Biochemical Properties of Purified Recombinant Human β-Carotene 15,15′-Monooxygenase
2002; Elsevier BV; Volume: 277; Issue: 26 Linguagem: Inglês
10.1074/jbc.m202756200
ISSN1083-351X
AutoresAnnika Lindqvist, Stefan Andersson,
Tópico(s)Photosynthetic Processes and Mechanisms
Resumoβ-Carotene 15,15′-monooxygenase (BCO), formerly known as β-carotene 15,15′-dioxygenase, catalyzes the first step in the synthesis of vitamin A from dietary carotenoids. We have biochemically and enzymologically characterized the purified recombinant human BCO enzyme. A highly active BCO enzyme was expressed and purified to homogeneity from baculovirus-infectedSpodoptera frugiperda 9 insect cells. TheK m and V max of the enzyme for β-carotene were 7 μm and 10 nmol retinal/mg × min, respectively, values that corresponded to a turnover number (k cat) of 0.66 min−1 and a catalytic efficiency (k cat/K m ) of ∼105m−1·min−1. The enzyme existed as a tetramer in solution, and substrate specificity analyses suggested that at least one unsubstituted β-ionone ring half-site was imperative for efficient cleavage of the carbon 15,15′-double bond in carotenoid substrates. High levels of BCO mRNA were observed along the whole intestinal tract, in the liver, and in the kidney, whereas lower levels were present in the prostate, testis, ovary, and skeletal muscle. The current data suggest that the human BCO enzyme may, in addition to its well established role in the digestive system, also play a role in peripheral vitamin A synthesis from plasma-borne provitamin A carotenoids. β-Carotene 15,15′-monooxygenase (BCO), formerly known as β-carotene 15,15′-dioxygenase, catalyzes the first step in the synthesis of vitamin A from dietary carotenoids. We have biochemically and enzymologically characterized the purified recombinant human BCO enzyme. A highly active BCO enzyme was expressed and purified to homogeneity from baculovirus-infectedSpodoptera frugiperda 9 insect cells. TheK m and V max of the enzyme for β-carotene were 7 μm and 10 nmol retinal/mg × min, respectively, values that corresponded to a turnover number (k cat) of 0.66 min−1 and a catalytic efficiency (k cat/K m ) of ∼105m−1·min−1. The enzyme existed as a tetramer in solution, and substrate specificity analyses suggested that at least one unsubstituted β-ionone ring half-site was imperative for efficient cleavage of the carbon 15,15′-double bond in carotenoid substrates. High levels of BCO mRNA were observed along the whole intestinal tract, in the liver, and in the kidney, whereas lower levels were present in the prostate, testis, ovary, and skeletal muscle. The current data suggest that the human BCO enzyme may, in addition to its well established role in the digestive system, also play a role in peripheral vitamin A synthesis from plasma-borne provitamin A carotenoids. β-carotene 15,15′-monooxygenase high performance liquid chromatography 1-S-octyl-β-d-thioglucopyranoside phosphate-buffered saline Tris(2-carboxyethyl)phosphine hydrochloride 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine monoclonal antibody Retinol, also referred to as vitamin A, is a fat-soluble polyisoprenoid essential for tissue development, growth, and vision. Retinol can be either ingested or synthesized within the body from dietary carotenoids. Preformed retinol is found almost exclusively in animals in the form of esters of fatty acids. It is hydrolyzed during the process of digestion, absorbed in the free form, re-esterified with fatty acids within the intestinal mucosa, and transported to the liver via the lymphatic route associated with chylomicrons (1Blaner W.S. Olson J.A. Sporn M.B. Roberts A.B. S. G.D. The Retinoids. Raven Press, New