Crystallization of a soluble form of the Kex1p serine carboxypeptidase from Saccharomyces cerevisiae.

A soluble form of the killer factor and prohormone-processing carboxypeptidase, "Kex1 delta p," from Saccharomyces cerevisiae, has been crystallized in 17-22% poly(enthylene glycol) methyl ether (average M(r) = 5,000), 100 mM ammonium acetate, 5% glycerol, pH 6.5, at 20 degrees C. A native data set (2.8 A resolution) and four derivative data sets (3.0-3.2 A resolution) were collected at the Photon Factory (lambda = 1.0 A). The crystals belong to space group P2(1)2(1)2(1) with a =56.6 A, b = 84.0 A, c = 111.8 A. Freezing a Kex1 delta p crystal has facilitated the collection of a 2.4-A data set using a rotating anode source (lambda = 1.5418 A). Molecular replacement models have been built based on the structures of wheat serine carboxypeptidase (CPDW-II; Liao DI et al., 1992, Biochemistry 31:9796-9812) and yeast carboxypeptidase Y.     

Relative stabilities of synthetic peptide homo- and heterodimeric troponin-C domains.

It has previously been shown that synthetic peptides corresponding to calcium-binding sites III (SCIII) and IV (SCIV) from troponin-C can undergo a calcium-induced dimerization to form the respective homodimers (Shaw GS, Hodges RS, Sykes BD, 1990, Science 249:280-283; Shaw GS et al., 1992a, J Am Chem Soc 114:6258-6259). In addition, an equimolar mixture of SCIII and SCIV has been shown to form preferentially the SCIII/SCIV heterodimer (Shaw GS et al., 1992a, J Am Chem Soc 114:6258-6259). The stabilities of these dimers have been investigated by using 1H-NMR and circular dichroism spectroscopies to follow temperature- and guanidine hydrochloride (GuHCl)-induced denaturations. It has been found that the most stable species, the SCIII/SCIV heterodimer (delta GuH2O = -64.8 kJ/mol), is about 13 kJ/mol more stable than the least stable species, the SCIV homodimer, while the SCIII homodimer is of intermediate stability. This trend of free energies agrees well with the trend of delta G0 values derived from the products of the dissociation constants for calcium binding and peptide association determined from earlier calcium-titration studies. These observations provide evidence that calcium affinity and the association of 2-calcium binding sites are tightly linked. However, it was noted that in all cases delta G0 was considerably more negative than delta GuH2O determined from GuHCl experiments. This difference increased as the stability of the peptide complex increased, providing evidence that linear extrapolation of GuHCl data for very stable proteins may significantly underestimate the value for delta G0.     

The expression of a catalytically active cholesterol 7 alpha-hydroxylase cytochrome P450 in Escherichia coli

We have recently cloned a full-length cDNA encoding the rat hepatic cholesterol 7 alpha-hydroxylase cytochrome P450 (P450c7) (Li, Y. C., Wang, D. P., and Chiang, J. Y. L. (1990) J. Biol. Chem. 265, 12012-12019), which catalyzes the rate-limiting reaction of bile acid synthesis in the liver. By using the polymerase chain reaction, we have designed two P450c7 cDNAs. One has the second Met codon deleted and the third Thr codon replaced with an Ala. The other lacks codons for the NH2-terminal hydrophobic sequence of amino acids 2-24 (P450c7 delta 2-24). The cDNAs were separately cloned into the expression vector pKK233-2 and transformed into Escherichia coli. After induction with isopropyl-beta-D-thiogalactopyranoside, bacteria harboring recombinant plasmids expressed a polypeptide which reacted with the antibody against cholesterol 7 alpha-hydroxylase in immunoblots. The slightly modified full-length enzyme was expressed to 0.2% of the total bacterial lysate and was located in the membrane fraction, whereas P450c7 delta 2-24 was expressed at a 10-fold higher level (2%), of which 85% was in the cytosol and the remaining associated with the membranes. We have purified P450c7 delta 2-24 which showed a typical reduced-CO difference spectrum of cytochrome P450 and reconstituted cholesterol 7 alpha-hydroxylase activity in the presence of NADPH-cytochrome P450 reductase. P450c7 delta 2-24 has a similar Km for cholesterol (24.6 microM) but a lower Vmax (0.10 nmol/min) and a lower turnover number (1.93 min-1) as compared with the enzyme isolated from rat liver microsomes. The purified P450c7 delta 2-24 has an unique hydrophilic NH2 terminus and contains monomers and dimers in equal amounts. This is the first report demonstrating that a genetically engineered cytochrome P450 enzyme lacking a typical NH2-terminal hydrophobic sequence is mainly cytosolic and catalytically active.     

