In both cases, the mass spectrometer was operated in the atmosphere pressure chemical ionization (APCI) positive ion mode and was tuned with authentic all- em trans- /em retinol. 38), and it has been possible to modulate the ratio of these reactions catalyzed by a bacterial P450 through site-directed mutagenesis (39, 40). In this work, we established the localization of human P450 27C1 in skin. Both 3- and 4-hydroxyretinol were characterized as minor products of P450 27C1. Kinetic isotope effect (KIE) studies with 3- and 4-deuterated retinol showed contributions of CCH bond breaking as a rate-limiting step in catalysis of desaturation and the Butein hydroxylations. Kinetic analysis of other actions in the reaction cycle indicated that P450 27C1 reduction by Adx also contributes in terms of rate limitation. Results Identification of P450 27C1 in skin by mass spectrometry and immunochemical methods MS profiling was done with human liver and skin samples, based on previous work showing P450 27C1 mRNA in the liver (23) and the demonstration of retinol desaturation activity in human skin epidermis (41). Mouse monoclonal to MUSK Because P450 27C1 is considered to be a mitochondrial protein (due to sequence similarity to P450s 27A1 and 27B1 and the exhibited reduction by Adx (24)), we utilized tissue homogenates instead of microsomal fractions. Human P450 27C1 is usually 43% identical to P450 27A1 (a liver enzyme (5)) and 38% identical to P450 27B1 (a kidney enzyme (5)), based on Uniprot.org alignment (http://www.uniprot.org/align).3 Purified recombinant P450 27C1 (see supplemental Fig. S1) was used to guide the selection of migration distances selected for analysis (the different individual) to define the Butein protein found in human skin. Open in a separate window Physique 3. Proteomic analysis of human skin tissue homogenate. drawn ((and of the gel Butein included 0.5, 0.25, 0.10, and 0.05 pmol of purified P450 27C1. The six around the in each include samples from five different humans (tissue homogenates). were further normalized using staining of the transferred proteins with SyPro Ruby dye. The estimated contents of P450 27C1 in the five skin samples were as follows(and supplemental Fig. S2) indicates that this is the correct start site. We are not exactly sure what the N terminus of the lower band is. We did identify the peptide SLAAMPGPR (amino acids 70C78 in Fig. 5and identified in the higher-and identified in the lower-and 267.2 (desaturation) (285.2 (hydroxylation products) (285.2 for a separate mixture of 3-OH and 4-OH all-of 5.6 nm (25). The rate of binding was measured using two different approaches. Equimolar concentrations of P450 27C1 and the substrate all-the substrate concentration (43) to obtain a of 7 4 m and gives a spectrally distinct complex with P450s (Type II) (44). The observed rate was 0.017 s?1, or 1 min?1 (compared with a rate of 1 1,200 240 min?1 for ketoconazole binding to P450 27C1 in the absence of dehydroretinol; results not shown). Open in a separate window Physique 7. Binding of (in seconds). and time (42) (at 37 C). = 3C7) as a function of substrate concentration to estimate a second-order rate of 1 1.6 105 m?1 s?1 (slope) and axis intercept) (= are measured with varying concentrations of protiated and deuterated substrates and used to calculate Dand D(= 1.1 0.2 m, = 3.7 0.7 m?1 min?1 (= 1.8 0.3 m, = 2.5 0.4 m?1 min?1 (= 1.6 0.3 m, = 1.8 0.4 m?1 min?1 (= 2.7 0.6 m, = 1.6 0.4 m?1 min?1 (285.2 all-286.2 (287.2 (blue line, 4-OH) products of 3,3-288.2 (289.2 (285.2 3-OH all-285.2 4-OH all-= 3, means S.D. (shows an expansion of shows an expansion of and P450 27A1 and 27B1 sequences. An alternative possibility is usually that mRNA is usually formed in several tissues but not translated into protein, which would not be surprising in light of the overall concordance between mRNA and protein expression (56). The possibility also exists that this mRNA could be alternatively spliced or transcribed in other tissues. In reviewing the Human Protein Atlas (http://www.proteinatlas.org),3 the highest level of mRNA expression of CYP27C1 is in skin (57). Some other sites in which higher than.
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