A comparison of the tryptic peptides of hemoglobin from Microtus pennsylvanicus tananaensis, mus musculus and man

Twenty-two tryptic peptides of the plus β chains of hemoglobin (Hb). taken from Microtuspennsylvanicus tananaensis, were identified as homologs of tryptic peptides from human Hb and account for 210 amino acids out of a total of 287 expected. Seven peptides could not be identified as homologs. With reference to the 210 amino acids identified in Microtus Hb: (a) there are 35 differences between Hb of man and Microtus (confined to 14 of the homologous peptides as compared to 28 differences between Hb of white mouse and man); (b) there are 22 differences between Hb of white mouse and Microtus.     

A comparison of hemoglobins in five species of Microtus

The composition of tryptic peptides was determined for Hb (major or fast electrophoretic component) from four additional species of Microtus; M. p. pennsylvanicus, M. abbreviatus, M. miurus, and M. oeconomus, The amino acids from the four Hb were compared with Hb from M. p. tananaensis, and, on the basis of the combination of analyses for the several Hb. Ca 95% of the overall amino acid composition was considered. The compositions of most of the homologous peptides were identical, and two deletions in the sequence of 21 amino acids β 41–61 are believed to characterize the Hb of all four species. From differences in peptide composition the following substitutions were inferred. In the β chain, M. pennsylvanicus (2 ssp) differed from other species at two positions, 8: Ser vs Thr and 12: Thr vs Ash. In the β chain M. pennsylvanicus (2 ssp) differed from other species at two positions, β45: Phe vs Leu, and β50: Ser-Glx vs Thr. M. p. pennsylvanicus differed from M. p. tananaensis at position β88: Val-Leu vs Leu. All species showed ambiguity among the amino acids Ala-Ser-Phe-Leu centred presumably in positions β129 and β130. On the basis of an incomplete examination, the slow electrophoretic component of M. abbreviatus Hb could not be seen to differ from the fast component in its peptide map or in the general composition of its and β peptides.     

Cytoprotective effects of the antioxidant phytochemical indicaxanthin in beta-thalassemia red blood cells

Antioxidant phytochemicals are investigated as novel treatments for supportive therapy in β-thalassemia. The dietary indicaxanthin was assessed for its protective effects on human β-thalassemic RBCs submitted in vitro to oxidative haemolysis by cumene hydroperoxide. Indicaxanthin at 1.0-10 μM enhanced the resistance to haemolysis dose-dependently. In addition, it prevented lipid and haemoglobin (Hb) oxidation, and retarded vitamin E and GSH depletion. After ex vivo spiking of blood from thalassemia patients with indicaxanthin, the phytochemical was recovered in the soluble cell compartment of the RBCs. A spectrophotometric study showed that indicaxanthin can reduce perferryl-Hb generated in solution from met-Hb and hydrogen peroxide (H₂O₂), more effectively than either Trolox or vitamin C.

Collectively our results demonstrate that indicaxanthin can be incorporated into the redox machinery of β-thalassemic RBC and defend the cell from oxidation, possibly interfering with perferryl-Hb, a reactive intermediate in the hydroperoxide-dependent Hb degradation. Opportunities of therapeutic interest for β-thalassemia may be considered.     

Conformationally restricted PACAP27 analogues incorporating type II/II′ IBTM β-Turn Mimetics. Synthesis, NMR Structure Determination, and Binding Affinity

To probe the importance of a proposed β-turn within residues S9-R12 of PACAP for recognition by VIP/PACAP receptors, compounds 1 and 2, two conformationally restricted analogues of PACAP27 incorporating respectively (S)- or (R)-IBTM as type II or II′ β-turn dipeptide mimetic at the Y10-S11 position, were synthesized. According to ¹H NMR conformational analyses in aqueous solution and 30% TFE, both PACAP27 and the [S-IBTM^10,11]PACAP27 analogue 1 adopt similar ordered structures. PACAP27 shows an N-terminal disordered region (residues H1-F6) and an -helical conformation within segment T7–L27. For residues S9–R12, our data seem more compatible with a segment of the -helix than with the β-turn previously proposed for this fragment. In compound 1 the -helix, also spanning T7–L27 residues, appears slightly distorted at the N-terminus relative to the native peptide. Although this distortion could lead to the marked decrease in binding affinity of this compound at the VIP/PACAP receptors, the lack of the Y10 side chain in analogues 1 and 2 could also significantly affect the binding of these compounds.     