Functional characterization of 102-amino acid-deleted form of human aromatase (delta102-aromatase).

A truncate form of human aromatase cDNA that corresponds to the recently identified rat cortical type aromatase mRNA variant (Yamada-Mouri et al., J. Steroid Biochem. Molec. Biol., 60: 325-329, 1997) has been generated, and the amino-terminus deleted form of the enzyme has been expressed in CHO cells. The resulting product lacking 102 residues from the N-terminus of aromatase (i.e. 102-aromatase) showed an extremely low enzyme activity using an 'In-cell' assay. A strong aromatase activity, however, was observed for the delta102-aromatase using an in vitro method on the solublized preparations. The in vitro activity was dependent on both incubation time and NADPH concentration as well as inclusion of NADPH-cytochrome P450 reductase in the assay mixture. The average turnover rate of aromatization of the reconstituted delta102-aromatase was 6.8 min(-1). The results of the immunosuppression assay suggested that delta102-aromatase still holds the epitope interactive to MAb3-2C2, a monoclonal antibody raised agaist human placental aromatase P450. Furthermore, the IC50 values of MAb3-2C2 were determined to be 24 and 23 microg/ml for the whole homogenate and the 105,000 x g precipitate fractions prepared from the truncated aromatase expressing cells, respectively, whereas an IC50 of 1.3 microg/ml was shown for the full-length human aromatase. These results indicate that the delta102-aromatase P450 can be expressed and is catalytically competent as the full-length enzyme, but the epitope structure for the monoclonal antibody MAb3-2C2 is altered from that of the native enzyme. In addition, the intracellular distribution of delta102-aromatase may be different from that of the wild-type enzyme, explaining why very low activity was measured using an 'In-cell' assay.     

Zinc-substituted cytochrome P450cam: characterization of protein conformers F420 and F450 by photoinduced electron transfer

Metal substitution of heme proteins is widely applied in the study of biologically relevant electron transfer (ET) reactions. It has been shown that many modified proteins remain in their native conformation and can provide useful insights into the molecular mechanism of electron transfer between the native protein and its substrates. We investigated ET reactions between zinc-substituted cytochrome P450(cam) and small organic compounds such as quinones and ferrocene, which are capable of accessing the protein's hydrophobic channel and binding close to the active site, like its native substrate, camphor. Following the substitution method developed by Gunsalus and co-workers [Wagner, G. C., et al. (1981) J. Biol. Chem. 256, 6262-6265], we have identified two dominant forms of the zinc-substituted protein, F450 and F420, that exhibit different photophysical and photochemical properties. The ET behavior of F420 suggests that hydrophobic redox-active ligands are able to penetrate the hydrophobic channel and place themselves in the direct vicinity of the Zn-porphyrin. In contrast, the slower ET quenching rates observed in the case of F450 indicate that the association is weak and occurs outside of the protein channel. Therefore, we conclude that F420 corresponds to the open structure of the native cytochrome P450(cam) while F450 has a closed or partially closed channel that is characteristic of the camphor-containing cytochrome P450(cam). The existence of two distinct conformers of Zn-bound P450(cam) is consistent with the findings of Goodin and co-workers [Lee, Y.-T., et al. (2010) Biochemistry 49, 3412-3419] and has significant consequences for future electron transfer studies on this popular metalloenzyme.     