York1994: 229-255Google Scholar). Τhe major substrate for the in vivo synthesis of retinol is the plant carotenoid β-carotene. Of the more than 600 different carotenoids isolated from nature, ∼50 possess biological activity; hence, these compounds are termed provitamin A carotenoids (2Bendich A. Olson J.A. FASEB J. 1989; 3: 1927-1932Crossref PubMed Scopus (487) Google Scholar, 3Olson J.A. J. Nutr. 1989; 119: 105-108Crossref PubMed Scopus (312) Google Scholar, 4Rock C.L. Pharmacol. Ther. 1997; 75: 185-197Crossref PubMed Scopus (223) Google Scholar). In vivo studies in humans show that the majority of the ingested β-carotene is cleaved at the central carbon 15,15′-double bond to form two molecules of retinal (retinaldehyde) (5Blomstrand R. Werner B. Scand. J. Clin. Lab. Invest. 1967; 19: 339-345Crossref PubMed Scopus (99) Google Scholar, 6Goodman D.S. Blomstrand R. Werner B. Huang H.S. Shiratori T. J. Clin. Invest. 1966; 45: 1615-1623Crossref PubMed Scopus (250) Google Scholar), and consequently, the enzyme that catalyzes this first step in vitamin A synthesis in the intestinal mucosa was named β-carotene 15,15′-dioxygenase (BCO)1(7Olson J.A. Hayaishi O. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1364-1370Crossref PubMed Scopus (259) Google Scholar). Although the enzyme activity was first described in the mid-1950s, it was not until 1965 that the research groups of Goodman (8Goodman D. Huang H. Science. 1965; 149: 879-880Crossref PubMed Scopus (184) Google Scholar) and Olson (7Olson J.A. Hayaishi O. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1364-1370Crossref PubMed Scopus (259) Google Scholar) independently demonstrated that the cleavage of β-carotene to retinal could be studied in vitro by using soluble enzyme preparations from the intestine and liver. Characterization of the native enzyme was performed with a 100,000 × gsupernatant fraction or an ammonium sulfate precipitate thereof. It was shown that the reaction is dependent on molecular oxygen and that nicotinamide dinucleotide cofactors are not required for catalysis. Also, the facts that the reaction was inhibited by the addition of iron chelating agents and that cyanide, which inhibits ferric protoporphyrin enzymes, did not attenuate the conversion of β-carotene to retinaldehyde suggested that BCO was a nonheme iron-containing enzyme. Furthermore, the native enzyme had the ability to cleave the 15,15′-double bond of a variety of carotenoids other than β-carotene, including α-carotene, β-apocarotenols, and β-apocarotenals (9Lakshmanan M.R. Pope J.L. Olson J.A. Biochem. Biophys. Res. Commun. 1968; 33: 347-352Crossref PubMed Scopus (36) Google Scholar, 10Lakshmanan M.R. Chansang H. Olson J.A. J. Lipid Res. 1972; 13: 477-482Abstract Full Text PDF PubMed Google Scholar, 11Singh H. Cama H.R. Biochim. Biophys. Acta. 1974; 370: 49-61Crossref PubMed Scopus (48) Google Scholar). The Michaelis-Menten constant (K m ) for the native enzyme of a variety of animal species relative to β-carotene has been determined to be in the 1–10 μm range. The pH optimum for the reaction was in the slightly alkaline range, and the enzyme was inhibited by sulfhydryl alkylating reagents such asp-chloromercuribenzoate and N-ethylmaleimide. BCO was either protected or activated by sulfhydryl reducing agents such as cysteine and glutathione (7Olson J.A. Hayaishi O. