Substrate specificity of monomeric and dimeric α‐sarcin

Abstract

The substrate specificity of monomeric and dimeric forms of c~-sarcin was investigated by membrane blotting procedures. Dimeric ~-sarcin fails to inactivate ribosomes as well as to hydrolyze mini-stem-loop RNA, whereas monomeric Separation ofdimer andmonomer ofoasarcin on PVDFmembraneMonomeric and dimeric a-sarcin were separated by electrophoresis oc-sarcin catalyzes both substrates. Both monomeric and dimeric on a 15% polyacrylamide gel containing SDS and electrophoretically cc-sarcin are effective ribonncleases that are displayed by in situ transferred onto PVDF membrane (Micron Separation Inc., MA, RNA-impregnated gel electrophoresis. The same purine base USA) [18]. The localization of monomers and dimers on the memspecificity was detected for both dimeric and monomeric forms, brane was visualized by Ponccau S staining (Merck Chem. Co.). Di-~-Sarcin is also an effective deoxyribonuclease to supercoiled mers and monomers were cut out from blotted membrane and washed DNA. The action of ~-sarcin as deoxyribonuclease and extensively with distilled water. They were stored for future use. The ribonuclease is inhibited by the presence of SDS (3.5 x 10 6 amounts of protein on the membrane were determined by amino acid M); the inhibition on ribonuclease, but not on deoxyribonuclease, analysis according to previous procedures [19]. The molar ratio of monomer and dimer (7 to 1) on the membrane was quantitatively is reversible if the proteins are renatured, calculated.In an exchange experiment, the electrophoretically blotted PVDF Rabbit reticulocyte ribosomes were used as the substrates. The assay RNA [6][7][8]. The action is specific; only one phosphodiester was done in appropriate amounts of protein for monomer, dimer, and bond in the sarcin domain of 23S 28S rRNA is hydrolyzed native ec-sarcin --1.2, 1.2, and 0.8 × 10 -8 M, respectively, a-fragments generated from large subunit 28S rRNA were scored by a composite [7,8]. It also cleaves a mini-stem-loop RNA that mimics the gel. sarcin domain [9][10][11]. At higher concentrations, the protein can act as a ribonuclease [12]. It carries purine-specific endo- Hydrolyzing mini-stem-loop RNA by membrane-bound ~-sarcinnuclease activity to single-and double-stranded RNA [12,13].The specific ribonucleolytic activities of dimers and monomers were directly examined by their abilities to cleave the mini-stem-loop RNA Besides the ribonuclease activity, a-sarcin has previously been that mimics sarcin domain of rat liver ribosomes. A eDNA template documented to have deoxyribonucleolytic activity [12]. Cleavthat carries T7 promoter and sequences complementary to the sarcin age of supercoiled DNA by ~-sarcin and other related ribodomain of 28S rRNA of rat liver ribosomes was synthesized chemitoxins were reported recently [14].cally using DNA synthesizer (API 470A). The template was further purified by electrophoresis on 20% polyacrylamide gel in TE buffer The dimeric form of a-sarcin is constantly observed and is a (89 mM Tris-borate, pH 8.3; 1 mM EDTA). Radioactive 35-mer un...

Identification of a biological inactive complex form of pokeweed antiviral protein

“…A dimeric form of α‐sarcin, a RIP with a ribonuclease activity, has been identified in a purified toxin preparation [19]. Recently, these dimers have been shown to be inactive and it has been suggested that a dimer formation could play a biological role in preventing the cytotoxicity of α‐sarcin [20]. Nevertheless, the existence of an unknown protein associated to PAP in a heterodimer cannot be excluded.…”

Homodimerization of pokeweed antiviral protein as a mechanism to limit depurination of pokeweed ribosomes

“…N ‐glycosidase), α‐sarcin forms a homodimer in solution that is stable during SDS‐PAGE (Sacco et al ., 1983), similar to the PAP dimer reported here. The α‐sarcin homodimer is present as a minor form, in a 1:7 ratio relative to the monomeric form, and is enzymatically inactive (Cheung et al ., 1996), characteristics shared with the PAP homodimer. Although the two proteins bear no sequence similarity to explain a common mechanism for dimerization, dimerization itself may represent a means by which cells protect their ribosomes from the toxic effects of these proteins during their synthesis.…”

The action mode of the ribosome-inactivating protein α-sarcin

“…Ribosome-inactivating proteins, besides catalyzing ribosomes, have been known to hydrolyze many substrates [5,8,10,11,28,36]. Two major actions, the cleavage of the phosphodiester bond on the sarcin domain of rRNA in ribosomes [8,10,11] and ribonuclease activity [5,9], were the primary targets of investigation in this study.…”

“…Two major actions, the cleavage of the phosphodiester bond on the sarcin domain of rRNA in ribosomes [8,10,11] and ribonuclease activity [5,9], were the primary targets of investigation in this study. Using combinatory genetic cloning and in vitro and in vivo analysis, the structural domains that are involved in hydrolyzing both substrates were therefore determined.…”