Cytochrome P-450-catalyzed dehydrogenation of 1,4-dihydropyridines

A variety of different 4-substituted 1,4-dihydropyridine Hantzsch esters are substrates for ring dehydrogenation by a cytochrome P-450 (P-450) enzyme (P-450 UT-A); the substitutent could be varied from a hydrogen to a naphthalenyl, but a pyrenyl derivative was not dehydrogenated. When a 4-alkyl group is present, both the P-450 which oxidizes the substrate and other P-450s can be inactivated (by putative alkyl radicals). P-450s did not discriminate with regard to removal of the 4-H atoms from an enantiomeric pair of dihydropyridines. Losses of the 4-proton and N-methyl from a N-methyl-1,4-dihydropyridine occur at similar rates. The calculated intrinsic kinetic hydrogen isotope effect (Dk) for dehydrogenation of 1,4-dihydro-2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid dimethyl ester was 2.9 in a reconstituted P-450 UT-A enzyme system. No significant kinetic hydrogen isotope effect was observed in microsomal incubations for the dehydrogenation of this compound or 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylic acid diethyl ester in a variety of competitive and noncompetitive experiments. In light of previous studies on the magnitude of kinetic hydrogen isotope effects in P-450 systems (e.g. Miwa et al., 1983 (Miwa, G. T., Walsh, J. S., Kedderis, G. L., and Hollenberg, P. F. (1983) J. Biol. Chem. 258, 14445-14449], the mechanistic proposals of Augusto et al., 1982 (Augusto, O., Beilan, H. S., and Ortiz de Montellano, P. R. (1982) J. Biol. Chem. 257, 11288-11295)) for enzyme inactivation by 4-alkyl-substituted Hantzsch pyridine esters, and other precedents for sequential electron transfer in amine oxidation by P-450s, we interpret these results as being consistent with P-450-mediated 1-electron oxidation of dihydropyridines followed by the facile loss of the 4-proton, with subsequent electron transfer to complete the reaction.     

Evidence for a family of four immunochemically related isozymes of cytochrome P-450 purified from untreated rats

Hepatic microsomal cytochromes P-450f, P-450g, P-450h, and P-450i have recently been purified to electrophoretic homogeneity from untreated adult rats and identified as distinct isozymes [Ryan et al. (1984) J. Biol. Chem. 259, 1239-1250; Haniu et al. (1984) Arch. Biochem. Biophys. 235, 304-311]. In Ouchterlony double-diffusion plates, as well as enzyme-linked immunosorbent assays and Western blots, purified immunoglobulin G (IgG) raised in rabbits against cytochromes P-450f or P-450g show strong cross-reactions with the heterologous protein, indicating apparent partial identity. Anti-P-450f and anti-P-450g also show strong cross-reactivity with cytochromes P-450h and P-450i but not with five previously characterized rat hepatic cytochromes, P-450 (i.e., P-450a-P-450e), indicating a high degree of immunochemical and structural relatedness among cytochromes P-450f, P-450g, P-450h, and P-450i.     

Nucleotide sequence of the gene for the delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni

The structural gene for the delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni has been sequenced by the dideoxy method. The sequence obtained confirms the amino acid (aa) sequence of Benson et al. [J. Biol. Chem. 246 (1971) 7514-7525] at all but 5 aa residues of the 125-aa polypeptide. Amino acid residues 22, 24, 33, and 38, reported to be asparagines by Benson et al., are found to be encoded by aspartic acid codons. Amino acid residue 77, reported to be a glutamine by Benson et al., is encoded by a glutamic acid codon. The identification of aa 38 as aspartic acid, coupled with its presence in the active site, as indicated by previous affinity and photoaffinity-labeling studies and confirmed independently by x-ray crystallographic studies, strengthens the hypothesis that Asp-38 is the aa responsible for the 4 beta to 6 beta proton transfer which is part of the enzymatic reaction.     