Alkaloids from Heimia salicifolia

Two alkaloids, 9β,2′-dihydroxy-4′′,5′′-dimethoxy-lythran-12-one or 9β-hydroxyvertine (1) and (2S,4S,10R)-4-(3-hydroxy-4-methoxyphenyl)-quinolizidin-2-acetate (2), as well as seven known alkaloids, lythrine (3), dehydrodecodine (4), lythridine (5), vertine (6), heimidine (7), lyfoline (8) and epi-lyfoline (9), were isolated from Heimia salicifolia. The structures of these compounds were elucidated by extensive spectroscopic techniques. Furthermore, the structures of 2, 3, and 6 were confirmed by X-ray crystallography, including absolute configuration determination of 2 and 6. Compounds 6 and 9 showed moderate antimalarial activity.     

High level expression of functional full length human thyroid hormone receptor β1 in insect cells using a recombinant baculovirus

We have cloned the human thyroid hormone receptor β1 (hThR β) from the human breast cancer cell line T47D using the PCR technique. A recombinant baculovirus transfer vector pVL1392/hThR β was constructed and the full length receptor was expressed in the insect cell line Spodoptera frugiperda (Sf9). Approx. 10–15 × 10⁶ receptors are expressed/ cell which implies a production level of 2.5–4.0 mg hThR β/1 of cell culture. The expressed hThR β displayed a single class of binding sites for T₃ with high affinity. Western blot analysis using a polyclonal antibody indicated that the molecular weight of the baculovirus expressed receptor is approx. 50 kDa. Crude nuclear extract of hThR β labeled with [¹²⁵I]T₃ sedimented as a 4 S peak on a glycerol gradient. No receptor could be detected in the cytoplasm indicating its proper translocation to the nuclear compartment. An oligonucleotide containing a palindromic thyroid hormone response element is specifically recognized and retarded in a gel-mobility-shift assay in the presence of nuclear extract of Sf9 cells expressing hThR β. These data suggest that hThR β expressed in Sf9 cells is functional and displays characteristics virtually indistinguishable from those of the thyroid hormone receptor (ThR) extracted from mammalian cells. Furthermore, the data indicate that the baculovirus expression system is adequate for large-scale production of receptor for detailed structural and functional studies.     

Anti-diabetic properties of the Canadian lowbush blueberry Vaccinium angustifolium Ait.

Incidence of type II diabetes is rapidly increasing worldwide. In order to identify complementary or alternative approaches to existing medications, we studied anti-diabetic properties of Vaccinium angustifolium Ait., a natural health product recommended for diabetes treatment in Canada. Ethanol extracts of root, stem, leaf, and fruit were tested at 12.5 μg/ml for anti-diabetic activity in peripheral tissues and pancreatic β cells using a variety of cell-based bioassays. Specifically, we assessed: (1) deoxyglucose uptake in differentiated C2C12 muscle cells and 3T3-L1 adipocytes; (2) glucose-stimulated insulin secretion (GSIS) in β TC-tet pancreatic β cells; (3) β cell proliferation in β TC-tet cells; (4) lipid accumulation in differentiating 3T3-L1 cells; (5) protection against glucose toxicity in PC12 cells. Root, stem, and leaf extracts significantly enhanced glucose transport in C2C12 cells by 15–25% in presence and absence of insulin after 20 h of incubation; no enhancement resulted from a 1 h exposure. In 3T3 cells, only the root and stem extracts enhanced uptake, and this effect was greater after 1 h than after 20 h; uptake was increased by up to 75% in absence of insulin. GSIS was potentiated by a small amount in growth-arrested β TC-tet cells incubated overnight with leaf or stem extract. However, fruit extracts were found to increase ³H-thymidine incorporation in replicating β TC-tet cells by 2.8-fold. Lipid accumulation in differentiating 3T3-L1 cells was accelerated by root, stem, and leaf extracts by as much as 6.5-fold by the end of a 6-day period. Stem, leaf, and fruit extracts reduced apoptosis by 20–33% in PC12 cells exposed to elevated glucose for 96 h. These results demonstrate that V. angustifolium contains active principles with insulin-like and glitazone-like properties, while conferring protection against glucose toxicity. Enhancement of proliferation in β cells may represent another potential anti-diabetic property. Extracts of the Canadian blueberry thus show promise for use as a complementary anti-diabetic therapy.     

