Amidation of salicyluric acid and gentisuric acid: a possible role for peptidylglycine alpha-amidating monooxygenase in the metabolism of aspirin.

Bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O2-dependent cleavage of C-terminal glycine-extended peptides, N-acylglycines, and the bile acid glycine conjugates to the corresponding amides and glyoxylate. Two known metabolites of aspirin, salicyluric acid and gentisuric acid, are also substrates for PAM, leading to the formation of salicylamide and gentisamide. The time course for O2 consumption and glyoxylate production indicates that salicylurate amidation is a two-step reaction. Salicylurate is first converted to N-salicyl-alpha-hydroxyglycine, which is ultimately dealkylated to salicylamide and glyoxylate. The enzymatically generated salicylamide and N-salicyl-alpha-hydroxyglycine were characterized by mass spectrometry and two-dimensional 1H-13C heteronuclear multiple quantum coherence NMR.

The enzymatic formation of novel bile acid primary amides.

Bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O(2)-dependent cleavage of C-terminal glycine-extended peptides and N-acylglycines to the corresponding amides and glyoxylate. The alpha-amidated peptides and the long-chain acylamides are hormones in humans and other mammals. Bile acid glycine conjugates are also substrates for PAM leading to the formation of bile acid amides. The (V(MAX)/K(m))(app) values for the bile acid glycine conjugates are comparable to other known PAM substrates. The highest (V(MAX)/K(m))(app) value, 3.1 +/- 0.12 x 10(5) M(-1) s(-1) for 3-sulfolithocholylglycine, is 6.7-fold higher than that for d-Tyr-Val-Gly, a representative peptide substrate. The time course for O(2) consumption and glyoxylate production indicates that bile acid glycine conjugate amidation is a two-step reaction. The bile acid glycine conjugate is first converted to an N-bile acyl-alpha-hydroxyglycine intermediate which is ultimately dealkylated to the bile acid amide and glyoxylate. The enzymatically produced bile acid amides and the carbinolamide intermediates were characterized by mass spectrometry and two-dimensional (1)H-(13)C heteronuclear multiple quantum coherence NMR.

Induction of peptidylglycine alpha-amidating monooxygenase in N(18)TG(2) cells: a model for studying oleamide biosynthesis.

The fatty-acid primary amide, oleamide, is a novel signaling molecule whose mechanism of biosynthesis is unknown. Recently, the N(18)TG(2) cell line was shown to synthesize oleamide from oleic acid, thereby demonstrating that these cells contain the necessary catalytic activities for generating the fatty-acid primary amide. The ability of peptide alpha-amidating enzyme, peptidylglycine-alpha-amidating monooxygenase (PAM; EC 1.14.17.3), to catalyze the formation of oleamide from oleoylglycine in vitro suggests this as a function for the enzyme in vivo. This investigation shows that N(18)TG(2) cells, in fact, express PAM and that cellular differentiation dramatically increases this expression. PAM expression was confirmed by the detection of PAM mRNA, PAM protein, and enzymatic activity that exhibits the functional characteristics of PAM isolated from mammalian neuroendocrine tissues. The regulated expression of PAM in N(18)TG(2) cells is consistent with the proposed role of PAM in the biosynthesis of fatty-acid primary amides and further establishes this cell line as a model for studying the pathway.

Dose-dependent mutation profile in the c-Ha-ras proto-oncogene of skin tumors in mice initiated with benzo[a]pyrene.