Characterization of two novel human retropseudogenes related to the calmodulin-encoding gene, CaMII

Two human genomic clones (lambda hg22 and lambda hg29), containing two novel calmodulin (CaM) retropseudogenes, were isolated and characterized. The two pseudogenes show high similarity with the human CaMII cDNA, hCE1 [SenGupta et al., J. Biol. Chem. 262 (1987) 16663-16670] and the CaMII-type retropseudogene, hCE2 (CaMII-psi 1) [SenGupta et al., Nucleic Acids Res. 17 (1989) 2868]. One of them, in clone lambda hg22 (CaMII-psi 2), shows all the characteristics of a processed pseudogene. In clone lambda hg29 (CaMII-psi 3), however, an Alu repetitive sequence was detected immediately upstream from the ancestral 5'-untranslated region. Downstream from the truncated 3'-untranslated region, three additional copies of Alu repetitive sequences flanking about 750 nucleotides of unknown origin were found. Such a processed retropseudogene flanked by multiple Alu repeats may be a target for further recombination events. The three retropseudogenes CaMII-psi 1, CaMII-psi 2 and CaMII-psi 3 are estimated to be about 49, 21 and 25 million years old, respectively.     

Proteolytic degradation of heme-modified hepatic cytochromes P450: A role for phosphorylation, ubiquitination, and the 26S proteasome?

The resident integral hepatic endoplasmic reticulum (ER) proteins, cytochromes P450 (P450s), turn over in vivo with widely varying half-lives. We and others (Correia et al., Arch. Biochem. Biophys. 297, 228, 1992; and Tierney et al., Arch. Biochem. Biophys. 293, 9, 1992) have previously shown that in intact animals, the hepatic P450s of the 3A and 2E1 subfamilies are first ubiquitinated and then proteolyzed after their drug-induced suicide inactivation. Our findings with intact rat hepatocytes and ER preparations containing native P450s and P450s inactivated via heme modification of the protein have revealed that the proteolytic degradation of heme-modified P450s requires a cytosolic ATP-dependent proteolytic system rather than lysosomal or ER proteases (Correia et al., Arch. Biochem. Biophys. 297, 228, 1992). Using purified cumene hydroperoxide-inactivated P450s (rat liver P450s 2B1 or 3A and/or a recombinant human liver P450 3A4) as models, we now document that these heme-modified enzymes are indeed ubiquitinated and then proteolyzed by the 26S proteasome, but not by its 20S proteolytic core. In addition, our studies indicate that the ubiquitination of these heme-modified P450s is preceded by their phosphorylation. It remains to be determined whether, in common with several other cellular proteins, such P450 phosphorylation is indeed required for their degradation. Nevertheless, these findings suggest that the membrane-anchored P450s are to be included in the growing class of ER proteins that undergo ubiquitin-dependent 26S proteasomal degradation.     

A small plasmid for recombination-based screening.

We reported recently the construction of the 4.4-kb R6K-derived pMAD1 plasmid carrying supF [Stewart et al., Gene 106 (1991) 97-101] that does not share nt sequences with ColE1 and therefore permits recombination-based screening of lambda libraries that contain ColE1 sequences. Here we describe the construction of the 2.5-kb R6K-derived plasmid, pMAD3, that lacks the pi-encoding pir gene required for R6K replication. To supply pi [Inuzuka and Helinski, Proc. Natl. Acad. Sci. USA 75 (1978) 5381-5385] in trans, we employed pPR1 delta 22pir116, referred to henceforth as pPR1 [McEachern et al., Proc. Natl. Acad. Sci. USA 86 (1989) 7942-7946; Dellis and Filutowicz, J. Bacteriol. 173 (1991) 1279-1286]. Plasmid pMAD3 is small enough to be amplified readily by PCR [Saiki et al., Science 230 (1985) 1350-1354]. This permits the insertion of larger fragments and the retrieval of larger lambda inserts, as well as the use of a simplified PCR-based cloning protocol which utilizes annealing rather than ligation to create recombinants in pMAD3 [Nisson et al., PCR Methods and Applications 1 (1991) 120-123].     

