Michiyo Imai

Molecular cloning of a cDNA encoding aldosterone synthase cytochrome P-450 in rat adrenal cortex

Using an oligonucleotide probe designed on the basis of the N‐terminal amino acid sequence of purified rat aldosterone synthase cytochrome P‐450 [(1989) J. Biol. Chem. 264 10935] we have isolated from rat adrenal cDNA library a 2687 base pair cDNA that encodes a protein of 500 amino acid residues. The deduced amino acid sequence contained the regions well conserved among all cytochrome P‐450s sequenced to date, and also a portion (residues 25–44) which was identical to the N‐terminal peptide sequence of rat aldosterone synthase cytochrome P‐450. These results indicate that the cDNA encodes a precursor form of rat aldosterone synthase cytochrome P‐450.

Structural differences in 5′-flanking regions of rat cytochrome P-450aldo and P-45011β genes

Two rat genomic clones, one for cytochrome P-450aldo and the other for P45011β, were isolated and characterized. The two genes, encoding structurally homologous proteins, were closely similar in their intron-exon organizations. Their 5′-flanking regions, however, contained only a few homologous regions. A putative cyclic AMP responsive element, TGACGTGA, was found in the P-450aldo gene, but this sequence was altered at two positions in the P-45011β gene. S1 nuclease protection assay revealed a single transcription initiation site for the P450aldo gene, while multiple sites were found for the P-45011β gene. These results suggest that transcriptional regulation of the rat P-450aldo and P-45011β genes is due to differences in the sequences of their 5′-flanking regions.

On the mechanism of heparin-induced potentiation of platelet aggregation.

The role of antithrombin III (AT III) in heparin-induced potentiation of platelet aggregation was investigated using purified AT III and AT III depleted plasma. When ADP or epinephrine was added to citrated platelet rich plasma (PRP) one minute after addition of heparin, marked enhancement of platelet aggregation was observed, compared with the degree of platelet aggregation in the absence of heparin. However, heparin exhibited no potentiating effect on ADP- or epinephrine-induced platelet aggregation when platelets were resuspended in AT III depleted plasma prepared by immunosorption using matrix-bound antibodies to AT III. When purified AT III was added to AT III depleted plasma at a concentration of 20 microgram/ml, potentiation of platelet aggregation by heparin was clearly demonstrated. These results suggest that the effect of heparin on platelet aggregation is also mediated by AT III.

The Role of Threonine 252 in the Oxygen Activation by Cytochrome P-450 cam: Mechanistic Studies by Site-directed Mutagenesis

Abstract Functional roles of threonine 252 located in the oxygen pocket of cytochrome P-450 cam were assessed by using site-directed mutants of the hemoprotein. The mutant proteins, in which the threonine 252 had been changed to alanine, valine, asparagine or serine, were expressed in Escherichia coli and were purified by conventional methods. All the mutant enzymes thus obtained exhibited high oxygen consuming activities in a reconstituted reaction system composed of NADH, NADH-putidaredoxin reductase and putidaredoxin in the presence of d -camphor. Their spectroscopic properties were indistinguishable from those of the native enzyme in their various redox and ligand-bound states. However, must of the oxygen consumed by the alanine and valine mutants were found to be converted to H 2 O 2 , the production of which was negligible in the reaction with native enzyme. Available evidence indicated that 2 electrons from putidaredoxin were directly transferred to O 2 giving H 2 O 2 . In contrast, a great majority of O 2 consumed by the serine and asparagine mutants was found to be incorporated into d -camphor to afford 5- exo -hydroxycamphor, indicating that the substitution of an amino acid with hydroxy or amide group for threonine retained the monooxygenase activity. These results indicate that uncoupling of O 2 consumption from the monooxygenase reaction was produced by the substitution of an amino acid without a proton donating group for the threonine. Thus the hydroxyl group of threonine 252 plays a crucial role in the O-O bond fission of O 2 in the cytochrome P-450 cam reaction.

Altered cytoskeletal structures of thrombasthenic platelets

The changes in cytoskeletal structures of thrombasthenic platelets were investigated following thrombin activation. Non-stimulated platelets from normal subjects and thrombasthenic patients yielded essentially the same amount of Triton-precipitated cytoskeletal proteins. When platelets were treated with thrombin at various concentrations, the amount of protein in Triton-precipitates was increased both in normal and thrombasthenic subjects; less degree in the latter. The Triton-insoluble materials from non-stimulated, normal and thrombasthenic platelets showed essentially similar polypeptide composition on SDSpolyacrylamide slab gel electrophoresis; three prominent bands of molecular weight 43,000, 200,000 and 250,000 respectively. A distinct difference was, however, observed in polypeptide composition of cytoskeletons between thrombinstimulated normal and thrombasthenic platelets. While polypeptides of molecular weight 43,000, 52,000, 56,000, 200,000 and 250,000 were increased after control platelets were stimulated with thrombin, cytoskeletal proteins of thrombinactivated thrombasthenic platelets showed no increase in the amount of polypeptides of molecular weight of 52, 000 and 56, 000. These findings may suggest the altered cytoskeletal structures of thrombasthenic platelets.