Eukaryotic precursor proteins are processed by Escherichia coli outer membrane protein OmpP.

A new specific endopeptidase that cleaves eukaryotic precursor proteins has been found in Escherichia coli K but not in E. coli B strains. After purification, protein sequencing and Western blotting, the endopeptidase was shown to be identical with E. coli outer membrane protein OmpP [Kaufmann, A., Stierhof, Y.-D. & Henning, U. (1994) J. Bacteriol. 176, 359-367]. Further characterization of enzymatic properties of the new peptidase was performed. Comparison of the cleavage specificities of the newly found endopeptidase and that of rat mitochondrial processing peptidase (MPP) showed that patterns of proteolytic cleavage on the investigated precursor proteins by both enzymes are similar. By using three mitochondrial precursor proteins, the specificity assigned to OmpP previously, a cleavage position between two basic amino-acid residues, was extended to a three amino-acid recognition sequence. Positions +1 to +3 of this extended recognition site consist of an amino-acid residue with a small aliphatic side chain such as alanine or serine, a large hydrophobic residue such as leucine or valine followed by an arginine residue. Additionally, structural motifs of the substrate seem to be required for OmpP cleavage.

Mutational analysis of both subunits from rat mitochondrial processing peptidase.

Rat liver mitochondrial processing peptidase (MPP) is the primary peptidase that cleaves leader peptides from nuclearly encoded mitochondrial proteins following their transport from the cytosol to the mitochondrial matrix. This enzyme consists of two nonidentical subunits that have overall similarity to each other and share certain amino acid motifs. These include the putative metal-ion binding HFLEH motif in the beta-subunit and the HFLEK motif of the alpha-subunit, as well as a possibly helical amino acid stretch bearing a high concentration of negatively charged residues about 70 amino acids downstream of these motifs in both subunits. In order to achieve a better understanding of the role of certain amino acids in rat MPP, we performed site-directed mutagenesis on both of its subunits. Our results show that whereas both histidines and the glutamate of the HFLEH motif in the beta-subunit are crucial for MPP function, this holds true only for the glutamate in the related HFLEK motif in the alpha-subunit. In addition, functionally important negatively charged residues in the region 70 amino acids downstream occur only in the beta-subunit and not in the alpha-subunit. This indicates a functional asymmetry between the subunits, with the beta-subunit containing a majority of residues participating in the active center.

Cloning and characterization of the MamI restriction-modification system from Microbacterium ammoniaphilum in Escherichia coli.

The genes encoding a class-IIN restriction-modification (R-M) system (MamI, sequence specificity [symbol: see text] from Microbacterium ammoniaphilum have been cloned in Escherichia coli. The vector used for cloning was plasmid pUC18 modified by the inclusion of three MamI recognition sites. Recombinant clones containing the mamIM gene in its genomic context became fully methylated in vivo and remained completely resistant against digestion with the R.MamI restriction endonuclease (ENase). Determination of the nucleotide (nt) sequence revealed three open reading frames with lengths of 1089 bp (ORF1), 276 bp (ORFc) and 927 bp (ORF2). On the basis of expression and deletion experiments, the 1089-bp ORF1 was assigned to mamIM encoding the M.MamI DNA methyltransferase (MTase). By amino acid sequencing of the N terminus of R.MamI and comparison of the deduced nt sequence with ORF2, the 927-bp ORF2 was identified as the mamIR gene encoding R.MamI. The 276-bp ORFc, located between mamIR and mamIM, is part of the DNA sequence downstream from mamIM shown to be necessary for controlled mamIM expression.

Novel specific endonuclease activity recognizing a 10-bp sequence.

We report here the generation of a novel restriction endonuclease (ENase) activity with the 10-bp recognition sequence, [symbol: see text]. This specificity could be achieved by first methylating a substrate DNA with M.MamI in vivo, followed by in vitro R.DpnI restriction.

MamI, a novel class-II restriction endonuclease from Microbacterium ammoniaphilum recognizing 5'-GATNN decreases NNATC-3'.

A new site-specific class-II restriction endonuclease, MamI, has been discovered in the nonsporulating Gram+ Microbacterium ammoniaphilum. MamI recognition sequence and cleavage positions were deduced using experimental and computer-assisted mapping and sequencing approaches. MamI cleavage specificity corresponds to: [formula: see text] The novel 43-kD enzyme recognizes a palindromic hexanucleotide interrupted by four ambiguous nucleotides. MamI cleavage positions are located in the center of the recognition sequence resulting in blunt-ended fragments after cleavage in the presence of Mg2+ ions. MamI is inhibited by N6-methyladenine residues. In case of overlapping sequences of MamI and Escherichia coli-coded DNA modification methyltransferase M.EcodamI (5'-[formula: see text]-3'), cleavage of DNA isolated from E. coli wild-type cells will be inhibited. By applying incubation conditions forcing star activity, relaxing of MamI sequence specificity is observed (MamI*).

[Biochemical analysis of free amino acids in human parotid secretions (author's transl)]. / Biochemische Analyse freier Aminosäuren im menschlichen Parotisspeichel.

Parotid secretion was obtained under standardized conditions from 17 male students by stimulation with citric acid. The average flow rate was 0.65 ml/min. The free amino acids in these samples were determined with the aid of two-dimensional thin layer microchromatography of the dansyl derivatives and TV densitometric determination of the blackening of photographic negatives. Apart from glutamic acid, only amino acids with nonpolar residues such as glycine, alanine, leucine, isoleucine, phenylalanine and proline occurred in all 17 samples. The mean values of the concentrations lay between 0.2 mumol/l for isoleucine and 1.2 mumol/l for proline. There were great interindividual variations. (SD between 40% ad 110%). In the present range of 0.3 to 1.1 ml/min, no dependence of the concentrations on the flow rate could be demonstrated. It was attempted to draw up a standard profile by elimination of greatly deviating samples.

[The use of an internal standard for the correction of deviations in densitometry of thin-layer chromatograms (author's transl)]. / Interne Standards zur Fehlerkorrektur bei Densitometrie von Dünnschichtchromatogrammen.

In order to obtain a better reproducibility in quantitative determination of amino acids in amounts of picomoles an internal standard was used. A mixture of ten amino acids was diluted and dansylated. 0.1 microliter of each solution was separated by two-dimensional thin-layer chromatography on polyamide sheets (3 x 3 cm). Each chromatogram was photographed with four different exposure times under ultraviolet light. The dark spots in the photographic negatives were measured by television densitometry. A number of factors influence the reproducibility of this method: loading volume, photographic exposure time and processing, and quenching of fluorescence. As an internal standard, which is influenced by all these factors, DANS-hydroxyproline was added to each solution. By using this standard, the variations in exposure time and fluorescence quenching can be corrected satisfactorily. The relative standard deviation of the mean value of four photographs of one chromatogram is 2%. The results on correction of the volume deviation were not so satisfying. The relative standard deviation of the mean of twelve different chromatograms can be reduced from 34% for the initially measured data, to 8% for the values corrected by the internal standard. It seems necessary to carry out three or more determinations of one solution. In this case the relative standard error of the mean is 2%.