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1364-1370Crossref PubMed Scopus (259) Google Scholar, 10Lakshmanan M.R. Chansang H. Olson J.A. J. Lipid Res. 1972; 13: 477-482Abstract Full Text PDF PubMed Google Scholar, 11Singh H. Cama H.R. Biochim. Biophys. Acta. 1974; 370: 49-61Crossref PubMed Scopus (48) Google Scholar, 12Goodman D. Huang H. Kanai M. Shiratori T. J. Biol. Chem. 1967; 242: 3543-3554Abstract Full Text PDF Google Scholar, 13Fidge N.H. Smith F.R. Goodman D.S. Biochem. J. 1969; 114: 689-694Crossref PubMed Scopus (51) Google Scholar). The human BCO cDNA encodes a hydrophilic protein of 547 amino acids with a predicted molecular weight of 62,637 (14Yan W. Jang G.-F. Haeseleer F. Esumi N. Chang J. Kerrigan M. Campochiaro M. Campochiaro P. Palczewski K. Zack D.J. Genomics. 2001; 72: 193-202Crossref PubMed Scopus (139) Google Scholar). Amino acid comparison of the human BCO with the mouse (15Wyss A. Wirtz G. Woggon W.-D. Brugger R. Wyss M. Friedlein A. Riss G. Bachmann H. Hunziker W. Biochem. J. 2001; 354: 521-529Crossref PubMed Scopus (111) Google Scholar), rat (GenBankTMaccession number NM_053648), chicken (16Wyss A. Wirtz G. Woggon W. Brugger R. Wyss M. Friedlein A. Bachmann H. Hunziker W. Biochem. Biophys. Res. Commun. 2000; 271: 334-336Crossref PubMed Scopus (198) Google Scholar), zebrafish (GenBankTM accession number AJ290390), andDrosophila (17von Lintig J. Vogt K. J. Biol. Chem. 2000; 275: 11915-11920Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar) enzymes show sequence identities of 85, 84, 67, 56, and 22%, respectively. Based on biochemical and amino acid sequence data, it has been proposed that BCO belongs to the nonheme iron-containing dioxygenase family. However, recently it was demonstrated that the reaction mechanism of enzymatic cleavage of the central carbon 15,15′-double bond in β-carotene involves a monooxygenase-type mechanism (18Leuenberger M.G. Engeloch-Jarret C. Woggon W.-D. Angew. Chem. Int. Ed. Engl. 2001; 40: 2614-2617Crossref Google Scholar). By using a partially purified chicken intestine BCO preparation and isomerically pure α-carotene as substrate, it was shown that both 17O2 and H218O were incorporated into the two retinal products. Thus, the BCO enzyme was renamed β-carotene 15,15′-monooxygenase (19Wirtz G. Bornemann C. Giger A. Muller R.K. Schneider H. Schlotterbeck G. Scheifer G. Woggon W.-D. Helv. Chim. Acta. 2001; 84: 2301-2315Crossref Scopus (28) Google Scholar). The human BCO gene structure and chromosomal localization were also recently reported (14Yan W. Jang G.-F. Haeseleer F. Esumi N. Chang J. Kerrigan M. Campochiaro M. Campochiaro P. Palczewski K. Zack D.J. Genomics. 2001; 72: 193-202Crossref PubMed Scopus (139) Google Scholar). However, no biochemical characterization of a purified human enzyme has been described. In the current study, we describe a detailed biochemical and enzymological characterization of the purified recombinant human BCO enzyme. Furthermore, a comprehensive analysis of tissue-specific expression is described. All-trans-β-carotene, lycopene, all-trans-retinal, all-trans-retinol, all-trans-retinoic acid, o-phenanthroline,N-ethylmaleimide, and p-chloromercuribenzoate were purchased from Sigma. Zeaxanthin and β-cryptoxanthin were obtained from Indofine (Somerville, NJ). α,α-Bipyridyl was from Aldrich. Acetonitrile was HPLC grade (Merck). Standard molecular biology techniques were used (20Sambrook J. Russell D.W. Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001Google Scholar). The complete coding sequence of human BCO cDNA was obtained from a liver cDNA pool (CLONTECH Laboratories, Inc., Palo Alto, CA) by PCR using the following primers: hBCOf2 (sense) 5′-AAATCGATCTCCCTCGGCACC-3′ and BacBCOr (antisense) 5′-CGGAATTCTTATCAGGTCAGAGGAGCCCCGTG-3′, corresponding to nucleotides 191–211 and 1863–1842, respectively, in the human cDNA sequence (GenBankTM accession number AK001592). The PCR product was first subcloned into the pCR2.1-TOPO vector (Invitrogen) using the extra deoxyadenosine added