The reactivity of alpha-hydroxyhaem and verdohaem bound to haem oxygenase-1 to dioxygen and sodium dithionite.

Recently we have shown that ferric alpha-hydroxyhaem bound to haem oxygenase-1 can be converted to ferrous verdohaem by approximately an equimolar amount of O2 in the absence of exogenous electrons [Sakamoto, H., Omata, Y., Palmer, G., and Noguchi, M. (1999) J. Biol. Chem.274, 18196-18200]. Contrary to those results, other studies have claimed that the conversion requires both O2 and an electron. More recently, Migita et al. have reported that the major reaction product of ferric alpha-hydroxyhaem with O2 is a ferric porphyrin cation radical that can be converted to ferrous alpha-hydroxyhaem with sodium dithionite [Migita, C. T., Fujii, H., Matera, K. M., Takahashi, S., Zhou, H., and Yoshida, T. (1999) Biochim. Biophys. Acta1432, 203-213]. To clarify the reason(s) for the discrepancy, we compared the reactions; i.e. alpha-hydroxyhaem to verdohaem and verdohaem to biliverdin, under various conditions as well as according to the procedures of Migita. We find that complex formation of alpha-hydroxyhaem with haem oxygenase may be small and a substantial amount of free alpha-hydroxyhaem may remain, depending on the reconstitution conditions; this could lead to a misinterpretation of the experimental results. We also find that ferrous verdohaem appears to be air-sensitive and is therefore easily converted to a further oxidized species with excess O2. Finally, we find that dithionite seems to be inappropriate for investigating the haem oxygenase reaction, because it reduces ferrous verdohaem to a further reduced species that has not been seen in the haem degradation system driven by NADPH-cytochrome P450 reductase.     

Copper mediates dityrosine cross-linking of Alzheimer's amyloid-beta

We have previously reported that amyloid Abeta, the major component of senile plaques in Alzheimer's disease (AD), binds Cu with high affinity via histidine and tyrosine residues [Atwood, C. S., et al. (1998) J. Biol. Chem. 273, 12817-12826; Atwood, C. S., et al. (2000) J. Neurochem. 75, 1219-1233] and produces H(2)O(2) by catalyzing the reduction of Cu(II) or Fe(III) [Huang, X., et al. (1999) Biochemistry 38, 7609-7616; Huang, X., et al. (1999) J. Biol. Chem. 274, 37111-37116]. Incubation with Cu induces the SDS-resistant oligomerization of Abeta [Atwood, C. S., et al. (2000) J. Neurochem. 75, 1219-1233], a feature characteristic of neurotoxic soluble Abeta extracted from the AD brain. Since residues coordinating Cu are most vulnerable to oxidation, we investigated whether modifications of these residues were responsible for Abeta cross-linking. SDS-resistant oligomerization of Abeta caused by incubation with Cu was found to induce a fluorescence signal characteristic of tyrosine cross-linking. Using ESI-MS and a dityrosine specific antibody, we confirmed that Cu(II) (at concentrations lower than that associated with amyloid plaques) induces the generation of dityrosine-cross-linked, SDS-resistant oligomers of human, but not rat, Abeta peptides. The addition of H2O2 strongly promoted Cu-induced dityrosine cross-linking of Abeta1-28, Abeta1-40, and Abeta1-42, suggesting that the oxidative coupling is initiated by interaction of H2O2 with a Cu(II) tyrosinate. The dityrosine modification is significant since it is highly resistant to proteolysis and is known to play a role in increasing structural strength. Given the elevated concentration of Cu in senile plaques, our results suggest that Cu interactions with Abeta could be responsible for causing the covalent cross-linking of Abeta in these structures.     

Unrestrained stochastic dynamics simulations of the UUCG tetraloop using an implicit solvation model.

