Isolation of the leucine aminopeptidase gene from Aeromonas proteolytica. Evidence for an enzyme precursor.

The leucine aminopeptidase of Aeromonas proteolytica (EC 3.4.11.10) is a monomeric metalloenzyme having the capacity to bind two Zn2+ atoms in the active site. Structural information of this relatively small aminopeptidase that could illuminate the catalytic mechanism of the metal ions is lacking; hence, we have obtained sequences from the purified enzyme, cloned the corresponding gene, and expressed the recombinant protein in Escherichia coli. The deduced primary amino acid sequence of this secreted protease suggests a potential signal peptide at the NH2 terminus. Expression of the recombinant and native proteins in E. coli and in extracts of culture media of A. proteolytica indicates that the aminopeptidase is secreted as an active and thermosensitive 43-kDa protein that is rapidly transformed to thermostable forms of 30 and 32 kDa. Comparison of the deduced amino acid sequence of the A. proteolytica leucine aminopeptidase with other Zn(2+)-binding metalloenzymes failed to show homologies to the consensus binding sequence His-Glu-X-X-His for the metal ion.     

Ubiquitous soluble Mg(2+)-ATPase complex. A structural study.

We have performed a detailed structural analysis of the soluble Mg(2+)-ATPase complex purified from Xenopus laevis ovary, which is an abundant and ubiquitous homo-oligomeric protein complex located in the nucleus and in the cytoplasm, belonging to a novel multigene-family of putative Mg(2+)-ATPases. Enzyme activity staining after non-denaturing polyacrylamide gel electrophoresis revealed that Mg(2+)-ATPase activity of the native protein is dependent on oligomerization and could not be detected in dissociated subunits. For the native protein a sedimentation coefficient of 15.3 S and a corresponding relative molecular mass of 612,000 was determined by analytical ultracentrifugation and a relative molecular mass of 590,000 was estimated from scanning transmission electron microscopy, supporting our previous conclusion that the oligomer comprises six 97,000 Mr subunits. Conventional electron microscopy of negatively stained specimens revealed the Mg(2+)-ATPase complex to be a hexagonal molecule in its favoured "end-on" projection and a double-banded molecule in its "side-on" projection (approx. 12 nm diameter; approx. 9 nm height). In addition, dimerized complexes could be observed in negatively stained specimens, yielding pronounced hexameric images and four-banded images in their end-on and side-on orientations, respectively (approx. 12 nm diameter; approx. 18.5 nm height). Two-dimensional (2D = mono-molecular) crystals have been produced from the dimerized complexes by the negative staining carbon film technique. Hexagonal crystals with a p6 plane group symmetry were obtained from molecules in their end-on orientation and longitudinal arrays with a p2 symmetry from complexes in their side-on orientation. A low-resolution molecular model of the native protein, derived from averages of these two 2D crystals, is presented. From our results we propose oligomerization as an inherent structural principle of organization for this whole newly defined Mg(2+)-ATPase multigene-family, that includes such seemingly diverse functionally defined proteins as mammalian and yeast "vesicle fusion" and "peroxisome assembly" proteins and the product of the yeast cell cycle gene CDC48.     

Characterization of two nisin-producing Lactococcus lactis subsp. lactis strains isolated from a commercial sauerkraut fermentation.

Two Lactococcus lactis subsp. lactis strains, NCK400 and LJH80, isolated from a commercial sauerkraut fermentation were shown to produce nisin. LJH80 was morphologically unstable and gave rise to two stable, nisin-producing (Nip+) derivatives, NCK318-2 and NCK318-3. NCK400 and derivatives of LJH80 exhibited identical morphological and metabolic characteristics, but could be distinguished on the basis of plasmid profiles and genomic hybridization patterns to a DNA probe specific for the iso-ISS1 element, IS946. NCK318-2 and NCK318-3 harbored two and three plasmids, respectively, which hybridized with IS946. Plasmid DNA was not detected in NCK400, and DNA from this strain failed to hybridize with IS946. Despite the absence of detectable plasmid DNA in NCK400, nisin-negative derivatives (NCK402 and NCK403) were isolated after repeated transfer in broth at 37 degrees C. Nisin-negative derivatives concurrently lost the ability to ferment sucrose and became sensitive to nisin. A 4-kbp HindIII fragment containing the structural gene for nisin (spaN), cloned from L. lactis subsp. lactis ATCC 11454, was used to probe genomic DNA of NCK318-2, NCK318-3, NCK400, and NCK402 digested with EcoRI or HindIII. The spaN probe hybridized to an 8.8-kbp EcoRI fragment and a 10-kbp HindIII fragment in the Nip+ sauerkraut isolates, but did not hybridize to the Nip- derivative, NCK402. A different hybridization pattern was observed when the same probe was used against Nip+ L. lactis subsp. lactis ATCC 11454 and ATCC 7962. These phenotypic and genetic data confirmed that unique Nip+ L. lactis subsp. lactis strains were isolated from fermenting sauerkraut.     

