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Biomedical subjects

N Chiang

Publications and source records attributed to N Chiang.

At least 19 recordsLinked to original sources
PubMed2005

RvE1 protects from local inflammation and osteoclast- mediated bone destruction in periodontitis.

Periodontitis is a well-appreciated example of leukocyte-mediated bone loss and inflammation that has pathogenic features similar to those observed in other inflammatory diseases such as arthritis. Resolvins are a new family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammatory signals. Because it is now increasingly apparent that local inflammation plays a critical role in many diseases, including cardiovascular disease, atherosclerosis, and asthma, experiments were undertaken to evaluate the actions of the newly described EPA-derived Resolvin E1 (RvE1) in regulation of neutrophil tissue destruction and resolution of inflammation. The actions of an aspirin-triggered lipoxin (LX) analog and RvE1 in a human disease, localized aggressive periodontitis (LAP), were determined. Results indicate that neutrophils from LAP are refractory to anti-inflammatory molecules of the LX series, whereas LAP neutrophils respond to RvE1. In addition, RvE1 specifically binds to human neutrophils at a site that is functionally distinct from the LX receptor. Consistent with these potent actions, topical application of RvE1 in rabbit periodontitis conferred dramatic protection against inflammation induced tissue and bone loss associated with periodontitis.

Administration, Topical↗
PubMed2001

Cutting edge: nociceptin stimulates neutrophil chemotaxis and recruitment: inhibition by aspirin-triggered-15-epi-lipoxin A4.

The nociceptin receptor (Noci-R) is a G protein-coupled receptor present in neural tissues and its activation by nociceptin is involved in the processing of pain signals. Here, we report that Noci-R is present and functional on peripheral blood polymorphonuclear leukocytes (PMN). Human PMN express mRNA for Noci-R, its nucleotide sequence determined, and specific binding with [(125)I]-labeled nociceptin gave an apparent K(d) approximately 1.5 nM for this PMN opioid receptor. Nociceptin evoked PMN chemotaxis with maximal activity at 100 pM, without intracellular Ca(2+) mobilization. When injected in murine air pouches, nociceptin elicited leukocyte infiltration in a concentration-dependent fashion. Nociceptin-stimulated PMN infiltration was inhibited by treating mice with a synthetic analog of the aspirin-triggered lipid mediator 15-epi-lipoxin A(4). The present results identify nociceptin as a potent chemoattractant and provide a novel link between the neural and immune systems that are blocked by aspirin-triggered lipid mediators and may be relevant in neurogenic inflammation.

Animals↗
PubMed2001

Selectivity of recombinant human leukotriene D(4), leukotriene B(4), and lipoxin A(4) receptors with aspirin-triggered 15-epi-LXA(4) and regulation of vascular and inflammatory responses.

Aspirin-triggered lipoxin A(4) (ATL, 15-epi-LXA(4)) and leukotriene D(4) (LTD(4)) possess opposing vascular actions mediated via receptors distinct from the LXA(4) receptor (ALX) that is involved in leukocyte trafficking. Here, we identified these receptors by nucleotide sequencing and demonstrate that LTD(4) receptor (CysLT(1)) is induced in human vascular endothelia by interleukin-1beta. Recombinant CysLT(1) receptor gave stereospecific binding with both [(3)H]-LTD(4) and a novel labeled mimetic of ATL ([(3)H]-ATLa) that was displaced with LTD(4) and ATLa ( approximately IC(50) 0.2 to 0.9 nmol/L), but not with a bioinactive ATL isomer. The clinically used CysLT(1) receptor antagonist, Singulair, showed a lower rank order for competition with [(3)H]-ATLa (IC(50) approximately 8.3 nmol/L). In contrast, LTD(4) was an ineffective competitive ligand for recombinant ALX receptor with [(3)H]-ATLa, and ATLa did not compete for [(3)H]-LTB(4) binding with recombinant LTB(4) receptor. Endogenous murine CysLT(1) receptors also gave specific [(3)H]-ATLa binding that was displaced with essentially equal affinity by LTD(4) or ATLa. Systemic ATLa proved to be a potent inhibitor (>50%) of CysLT(1)-mediated vascular leakage in murine skin (200 microg/kg) in addition to its ability to block polymorphonuclear leukocyte recruitment to dorsal air pouch (4 microg/kg). These results indicate that ATL and LTD(4) bind and compete with equal affinity at CysLT(1), providing a molecular basis for aspirin-triggered LXs serving as a local damper of both vascular CysLT(1) signals as well as ALX receptor-regulated polymorphonuclear leukocyte traffic.

