Elke Herfurth

Purification, characterization and partial amino acid sequences of a xylanase produced by Penicillium chrysogenum

An extracellular xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8, endo 1,4-beta-xylanase) was found to be the major protein in the culture filtrate of Penicillium chrysogenum when grown on 1% xylan. In contrast to other microorganism no xylanase multiplicity was found in P. chrysogenum under the conditions used. This enzyme was purified to homogeneity by high performance anion-exchange and size-exclusion chromatography. It had an M(r) of 35,000 as estimated by SDS-PAGE and was shown to be active as a monomer. No glycosylation of the protein could be detected neither by a sensitive glycostain nor by enzymatic deglycosylation studies. The enzyme hydrolyzed oat spelt and birchwood xylan randomly, yielding xylose and xylobiose as major end products. It had no cellulase, CMCase, beta-xylosidase or arabinogalactanase activity but acted on p-nitrophenylcellobioside. The pH and temperature optima for its activity were pH 6.0 and 40 degrees C, respectively. Eight peptides obtained after endoproteinase LysC digestion of xylanase have been sequenced, six of them showed considerable amino acid similarity to glucanases and high M(r)/acidic xylanases from different bacteria, yeasts and fungi.

COMPLETE AMINO ACID SEQUENCE OF RIBOSOMAL PROTEIN S14 FROM BACILLUS STEAROTHERMOPHILUS AND HOMOLOGY STUDIES TO OTHER RIBOSOMAL PROTEINS

The complete amino acid sequence of protein S14 from the small subunit of Bacillus stearothermophilus was determined by N‐terminal sequence analysis and by sequencing of overlapping peptides obtained from enzymatic digestions. Protein S14 consists of 60 amino acid residues with a molecular mass of 7148 Da. It has a high content of basic amino acids and a predicted isoelectric point of 11.46. Protein S14 contains two pairs of cysteines in the carboxyl‐terminal region, presumably linked by two sulphur bridges. A comparison between protein S14 of B. stearothermophilus and homologous proteins from other organisms revealed highly conserved carboxyl‐termini for this protein in eubacteria, archaebacteria and eukaryotes.

Amino acid sequence of the ribosomal protein HS23 from the halophilic Haloarcula marismortui and homology studies to other ribosomal proteins.

The ribosomal protein HS23 from the 30S subunit of the extreme halophilicHaloarcula marismortui, belonging to the group of archaea, was isolated either by RP-HLPLC or two-dimensional polyacrylamide gel electrophoresis. The complete amino acid sequence was determined by automated N-terminal microsequencing. The protein consists of 123 residues with a corresponding molecular mass of 12,552 Da as determined by electrospray mass spectroscopy; the pI is 11.04. Homology studies reveal similarities to the eukaryotic ribosomal protein S8 fromHomo sapiens, Rattus norvegicus, Leishmania major, andSaccharomyces cerevisiae.

Towards Ribosomal Structure at Peptide Level: Use of Crosslinking, Antipeptide Antibodies and Limited Proteolysis

Although remarkable progress has been achieved in the past decades towards an understanding of the structure and function of the ribosome, the interactions of nucleic acids and proteins involved in protein biosynthesis remain largely unresolved. It has convincingly been demonstrated that ribosomal RNA is essential for ribosomal function (e.g. Schulze and Nierhaus, 1982; Dahlberg, 1989; Noller et al., 1992), yet there can be no doubt that in contemporary ribosomes complexes of RNA and proteins constitute the functional units.Models of the tertiary structure of the 16S RNA have been derived from footprinting and crosslinking experiments (Stern et al., 1988; Brimacombe et al., 1988; Nagano et al., 1988). In addition, functionally important domains and even nucleotides were identified by affinity labelling and site-directed mutagenesis.