by the Taqpolymerase and then subcloned in the pCMV-Sport2.0 vector (Invitrogen) using EcoRI. The insert in the pCMV-hBCO vector was subcloned into pBluescript SK− (Stratagene, La Jolla, CA) using KpnI andXbaI, and the 3′ part of the hBCO coding insert was removed by digestion with SalI and AccI. A 494-bp fragment containing the corresponding coding region part but with six histidine codons inserted before the stop codon was obtained by PCR using primers CHis5 (sense) 5′-AAGTCTACTGCCAGCCGGAATTTCTTTATGAAGGC-3′ and CHis3 (antisense) 5′-TTGTCGACTATCAATGATGGTGATGATGGTGGGTCAGAGGAGCCCCGTGGC-3′. The fragment was then digested with SalI and AccI and subcloned into the SalI/AccI-digested pBluescript-hBCO vector, resulting in pBluescript-hBCO-His. The pFastBac1-hBCO-His donor plasmid for subsequent production of recombinant baculovirus was constructed by transfer of the pBluescript vector insert into pFastBac1 (Invitrogen) using EcoRI andSalI. Spodoptera frugiperda 9 cells were maintained in monolayer cultures with Sf-900 II SFM (catalog number 10902-096; Invitrogen), or Grace's insect medium (catalog number 11605-094; Invitrogen) supplemented with 10% fetal calf serum. Using the Bac-To-BacTM baculovirus expression system (Invitrogen) and the pFastBac1-hBCO-His plasmid (above), recombinant baculovirus was obtained. Expression of the C-terminally histidine-tagged recombinant human BCO protein was performed by infecting S. frugiperda 9 cells at a multiplicity of infection of 10 and harvesting the cells after 3 days culture. The cells from three 225-cm2 flasks were detached and collected by centrifugation 5 min at 500 ×g, then resuspended in 2.7 ml of chilled 50 mmsodium phosphate buffer (pH 7.0), kept on ice 5 min, and subsequently homogenized by 20 strokes in a glass tissue grinder. After centrifugation 30 min at 10,000 × g at 4 °C, the supernatant (S10) was collected, and sodium chloride was added to a final concentration of 100 mm. The S10 was then immediately incubated with 0.5 ml of Talon CellThru resin (CLONTECH) with gentle agitation for 30 min at 4 °C. The resin was washed three times with 10 column volumes of 50 mm sodium phosphate buffer (pH 7.0) containing 100 mm sodium and 5 mm imidazole (wash/extraction buffer), and then bound protein was eluted by two 15-min incubations with one column volume of wash/extraction buffer containing 300 mm sodium chloride and 150 mm imidazole each time. The slurry was centrifuged at 500 × g for 2 min after each wash and elution step, and the supernatant was collected. The purified hBCO-His enzyme was stored on ice until used. All of the reactions were performed in a volume of 100 μl in an assay buffer consisting of 100 mm Tricine-KOH (pH 8.0), 125 mm sodium chloride, 10 μmFe2SO4, 5 mmTris(2-carboxylethyl)phosphine hydrochloride (TCEP) (Pierce), and 1% (w/v) 1-S-octyl-β-d-thioglucopyranoside (OTG) (Pierce). The carotenoid concentration in the reactions varied between 0.125 and 128 μm, and the hBCO-His enzyme amount varied between 60 and 1000 ng/reaction. The assays were set up and run under subdued light as follows; carotenoid in hexane was added to 25 μl of 4% (w/v) OTG in 2-ml Eppendorf tubes, and the solvent was evaporated. The assay buffer and enzyme, in some cases preincubated with various potentially inhibitory compounds for 30 min on ice, were then added to the carotenoid/detergent mix, and the tubes were incubated for 5 min to 1 h at 37 °C with gentle agitation (70 rpm). HPLC analyses of reaction products were performed essentially as described by Duringet al. (21During A. Nagao A. Hoshino C. Terao J. Anal. Biochem. 