Site-directed mutants of cytochrome P-450cam (the cytochrome P-450 that acts as the terminal monooxygenase in the d-camphor monooxygenase system), in which threonine-252 had been changed to alanine, valine, or serine, were employed to study the role of the hydroxy amino acid in the monooxygenase reaction. The mutant enzymes were expressed in Escherichia coli and were purified by a conventional method. All the mutant enzymes in the presence of d-camphor exhibited optical absorption spectra almost indistinguishable from those of the wild-type enzyme in their ferric, ferrous, oxygenated, and carbon monoxide ferrous forms. In a reconstituted system with putidaredoxin and its reductase, the alanine enzyme consumed O2 at a rate (1100 per min per heme) comparable to that of the wild-type enzyme (1330 per min per heme), whereas the amount of exo-5-hydroxycamphor formed was less than 10% of that formed by the wild-type enzyme. About 85% of the O2 consumed was recovered as H2O2. The valine enzyme also exhibited an oxidase activity to yield H2O2 accompanied by a relative decrease in the monooxygenase activity. On the other hand, the serine enzyme exhibited essentially the same monooxygenase activity as that of the wild-type enzyme. Thus, uncoupling of O2 consumption from the monooxygenase function was produced by the substitution of an amino acid without a hydroxyl group. When binding of O2 to the ferrous forms was examined, the alanine and valine enzymes formed instantaneously an oxygenated form, which slowly decomposed to the ferric form with rates of 5.5 and 3.2 x 10(-3) sec-1 for the former and latter enzymes, respectively. Since these rates were too slow to account for the overall rates of O2 consumption, the formation of H2O2 was considered to proceed not by way of this route but through the decomposition of a peroxide complex formed by reduction of the oxygenated form by reduced putidaredoxin. Based on these findings, a possible mechanism for oxygen activation in this monooxygenase reaction has been discussed.

Pierisin-1 is a potent apoptosis-inducing protein derived from the cabbage butterfly, Pieris rapae. It has been shown that pierisin-1 has an A⅐B structure-function organization like cholera or diphtheria toxin, where the ''A'' domain (N-terminal) exhibits ADPribosyltransferase activity. The present studies were designed to identify the target molecule for ADP-ribosylation by pierisin-1 in the presence of ␤-[adenylate-32 P]NAD, and we found DNA as the acceptor, but not protein as is the case with other bacteria-derived ADP-ribosylating toxins. ADP-ribosylation of tRNAs from yeast was also catalyzed by pierisin-1, but the efficiency was around 1 ⁄10 of that for calf thymus DNA. Pierisin-1 efficiently catalyzed the ADPribosylation of double-stranded DNA containing dG⅐dC, but not dA⅐dT pairs. The ADP-ribose moiety of NAD was transferred to the amino group at N 2 of 2 -deoxyguanosine to yield N 2 -(␣-ADP-ribos-1-yl)-2 -deoxyguanosine and its ␤ form, which were determined by several spectral analyses including 1 H-and 13 C-NMR and mass spectrometry. The chemical structures were also ascertained by the independent synthesis of N 2 -(D-ribos-1-yl)-2 -deoxyguanosine, which is the characteristic moiety of ADP-ribosylated dG. Using the 32 P-postlabeling method, ADP-ribosylated dG could be detected in DNA from pierisin-1-treated HeLa cells, in which apoptosis was easily induced. Thus, the targets for ADP-ribosylation by pierisin-1 were concluded to be 2 -deoxyguanosine residues in DNA. This finding may open a new field regarding the biological significance of ADP-ribosylation. P ierisin-1, a 98-kDa protein, is abundantly present in the fifth instar larvae and the early phase of pupae in the cabbage butterfly, Pieris rapae (1, 2). This protein has potent cytotoxic activity against various human cancer cell lines with a wide range of IC 50 values, inducing typical apoptotic cell death with characteristic morphological features, DNA fragmentation, and cleavage of poly(ADP-ribose) polymerase (2, 3). The N-terminal region of pierisin-1 has a partial regional sequence similarity with ADP-ribosylating toxins such as the A-subunit of cholera toxin, and disruption of this possible NAD-binding site by site-directed mutagenesis abolishes its apoptosis-inducing activity (4). Inhibitors of ADP-ribosyltransferase also significantly reduce its ability to induce apoptosis of cancer cells. Recently, we found that pierisin-1 is likely to have an A⅐B structure-function organization similar to that of cholera and diphtheria toxins, the ''A'' domain featuring the ADP-ribosyltransferase activity and the ''B'' domain binding to receptors in cell membranes, thereby incorporating the A domain into cells for ADP-ribosylation of intracellular target molecules. It is also suggested that the glycolipid in the membrane was a possible candidate for the receptor of pierisin-1, and the sensitivity of cancer cells to pierisin-1 depended on the presence of the receptor in cell membrane. Moreover, treatment using either the N-terminal polypeptide (amino acids...