Three unrestrained stochastic dynamics simulations have been carried out on the RNA hairpin GGAC[UUCG] GUCC, using the AMBER94 force field (Cornell et al., 1995. J. Am. Chem. Soc. 117:5179-5197) in MacroModel 5.5 (Mohamadi et al., 1990. J. Comp. Chem. 11:440-467) and either the GB/SA continuum solvation model (Still et al., 1990. J. Am. Chem. Soc. 112:6127-6129) or a linear distance-dependent dielectric (1/R) treatment. The linear distance-dependent treatment results in severe distortion of the nucleic acid structure, restriction of all hydroxyl dihedrals, and collapse of the counterion atmosphere over the course of a 5-ns simulation. An additional vacuum simulation without counterions shows somewhat improved behavior. In contrast, the two GB/SA simulations (1.149 and 3.060 ns in length) give average structures within 1.2 A of the initial NMR structure and in excellent agreement with results of an earlier explicit solvent simulation (Miller and Kollman, 1997. J. Mol. Biol. 270:436-450). In a 3-ns GB/SA simulation starting with the incorrect UUCG tetraloop structure (Cheong et al., 1990. Nature. 346:680-682), this loop conformation converts to the correct loop geometry (Allain and Varani, 1995. J. Mol. Biol. 250:333-353), suggesting enhanced sampling relative to the previous explicit solvent simulation. Thermodynamic effects of 2'-deoxyribose substitutions of loop nucleotides were experimentally determined and are found to correlate with the fraction of time the ribose 2'-OH is hydrogen bonded and the distribution of the hydroxyl dihedral is observed in the GB/SA simulations. The GB/SA simulations thus appear to faithfully represent structural features of the RNA without the computational expense of explicit solvent.     

Distribution of human rotaviruses, especially G9 strains, in Japan from 1996 to 2000

A 4-year (1996-2000) survey of rotavirus infection involving 2,218 diarrheal fecal specimens of children collected from five regions of Japan was conducted. A total of 642 (28.9%) specimens were found to be rotavirus positive. A changed prevalence pattern of rotavirus G serotype was found with an increase of G9 and G2 and a decrease of G1, although G1 remained the prevailing serotype. Serotype G9 was unexpectedly determined to be the prevailing serotype in Sapporo (62.5%) and Tokyo (52.9%) in 1998-1999, and in Saga (78.4%) in 1999-2000. G9 strains isolated from 1998-1999 belonged to the P[8]-NSP4-Wa-group with long RNA pattern, while, G9 strains isolated from 1999-2000 belonged to three groups, the P[8]-NSP4-Wa-group with long RNA pattern, the P[4]-NSP4-KUN-group with short RNA pattern and a mixed-type group (P[4]/P[8]-NSP4-KUN/Wa-group with long RNA pattern). Both sequence and immunological analysis of VP7 revealed that the G9 strains from 1999-2000 were much more closely related to the G9 strains isolated worldwide in the 1990s, including G9 strains found in Thailand in 1997. However, the G9 strains from 1998-1999 were distinct from these and more closely related to the G9 prototype strains F45, AU32 and WI61 discovered in Japan and the US in the 1980s. Thus the G9 strains isolated in 1998-1999 had progenitors common to the G9 prototype strains, while the strains isolated in 1999-2000 did not directly evolve from them but were related to global G9 strains that have emerged in recent years. These data supported our previous report that G9 rotavirus might exist as two or more subtypes with diverse RNA patterns, P-genotype and NSP4 genogroup combinations (Y.M. Zhou et al., J. Med. Virol. 65: 619-628, 2001) and suggested that G9 rotavirus prevalent in Japan during two successive years belonged to different subtypes. The nucleotide sequences presented in this paper were submitted to DDBJ, EMBL and GenBank nucleotide sequence databases. The accession numbers are: 00-Ad2863VP7 (AB091746), 00-OS2986VP7 (AB091747), 00-SG2509VP7 (AB091748), 00-SG2518VP7 (AB091749), 00-SG2541 (AB091750), 00-SG2864 (AB091751), 00-SP2737VP7 (AB091752), 99-SP1542VP7 (AB091753), 99-SP1904VP7 (AB091754), 99-TK2082VP7 (AB091755) and 99-TK2091VP7 (AB091756).     