1996; 241: 199-205Crossref PubMed Scopus (78) Google Scholar). Briefly, 25 μl of 37% (v/v) formaldehyde was added, and the incubation was continued for 10 min at 37 °C. For extraction of the products, 250 μl of acetonitrile was added, and the tube was vortexed and kept on ice for 5 min. After centrifugation for 10 min at 10,000 × g at 4 °C, the supernatant was separated on a 4.6 × 150-mm Phenomenex LUNA 3μ C18 column (Phenomenex, Torrance, CA) or a 4.6 × 150-mm XTerra MS C18 3.5 μm column (Waters, Milford, MA) in a mobile phase consisting either of 90% acetonitrile, 10% water, 0.1% (w/v) ammonium acetate or of 85% acetonitrile, 15% water, 0.1% (w/v) ammonium acetate with a flow rate of 1 ml/min (Waters 501 HPLC pump) and UV detection (Waters 484 tunable absorbance detector) at 380 nm. The enzyme kinetics were calculated using GraFit Version 5.0.1 (Erithacus Software Limited, Horley, UK). This program fits the data to the Michaelis-Menten equation using nonlinear regression analysis. For spectral scan analyses of the products, the 4.6 × 150-mm XTerra MS C18 3.5 μm column was used under the same conditions as above, but with a LC-10AT liquid chromatograph, SPD-M10AVP diode array detector, and Class-VP chromatograph data system, version 4.2 (Shimadzu, Columbia, MD). Retinal formed during the reactions was quantified from its peak height by using a standard curve obtained by incubating 1–50 pmol of all-trans-retinal in a 100 mm Tricine-KOH (pH 8.0) buffer, containing 125 mm NaCl, 10 μmFe2SO4, 5 mm TCEP, 1% (w/v) OTG, and 250 ng of heat-inactivated BCO for 15 min at 37 °C with gentle agitation (70 rpm). The samples were then treated the same way as described above for the BCO assay samples and were separated on a 4.6 × 150-mm Phenomenex LUNA 3μ C18 column in a mobile phase consisting of 90% acetonitrile, 10% water, and 0.1% (w/v) ammonium acetate. The synthetic peptide [C]RNRKEQLEPVRAKVTGK, corresponding to amino acid residues 7–23 in human BCO, was coupled to keyhole limpet hemocyanin usingm-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Pierce) (22Harlow E. Lane D. Antibodies: A Laboratory Manual. 1st Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 82-83Google Scholar), and used for immunization of mice as described previously (23Moghrabi N. Head J.R. Andersson S. J. Clin. Endocrinol. Metab. 1997; 82: 3872-3878Crossref PubMed Google Scholar). Hybridomas were established and screened by an enzyme-linked immunosorbent assay using the synthetic peptide as antigen as described previously (23Moghrabi N. Head J.R. Andersson S. J. Clin. Endocrinol. Metab. 1997; 82: 3872-3878Crossref PubMed Google Scholar). The anti-BCO monoclonal antibody, designated mAb-1-11, was determined to belong to the IgG1/κ subclass as revealed by an enzyme-linked immunosorbent assay-based mouse typer subisotyping kit (Bio-Rad). SDS-polyacrylamide electrophoresis was performed according to the method of Laemmli (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207192) Google Scholar), and the proteins were either detected in the gel with Coomassie Brilliant Blue R or with silver staining using a Silver Stain Plus kit (Bio-Rad). Alternatively, the proteins were transferred onto an Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA) using a previously published method (25Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44918) Google Scholar). For immunodetection of BCO proteins, mAb-1-11 containing tissue culture medium was incubated 1 h at room temperature, followed by a secondary horseradish peroxidase-conjugated goat anti-mouse IgG polyclonal antibody (catalog number 170-6516; Bio-Rad) diluted 1:10,000, and incubated for 1 h at room temperature. Antibody binding was detected by chemiluminescence using an ECL kit (Amersham Biosciences). The protein concentrations were determined using a Coomassie Plus protein assay reagent (Pierce). The purified enzyme (50 μg) was applied on a Sephacryl S-300 (Amersham Biosciences) column (7 × 300 mm) equilibrated with 100 mm Tricine-KOH buffer (pH 8.0), containing 125 mm NaCl, 10 μmFe2SO4, 5 mm TCEP, and 1% (w/v) OTG with a flow rate of 17.5 ml/h, and 0.3-ml fractions were collected. 