Novel paired starter culture system for sauerkraut, consisting of a nisin-resistant Leuconostoc mesenteroides strain and a nisin-producing Lactococcus lactis strain.

Nisin-resistant Leuconostoc mesenteroides NCK293 and nisin-producing Lactococcus lactis subsp. lactis NCK401 were evaluated separately and in combination for growth and nisin production in a model sauerkraut fermentation. Strains were genetically marked and selectively enumerated by using antibiotic-containing media. The growth and survival of L. mesenteroides were similar in the presence and absence of Lactococcus lactis subsp. lactis. The growth of Lactococcus lactis subsp. lactis was not inhibited, although the maximum cell density was reduced and the population decline was more pronounced in the presence of L. mesenteroides. Nisin was detected within 24 h, and levels were relatively constant over the 12-day test period. The maximum cell populations and nisin level achieved could be altered by changing the initial cell ratios of L. mesenteroides and lactococcus lactis subsp. lactis. Isogenic nisin-producing and nisin-negative Lactococcus lactis subsp. lactis derivatives were used in combination with nisin-resistant L. mesenteroides to demonstrate that nisin levels produced in mixed culture were sufficient to retard the onset of the growth of nisin-sensitive, homofermentative Lactobacillus plantarum ATCC 14917.     

Binding of phosphonate chelating agents and pyrophosphate to apotransferrin.

Difference ultraviolet spectroscopy has been used to monitor the binding of a series of phosphonate ligands to human apotransferrin. The ligands consist of pyrophosphate as well as the phosphonic acids (aminomethyl)phosphonic acid (AMPA), (hydroxymethyl)phosphonic acid (HMP), (phosphonomethyl)-iminodiacetic acid (PIDA), N,N-bis(phosphonomethyl)glycine (DPG), and nitrilotris(methylenephosphonic acid) (NTP). Equilibrium constants have been measured for the sequential binding of two ligands per molecule of apotransferrin. In addition, site-specific equilibrium constants have been measured for the binding of AMPA, HMP, and PIDA to the vacant binding site of both forms of monoferric transferrin. Since titrations of diferric transferrin produce no difference UV spectrum, it is proposed that the primary binding site for phosphonic acids includes the protein groups that bind the synergistic bicarbonate anion that is required for formation of a stable ferric transferrin complex. It is further proposed that those ligands with two phosphonate groups can simultaneously bind to cationic amino acid side chains that extend into the cleft between the two domains of each lobe of transferrin. From an inspection of the ferric transferrin crystal structure, the most likely anion binding residues in the cleft are Arg-632 and Lys-534 in the C-terminal lobe and Lys-206 and Lys-296 in the N-terminal lobe.     

Tissue plasminogen activator has an O-linked fucose attached to threonine-61 in the epidermal growth factor domain

An unusual type of glycosylation has been observed for tissue plasminogen activator (t-PA). The monosaccharide fucose is glycosidically linked to threonine-61 in the epidermal growth factor region of t-PA. The presence of O-linked fucose was demonstrated by carbohydrate analysis and mass spectrometry of tryptic and chymotryptic peptides that contain this site. The susceptibility of the fucose residue to alpha-fucosidase indicated that it was in the alpha-anomeric configuration. Fucosylation of threonine-61 was observed in t-PA isolated from the Bowes melanoma cell line and from recombinant expression systems using Chinese hamster ovary or human embryonic kidney cells. Fucosylation of the homologous residue in prourokinase has also been reported recently. Our results indicate that this novel type of glycosylation may be common to the epidermal growth factor domains found in coagulation and fibrinolytic proteins and, therefore, suggest that the modification may have functional significance.     