Female CD-1 mice were treated topically with a low (25-50 nmol) or high (800 nmol) dose of benzo[a]pyrene (BP) or acetone vehicle, followed by 5 nmol 12-O-tetradecanoylphorbol 13-acetate (TPA) twice a week for 26 weeks. Selective UV radiation fractionation followed by PCR methods were used to analyze histologically defined subsets of cells (approximately 100-200 cells) on formalin-fixed, paraffin-embedded and H&E stained microscope sections. DNA samples from normal-appearing, hyperplastic or tumor regions from the skin of animals from each treatment group were isolated and amplified by PCR with c-Ha-ras-specific primers. Single-strand conformation polymorphism (SSCP) analyses were performed on both exon 1 and 2 products from each sample. DNA extracted from each aberrant band of SSCP analyses was amplified by PCR for further sequence analysis. The data indicate that c-Ha-ras mutations can be detected in normal-looking and hyperplastic epidermal cells as well as in tumor cells obtained from mice initiated with BP and promoted with TPA. The frequencies of c-Ha-ras mutations for normal-looking, hyperplastic and tumor samples were 3/20 (15%), 8/17 (47%) and 58/68 (85%), respectively, for the low dose group and 8/18 (44%), 10/20 (50%) and 64/86 (74%), respectively, for the high dose group. These observations indicate that there were no dose dependencies in the mutation frequencies for normal-looking, hyperplastic and tumor samples. For combined high dose and low dose samples, differences in mutation frequencies of the c-Ha-ras gene between the normal-looking, hyperplastic and tumor samples were highly significant (P < 0.0001, Fisher's exact test). All mutations detected were located at codons 12, 13 and 61 of the c-Ha-ras gene. With the numbers in parentheses indicating the nucleotide position in the coding sequence of the c-Ha-ras proto-oncogene, the distributions of mutations for G-->A (35), G-->T (35), G-->C (37), G-->T (38), C-->A (181), A-->T (182) and A-->G (182) in the low dose tumors were 5, 2, 11, 74, 0, 7 and 2%, respectively, and the distribution of mutations in tumors from animals treated with a high dose of BP were 3, 7, 13, 61, 15, 1 and 0%, respectively. Differences in the global mutation spectra (site and kind of all mutations) for the c-Ha-ras gene between the high and low dose group tumors were statistically significant (P < 0.004, Fisher's exact test) and the major difference between these two groups was C-->A (181) base substitutions. In summary, our data indicate that: (i) 79% of the BP/TPA skin tumors in CD-1 mice had c-Ha-ras mutations for the combined data for high dose and low dose tumors; (ii) the major mutations detected in BP/TPA skin tumors were G-->T transversions; (iii) the global mutation profile in the c-Ha-ras proto-oncogene in skin tumors obtained after initiation with a low dose of BP was different from that obtained after initiation with a high dose of BP.

N-acylglycine amidation: implications for the biosynthesis of fatty acid primary amides.

Bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the O2-dependent conversion of C-terminal glycine-extended prohormones to the active, C-terminal alpha-amidated peptide and glyoxylate. We show that alpha-AE will also catalyze the oxidative cleavage of N-acylglycines, from N-formylglycine to N-arachidonoylglycine. N-Formylglycine is the smallest amide substrate yet reported for alpha-AE. The (V/K)app for N-acylglycine amidation varies approximately 1000-fold, with the (V/K)app increasing as the acyl chain length increases. This effect is largely an effect on the KM,app; the KM,app for N-formylglycine is 23 +/- 0.88 mM, while the KM,app for N-lauroylglycine and longer chain N-acylglycines is in the range of 60-90 microM. For the amidation of N-acetylglycine, N-(tert-butoxycarbonyl)glycine, N-hexanoylglycine, and N-oleoylglycine, the rate of O2 consumption is faster than the rate of glyoxylate production. These results indicate that there must be the initial formation of an oxidized intermediate from the N-acylglycine before glyoxylate is produced. The intermediate is shown to be N-acyl-alpha-hydroxyglycine by two-dimensional 1H-13C heteronuclear multiple quantum coherence (HMQC) NMR.

The ratio of deoxyadenosine to deoxyguanosine adducts formed by (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene in purified calf thymus DNA and DNA in V-79 cells is independent of dose.

The hypothesis that the decrease in the proportion of mutations at AT base pairs in Chinese hamster V-79 cells treated with increasing doses of (+)-(R,S,S,R)-benzo[a]pyrene diol epoxide ((+)-BPDE) is due to saturation of A for adduct formation was investigated by comparing the ratio of dA to dG adducts formed at high (0.48 microM) and low (0.04 microM) doses of [3H]-labeled (+)-BPDE. The dA to dG adduct ratio was similar in both calf thymus DNA and the genomic DNA in V-79 cells, and did not change with dose. For the V-79 cells, this ratio was also unaffected by a 24-h post treatment repair incubation.

Dose-dependent differences in the mutational profiles of (-)-(1R,2S,3S,4R)-3,4-dihydroxy-1, 2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene and its less carcinogenic enantiomer.

