The site of action of the A-chain of mistletoe lectin I on eukaryotic ribosomes. The RNA N-glycosidase activity of the protein

The site of action of the A-chain of mistletoe lectin (ML-A) from Viscum album on eukaryotic ribosomes was studied. Treatment of rat liver ribosomes with ML-A, followed by treatment of the isolated rRNA with aniline, caused the release of a fragment with about 450 nucleotides from 28 S rRNA. Further analysis of nucleotide sequences of this fragment revealed that the aniline-sensitive site of phosphodiester bond was between positions A-4324 and G-4325 in 28 S rRNA. These results indicate that ML-A inactivates the ribosomes by cleaving a N-glycosidic bond at A-4324 of 28 S rRNA in the ribosomes as ricin A-chain does.     

Carbohydrate structures of acetylcholine receptor from Torpedo californica and distribution of oligosaccharides among the subunits

The structure of carbohydrates in acetylcholine receptor (AChR) from Torpedo californica is reported. Oligosaccharides released quantitatively from the whole molecule by N-oligosaccharide glycopeptidase digestion were fractionated by thin-layer chromatography and further purified by high-performance liquid chromatography. We show that more than 70% of the total oligosaccharide chains in Torpedo AChR are of the high-mannose type with the structures (Man)8(GlcNAc)2 and (Man)9(GlcNAc)2. The structure of these oligosaccharides were determined by proton nuclear magnetic resonance spectroscopy. These two types of oligosaccharides were shown to be distributed different proportions in all subunits of Torpedo AChR. We also show that several kinds of complex-type oligosaccharides comprising the rest of the carbohydrate in the protein exist mainly in the gamma and delta subunits. The structure of the carbohydrate moiety that is distributed on the four subunits of AChR was also examined by susceptibility to endo-beta-N-acetylglucosaminidase and sialidase and by binding affinity to lectins, e.g. concanavalin A, leucoagglutinating phytohemagglutinin, and wheat germ agglutinin.     

Probing stability and dynamics of proteins by protease digestion. I: Comparison of protease susceptibility and thermal stability of cytochromes c

Protease susceptibility of homologous proteins in their native conformations was studied. This work aims to establish a broad and quantitative basis for the utilization of protease digestion to analyze the local stability of native proteins. Using high-performance liquid chromatography (HPLC) the time course of the proteolytic degradation of intact proteins was quantitatively traced. Rapid separation of peptide fragments with HPLC made possible the elucidation of sequential digestion originating from the cleavage at a very few sites which are locally unstable in the protein structure. Using four serine proteases, chymotrypsin, trypsin, elastase and subtilisin BPN', we found some common trends in proteolysis for a group of proteins of the cytochrome c family. By comparing of the proteolysis and thermal denaturation with ten homologous cytochromes c extracted from horse, beef, Candida krusei, Saccharomyces cerevisiae, chicken, tuna, pigeon, rabbit, dog and rat, protease susceptibility was related to locally unfolding states intrinsic to the native conformation.     

An 18K protein from ascites hepatoma cell depolymerizes actin filaments rapidly

Several actin binding proteins were isolated from ascites hepatoma cells AH7974 by DNase I affinity chromatography. Among them, a protein having a molecular weight of 18,000 was further purified by DEAE cellulose and hydroxyapatite column chromatographies and gel filtration on a Sephadex G-75 column. The 18K protein not only inhibits actin polymerization but also depolymerizes actin filaments. This conclusion was supported by viscosity and fluorescence intensity measurements and the DNase I inhibition assay. A chemical cross-linking experiment suggested that the 18K protein binds to monomeric actin and forms and 18K-actin 1:1 complex. The net depolymerization rate by the 18K protein measured by the DNase I inhibition assay was slower than the rapid reduction of the fluorescence intensity of pyrene-labeled F-actin upon addition of the 18K protein. This result suggests that the 18K protein not only binds to monomeric actin but also binds to actin filaments directly. The sedimentation assay showed that a part of the 18K protein was cosedimented with actin filaments. Electron microscopic observations demonstrated that the 18K protein decreased the amount of actin filaments and the remaining filaments appeared to be decorated and distorted by the 18K protein. The 18K protein had no Ca2+ ion sensitivity and exhibited the same effect on both this tumor actin and muscle actin.     

Differential expression and distribution of chicken skeletal- and smooth-muscle-type alpha-actinins during myogenesis in culture

Antibodies to chicken fast skeletal muscle (pectoralis) alpha-actinin and to smooth muscle (gizzard) alpha-actinin were absorbed with opposite antigens by affinity chromatography, and four antibody fractions were thus obtained: common antibodies reactive with both pectoralis and gizzard alpha-actinins ([C]anti-P alpha-An and [C]anti-G alpha-An), antibody specifically reactive with pectoralis alpha-actinin ([S]anti-P alpha-An), and antibody specifically reactive with gizzard alpha-actinin ([S]anti-G alpha-An). In indirect immunofluorescence microscopy, (C)anti-P alpha-An, (S)anti-P alpha-An, and (C)anti-G alpha- An stained Z bands of skeletal muscle myofibrils, whereas (S)anti-G alpha-An did not. Although (S)anti-G alpha-An and two common antibodies stained smooth muscle cells, (S)anti-P alpha-An did not. We used (S)anti-P alpha-An and (S)anti-G alpha-An for immunofluorescence microscopy to investigate the expression and distribution of skeletal- and smooth-muscle-type alpha-actinins during myogenesis of cultured skeletal muscle cells. Skeletal-muscle-type alpha-actinin was found to be absent from myogenic cells before fusion but present in them after fusion, restricted to Z bodies or Z bands. Smooth-muscle-type alpha- actinin was present diffusely in the cytoplasm and on membrane- associated structures of mononucleated and fused myoblasts, and then confined to membrane-associated structures of myotubes. Immunoblotting and peptide mapping by limited proteolysis support the above results that skeletal-muscle-type alpha-actinin appears at the onset of fusion and that smooth-muscle-type alpha-actinin persists throughout the myogenesis. These results indicate (a) that the timing of expression of skeletal-muscle-type alpha-actinin is under regulation coordination with other major skeletal muscle proteins; (b) that, with respect to expression and distribution, skeletal-muscle-type alpha-actinin is closely related to alpha-actin, whereas smooth-muscle-type alpha- actinin is to gamma- and beta-actins; and (c) that skeletal- and smooth- muscle-type alpha-actinins have complementary distribution and do not co-exist in situ.     

The sequence of the nucleotides at the alpha-sarcin cleavage site in rat 28 S ribosomal ribonucleic acid

The sequence of the 521 nucleotides at the 3' end of a rat 28 S rRNA gene was determined. The region encompasses the site of cleavage of 28 S rRNA by the cytotoxin alpha-sarcin. The toxin hydrolyzes a phosphodiester bond on the 3' side of a guanine residue 393 nucleotides from the 3' end. The alpha-sarcin domain is composed of a purine-rich sequence of 14 highly conserved nucleotides.