Animals↗
PubMed2000

Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing.

Aspirin therapy inhibits prostaglandin biosynthesis without directly acting on lipoxygenases, yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins (LXs) epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered LX [ATL]). Here, we report that inflammatory exudates from mice treated with omega-3 polyunsaturated fatty acid and aspirin (ASA) generate a novel array of bioactive lipid signals. Human endothelial cells with upregulated COX-2 treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Each was used by polymorphonuclear leukocytes to generate separate classes of novel trihydroxy-containing mediators, including 5-series 15R-LX(5) and 5,12,18R-triHEPE. These new compounds proved to be potent inhibitors of human polymorphonuclear leukocyte transendothelial migration and infiltration in vivo (ATL analogue > 5,12,18R-triHEPE > 18R-HEPE). Acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on hematologic cells. These findings establish new transcellular routes for producing arrays of bioactive lipid mediators via COX-2-nonsteroidal antiinflammatory drug-dependent oxygenations and cell-cell interactions that impact microinflammation. The generation of these and related compounds provides a novel mechanism(s) for the therapeutic benefits of omega-3 dietary supplementation, which may be important in inflammation, neoplasia, and vascular diseases.

Acetaminophen↗
PubMed2000

Oxidoreductases in lipoxin A4 metabolic inactivation: a novel role for 15-onoprostaglandin 13-reductase/leukotriene B4 12-hydroxydehydrogenase in inflammation.

The lipoxins (LX) are autacoids that act within a local inflammatory milieu to dampen neutrophil recruitment and promote resolution. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) and 15-oxoprostaglandin 13-reductase, also termed leukotriene B(4) 12-hydroxydehydrogenase (PGR/LTB(4)DH), are two enzymatic activities appreciated for their roles in the metabolism of prostaglandins and LTB(4). Here, we determined whether these oxidoreductases also catalyze the conversion of lipoxin A(4) (LXA(4)) and assessed the activities of these LXA(4) metabolites. 15-Oxo-LXA(4) was generated by incubating LXA(4) with 15-PGDH and NAD(+) for studies of its further conversion. PGR/LTB(4)DH catalyzed the NADH-dependent reduction of 15-oxo-LXA(4) to yield 13,14-dihydro-15-oxo-LXA(4). With NADH as a cofactor, 15-PGDH acted as a 15-carbonyl reductase and catalyzed the conversion of 13,14-dihydro-15-oxo-LXA(4) to 13, 14-dihydro-LXA(4). Human polymorphonuclear leukocytes (PMN) exposed to native LXA(4), 15-oxo-LXA(4), or 13,14-dihydro-LXA(4) did not produce superoxide anions. At concentrations where LXA(4) and a metabolically stable LXA(4) analog potently inhibited leukotriene B(4)-induced superoxide anion generation, the further metabolites were devoid of activity. Neither 15-oxo-LXA(4) nor 13, 14-dihydro-LXA(4) effectively competed with (3)H-labeled LXA(4) for specific binding to recombinant LXA(4) receptor (ALXR). In addition, introducing recombinant PGR/LTB(4)DH into a murine exudative model of inflammation increased PMN number by approximately 2-fold, suggesting that this enzyme participates in the regulation of PMN trafficking. These results establish the structures of LXA(4) further metabolites and indicate that conversion of LXA(4) to oxo- and dihydro- products represents a mode of LXA(4) inactivation in inflammation. Moreover, they suggest that these eicosanoid oxidoreductases have multifaceted roles controlling the levels of specific eicosanoids involved in the regulation of inflammation.

15-Oxoprostaglandin 13-Reductase↗
PubMed2000

Activation of lipoxin A(4) receptors by aspirin-triggered lipoxins and select peptides evokes ligand-specific responses in inflammation.

