Tauropine dehydrogenase from the sandwormArabella iricolor (Polychaeta: Errantia): Purification and characterization

This is the first report of the purification of tauropine dehydrogenase (NAD: tauropine oxidoreductase) from a polychaete worm. In the sandwormArabella iricolor Montagu (Polychaet: Errantia), two forms of TaDH were detected: major component (pl = 7.5) and minor one (pI = 6.4). The major TaDH component was purified to homogeneity by means of (NH₄)₂SO₄ precipitation, anion-exchange, affinity, chromatofocusing and hydrophobic chromatography, and characterized. From the molecular mass of 43.7 kDa obtained by rapid gel-filtration and that of 43.5 kDa by SDS-PAGE, the sandworm enzyme appeared to be a monomeric protein. Maximum rates of reduction of pyruvate and oxidation of tauropine were observed at pH 7.0 and 8.5, respectively. Pyruvate and taurine were preferred substrate for the enzyme. Apparent Km values determined using constant co-substrate concentrations were: 35.7 mM, 0.34 mM, and 0.036 mM for taurine, pyruvate and NADH, respectively, in the tauropine synthesizing reaction; and 4.8 mM and 0.051 mM for tauropine and NAD⁺, respectively, in the tauropine oxidizing reaction. The tauropine synthesizing reaction was subject to substrate inhibition by pyruvate: maximum rate was observed at 2.5–3.0 mM (inhibitory range of pyruvate concentration producing half-maximal rate was 26.8 mM).     

New and interesting species ofEmericella from Chinese soil

Emericella miyajii, a new species isolated from Chinese soil, is described and illustrated. It is characterized by pale orange to brownish orange colonies on malt extract agar, subglobose to broadly elliptical ascospores with defective four equatorial crests and smooth convex walls, and with anAspergillus anamorph.Emericella undulata is also described as an uncommon species from Chinese soil.     

Probing Novel Elements for Protein Splicing in the Yeast Vma1 Protozyme: A Study of Replacement Mutagenesis and Intragenic Suppression

Protein splicing is a compelling chemical reaction in which two proteins are produced posttranslationally from a single precursor polypeptide by excision of the internal protein segment and ligation of the flanking regions. This unique autocatalytic reaction was first discovered in the yeast Vma1p protozyme where the 50-kD site-specific endonuclease (VDE) is excised from the 120-kD precursor containing the N-and C-terminal regions of the catalytic subunit of the vacuolar H(+)-ATPase. In this work, we randomized the conserved valine triplet residues three amino acids upstream of the C-terminal splicing junction in the Vma1 protozyme and found that these site-specific random mutations interfere with normal protein splicing to different extents. Intragenic suppressor analysis has revealed that this particular hydrophobic triplet preceding the C-terminal splicing junction genetically interacts with three hydrophobic residues preceding the N-terminal splicing junction. This is the first evidence showing that the N-terminal portion of the V-ATPase subunit is involved in protein splicing. Our genetic evidence is consistent with a structural model that correctly aligns two parallel β-strands ascribed to the triplets. This model delineates spatial interactions between the two conserved regions both residing upstream of the splicing junctions.     

Purification and characterization of M3 protein expressed on the surface of group A streptococcal type 3 strain C203

Abstract Monoclonal antibodies (mAbs) have been produced by immunizing BALB/C mice with whole M⁺ bacteria in incomplete Freund adjuvant and the resulting mAbs for M3 protein have been selected by an indirect immuno-fluorescent technique using formaldehyde-fixed M⁺ and M⁻ bacteria. Four mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M⁺ bacteria after treatment with N -acetyl muramidase and lysozyme. The purified 65 kDa protein neutralized the phagocytic activity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The evidence indicates that the 65 kDa protein is M3 protein. The M3 protein bound not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromatography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the intact molecule appeared. N-terminal amino acid sequence analysis showed that 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, being identical at positions 1–5 and 198–202 to the M3 gene derived protein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed to be cleavage products, may be derived from the C-terminal part and N-terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human blood in the presence of M⁻ bacteria was investigated, the M3 protein was responsible for the organism's resistance to attack by phagocytic cells.     

Nitrobacter agilis Cytochrome c-550: Isolation, Physicochemical and Enzymatic Properties, and Primary Structure

Nitrobacter agilis cytochrome c-550 was purified to an electrophoreticallyhomogeneous state, and some of its properties were determined.The cytochrome showed an absorption peak at 410 nm in the oxidizedform, and peaks at 416, 521 and 550 nm in the reduced form.Its isoelectric point was 8.1 at 5?C. Analysis of the aminoacid composition showed that the cytochrome molecule was composedof 108 amino acid residues, 16 of which were lysine residues. The cytochrome reacted rapidly with N. agilis cytochrome c oxidaseand yeast cytochrome c peroxidase and more slowly with Pseudomonasaeruginosa nitrite reductase and bovine cytochrome c oxidase.The reactivities with these redox enzymes suggested that thecytochrome might be an evolutionary stage between bacterialand eukaryotic cytochromes c. The primary structure of the cytochrome from the N-terminusto the 85th residue was determined. The N-terminal sequencewas homologous to the corresponding portion of the primary structureof horse cytochrome c. ¹ Present adress: Department of Chemistry, Faculty of Science,Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo,152, Japan. (Received December 3, 1981; Accepted January 28, 1982)