Chloride channel antagonists perturb growth and morphology of Neurospora crassa.

The chloride channel antagonists anthracene-9-carboxylic acid, ethacrynic acid and niflumic acid were found to be fungistatic and morphogenic when tested against the ascomycete Neurospora crassa. Potency increased with decreasing pH, suggesting that the protonated forms of the compounds were active. Niflumic acid produced the most pronounced growth aberrations which may reflect an ability to acidify the cytoplasm and block the plasma membrane anion channel of N. crassa.

Pituitary fibroblast growth factors: immunocharacterization of an acid component and N-terminal sequence analysis of a truncated basic component.

Extraction of bovine pituitaries at pH 7.0, in the presence or absence of protease inhibitors (PMSF, leupeptin, pepstatin A and EDTA) yielded both basic and acidic FGF components that were characterized by Western blotting and sequence analysis. Basic FGF comprised several components: an 18 KDa form that is similar, if not identical, to the basic FGF (1-146) already described; a 17 KDa form that is likely to be a new truncated molecular species (11-146) and a group of immunoreactive components of about 29 KDa. Acidic FGF showed several active components of pI 4.5-6.5. The most active component has a pI of approximately 5.0; molecular weight of 17 KDa and is shown, by Western blotting, to be similar to a truncated form of bovine brain acidic FGF. The biological activity of the latter component is shown to be neutralized by anti-brain acidic FGF antiserum.

Structure of C3f, a small peptide specifically released during inactivation of the third component of complement.

C3f, a peptide presumed to be generated by the combined actions of factors I and H on fluid-phase C3b, has been isolated and sequenced. The peptide is 17 residues long and has a molecular weight of 1,847 daltons. The amino-terminal sequence is, with the exception of a single residue, identical to that deduced for the 46-kilodalton polypeptide seen transiently in the generation of iC3b from C3b, and is in full agreement with the sequence deduced from cDNA analysis. In addition, high-pressure liquid chromatography of the digestion of C3b by factor I has shown that C3f is the sole peptide released during iC3b generation.

The N-terminal amino acid sequences of the heavy and light chains of human cathepsin L. Relationship to a cDNA clone for a major cysteine proteinase from a mouse macrophage cell line.

Human liver cathepsin L consists of a heavy chain and a light chain with Mr values of 25,000 and 5000 respectively. The chains have been purified and their N-terminal amino acid sequences have been determined. The 40 amino acids determined from the heavy chain and 42 amino acids sequenced in the light chain are homologous with the N-terminal and C-terminal regions respectively of the superfamily of cysteine proteinases. Therefore it is likely that the two chains of cathepsin L are derived by proteolysis of a single polypeptide precursor. Of the amino acids sequenced, 81% are identical with the homologous portions of a protein sequence for a major cysteine proteinase predicted from a cDNA clone from a mouse macrophage cell line. This is the closest relative amongst the known sequences in the superfamily and strongly indicates that the protein encoded by this mRNA is cathepsin L. The mouse protein is also probably the major excreted protein of a transformed cell line [Gal & Gottesman (1986) Biochem. Biophys. Res. Commun. 139, 156-162]. The heavy chain is identical in only 71% of its residues with the sequence of ox cathepsin S, providing further evidence that this latter enzyme is probably not a species variant of cathepsin L. The relationship with a second unidentified cathepsin cDNA clone from a bovine library is much weaker (41% identity), and so this clone remains unidentified.

Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria.

The enzyme complex F1-ATPase has been isolated from bovine heart mitochondria by gel filtration of the enzyme released by chloroform from sub-mitochondrial particles. The five individual subunits alpha, beta, gamma, delta and epsilon that comprise the complex have been purified from it, and their amino acid sequences determined almost entirely by direct protein sequence analysis. A single overlap in the gamma-subunit was obtained by DNA sequence analysis of a complementary DNA clone isolated from a bovine cDNA library using a mixture of 32 oligonucleotides as the hybridization probe. The alpha, beta, gamma, delta and epsilon subunits contain 509, 480, 272, 146 and 50 amino acids, respectively. Two half cystine residues are present in the alpha-subunit and one in each of the gamma- and epsilon-chains; they are absent from the beta- and delta-subunits. The stoichiometry of subunits in the complex is estimated to be alpha 3 beta 3 gamma 1 delta 1 epsilon 1 and the molecular weight of the complex is 371,135. Mild trypsinolysis of the F1-ATPase complex, which has little effect on the hydrolytic activity of the enzyme, releases peptides from the N-terminal regions of the alpha- and beta-chains only; the C-terminal regions are unaffected. Sequence analysis of the released peptides demonstrates that the N terminals of the alpha- and beta-chains are ragged. In 65% of alpha-chains, the terminus is pyrrolidone carboxylic acid; in the remainder this residue is absent and the chains commence at residue 2, i.e. lysine. In the beta-subunit a minority of chains (16%) have N-terminal glutamine, or its deamidation product, glutamic acid (6%), or the cyclized derivative, pyrrolidone carboxylic acid (5%). A further 28% commence at residue 2, alanine, and 45% at residue 3, serine. The delta-chains also are heterogeneous; in 50% of chains the N-terminal alanine residue is absent. The sequences of the alpha- and beta-chains show that they are weakly homologous, as they are in bacterial F1-ATPases. The sequence of the bovine delta-subunit of F1-ATPase shows that it is the counterpart of the bacterial epsilon-subunit. The bovine epsilon-subunit is not related to any known bacterial or chloroplast H+-ATPase subunit, nor to any other known sequence. The counterpart of the bacterial delta-subunit is bovine oligomycin sensitivity conferral protein, which helps to bind F1 to the inner mitochondrial membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural comparisons of superoxide dismutases.

The amino-terminal sequences of superoxide dismutase isolated from seven microorganisms have been determined. These include the first sequences of enzyme from anaerobic phototrophes. Five enzymes contain iron and two manganese. The enzymes are all related to each other but not to the Cu/Zn family of superoxide dismutases. These sequences, taken with six others from the same family, show that there is no clear distinction in sequence between Fe and Mn types. Moreover it demonstrates a wide variation between enzymes from different bacteria. Also enzymes from anaerobes do not seem to be a particularly closely related group and are not more closely related to each other than to enzymes from aerobes. Two histidine residues are conserved in all proteins and secondary structure predictions suggest they are in close proximity in the same alpha-helix. These residues may provide ligands for the bound metal.