric elution pattern, consisting of a continuous irregular broad peak with several shoulders distributed along the whole chromatogram. Selected fractions containing shoulders were rechromatographed and finally six symmetrical homogeneous peaks with different retention times were obtained from each protein HC preparation. The direct automatic absorption spectra analyses at each peak maximum, indicated that all of the homogeneous peaks seemed to be protein HC, all of them associated to the same chromophore although with different stoichiometry ratios. Isoelectric focusing showed that each peak was composed of a limited number of subpopulations of protein HC with different isoelectric points. Size microheterogeneity has been also demonstrated in both urinary and amniotic protein HC preparations by a combination of size-exclusion HPLC on a TSK 3000 SW6 column and photodiode array detection. Partial fractionation of human albumin on an analytical anion-exchange Mono-Q PC 1.6/5 column, has allowed the identification of heterogeneous chromophore-containing populations displaying significant absorption in the visible region in resemblance to that of protein HC.

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.