Lipoxin (LX) A(4) and aspirin-triggered LX (ATL) are endogenous lipids that regulate leukocyte trafficking via specific LXA(4) receptors (ALXRs) and mediate antiinflammation and resolution. ATL analogues dramatically inhibited human neutrophil (polymorphonuclear leukocyte [PMN]) responses evoked by a potent necrotactic peptide derived from mitochondria as well as a rogue synthetic chemotactic peptide. These bioactive lipid analogues and small peptides each selectively competed for specific (3)H-LXA(4) binding with recombinant human ALXR, and its N-glycosylation proved essential for peptide but not LXA(4) recognition. Chimeric receptors constructed from receptors with opposing functions, namely ALXR and leukotriene B(4) receptors (BLTs), revealed that the seventh transmembrane segment and adjacent regions of ALXR are essential for LXA(4) recognition, and additional regions of ALXR are required for high affinity binding of the peptide ligands. Together, these findings are the first to indicate that a single seven-transmembrane receptor can switch recognition as well as function with certain chemotactic peptides to inhibitory with ATL and LX (lipid ligands). Moreover, they suggest that ALXR activation by LX or ATL can protect the host from potentially deleterious PMN responses associated with innate immunity as well as direct effector responses in tissue injury by recognition of peptide fragments.

Animals↗
PubMed2000

Lipoxins, aspirin-triggered 15-epi-lipoxin stable analogs and their receptors in anti-inflammation: a window for therapeutic opportunity.

LXs and 15-epimer LXs are generated during cell-cell interactions that occur during multicellular host response to inflammation, tissue injury or host defense. Results indicate that they are present in vivo during human illness and carry predominantly counter-regulatory biological actions opposing the action of well-characterized mediators of inflammation that appear to lead to resolution of the inflammatory response or promotion of repair and wound healing. The first selective receptor of LXA4 was identified by direct ligand binding and was cloned and characterized. Its signaling involves a novel polyisoprenyl-phosphate pathway that directly regulates PLD (Levy et al. 1999a). LX- and 15-epimer-LX-stable analogs that resist metabolic inactivation were designed, synthesized and shown to be potent LX mimetics and novel topically active anti-inflammatory agents in animal models. These new investigational tools enable structure-function studies of LX signal transduction, further elucidation of the role of LX and 15-epimer LX in host responses and exploitation of their potent bioactions in the design of novel pharmacologic agents.

Animals↗
PubMed2000

Aspirin-tolerant asthmatics generate more lipoxins than aspirin-intolerant asthmatics.

Asthma is characterized by chronic airway inflammation resulting from overproduction of pro-inflammatory mediators, such as leukotrienes (LT). The authors questioned the biosynthetic capacity of asthmatic patients for lipoxins (LX) and 15-epimer lipoxins (15-epi-LX), endogenous regulators of inflammatory responses that inhibit pro-inflammatory events. Levels of LXA4, 15-epi-LXA4 and LTC4 were determined in 14 clinically characterized aspirin-intolerant asthmatics (AIA), 11 aspirin-tolerant asthmatics (ATA) and eight healthy volunteers using a stimulated whole blood protocol. Both LXA4 and 15-epi-LXA4 were generated in whole blood activated by the divalent cation ionophore, A23187. Higher levels of LXA4 were produced in ATA than either AIA or healthy volunteers. Exposure of AIA whole blood to interleukin-3 prior to A23187 did not elevate their reduced capacity to generate LXA4. Generation of a bronchoconstrictor, LTC4, was similar in both AIA and ATA. Consequently, the ratio of LXA4:LTC4 quantitatively favoured the bronchoconstrictor for AIA and differed from both ATA and healthy subjects. In addition, the capacity for 15-epi-LXA4 generation was also diminished in AIA, since whole blood stimulated in the presence of aspirin gave increased levels only in samples from ATA. The present results indicate that asthmatics possess the capacity to generate both lipoxins and 15-epimer-lipoxins, but aspirin-intolerant asthmatics display a lower biosynthetic capacity than aspirin-tolerant asthmatics for these potentially protective lipid mediators. This previously unappreciated, diminished capacity for lipoxin formation by aspirin-intolerant asthmatic patients may contribute to their more severe clinical phenotype, and represents a novel paradigm for the development of chronic inflammatory disorders.