Non-radioactive techniques for the labelling of nucleic acids

Non-radioactively labelled probes potentially have several advantages over radioactively labelled ones, such as increased stability and reduced hazard. As yet, no non-radioactive methods of labelling are as robust or produce as sensitive a probe as 32P. However, there are many options, some of which use approaches familiar to 32P users (nick translation, random priming, tailing). The majority of methods, whether enzymatic or chemical, direct or indirect, ultimately require the detection of an enzyme by a colourimetric or chemiluminescent substrate. Such detection can be achieved variously by direct visualisation (e.g. of a colourimetrically stained blot), by film, by microtitre plate reader or by CCD camera, depending on the choice of assay format and the degree of quantification required.     

Evolutionarily conserved regions in Caenorhabditis transposable elements deduced by sequence comparison.

In this paper we present the sequence of an intact Caenorhabditis briggsae transposable element, Tcb2. Tcb2 is 1606 base pairs in length and contains 80 base pair imperfect terminal repeats and a single open reading frame. We have identified blocks of T-rich repeats in the regions 150-200 and 1421-1476 of this element which are conserved in the Caenorhabditis elegans element Tc1. The sequence conservation of these regions in elements from different Caenorhabditis species suggests that they are of functional importance. A single open reading frame corresponding to the major open reading frame of Tc1 is conserved among Tc1, Tcb1, and Tcb2. Comparison of the first 550 nucleotides of the sequence among the three elements has allowed the evaluation of a model proposing an extension of the major open reading frame. Our data support the suggestion that Tc1 is capable of producing a 335 amino acid protein. A comparison of the sequence coding for the amino and carboxy termini of the 273 amino acid transposase from Caenorhabditis Tc1-like elements and Drosophila HB1 showed different amounts of divergence for each of these regions, indicating that the two functional domains have undergone different amounts of selection. Our data are not compatible with the proposal that Tc1-related sequences have been acquired via horizontal transmission. The divergence of Tc1 from the two C. briggsae elements, Tcb1 and Tcb2, indicated that all three elements have been diverging from each other for approximately the same amount of time as the genomes of the two species.     

5,8,11,14-Eicosatetraynoic acid-induced destruction of mitochondria in human prostate cells (PC-3)

Summary Culturing human prostate PC-3 cells for 4, 24, or 72 h in the presence of 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of arachidonic acid metabolism and cholesterol biosynthesis, markedly altered the morphology and reduced the number of mitochondria in the treated cells. Using quantitative electron microscopic morphometry, we documented changes in the number, form, area, matrix density, and integrity of the cristae and limiting membranes of mitochondria in cells cultured with ETYA. The inhibition of cholesterol synthesis or the substitution of ETYA for polyunsaturated fatty acids in the inner membrane may participate in the disruption of the mitochondria, which resembles the morphologic sequelae of oxidative stress. If sufficiently extensive, these changes could contribute to the inhibition of cellular proliferation by ETYA.     

Mus308 Mutants of Drosophila Exhibit Hypersensitivity to DNA Cross-Linking Agents and Are Defective in a Deoxyribonuclease

Mutagen-sensitive strains that identify 16 different Drosophila genes have been screened for alterations in DNA metabolic enzymes. A characteristic defect in an acid-active deoxyribonuclease was observed in strains carrying the six available mutant alleles of the mus308 gene. Since that enzyme is detected at normal levels in a mutant strain that is deficient in the previously identified enzymes DNase 1 and DNase 2, it represents a new Drosophila nuclease that is designated Nuclease 3. The mus308 mutants were originally distinguished from all other mutagen-sensitive mutants of Drosophila because they exhibit hypersensitivity to the DNA cross-linking agent nitrogen mustard without expressing a concurrent sensitivity to the monofunctional agent methyl methanesulfonate. Further observations of hypersensitivity to the mutagens trimethylpsoralen, diepoxybutane and cis-platinum now establish a more general sensitivity of these mutants to agents capable of generating DNA cross-links. In spite of the hypersensitivity of the mus308 mutants to DNA cross-linking agents, the initial incision step of DNA cross-link repair is normal in mus308 cells as assayed by the alkaline elution procedure. The Drosophila mus308 mutants show promise of providing a useful model for analogous defects in other organisms including man.