Human milk lactoferrin binds two DNA molecules with different affinities.

Evidence is presented that lactoferrin (LF), an Fe3+-binding glycoprotein, possesses two DNA-binding sites with different affinities for specific oligonucleotides (ODNs) (Kdl = 8 nM; Kd2 approximately 0.1 mM). The high affinity site became labeled after incubation with affinity probes for DNA-binding sites; like the antibacterial and polyanion-binding sites, this site was shown to be located in the N-terminal domain of LF. Interaction of heparin with the polyanion-binding site inhibits the binding of ODNs to both sites. These data suggest that the DNA-binding sites of LF coincide or overlap with the known polyanion and antimicrobial domains of the protein.     

Kinetics of elementary steps in the cytochrome P-450 reaction sequence. III. NADPH reduction of cytochrome P-450LM at different integrational levels.

The aerobic NADPH reduction of cytochrome P-450LM has been investigated on microsomes, as well as on the solubilized enzyme system in the associated, disintegrated, and reconstituted state, respectively. P-450 exhibits biphasic reduction kinetics of about 70/30% phase distribution and rate constants differing 10-fold. The partial reactions are due to organizational asymmetries, the cytochrome being either incorporated into P-450/reductase associates (cluster) or localized outside (randomly distributed, homoassociated, weakly cluster-associated). Triton N-101 disintegrates the different associate structures, consequently followed by the disappearance of the rapid reaction phase. The enzyme system can be reconstituted; at microsomal stoichiometry the respective standard parameters are approached, depending on the composition and structural organization of the phospholipid. The reorganization without any membrane matrix is obviously thermodynamically determined.     

Cloning and nucleotide sequence of the isoamylase gene from Pseudomonas amyloderamosa SB-15

The gene (iam) coding for isoamylase (glycogen 6-glucanohydrolase) of Pseudomonas amyloderamosa SB-15 was cloned. Its nucleotide sequence contained an open reading frame of 2313 nucleotides (771 amino acids) encoding a precursor of secreted isoamylase. The precursor contained a signal peptide of 26 amino acid residues at its amino terminus and three regions homologous with those conserved in alpha-amylases (1,4-alpha-D-glucan 4-glucanohydrolase) of species ranging from prokaryotes to eukaryotes. These homologous regions were also found in another debranching enzyme, pullulanase (pullulan 6-glucanohydrolase) from Klebsiella aerogenes. Sequences of the isoamylase also showed significant homology with those between positions 300 and the carboxyl terminus of pullulanase. The regions required for the specificity of isoamylase were discussed on the basis of a comparison of its amino acid sequence with those of alpha-amylases, cyclomaltodextrin glucanotransferases, and pullulanase.     

Isolation of adenosine 5'-diphosphate-L-glycero-D-mannoheptose, the assumed substrate of heptose transferase(s), from Salmonella minnesota R595 and Shigella sonnei Re mutants

From heptose transferase-less Re mutants of Salmonella minnesota and Shigella sonnei, a mixture of nucleotide-linked heptoses was isolated. After paper chromatography in different solvent systems, ADP derivatives of D-glycero-D-mannoheptose and L-glycero-D-mannoheptose could be isolated in pure form. The structure of ADP-L-glycero-D-mannoheptose was verified by analytical methods and by transformation of ADP-D-glycero-D-mannoheptose with ADP-D-glycero-D-mannoheptose-6-epimerase.