Adult↗
PubMed2000

Epitope location on tissue factor determines the anticoagulant potency of monoclonal anti-tissue factor antibodies.

Tissue factor (TF), the cellular cofactor for the serine protease factor VIIa (F.VIIa), triggers blood coagulation and is involved in the pathogenesis of various thrombosis-related disorders. Therefore, agents which specifically target tissue factor, such as monoclonal antibodies, may provide promising new antithrombotic therapy. We mapped the epitopes of several anti-TF antibodies using a panel of soluble TF mutants. They bound to three distinct TF regions. The epitope of the 7G11 antibody included Phe50 and overlapped with a TF-F.VIIa light chain contact area. The common epitope of the antibodies 6B4 and HTF1 included residues Tyr94 and Phe76 both of which make critical contacts to the catalytic domain of F.VIIa. The antibodies D3 and 5G6 had a common epitope outside the TF-F.VIIa contact region. It included residues Lys 165, Lys 166, Asn199, Arg200 and Lys201 and thus overlapped with the substrate interaction region of tissue factor. The antibodies 5G6 and D3 were potent anticoagulants when infused to flowing human blood in an ex-vivo thrombosis model. Plasma fibrinopeptide A levels and fibrin deposition were completely inhibited. In contrast, 6B4 was a weak inhibitor in this ex-vivo thrombosis model, and HTF1 displayed no inhibition at all. These disparate activities were also reflected in TF-dependent F.X activation assays performed with human plasma. The potency differences could neither be explained by the determined binding affinities nor by the on-rates of antibodies. Therefore, the results suggest that antibody binding epitope and hence the particular mechanism of inhibition, is the main determinative factor of anticoagulant potency of anti-TF antibodies.

Antibodies, Monoclonal↗
PubMed2000

Anti-microinflammatory lipid signals generated from dietary N-3 fatty acids via cyclooxygenase-2 and transcellular processing: a novel mechanism for NSAID and N-3 PUFA therapeutic actions.

Aspirin therapy inhibits prostaglandin biosynthesis; yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered lipoxin or ATL). Here, we review our findings indicating that inflammatory exudates from mice treated with omega-3 PUFA and aspirin (ASA) generate a novel array of bioactive lipid signals. Also, human endothelial cells, both HUVEC and microvascular, with upregulated COX-2 and treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Human PMN activated with serum treated zymosan (STZ) utilized each of these R-HEPEs to generate novel classes of trihydroxy-containing mediators including 5-series 15R-LX and 5,12,18R-triHEPE. The novel products were potent inhibitors of human PMN transendothelial migration and infiltration of PMN in dorsal air pouches in vivo. In addition to ASA, both acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant human COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on leukocytes, platelets and endothelial cells. These findings establish new transcellular routes for producing arrays of lipid mediators via COX-2-NSAIDs and cell-cell interactions that impact microinflammation. Moreover, they provide novel mechanism(s) that could underlie the many reported therapeutic benefits of omega-3 dietary supplementation of interest in inflammation, cancer, and vascular disorders.

Animals↗
PubMed1999

Hepatocytes are a rich source of novel aspirin-triggered 15-epi-lipoxin A(4).

Novel aspirin (ASA)-triggered 15-epi-lipoxins (ATL) comprise new potent bioactive eicosanoids that may contribute to the therapeutic effect of this drug. ATL biosynthesis is initiated by ASA acetylation of cyclooxygenase (COX)-2 and was originally identified during the interaction of leukocytes with either endothelial or epithelial cells. Here, we examined ATL biosynthesis in rat hepatocytes either alone or in coincubation with nonparenchymal liver cells (NPC) and in liver homogenates from ASA-treated rats. Rat hepatocytes and CC-1 cells, a rat hepatocyte cell line, displayed COX-1 but not COX-2 mRNA expression and predominantly produced thromboxane A(2) (TXA(2)) and 15-hydroxyeicosatetraenoic acid (15-HETE). In these cells, ASA shifted the arachidonic acid metabolism from TXA(2) to 15-HETE in a concentration-dependent manner. In contrast, neither indomethacin, ibuprofen, valeryl salicylate, nor nimesulide was able to trigger 15-HETE biosynthesis. SKF-525A, a cytochrome P-450 inhibitor, significantly reduced the effect of ASA on 15-HETE biosynthesis. Furthermore, phenobarbital, a potent inducer of cytochrome P-450 activity, further increased ASA-induced 15-HETE production. ASA treatment of hepatocyte-NPC coincubations resulted in the generation of significant amounts of ATL. In addition, in vivo experiments demonstrated augmented hepatic levels of 15-epi-lipoxin A(4) in ASA-treated rats. Taken together and considering that ASA is hydrolyzed on its first pass through the portal circulation, these data indicate that, during ASA's consumption, liver tissue generates biologically relevant amounts of ATL by COX-2-independent mechanisms.