Chinese hamster V-79 cells were treated with high cytotoxic or low noncytotoxic concentrations of the highly carcinogenic and mutagenic (-)-(1R,2S,3S,4R)-3,4-dihydroxy-1, 2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene [(-)-B[c]PhDE; fjord-region diol epoxide] or its biologically less active (+)-(1S,2R,3R,4S) enantiomer [(+)-B[c]PhDE]. The benzylic 4-hydroxyl group and the epoxide oxygen are trans in both enantiomers. Independent 8-azaguanine-resistant clones were isolated. The coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene was amplified by reverse transcription-PCR and sequenced. For (-)-B[c]PhDE, mutation frequencies were 10- or 356-fold above background for the low (0.01-0.1 microM; 97% cell survival) or high (1.0-1.25 microM; 26% cell survival) doses, respectively. For the high dose group, 20 of 64 base substitutions occurred at GC base pairs (31%) and 44 at AT base pairs (69%). For the low-dose group, 6 of 55 base substitutions were at GC base pairs (11%), and 49 were at AT base pairs (89%). For the less active (+)-B[c]PhDE, mutation frequencies were 17- or 372-fold above background for the low (0.12-0.5 microM; 95% cell survival) or high (2.0-3.0 microM; 31% cell survival) doses, respectively. In contrast to the results with the (-)-B[c]PhDE, both the high- and the low-dose groups for (+)-B[c]PhDE gave a 50:50 distribution of base substitution at GC versus AT base pairs. Our data indicate that: (a) transversions were the predominant base substitutions observed for both the (+)- and (-)-enantiomers of B[c]PhDE; (b) (-)-B[c]PhDE showed high selectivity for causing AT --> TA transversions, whereas considerably less selectivity was observed for (+)-B[c]PhDE; (c) (-)-B[c]PhDE had a different hot spot profile for base substitutions than did (+)-B[c]PhDE, but some common hot spots were observed for both compounds; and (d) decreasing the dose of (-)-B[c]PhDE increased the proportion of mutations at AT base pairs and decreased those at GC base pairs, but this was not observed for (+)-B[c]PhDE.

Fatty acid amide biosynthesis: a possible new role for peptidylglycine alpha-amidating enzyme and acyl-coenzyme A: glycine N-acyltransferase.

Fatty acid primary amides have recently been recognized as mammalian hormones [Cravatt et al. (1995) Science 268, 1506-1509]. The route to their biosynthesis is unknown. Many mammalian peptide hormones also possess a C-terminal alpha-amide moiety that arises from the posttranslational oxidative cleavage of a C-terminal glycine-extended precursor. The enzyme that catalyzes this reaction is peptidylglycine alpha-amidating enzyme, which is known to preferentially amidate peptide substrates containing a penultimate, hydrophobic amino acid [Tamburini et al. (1990) Int. J. Pept. Protein Res. 35, 153-156]. We show that N-myristoylglycine is a substrate for peptidylglycine alpha-amidating enzyme with a (V/K)app that is 55 +/- 4% of the value measured for D-Tyr-Val-Gly. N-Fatty acylglycines are enzymatically produced in mammals from fatty acyl-coenzyme A (CoAs) and glycine by acyl-CoA:glycine N-acyltransferase. The sequential actions of acyl-CoA:glycine N-acyltransferase and peptidyl-glycine alpha-amidating enzyme would lead to the biosynthesis of fatty acid amides.

Mutagenic selectivity at the HPRT locus in V-79 cells: comparison of mutations caused by bay-region benzo[a]pyrene 7,8-diol-9,-10-epoxide enantiomers with high and low carcinogenic activity.

