Evidence on the conformation of HeLa-cell 5.8S ribosomal ribonucleic acid from the reaction of specific cytidine residues with sodium bisulphite. (1/370)

The reaction of HeLa-cell 5.8S rRNA with NaHSO3 under conditions in which exposed cytidine residues are deaminated to uridine was studied. It was possible to estimate the reactivities of most of the 46 cytidine residues in the nucleotide sequence by comparing 'fingerprints' of the bisulphite-treated RNA with those of untreated RNA. The findings were consistent with the main features of the secondary-structure model for mammalian 5.85S rRNA proposed by Nazar, Sitz, & Busch [J. Biol. Chem (1975) 250, 8591--8597]. Five out of six regions that are depicted in the model as single-stranded loops contain cytidine residues that are reactive towards bisulphite at 25 degrees C (the other loop contains no cytidine). The cytidine residue nearest to the 3'-terminus is also reactive. Several cytidines residues that are internally located within proposed double-helical regions show little or no reactivity towards bisulphite, but the cytidine residues of several C.G pairs at the ends of helical regions show some reactivity, and one of the proposed loops appears to contain six nucleotides, rather than the minimum of four suggested by the primary structure. Two cytidine residues that are thought to be 'looped out' by small helix imperfections also show some reactivity.  (+info)

A simple and sensitive fluorometric assay method for taurine using high-voltage paper electrophoresis. (2/370)

A simple and sensitive fluorometric assay method for taurine (2-aminoethanesulfonic acid) has been developed. For the separation of taurine, high voltage paper electrophoresis subsequent to column chromatographic procedures was employed. Fluorescent product of taurine was yielded by spraying fluorescamine (4-phenylspiro [furan-2(3H), 1'-phthalan]-3, 3'-dione) and borate buffer on the paper, and the fluorescence was assayed spectro-fluorometrically after eluting with 50% ethanol. The linear relationship between the concentration of taurine and fluorescence developed was achieved over the concentration ranges of 0.5-10 nmoles, and the recoveries obtained were 90-100%. The specificity of this method for taurine was satisfactory and structural analogues involved in the metabolic pathway of taurine did not interfere with the assay. Examples for tissue levels of taurine in various organs of the rat as determined by this new method are also presented.  (+info)

Bovine liver phosphoamidase as a protein histidine/lysine phosphatase. (3/370)

A 13-kDa phosphoamidase was isolated as a single band on SDS-PAGE from bovine liver. Its Stokes' radius, sedimentation coefficient, molecular mass, and optimal pH were estimated to be 1.6 nm, 1.8 s, 13 kDa, and 6.5, respectively. The enzyme released P(i) from 3-phosphohistidine, 6-phospholysine, and amidophosphate at rates of 0.9, 0.6, and 2.6 micromol/min/mg protein, respectively. However, it did not dephosphorylate phosphocreatine, N(omega)-phosphoarginine, imidodiphosphate, or O-phosphorylated compounds including inorganic pyrophosphate. It also dephosphorylated succinic thiokinase and nucleoside diphosphate kinase autophosphorylated at His residues, indicating that it works as a protein histidine phosphatase. A thiol reagent, 30 microM N-ethylmaleimide, depressed the activity by half, while a thiol compound, 2-mercaptoethanol, protected the enzyme from heat-inactivation. Five millimolar divalent cations, such as Mg2+ and Mn2+, and 5 mM EDTA, had no effect on the activity.  (+info)

5'-Terminal m-7G(5')ppp(5')G-m-p in vivo: identification in reovirus genome RNA. (4/370)

Methylated reovirus mRNA was synthesized in vitro in the presence of S-adenosyl-L-[methyl-3H]-methionine. Viral genome double-stranded RNA that was uniformly labeled with 32-P was isolated from purified virions. The RNAs were mixed and their 5'-terminal structures compared by electrophoretic and chromatographic analyses after enzymatic digestion. Both the mRNA and the corresponding strand in the genome RNA contain m-7G(5')ppp(5')G-m-pCp, indicating that infected cells synthesize viral RNA with blocked, methylated 5' termini.  (+info)

In yeast the export of small glycopeptides from the endoplasmic reticulum into the cytosol is not affected by the structure of their oligosaccharide chains. (5/370)