-Subunits of Ns are released from the plasma membrane following cholera toxin activation

Cholera toxin (CT) and islet-activating protein (IAP, a Bordetella pertussis toxin) were employed to test the hypothesis that GTP-binding regulatory proteins are released from plasma membranes to a greater extent when ‘activated’ than when ‘inactivated’. CT, which activates N_s (the stimulatory GTP-binding regulatory protein of the adenylate cyclase system), catalyzed the incorporation of radioactivity from [³²P]NAD into 45 and 47.5 kDa peptides associated with rat liver plasma membranes. Following ADP-ribosylation and centrifugation at 100000 × g for 1 h, approx. 30–35% of these CT-labelled peptides were no longer associated with the plasma membranes, but were recovered from the supernatant fraction. IAP, which inactivates N_i (the inhibitory GTP-binding regulatory protein of the adenylate cyclase system) catalyzed the incorporation of radioactivity from [³²P]NAD into a 41 kDa peptide associated with the membranes. However, in contrast to the CT-labelled peptides, typically less than 5% of the lAP-labelled peptide was found in the 100000 × g supernatant fraction, but rather was almost exclusively associated with the membrane pellet. The data indicate that the -subunits of N_s are released from the plasma membrane following activation, and support the hypothesis that the βγ-subunits act to anchor the -subunits to the plasma membrane. Cholera toxin Islet-activating protein GTP-binding protein     

Use of precision cut human liver slices for studying the metabolism and genotoxic potential of xenobiotics by means of the 32P-postlabelling technique: steps towards method validation using testosterone and 2-aminofluorene

In the present study, a new in vitro model combining the short-term incubation of precision-cut human liver slices with DNA-adduct analysis by the ³²P-postlabelling technique is proposed for investigation of the genotoxic potential of xenobiotics. For method validation, the metabolic turnover of testosterone (TES) and the DNA-adduct inducing potential of 2-aminofluorene (2-AF) were used. Precision-cut human liver slices were prepared from a total of 12 human liver samples which were freshly obtained as parts of resectates from liver surgery. The slices were incubated as submersion cultures with TES and 2-AF for up to 6 h in 12-well tissue culture plates at concentrations of 10-50 and 0.06-28 μM, respectively. Slices recovered from the slicing procedure in the 4 °C cold Krebs-Henseleit buffer as indicated by intracellular potassium concentrations which increased for 2 h and then remained stable until the end of the incubation. TES was extensively metabolized by human liver slices with a similar metabolite pattern as observed in vivo. Almost 90% of the metabolites were conjugates. Major phase-I metabolites were androstendione, 6β-OH-androstendione, 6β-OH-TES, and 15β-OHTES. After incubation with 2-AF, substance related DNA-adducts were detected which increased dose-dependently from 12 to 1146 adducts per 10⁹ nucleotides. The adduct pattern consisted of one major adduct spot, A, representing 80-90% of the total adduct level and up to four minor adduct spots, B-E. In summary, the present data demonstrate that precision-cut liver slices are a valuable alternative in vitro system for DNA-adduct determination to screen chemicals for potential genotoxicity in humans.     

Transport of micro-opioid receptor agonists and antagonist peptides across Caco-2 monolayer

Milk is the source of β-casomorphins – biologically active peptides with opioid activity – which are suspected to play various roles in the human body. The local influence of exogenous opioid peptides on gastrointestinal functions has been widely reported. After passing the gut barrier, β-casomorphins may affect the functions of immunological system, as well as dopaminergic, serotoninergic and GABA-ergic systems in brain, regulate the opioid receptor development and elicit behavioral effects. However, possibilities and mechanisms of the intestinal transport of β-casomorphins in human body in vivo have not been reported so far. In our research, the transepithelial transport of μ-opioid receptor agonists – human β-casomorphin-5 and 7(BCM5, BCM7) and antagonist – lactoferroxin A (LCF A) have been investigated using Caco-2 monolayer. In order to determine the pathway of investigated peptide transport across Caco-2 monolayer, two directions of the transport (apical to basolateral and basolateral to apical) have been studied. All investigated peptides were transported across the human intestinal cell line Caco-2 and the curves of cumulative amount of transported peptides in time were linear in each case. In addition, the hydrolysis of β-casomorphins during 60 min of experiment by dipeptidyl peptidase IV was observed. The data suggest the possibility of transport of opioid peptides derived from food across human intestinal mucosa.     

