Aminopeptidase from Sphingomonas capsulata.

A novel aminopeptidase with unique substrate specificity was purified from a culture broth of Sphingomonas capsulata. This is the first reported aminopeptidase to demonstrate broad substrate specificity and yet release glycine and alanine with the highest efficacy. On a series of pentapeptide amides with different N-terminal amino acids, this enzyme efficiently releases glycine, alanine, leucine, proline, and glutamate with the lowest turnover value of 370 min(-1) for glutamate. At pH 7.5 (pH optimum) and 25 degrees C, the kinetic parameters for alanine para-nitroanilide were found to be k(cat) = 7600 min(-1) and K(m) = 14 mm. For alanine beta-naphthylamide, they were k(cat) = 860 min(-1) and K(m) = 6.7 mm. Polymerase chain reaction primers were designed based upon obtained internal sequences of the wild type enzyme. The subsequent product was then used to acquire the full-length gene from an S. capsulata genomic library. An open reading frame encoding a protein of 670 amino acids was obtained. The translated protein has a putative signal peptide that directs the enzyme into the supernatant. A search of the amino acid sequence revealed no significant homology to any known aminopeptidases in the available data bases.

Cytochrome P-450 transfer from adrenocortical submitochondrial particles to liposome membranes.

The transfer of cytochrome P-450 from bovine adrenocortical submitochondrial particles (smp) to unilamellar liposome membranes was investigated using a table top ultracentrifuge. Submitochondrial particles were incubated with liposome membranes at 25 degrees C and precipitated by ultracentrifugation at 200000g for a few minutes at 25 degrees C. All liposome vesicles were recovered in the supernatant. Almost no proteins were detected in the supernatant when only smp were incubated and centrifuged. SDS-PAGE revealed one main protein band for the supernatant when smp were incubated with liposome vesicles at 25 degrees C. This band was reactive to anti-P-450scc IgG. Inaccuracy in time for kinetic studies of the transfer was less than 0.5 min. Transfer of P-450scc from smp to liposome membranes was further demonstrated by the decrease in side-chain cleavage activity of smp for endogenous cholesterol after incubation. Cytochromes P-450 accounted for about 70% of the transferred proteins in the liposome membranes, the amount of which increased exponentially with the incubation time. The inverse value of the relaxation time of the transfer increased linearly with the smp concentration and decreased hyperbolically with the liposome concentration. These results coincide with a mechanism by which cytochrome P-450 dissociates from smp membranes, diffuses, and binds to the liposome membranes. In the transfer of cytochrome P-450, the dissociation from smp membranes was deduced to be the rate-limiting step.