Animals↗
PubMed1999

Leukotriene B4 receptor transgenic mice reveal novel protective roles for lipoxins and aspirin-triggered lipoxins in reperfusion.

Polymorphonuclear neutrophil (PMN) activation is pivotal in acute inflammation and injury from reperfusion. To elucidate components controlling PMNs in vivo, we prepared novel transgenic mice with the human leukotriene (LT) B4 receptor (BLTR) for functional characterization. Overexpression of BLTR in leukocytes dramatically increased PMN trafficking to skin microabscesses and lungs after ischemia-reperfusion, whereas mice deficient in 5-lipoxygenase (5-LO) showed diminished PMN accumulation in reperfused lungs. Hence, both BLTR expression and LT biosynthesis are critical for PMN infiltration in reperfusion-initiated second-organ injury. Also, in BLTR transgenic mice, 5-LO expression and product formation were selectively increased in exudates, demonstrating that receptor overexpression amplifies proinflammatory circuits. Endogenous lipoxin (LX) A4 was produced in ischemic lungs and elevated by reperfusion. Because LXA4 and aspirin-triggered 15-epimeric LXA4 (ATL) selectively regulate leukocyte responses, they were tested in BLTR transgenic mice. Despite excessive PMN recruitment in BLTR transgenic mice, intravenous injection of ATL sharply diminished reperfusion-initiated PMN trafficking to remote organs, and topical application of LX was protective in acute dermal inflammation. These results demonstrate a direct role for BLTR with positive feedback, involving BLTR and 5-LO signaling in controlling PMNs. Moreover, LXA4 and ATL counter BLTR-amplified networks, revealing a novel protective role for LX and ATL in stress responses that has applications in perioperative medicine.

Animals↗
PubMed1999

Lipoxin and aspirin-triggered 15-epi-lipoxin cellular interactions anti-inflammatory lipid mediators.

Eicosanoids are known to play important roles in inflammation. Recent findings have given rise to several new concepts regulating the generation of eicosanoids, illustrated in Figure 1. Lipoxins (LX) are trihydroxytetraene-containing eicosanoids that are generated within vascular lumen by platelet-leukocyte interactions and at mucosal surfaces by leukocyte-epithelial cell interactions. During these cell-cell interactions, transcellular biosynthetic pathways are used as major routes, and thus, in humans, LX are formed in vivo during multicellular responses such as inflammation, atherosclerosis, and thrombosis. This branch of the eicosanoid cascade generates specific tetraene-containing products that appear to function as stop signals, since they inhibit key steps in leukocyte-mediated inflammation. Of special interest, it appears that aspirin also functions in part via production of novel epimers of lipoxins or 15-epi-lipoxins (Figure 1). Here, we review recent developments on the cellular interactions of these novel anti-inflammatory mediators.

Amino Acid Sequence↗
PubMed1998

The role of N-glycosylation of human thromboxane A2 receptor in ligand binding.

Thromboxane A2 receptor (TXA2R) was expressed in insect Sf21 cells and demonstrated to interact with 8-iso-PGF2 alpha and 9 alpha, 11 beta-PGF2 alpha with a potency similar to that of TXA2 agonist U46619. TXA2R was shown to be a glycoprotein. The role of N-glycosylation of TXA2R in ligand binding was investigated in the insect cells over-expressed with recombinant TXA2R. Deletion of the carbohydrate moiety by adding tunicamycin during infection of Sf21 cells or mutation of both potential N-glycosylation sites (Asn-4 and Asn-16) abolished the ligand binding of TXA2R, suggesting that N-glycosylation is crucial for binding function. Mutation of either Asn-4 or Asn-16 to a leucine did not have much effect on maximal binding. However, the mutant receptors possess lower binding affinity toward TXA2R antagonist [3H]SQ29548. Furthermore, the binding specificity of the mutant receptors was shown to be altered. Our data suggest that both Asn-4 and Asn-16 are glycosylated and glycosylation on either site is sufficient for ligand recognition. However, glycosylation on both sites is required to maintain binding affinity and specificity.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1998

Aspirin-triggered 15-epi-lipoxin A4 (ATL) generation by human leukocytes and murine peritonitis exudates: development of a specific 15-epi-LXA4 ELISA.