75 μl of each fraction was used directly in BCO enzyme assays, and 7.5 μl of each fraction was analyzed by immunoblotting. The columns were calibrated with proteins of known molecular mass: albumin (67 kDa), aldolase (158 kDa), catalase (232 kDa), and blue dextran (∼2 × 106 Da). The human hepatocellular carcinoma cell line, HepG2 (ATCC HB-8065), maintained in Dulbecco's modified Eagle's medium (catalog number 11995-065; Invitrogen) supplemented with 10% fetal calf serum, 10 mm HEPES, and 1% penicillin/streptomycin was cultured on 12-mm diameter microscope glass covers. The cells were transiently transfected with pCMV-hBCO using Fugene 6 (Roche Diagnostics Corporation, Indianapolis, IN) according to the manufacturer's instructions. 36 h after transfection, the cells were rinsed twice with cold PBS (10 mm sodium phosphate, pH 7.4, 150 mm sodium chloride), fixated with cold methanol for 10 min at −20 °C, then rinsed twice with cold PBS, and blocked 1 h at room temperature with PBS containing 1% (w/v) bovine serum albumin. The cells were then incubated 1 h at room temperature with undiluted mAb-1-11 monoclonal antibody containing tissue culture medium, rinsed three times with PBS, and then incubated 40 min at room temperature with fluorescein isothiocyanate-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) diluted 100 times in PBS containing 1% (w/v) bovine serum albumin. The samples were analyzed by confocal microscopy using a Zeiss axioplan research microscope with a Bio-Rad MRC600 laser scanning head. A human digestive system 12-lane MTN® blot (catalog number 7782-1), a human 12-lane MTN® blot (catalog number 7780-1) containing ∼1 μg of poly(A)+ RNA/lane, and a human MTN® blot II (catalog number 7759-1) containing ∼2 μg of poly(A)+RNA/lane were purchased from CLONTECH. A 552-bp fragment, corresponding to residues 714–1265 in the human BCO cDNA (GenBankTM accession number AK001592) was obtained by PCR and radiolabeled with [α-32P]dCTP (AmershamBiosciences) using a RediprimeTM II kit (AmershamBiosciences). ExpressHybTM hybridization solution (CLONTECH) was used for 2 h before hybridization at 68 °C and for overnight hybridization at 68 °C with 2 × 106 cpm probe/ml. Washing was performed according to the blot manufacturer. Autoradiography was carried out at −80 °C with HyperfilmTM MP (Amersham Biosciences) and Quanta Rapid intensifying screens (DuPont) for 3 days. The same filters were subsequently hybridized with a human β-actin probe and exposed at room temperature for 1 h. Using the Drosophila BCO as query sequence (17von Lintig J. Vogt K. J. Biol. Chem. 2000; 275: 11915-11920Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar), we performed BLAST searches of the GenBankTM data base and identified a human protein (GenBankTM accession number AK001592) with a 22% sequence identity to theDrosophila protein. To investigate whether this was the human BCO homologue, we isolated the corresponding cDNA from human liver cDNA by the PCR. DNA sequencing of multiple independently amplified cDNAs revealed an aspartate instead of a glycine in position 302 of the BCO protein. These results were in concordance with the human genomic BCO sequences present in the Celera data base, and with the recently published gene sequence (GenBankTMaccession number NP_059125) (14Yan W. Jang G.