maf is a family of oncogenes originally identi®ed from avian oncogenic retrovirus, AS42, encoding a nuclear bZip transcription factor. We have isolated two maf related cDNA clones, maf-1 and maf-2, from a rat liver cDNA library. Comparison of the sequence homologies of the proteins encoded by maf-1 and maf-2 with those of c-maf and chicken mafB indicated that maf-1 and maf-2 are the rat homologues of mafB and c-maf, respectively. Both genes are expressed at low levels in a wide variety of rat tissues, including spleen, kidney, muscle and liver. Immunohistochemical studies and in situ hybridization analyses show that maf-1 and maf-2 are strongly expressed in the late stages of chondrocyte development in the femur epiphysis and the rib and limb cartilage of 15 day old (E15) embryo in rat. Cartilage cells, induced by subcutaneous implantation of bone morphogenic protein, also expressed maf-1 and maf-2. In situ hybridization analyses of E15 embryos show that both genes are expressed in the eye lens and the spinal cord as well as the cartilage. However, the expression patterns of maf-1 and maf-2 in lens and spinal cord are dierent.

We have previously reported that the cabbage butterf ly, Pieris rapae, contains a 98-kDa protein, named pierisin, that induces apoptosis in a variety of human cancer cell lines. In the present study, sequencing and cloning of a cDNA encoding pierisin was accomplished. PCR-direct sequencing showed that the gene encodes an 850-amino acid protein with a calculated molecular weight of 98,081. An intact clone at the amino acid level encompassing the entire coding region was obtained by recombination of two independent clones, and the molecular mass of its in vitro expressed protein was about 100 kDa on SDS͞PAGE, the same as that of purified native pierisin. The expressed protein induced apoptosis in human gastric carcinoma TMK-1 and cervical carcinoma HeLa cells, like the native protein, indicating functional activity. The deduced amino acid sequence of pierisin showed 32% homology with a 100-kDa mosquitocidal toxin from Bacillus sphaericus SSII-1. In addition, pierisin showed regional sequence similarities with ADP-ribosylating toxins, such as the A subunit of cholera toxin. A glutamic acid residue at the putative NAD-binding site, conserved in all ADPribosylating toxins, was also found in pierisin. Substitution of another amino acid for glutamic acid 165 resulted in a great decrease in cytotoxicity and induction of apoptosis. Moreover, inhibitors of ADP-ribosylating enzymes reduced pierisininduced apoptosis. These results suggest that the apoptosisinducing protein pierisin might possess ADP-ribosylation activity that leads to apoptosis of the cells.

Cyclooxygenase‐2 (COX‐2) plays an important role in carcinogenesis. Investigation of the suppressive action of twelve flavonoids of different chemical classes on the transcriptional activity of the COX‐2 gene in human colon cancer DLD‐1 cells using a reporter gene assay have revealed quercetin to be the most potent suppressor of COX‐2 transcription (IC50=10.5 μM), while catechin and epicatechin showed weak activity (IC50=415.3 μM). Flavonoids have three heterocyclic rings as a common structure. A structure‐activity study indicated that the number of hydroxyl groups on the B ring and an oxo group at the 4‐position of the C ring are important in the suppression of COX‐2 transcriptional activity. A low electron density of the oxygen atom in the hydroxyl group of the A ring was also important. Further examination of the role of the hydroxyl group in the A ring showed that bromination of resacetophenone to give 3,5‐dibromo‐2,4‐dihydroxyacetophenone resulted in a 6.8‐fold increase in potency for suppressing COX‐2 promoter activity. These results provide a basis for the design of improved suppressors of COX‐2 transcriptional activity.