In vitro import of cytochrome c peroxidase into the intermembrane space: release of the processed form by intact mitochondria.

Cytochrome c peroxidase (CCP) is a nuclearly encoded hemoprotein located in the intermembrane space (IMS) of Saccharomyces cerevisiae mitochondria. Wild-type preCCP synthesized in rabbit reticulocyte lysates, however, was inefficiently translocated into isolated mitochondria and was inherently resistant to externally added proteases. To test whether premature heme addition to the apoprecursor was responsible for the protease resistance and the inability to import preCCP, site-directed mutagenesis was used to replace the axial heme ligand (His175) involved in forming a pseudo-covalent link between the heme iron and CCP. Mutant proteins containing Leu, Arg, Met, or Pro at residue 175 of mature CCP were sensitive to proteolysis and were imported into isolated mitochondria as judged by proteolytic processing of the precursor. The inhibition of wild-type CCP translocation across the outer membrane may result from the inability of the heme-containing protein to unfold during the translocation process. Although the protease responsible for cleaving preCCP to its mature form is believed to be located in the IMS, most of the processed CCP was located in the supernatant rather than the mitochondrial pellet. Since the outer membranes were shown to be intact, the anomalous localization indicated that preCCP may not have been completely translocated into the IMS before proteolytic processing or that conformationally labile proteins may not be retained by the outer membrane. Proteolytic maturation of preCCP also occurred in the presence of valinomycin, suggesting that the precursor may be completely or partially translocated across the outer mitochondrial membrane independent of a potential across the inner mitochondrial membrane.

L-aspartate of erythromycin A cyclic 11,12-carbonate, a new semisynthetic erythromycin derivative.

Erythromycin A cyclic 11,12-carbonate, a compound with high antibacterial activity, forms with L-aspartic acid a salt possessing valuable properties as a potential chemotherapeutic agent. The L-aspartate of erythromycin A cyclic 11,12-carbonate exhibits strong anti-bacterial activity, especially against Gram-positive bacteria and shows low toxicity. The serum and the lung tissue levels of the discussed salt after a single dose administration to a rat were measured in comparison with those of erythromycin, its L-aspartate, erythromycin cyclic 11,12-carbonate and its L-glutamate. The new erythromycin derivative showed definitely superior characteristics to those of the other substances tested. The activity of the L-aspartate of erythromycin A cyclic 11,12-carbonate in chemotherapy of experimental staphylococcal infection and experimental pneumococcal bronchopneumonia in mice is superior to that of the parent carbonate and erythromycin itself.