Structural rationalization of novel drug metabolizing mutants of cytochrome P450 BM3

Three newly discovered drug metabolizing mutants of cytochrome P450 BM3 (van Vugt-Lussenburg et al., Identification of critical residues in novel drug metabolizing mutants of Cytochrome P450 BM3 using random mutagenesis, J Med Chem 2007;50:455-461) have been studied at an atomistic level to provide structural explanations for a number of their characteristics. In this study, computational methods are combined with experimental techniques. Molecular dynamics simulations, resonance Raman and UV-VIS spectroscopy, as well as coupling efficiency and substrate-binding experiments, have been performed. The computational findings, supported by the experimental results, enable structural rationalizations of the mutants. The substrates used in this study are known to be metabolized by human cytochrome P450 2D6. Interestingly, the major metabolites formed by the P450 BM3 mutants differ from those formed by human cytochrome P450 2D6. The computational findings, supported by resonance Raman data, suggest a conformational change of one of the heme propionate groups. The modeling results furthermore suggest that this conformational change allows for an interaction between the negatively charged carboxylate of the heme substituent and the positively charged nitrogen of the substrates. This allows for an orientation of the substrates favorable for formation of the major metabolite by P450 BM3.     

Avian kidney mitochondrial hemeprotein P-4501 alpha: isolation, characterization and NADPH-ferredoxin reductase-dependent activity

We describe the isolation of cytochrome P-4501 alpha from chick-kidney mitochondria. Although, gel permeation HPLC yielded 41% of the total amount of P-450 present in cholate-solubilized hemeproteins, it produced a highly purified mixture from which the P-4501 alpha could be purified to homogeneity in a final detergent-free state by a single-step application of hydrophobic interaction HPLC using hydroxypropyl silica. The purified P-4501 alpha traveled as a single band in SDS gel electrophoresis with an apparent Mr = 57,000. The absolute spectrum of the P-4501 alpha (Fe3+) form gave a lambda max at 403 nm. This characteristic lends support to the anomalous high-spin heme electron paramagnetic resonance spectrum and the heme structure of P-4501 alpha which we have previously reported (Ghazarian et al. (1980) J. Biol. Chem. 255, 8275-8281; Pedersen et al. (1976) J. Biol. Chem. 251, 3933-3941). In reconstitution experiments with ferredoxin-dependent NADPH-cytochrome c (P-450) reductase complexes, P-4501 alpha catalyzed the hydroxylation of 25-hydroxy-9,10-secocholesta-5,7,10(19)-trien-3 beta-ol at the C-1 position exclusively with a turnover number of 0.03 min-1. This number is identical to that obtained from measurements of the catalytic activity in intact mitochondria, indicating that only one major species of cytochrome P-450 occurs in chick-kidney mitochondria. The complete responsiveness of cytochrome P-450 concentrations in intact mitochondria to the vitamin D status of chicks provided additional evidence that the major cytochrome P-450 species present in renal mitochondria is uniquely associated with vitamin D metabolism.     

Intracellular singlet oxygen generation by phagocytosing neutrophils in response to particles coated with a chemical trap.