Earlier studies from our laboratories characterized the mutation profile of the optically active (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-BPDE--the ultimate carcinogenic metabolite of benzo[a]pyrene] in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene of Chinese hamster V-79 cells. In the present study, we evaluated the mutation profile of (-)-7S,8R-dihydroxy-9R, 10S-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-BPDE-a weakly carcinogenic or inactive enantiomer] and compared its mutation profile with that of (+)-BPDE. In both diol epoxide enantiomers, the benzylic 7-hydroxy group and epoxide oxygen are trans. The mutation frequency for V-79 cells treated with DMSO vehicle or with a low, non-cytotoxic dose (0.5 microM) or a high cytotoxic dose (2.0 microM) of (-)-BPDE was 1, 25 or 185 8-azaguanine-resistant colonies/10(5) survivors, respectively. Independent 8-azaguanine-resistant clones were isolated, and complementary DNAs were prepared by reverse transcription. The coding region of the HPRT gene was amplified by the polymerase chain reaction and sequenced. Altogether, 92 (-)-BPDE-induced mutant clones were examined. At both doses, base substitutions were the most prevalent mutations observed (present in approximately 7% of the mutant clones), followed by exon deletions (present in approximately 22% of the mutant clones) and frame shift mutations (present in approximately 6% of the mutant clones) in the cDNAs analyzed. At the high cytotoxic dose, 5 out of 36 base substitutions occurred at AT base pairs (14%) and 31 at GC base pairs (86%). At the low, non-cytotoxic dose, 7 out of 34 base substitutions were at AT base pairs (21%) and 27 were at GC base pairs (79%). Although there was a trend towards an increase in the proportion of mutations at AT base pairs when the dose of (-)-BPDE was decreased, this trend was not statistically significant. The data also indicated no dose-dependent differences in the kinds of base substitutions or exon deletions in cDNAs induced by (-)-BPDE. Ninety-one per cent of the (-)-BPDE-induced mutations that occurred at guanine were on the non-transcribed strand of DNA and 9% were on the transcribed strand. In contrast to these results, 50% of the (-)-BPDE-induced mutations that occurred at adenine were on the transcribed strand and 50% on the non-transcribed strand.(ABSTRACT TRUNCATED AT 400 WORDS)

Dose-dependent differences in the profile of mutations induced by (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene in Chinese hamster V-79 cells.

Chinese hamster V-79 cells were exposed to a high dose (0.30-0.48 microM; 32% cell survival), an intermediate dose (0.04-0.10 microM; 100% cell survival) or a low dose (0.01-0.02 microM; 97% cell survival) of (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+)-BPDE] which is the ultimate carcinogenic metabolite of benzo(a)pyrene. The mutation frequency for cells treated with dimethyl sulfoxide vehicle or with low, intermediate or high dose of (+)-BPDE were 1, 10, 52 or 514 8-azaguanine-resistant colonies/10(5) survivors, respectively. Independent 8-azaguanine-resistant clones were isolated, and complementary DNAs were prepared by reverse transcription. The coding region of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene was amplified by the polymerase chain reaction and sequenced. Altogether, 368 (+)-BPDE-induced mutant clones were examined. At all doses, base substitutions were the most prevalent mutations observed (about 72% of the mutant clones), followed by exon deletions (about 26% of the mutant clones) and frame-shift mutations (about 6% of the mutant clones). At the high cytotoxic dose, 7 of 120 base substitutions occurred at AT base pairs (6%) and 113 at GC base pairs (94%). At the intermediate noncytotoxic dose, 20 of 82 base substitutions occurred at AT base pairs (24%) and 62 at GC base pairs (76%). At the low noncytotoxic dose, 27 of 76 base substitutions were at AT base pairs (36%) and 49 were at GC base pairs (64%). The results indicated that decreasing the dose of (+)-BPDE decreased the proportion of mutations at GC base pairs and increased the proportion of mutations at AT base pairs. At the dose of (+)-BPDE was decreased, there was a dose-dependent decrease in the proportion of GC-->TA transversions (from 69% to 42% of the base substitutions) and a dose-dependent increase in the proportion of AT-->CG transversions (from 1% to 25% of the base substitutions). The data also indicated dose-dependent differences in (+)-BPDE-induced exon deletions and hot spots for base substitutions at GC and AT base pairs. Although more than 99% of the (+)-BPDE-induced mutations at guanine occurred on the nontranscribed strand of DNA, (+)-BPDE-induced mutations at adenine occurred on both the transcribed and nontranscribed strands. The ratio of mutations at adenine on the transcribed strand to mutations at adenine on the nontranscribed strand was 35:19 in (+)-BPDE-treated V-79 cells.(ABSTRACT TRUNCATED AT 400 WORDS)