Partition of porphyrins between cyclohexanone and aqueous sodium acetate as a function of pH. Determination of uroporphyrin and of hydrophilic porphyrin conjugates

1. The partition of uroporphyrins I and III, coproporphyrins I and III, haematoporphyrin IX, porphyrin c and a hydrophilic porphyrin–peptide fraction from variegate-porphyria faeces has been studied in systems of equal volumes of cyclohexanone and sodium acetate buffers of varying pH and concentration. 2. The concentration of acetate in the aqueous phase has little effect on the partition of porphyrin c, but markedly influences that of uroporphyrin. At 50% acetate saturation and pH4·5, only 5% enters the cyclohexanone phase whereas 60% of porphyrin c is extracted under similar conditions. 3. This circumstance forms the basis of a method for the determination of hydrophilic porphyrin–peptides in variegate-porphyria urine. Its reliability has been checked in model experiments. 4. At pH1·5 and an aqueous phase half-saturated with sodium acetate, an equal volume of cyclohexanone removes 95–97% of uroporphyrin and about 55% of porphyrin c. Uroporphyrin may therefore be determined as a second step in the method. 5. For the routine determination of uroporphyrin in systems free from other hydrophilic porphyrins, cyclohexanone extraction may be performed at any pH in the range 1·0–3·0.     

Purification and properties of coproporphyrinogenase

1. Coproporphyrinogenase has been prepared from rat-liver mitochondria and its properties have been studied. The isoelectric point was found to be around pH5.0 and the molecular weight to be 80000+/-8000. The pH optimum of the enzymic reaction was 7.4 and the apparent K(m) was of the order 0.03mm. The enzyme was destroyed by boiling and irreversible inactivation occurred below pH3.5. It could be stored at -10 degrees without loss of activity. The enzyme acts specifically on coproporphyrinogen III and does not form protoporphyrinogen from trans-2,4-diacrylicdeuteroporphyrin or its porphyrinogen. It was unaffected by prolonged dialysis and no cofactor requirement could be demonstrated. 2. Column chromatography of a partially purified enzyme preparation on Sephadex G-200 was found to be an improved method of purification, which gave a coproporphyrinogenase 58-fold purified. The purified enzyme was studied electrophoretically but no evidence was obtained to suggest that more than one enzyme was involved in the reaction. 3. The action was studied of various compounds added to the system. The presence of monothiol groups in the enzyme system was indicated, whereas vicinal dithiol groups were not involved in the reaction. Metal-chelating agents did not inhibit the reaction and no requirement for the presence of any essential metal has been found. All attempts to demonstrate the presence of a prosthetic group, in particular flavines, failed. Neither pyridoxal phosphate nor ATP was involved in the reaction, nor was a mitochondrial electron-transport chain required for the activity of the enzyme. Some circumstantial evidence was obtained to suggest that cis-2,4-diacrylicdeuteroporphyrin is an intermediate in the reaction.     

Construction, transfer and properties of a novel temperature-sensitive integrable plasmid for genomic analysis of Staphyiococcus aureus

As an alternative approach to genetic transfer and analysis, a novel integrable plasmid system was developed that should prove useful for mapping and cloning various genes in Staphylococcus aureus and other Gram-positive bacteria. The use of a restriction-deficient recipient strain and an improved protocol for protoplast plasmid transformation facilitated direct cloning of a recombinant plasmid (pPQ126) in S. aureus NCTC 8325-4. Plasmid pPQ126 (13.6 kb) is a novel, temperature-sensitive integrable plasmid containing genes encoding resistance to erythromycin and chloramphenicol (from plasmid pTV1ts), and resistance to gentamicin (from transposon Tn4001). When introduced into an appropriate recipient strain at the permissive temperature (30 degrees C), pPQ126 replicates autonomously. Integration of pPQ126 is directed into homologous chromosomal target sequences (chromosomal insertions of Tn551 or Tn4001) by growing a population of cells containing autonomous pPQ126 in the presence of gentamicin, erythromycin, and chloramphenicol at 39 degrees C (nonpermissive temperature). Elevated temperature both selects for and maintains pPQ126 as an integrated replicon. Integration of pPQ126 occurs at significantly reduced frequency in a recombination-deficient host, and does not occur in the absence of host chromosomal homology. Integrated pPQ126 excises from the chromosome under permissive conditions (30 degrees C), and excision results in derivatives of pPQ126 that harbour DNA of chromosomal origin.     

Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination

Summary Recombinant plasmids carrying ruvA, ruvB, or both were constructed and used to investigate the genetic defects in a collection of UV-sensitive ruv mutants. The results revealed that efficient survival of UV-irradiated cells depends on both ruvA and ruvB, and on a third gene, ruvC, located upstream of the ruvAB operon. Southern blotting analysis was used to locate insertions in ruv and to examine putative deletion mutants. Two Tn10 insertions were located to the region encoding ruvA. Since these insertions caused a deficiency in the activities of both ruvA and ruvB, we concluded that they must exert a polar effect on ruvB. Two putative ruv deletion mutants were shown to be the result of deletion-inversion events mediated during imprecise excision of Tn10. The relevant inversion breakpoints in these mutants were located to ruvA and ruvC.     

Effect of cystine loading and cystine dimethylester on renal brushborder membrane transport

The effect of loading renal tubule cells with cystine was studied by incubating them with cystine dimethylester. Proline uptake into brushborder membrane vesicles isolated from the cystine loaded cells was not different from that observed into brushborder vesicles isolated from tubules incubated in buffer alone. Incubating brushborder membranes with 2 mM cystine dimethylester for 10 minutes reduced the uptake of proline by 27% after 15 seconds of incubation and by 21% after 60 seconds of incubation. There was no effect after 20 minutes of incubation. Pre-incubating brushborder membrane vesicles with cystine dimethylester had no statistically significant effect on the affinity of priline for the carrier, but did reduce the maximal rate of proline uptake by 49%.     

GenBank.

The GenBank sequence database continues to expand its data coverage, quality control, annotation content and retrieval services. GenBank is comprised of DNA sequences submitted directly by authors as well as sequences from the other major public databases. An integrated retrieval system, known as Entrez, contains data from GenBank and from the major protein sequence and structural databases, as well as related MEDLINE abstracts. Users may access GenBank over the Internet through the World Wide Web and through special client-server programs for text and sequence similarity searching. FTP, CD-ROM and e-mail servers are alternate means of access.