A "quality control" system associated with the endoplasmic reticulum (ER) that discriminates between misfolded proteins and correctly folded proteins is present in a variety of eukaryotic cells, including yeast. Recently, it has been shown that misfolded proteins that are N -glycosylated in the lumen of the ER are transported out of the ER, de-N-glycosylated by a soluble peptide: N -glycanase (PNGase) and degraded by action of the proteasome. It also has been shown that small N -glycosylatable peptides follow a fate similar to that of misfolded proteins, i.e., glycosylation in the lumen of the ER, transport out of the ER, and de- N -glycosylation in the cytosol. These processes of retrograde glycopeptide transport and de- N -glycosylation have been observed in mammalian cells, as well as in yeast cells. However, little is known about the mechanism involved in the movement of glycopeptides from the ER to the cytosol. Here we report a simple method for assaying N -glycosylation/de- N -glycosylation by simple paper chromatographic and electrophoretic techniques using an N -glycosylatable(3)H-labeled tripeptide as a substrate. With this method, we confirmed the cytosolic localization of the de- N -glycosylated peptide, which supports the idea that de- N -glycosylation occurs after the export of the glycopeptide from the lumen of the ER to the cytosol. Further, we found that the variations in the structure of the oligosaccharide chain on the glycopeptide did not cause differences in the export of the glycopeptide. This finding suggests that the mechanism for the export of small glycopeptides may differ from that of misfolded (glyco)proteins.  (+info)

Primary structure of the L chain from a rabbit homogeneous antibody to streptococcal carbohydrate. II. Sequence determination of peptides from tryptic and peptic digests. (6/370)

A complete amino acid sequence containing 210 residues of a rabbit light chain of a homogeneous Group C streptococcal antibody has been determined as: See journal for formula. This light chain is allotype b4 and is of the V-K1 subgroup.  (+info)

Glyceraldehyde phosphate at the reducing terminus of Salmonella Q haptens. Salmonella montevideo. (7/370)

The O antigen polysaccharide of Salmonella montevideo was isolated from a core-defective mutant by the phenol/water procedure, and was suspected to contain phosphomonester and cyclic phosphodiester at its reducing end in anology to the O hapten from Salmonella typhimurium (Kent and Obsborn, 1968. Therefore, it was chromatographed on a DEAE-cellulose column. Whereas one part eluted with water the other part of the polysaccharide could only be eluted with buffer. Both fractions were further purified on Sephadex G100 and contained mannose, glucose, N-acetylglucosamine and phosphate in a molar ratio of 4:1:1: less than 0.1. In order to specifically label the reducing end phosphate was removed enzymatically, or the presumed cyclic diester was cleaved by mild hydrolysis, and the fractions were reduced with sodium horo[3H]hydride. Both fractions yield mainly [3H]glycerol after hydrolysis and paper chromatogaphy. In addition, [3H]mannitol and [H]monohydroxyacetone could be identified by paper chromatography and were concluded to be the result of phosphate migration and beta-elimination reactions taking place during the isolation procedure and the various treatments prior to sodium boro[3H]hydride reduction. These findings in addition to periodate oxidation studies indicated that the O antigen polysaccharide of Salmonella montevideo had glyceraldehyde phosphate at its reducing end. From the incorporation of 3H into the polysaccharide the O antigen was calculated to consist of about 19 repeating units of 6 sugar residues each.  (+info)

Specialized peptide transport system in Escherichia coli. (8/370)

Trileucine is utilized as a source of leucine for growth of strains of Escherichia coli K-12 that are deficient in the oligopeptide transport system (Opp). Trithreonine is toxic to E. coli K-12. Opp- mutants of E. coli K-12 retain complete sensitivity to this tripeptide. Moreover, E. coli W, which is resistant to trithreonine, can utlize this tripeptide as a threonine source and this capability is fully maintained in E. coli W (Opp-). A spontaneous trithreonine-resistant mutant of E. coli K-12 (Opp-) has been isolated that has an impaired growth response to trileucine and is resistant to trithreonine. Trileucine competes with the uptake of trithreonine as measured by its ability to relieve trithreonine toxicity in E. coli K-12. It is concluded that trileucine as well as trithreonine are transported into E. coli K-12 or W by a common uptake system that is distinct from the Opp system. Trimethionine can act as a competitor of trileucine or trithreonine-supported growth and as an antagonist of trithreonine toxicity in Opp- mutants. It is concluded that trimethionine is recognized by the trileucine-trithreonine transport system. Trithreonine, trimethionine, and trileucine are also transported by the Opp system, as they all relieve triornithine toxicity towards E. coli W and compete with tetralysine utilization as lysine source for growth of a lysine auxotroph of this strain.  (+info)