-F. Haeseleer F. Esumi N. Chang J. Kerrigan M. Campochiaro M. Campochiaro P. Palczewski K. Zack D.J. Genomics. 2001; 72: 193-202Crossref PubMed Scopus (139) Google Scholar). The cDNA was subcloned into a FastBac vector with a hexahistidine tag added to the C terminus of the protein. The recombinant protein was expressed in S. frugiperda 9 insect cells and purified to homogeneity by Co2+ column chromatography. Fig.1 A shows a Coomassie-stained polyacrylamide gel of uninfected (lane 1) and infected (lane 2) insect cell homogenates, and 100 ng of the purified histidine-tagged protein of ∼64 kDa (lane 3), consistent with the predicted molecular weight of 63,460 (including the six histidines). To further examine the purified protein, 650 ng of the protein was analyzed by SDS-polyacrylamide gel electrophoresis followed by silver staining. The purified protein showed a major band of ∼64 kDa (lane 4). Fig 1 B shows an immunoblot with a monoclonal antibody (mAb-1-11) against a synthetic peptide derived from the putative human BCO protein. Taken together, these data show that isolation of a highly purified recombinant protein was achieved. Fig. 2 shows the results of an in vitro time course experiment in which 2.5 μmβ-carotene was used as substrate with 60 ng of purified BCO protein. The products at the different time points were analyzed by reverse-phase HPLC and found to migrate with the same retention time as an all-trans-retinal standard, and the formation of product was linear up to 20 min. Preincubation of the purified protein at 95 °C for 5 min eliminated enzyme activity (data not shown). Fig.3 shows the spectral properties of the enzyme reaction products by photodiode array detector on-line analysis after separation by HPLC. The enzyme dependent product after incubation with β-carotene (Fig. 3, solid line) has an absorbance spectrum virtually identical to that of authentic all-trans-retinal (Fig. 3, dashed line C). Identical absorbance spectrum and retention time were seen for one of the enzyme-dependent reaction products when β-cryptoxanthin was used as substrate, indicating that all-trans-retinal is one of the two reaction products produced from this substrate (data not shown). Taken together, these data showed that the purified recombinant protein catalyzed the central cleavage of β-ionone ring containing carotenoids, suggesting that the PCR-amplified cDNA encoded a human homologue of theDrosophila BCO.Figure 3Absorbance spectrum of BCO reaction product. Photodiode array detector on-line analysis of BCO reaction products after separation on HPLC was performed as described under "Experimental Procedures." A, the absorbance spectrum of authentic all-trans-retinol. B, the absorbance spectrum of all-trans-retinoic acid.C, the enzyme-dependent product after incubation with β-carotene (solid line) has a virtually identical absorbance spectrum to that of authentic all-trans-retinal (dashed line). The scale of the left y axisrepresents absorbance units of retinoid standards; the scale of theright y axis represents absorbance units of enzyme dependent product. mAU, milliabsorbance units.