We found a novel protein in the postmitochondria supernatant fraction of rat liver, which is soluble in 5% perchloric acid and strongly inhibits protein synthesis in a rabbit reticulocyte lysate system. The protein extracted from the supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The protein was shown to consist of two identical subunits with a molecular mass of 14 kDa. By immunoscreening with the rabbit antisera against the protein, a cDNA encoding the protein was cloned and sequenced. The cDNA contained an open reading frame of 411 base pairs encoding a 136-amino acid protein with a molecular mass of 14,149 Da. The deduced amino acid sequence was completely identical with that constructed from all of the above peptides. Interestingly, the perchloric acid-soluble protein inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner from RNase A. The protein is likely to inhibit an initiation stage of cell-free protein synthesis. Among the rat tissues tested, the protein was located only in liver and kidney. These findings are the first report on a new inhibitor that may be involved in the regulation of protein synthesis in those tissues.

No abstract

The protein toxin restrictocin, isolated from the mould Aspergillus restrictus, inactivates protein synthesis in eukaryotic cells by blocking the ribosome elongation cycle. This protein acts as a specific nuclease that cuts off a small fragment from the 28-S rRNA. Biochemical and biological characterization of this toxin indicated that it is a non-glycosylated polypeptide of M , 16836, exhibiting in cell-free systems a protein synthesis inhibition capacity similar to that of the ricin A chain. This polypeptide seemed unable to penetrate most of the cancer cell lines tested, as measured by its low in vitro cytotoxicity. In addition in vivo studies in BALB/c mice demonstrated that restrictocin toxicity was very low and that in rabbits, after intravenous injection 15% of the toxin was still present in the blood stream 24 h later.After derivatization with N-succinimidyl 3-(2-pyridyldithio)propionate and reduction by dithiothreitol, the restrictocin maintained its protein synthesis inhibitory activity, as assayed in a cell-free system. This derivatized toxin was then coupled to monoclonal antibodies (MBrl, MLuC1, MLuC2, MOv17, MOv18, MOv19) which exhibited a restricted spectrum of reactivity against human carcinomas. The biochemical and biological characterization of the immunoconjugates indicated that (a) when restrictocin was coupled to monoclonal antibodies with an average molar ratio of about 2, the immunoconjugates maintained the binding activity of the antibody and protein synthesis inhibition activity of the toxin; (b) four immunoconjugates were tested for cytotoxicity and three of them obtained with the MBrl, MLuCl and MOvl7 monoclonal antibodies exhibited a good level of cytotoxicity for relevant target cells and low or no toxicity for the irrelevant cell lines. The MLuC2 monoclonal antibody which gave rise to a completely ineffective immunoconjugate, induced internalization of less than one tenth of the antigenic sites whereas the MBrl, MLuCl and MOv17 monoclonal antibodies exhibited about one third of the antigenic sites internalized. From these data it is concluded that, providing an appropriate target antigen and coupling procedure are selected, restrictocin can be considered a suitable toxin for immunoconjugate generation.

The effect of vitamin B6 on the growth of a human hepatoma cell line HepG2 in culture was studied. The growth of HepG2 cells and protein synthesis were almost completely inhibited in medium supplemented with 5 mM pyridoxine. Pyridoxal was as effective as pyridoxine, but pyridoxamine showed no inhibitory action. The growth inhibition of HepG2 cells by pyridoxine was accompanied by a marked inhibition of secretion of plasma proteins, particularly albumin. Northern blot analysis of albumin mRNA showed that pyridoxine caused a rapid decrease in the expression of albumin gene. The electron-microscopic examination of pyridoxine-treated HepG2 cells revealed a smoothing of nuclear membrane, a decrease in the number of nucleoli, and an appearance of aggregated heterochromatin structures. These morphological features are compatible with the depressed transcriptional activity in the pyridoxine-treated cells. The mechanism by which vitamin B6 exerts its inhibitory effect was discussed in terms of our recent finding that vitamin B6 modulates expression of albumin gene by inactivating tissue-specific DNA-binding proteins. Binding of pyridoxal phosphate with tissue-specific transcription factors may reduce the capacity of these factors to interact with the regulatory region of albumin gene, resulting in the inhibition of the gene expression.

A protein has been isolated from a culture medium of Aspergillus giganteus I F 0 5818 (gigantin) and purified by ion-exchange chromatography successively on DEAE-cellulose and carboxymethylcellulose, and gel-filtration chromatography on Biogel P10. With a high purity, gigantin was found to be a non-glycosylated basic protein with a relative molecular mass of 17000 2 200 determined in PAGEEDS. Gigantin was able to digest the synthetic homopolymers of nucleic acids poly(A), poly(I), poly(C) and poly(U). The catalytic action has an optimal pH around 7.0, an optimal temperature at 45-55°C and can be inhibited by cations. Gigantin activity, analyzed as its capacity to hydrolyze RNA from yeast, was comparable to that of alphasarcin, a similar biochemical protein produced by the strain A. giganteus MDH 18894. A study of the substrate specificity for alphasarcin indicated a preference for poly(A) and poly(I), while gigantin had greater activity on poly(C) and poly(U). The cross reaction of gigantin with a rabbit antiserum to alphasarcin suggests a high sequence similarity between both proteins. However, gigantin is immunologically distinguishable from alphasarcin as alphasarcin antiserum detects epitopes in alphasarcin that are not present in gigantin.Several ribonucleases with guanyl-specific or purine-specific activities have been isolated from different fungal species, many of them from Aspergillus [ 11. They are extracellular proteins secreted by the fungus to the culture medium and can be divided into three families on the basis of their biochemical characteristics. The first, of approximately 100 amino acids, is represented by ribonuclease T1 (RNase T1) with alphasarcin [lo]. These three proteins were originally characterized as potent cytotoxic agents able to kill tumour cells [4,8, 91.The basis of the action of alphasarcin, whose activity has been most studied, is the result of a RNase activity that produces cleavage of a phosphodiester bond on the 3' side of G4325 in a universal purine-rich sequence of the large ribosomal subunit, with the formation, from the 3' end of the large ribosomal ribonucleic acid (rRNA), of a fragment 240-500 nucleotides in length, depending on the species [ 11,121. Although alphasarcin cleaves only a single phosphodiester bond when ribosomes are the substrate, the toxin causes extensive progressive digestion of the nucleic acids when 28s rRNA or other ribonucleic acids are the substrates The family of alphasarcin ribonucleases that we have generically called aspergillins [14-161, shows a low toxicity in cells related to the absence of a B-chain-like activity that brings the toxin into the cell [17]. The toxic effect can be present in cells infected by virus that change the membrane cell permeability [18]. Their low cytotoxicity and their low molecular mass are favourable factors for their use in the generation of immunoconjugates for clinical applications [19] ; two members of the group, restrictocin and alphasarcin, have been used conjugated with monoclonal antibodies against tumour...