Aspirin (ASA) triggers the formation of 15-epi-lipoxins (15-epi-LXs or ATL [ASA-triggered LX]), which are potent bioactive eicosanoids that may contribute to the therapeutic impact of ASA. To elucidate the role of these new compounds in vivo, it is essential to establish quick and sensitive detection methods. To this end, we prepared an enzyme-linked immunosorbent assay specific for 15-epi-LXA4 that proved to be highly sensitive (IC50 approximately 50 pg, minimum detection approximately 3.5 pg) and stereoselective. The amounts of 15-epi-LXA4 generated by human neutrophils from peripheral blood of healthy volunteers using this enzyme-linked immunosorbent assay were in agreement with those values obtained by liquid chromatography. Formation of 15-epi-LXA4 was cell ratio-dependent during THP-1 (a monocytic leukemia cell line)-neutrophil interactions with ASA-treated cells, and 15-epi-LXA4 was not detected with either cell type alone. Generation of 15-epi-LXA4 was also examined in murine peritonitis with ASA administration. Exudates from ASA-treated mice showed increased production of 15-epi-LXA4 that was diminished by indomethacin, a blocker of ASA-dependent acetylation of prostaglandin G/H synthase. A cytochrome P450 inhibitor administered in the presence of ASA did not prevent 15-epi-LXA4 formation, which suggests that P450 does not significantly contribute to formation of 15-epi-LXA4 in this murine model. These results indicate that the new enzyme-linked immunosorbent assay is both sensitive and selective for 15-epi-LXA4 and that 15-epi-LXA4 is produced by human leukocyte-leukocyte interactions. In addition, 15-epi-LXA4 is generated by inflammatory exudates when ASA is administered during murine peritonitis and when prostaglandin G/H synthase is upregulated and acetylated. This assay should provide rapid means to investigate 15-epi-LXA4 generation in both cellular and animal models.

Animals↗
PubMed1997

Expression of rat steroid 5 alpha-reductase (isozyme-1) in Spodoptera frugiperda, SF21, insect cells: expression of rat steroid 5 alpha-reductase.

The enzyme steroid 5 alpha-reductase (5 alpha R) catalyzes the reduction of testosterone (T) to 5 alpha-dihydrotestosterone (DHT). In this study, the baculovirus expression system was used to overexpress rat 5 alpha R type I isozyme (r5 alpha R 1). The full length of r5 alpha R1 cDNA was inserted into the Autographa californica nuclear polyhedrosis virus (Ac-MNPV) genome and expressed in Spodoptera frugiperda, Sf 21, insect cells. The expressed recombinant r5 alpha-R1 showed maximal enzymatic activity when the infected cells were harvested on day 3 of post-transfection. The K(m) values for NADPH and T were 17 microM and 2.7 microM, respectively. Inhibition of the recombinant r5 alpha R1 by N,N diethyl-4-aza-4-methyl-3-oxo-5 alpha-androstane-17 beta-carboxamide (4MA) was competitive with respect to the substrate (T), and a Ki of 3 nM was obtained. The enzyme was located primarily in the nuclear fraction, and the maximum velocity for the recombinant r5 alpha R1 in this fraction was 60 nmoles DHT/min/mg. Immunoblot analysis indicated a single immunoreactive band at 26 kDa, which corresponds to the molecular weight of r5 alpha R1. Photoaffinity labeling by [2'-32P]-2-azido-NAD P+ ([2'-32P]2N3-NAD P+) and [1,2(3)H] N-(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5 alpha androstane-17 beta-carboxamide ([3H]-4MABP) also showed a labeled protein band at 26 kDa.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