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The biochemical properties of the purified protein were investigated by performing enzyme assays, gel filtration chromatography, and immunocytochemistry. Table I summarizes data on the effect of seven different detergents on BCO enzyme activity. The data show that maximal enzyme activity was achieved when OTG was used at a final concentration of 1% (w/v). TableII shows the effect of four different sulfhydryl reducing agents on enzyme activity. The data are consistent with the observations that sulfhydryl reducing agents are imperative for maximal enzyme activity in vitro (11Singh H. Cama H.R. Biochim. Biophys. Acta. 1974; 370: 49-61Crossref PubMed Scopus (48) Google Scholar, 12Goodman D. Huang H. Kanai M. Shiratori T. J. Biol. Chem. 1967; 242: 3543-3554Abstract Full Text PDF Google Scholar, 13Fidge N.H. Smith F.R. Goodman D.S. Biochem. J. 1969; 114: 689-694Crossref PubMed Scopus (51) Google Scholar) and that TCEP was the most effective agent when used at concentrations above 0.5 mm. In contrast to the native enzyme (11Singh H. Cama H.R. Biochim. Biophys. Acta. 1974; 370: 49-61Crossref PubMed Scopus (48) Google Scholar), inclusion of ferrous iron in the enzyme assay buffer was not essential for maximal activity of the recombinant human enzyme (data not shown).Table IEffect of different detergents on human BCO activityDetergentConcentrationRelative activity % %1-S-Octyl-β-d-thioglucopyranoside0.51001-a100% activity defined as the activity seen with 65 ng of BCO and 25 μm β-carotene in a 1-h reaction, 5.6 nmol/mg × min.Triton X-1000.516Sodium dodecyl sulfate0.54Tween 200.54CHAPS0.53Octyl-β-glucoside0.529Sodium cholate0.5121-S-Octyl-β-d-thioglucopyranoside0.05120.1160.25290.7514911511-a 100% activity defined as the activity seen with 65 ng of BCO and 25 μm β-carotene in a 1-h reaction, 5.6 nmol/mg × min. Open table in a new tab Table IIEffect of different reducing agents on human BCO activityReducing agentConcentrationRelative activity mm %None019Tris(2-carboxyethyl)phosphine hydrochloride51002-a100% activity defined as the activity seen with 1 μg of BCO and 25 μm β-carotene in a 15-min reaction, 1.35 nmol/mg × min.Reduced glutathione523Dithiothreitol532β-Mercaptoethanol526Tris(2-carboxyethyl)phosphine hydrochloride0.05220.251080.51392.51052-a 100% activity defined as the activity seen with 1 μg of BCO and 25 μm β-carotene in a 15-min reaction, 1.35 nmol/mg × min. Open table in a new tab The data shown in Fig. 4 indicate that the enzyme demonstrates a slightly alkaline pH optimum, similar to the activity present in intestinal homogenates of rat (12Goodman D. Huang H. Kanai M. Shiratori T. J. Biol. Chem. 1967; 242: 3543-3554Abstract Full Text PDF Google Scholar), rabbit (10Lakshmanan M.R. Chansang H. Olson J.A. J. Lipid Res. 1972; 13: 477-482Abstract Full Text PDF PubMed Google Scholar), hog (13Fidge N.H. Smith F.R. Goodman D.S. Biochem. J. 1969; 114: 689-694Crossref PubMed Scopus (51) Google Scholar), and guinea pig (11Singh H. Cama H.R. Biochim. Biophys. Acta. 1974; 370: 49-61Crossref PubMed Scopus (48) Google Scholar). The experiments of Fig.5 show that the enzyme is sensitive to the metal chelating agents α,α-bipyridyl ando-phenanthroline, as well as the sulfhydryl alkylating agents N-ethylmaleimide andp-chloromercuribenzoate. These findings are consistent with data regarding the biochemical properties of the intestinal BCO activity from different species. These results suggested that the purified recombinant human BCO enzyme possessed similar biochemical properties as compared with the partially purified native enzyme from a variety of animal species.Figure 5Inhibition of BCO enzyme activity by metal chelating and sulfhydryl alkylating agents. A,o-phenanthroline; B, α,α-bipyridyl;C, N-ethylmaleimide; D,p-chloromercuribenzoate. The assays were performed in duplicate in the presence of inhibitors at the indicated concentrations with 500 ng of enzyme and 8 μm β-carotene for 30 min at 37 °C as described under "Experimental Procedures." 100% equals 0.9 nmol/mg × min. Each value represents the average of two independent experiments.View Large Image Figure ViewerDownload Hi-res image
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