To determine if singlet oxygen (O2(1 delta g)) is produced by neutrophils (PMNs) during the process of phagocytosis, glass beads were coated with a specific chemical trap for O2(1 delta g), 9,10-diphenylanthracene (DPA). Singlet oxygen, but not other reactive oxygen species, reacts rapidly with DPA at a rate of kr = 1.3 x 10(6) M-1 s-1 to form a stable product, DPA-endoperoxide (Corey, E. J., and Taylor, W. C. (1964) J. Am. Chem. Soc. 86, 3881-3882; Wasserman, H. H., Scheffer, J. R., and Cooper, J. L. (1972) J. Am. Chem. Soc. 94, 4991-4996; Turro, N. J., Chow, M.-F., and Rigaudy, J. (1981) J. Am. Chem. Soc. 103, 7218-7224). The production of DPA-endoperoxide was determined by ultraviolet spectroscopy as a decrease in DPA absorbance at 355 nm. The absorbance of DPA was normalized to the absorbance of perylene, which was included in the coating on the beads as a nonreactive, internal standard. In the present study, DPA- and perylene-coated beads were initially allowed to adhere to fibronectin-coated coverslips. PMNs were then added to the bead-coated coverslips and allowed to adhere and phagocytose the beads for 1 h at 37 degrees C. In some experiments, 4B-phorbol-12-myristate-13-acetate (PMA) (1 ng/2.5 x 10(7) cells/ml), a known activator of the PMN NADPH-oxidase, was added as a co-stimulant. The amount of O2(1 delta g) produced by phagocytically stimulated PMNs was calculated to be 11.3 +/- 4.9 nmol of O2(1 delta g)/1.25 x 10(6) cells. Low dose PMA co-stimulation increased the production of O2(1 delta g) to 14.1 +/- 4.1 nmol/1.25 x 10(6) cells. Averaged together these amounts represent approximately 19 +/- 5.0% of the total oxygen consumed by PMNs in response to DPA- and perylene-coated beads. The specificity of the DPA reaction with O2(1 delta g) was confirmed by warming to 120 degrees C, which releases O2(1 delta g) from the DPA-endoperoxide, regenerating the parent DPA compound (Wasserman et al., 1972; Turro et al., 1981) and the absorbance at 355 nm. In addition, beta-carotene, an avid quencher of O2(1 delta g), was included in the coating of some bead preparations; assays in which these beads were used showed no change in the absorbance at 355 nm. Singlet oxygen production by myeloperoxidase was also measured using the coated bead assay and the results suggest that this is a major pathway by which singlet oxygen is generated in phagocytically stimulated PMNs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two forms of DNA polymerase delta from mouse cells. Purification and properties

A procedure is described for the purification from cultured mouse cells of two DNA polymerase "delta-like" enzymes, as defined by intrinsic 3'-exonuclease activity, inhibition by aphidicolin, and relative insensitivity to N2-(p-n-butylphenyl)-dGTP. One of the two enzymes has been purified to near homogeneity and, similar to the DNA polymerase delta from calf thymus described by Lee et al. (Lee, M. Y. W. T., Tan, C. K., Downey, K. M., and So, A. G. (1984) Biochemistry 23, 1906-1913), it has a total molecular mass of 178 kDa (from sedimentation velocity of 8.0 S and Stokes radius of 54 A) and is composed of one each of 125- and 50-kDa polypeptides. It also resembles the DNA polymerase delta of Lee et al. in being stimulated by proliferating cell nuclear antigen (PCNA). It is the first clear structural and functional counterpart of the calf thymus enzyme. The major difference between the mouse DNA polymerase delta and the calf thymus enzyme of Lee et al. is that, under specific conditions, the mouse enzyme is active with poly(dA).oligo(dT) in the absence of PCNA, whereas the activity of the calf thymus enzyme with this template is reported to be completely dependent on PCNA. The reason for this difference is not known at this time. The second mouse cell enzyme has a molecular mass of 112 kDa (from sedimentation velocity of 6.3 S and Stokes radius of 43.0 A) and consists of a single polypeptide of 123-125 kDa in denaturing gels (p125). On the basis of its apparent formation by dissociation of DNA polymerase delta, and multiple similarities with DNA polymerase delta in enzymatic properties, the p125 is provisionally identified as the 125-kDa polypeptide of DNA polymerase delta. The p125 does not respond to PCNA, suggesting that the 50-kDa polypeptide is required for the stimulation of DNA polymerase delta by PCNA. The presence of the p125 in cell extracts would explain reports that DNA polymerase delta consists of a single polypeptide of approximately 125 kDa and/or thast it has a smaller molecular mass than DNA polymerase delta of Lee et al. and is not affected by PCNA (this does not apply to PCNA-independent DNA polymerase delta-like enzymes with higher molecular mass than the polymerase delta of Lee et al., which have recently been named DNA polymerases epsilon).