Tachykinins (Substance P, Neurokinin A and Neuropeptide γ) and Neurotensin from the Intestine of the Burmese Python, Python molurus

Conlon, J. M., T. E. Adrian and S. M. Secor. Tachykinins (substance P, neurokinin A and neuropeptide γ,) and neurotensin from the intestine of the burmese python, Python molurus. Peptides 18(10) 1505–1510, 1997.—Peptides with substance P-like immunoreactivity, neurokinin A-like immunoreactivity and neurotensin-like immunoreactivity were isolated in pure form from an extract of the intestine of the Burmese python (Python molurus). The primary structure of python substance P (Arg-Pro-Arg-Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH₂) shows one amino acid substitution (Phe⁸ → Tyr) compared with chicken/alligator substance P and an additional substitution (Lys³ → Arg) as compared with mammalian substance P. The neurokinin A-like immunoreactivity was separated into two components. Python neuropeptide γ (Asp-Ala-Gly-Tyr-Ser-Pro-Leu-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH₂ shows three substitutions (Gly⁵ → Ser, Gln⁶ → Pro and Ile⁷ → Leu) compared with alligator neuropeptide γ and an additional substitution (His⁴ → Tyr) compared with mammalian neuropeptide γ. Python neurokinin A (His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met.NH₂) is identical to human/chicken/alligator neurokinin A. Python neurotensin (pGlu-Leu-Val-His-Asn-Lys-Ala-Arg-Pro-Tyr-Ile-Leu) is identical to chicken/alligator neurotensin. The data are indicative of differential evolutionary pressure to conserve the amino acid sequences of reptilian gastrointestinal peptides.     

Analytical Extraction of Regulatory Peptides from Rat Lung Tissue

Kraiczi, H., G. Karlsson and R. Ekman. Analytical extraction of regulatory peptides from rat lung tissue. Peptides 18(10) 1597–1601, 1997.—We evaluated protocols for the extraction of calcitonin gene-related peptide, neuropeptide Y, substance P, peptide YY and β-endorphin from rat lung tissue for subsequent radioimmunoassay. The effects of varying acidity of the extraction solution and repeating extraction on the recovery of peptide immunoreactivity and non-specific tracer-binding were compared by analysis of variance. Moreover, variability of immunoreactivity was quantified for comparison. Considering all three criteria, the optimal acidity for extraction was: 0.1 M or 1 M acetic acid for CGRP and β-endorphin, 0.1 M acetic acid for NPY, 1 M acetic acid for substance P and phosphate buffer for peptide YY. Double or combined extraction unambiguously improved assay results only for substance P. Reversed-phase high-performance liquid chromatography of CGRP-, NPY- and SP-immunoreactivity obtained from selected extracts suggested that differences in recovery of these peptides are not explainable by differential peptide fragmentation during extraction.     

New ultra-micro high-performance liquid chromatographic method for determining the γ chain composition of hemoglobin F in normal adults

The difficulty in isolating the minute quantity of Hb F (<1%) present in the red blood cells of normal adults greatly complicates the determination of its γ chain composition. We have developed a rapid ultra-micro high-performance liquid chromatographic (HPLC) method and used it to analyze the γ chain composition of Hb F in 47 adults with Hb F levels between 0.1–3.4%. The method involves the isolation of Hb F from as little as 50 μl of whole blood on an analytical size cation-exchange HPLC column, followed by concentration in a Centricon micro concentrator unit and by reversed-phase HPLC analysis. The entire procedure can be completed in one day and 3–4 analyses can be made simultaneously. We reanalyzed the blood samples from 22 subjects with known β-globin gene cluster haplotypes, and confirmed the association of high, low, and very low ^Gγ levels with haplotypes A, B, and C, respectively. Also included are the results of DNA sequence analyses of the ^Gγ and β promoters, and of the locus-control-region hypersensitive site-2 (LCR-HS-2) of the β-globin gene cluster in five subjects homozygous for haplotypes A, B or C; the data obtained failed to provide a satisfactory explanation for all the variations in the ^Gγ levels that have been observed.     

Synthesis and characterization of SrCu2(O2CR)3(bdmap)3 and Bi2(O2CCH3)4(bdmap)2(H2O) (R = CH3, CF3; bdmap = 1,3-bis(dimethylamino)-2-propanolato)

New mixed metal complexes SrCu₂(O₂CR)₃(bdmap)₃ (R = CF₃ (1a), CH₃ (1b)) and a new dinuclear bismuth complex Bi₂(O₂CCH₃)₄(bdmap)₂(H₂O) (2) have been synthesized. Their crystal structures have been determined by single-crystal X-ray diffraction analyses. Thermal decomposition behaviors of these complexes have been examined by TGA and X-ray powder diffraction analyses. While compound 1a decomposes to SrF₂ and CuO at about 380°C, compound 1b decomposes to the corresponding oxides above 800°C. Compound 2 decomposes cleanly to Bi₂O₃ at 330°C. The magnetism of 1a was examined by the measurement of susceptibility from 5–300 K. Theoretical fitting for the susceptibility data revealed that 1a is an antiferromagnetically coupled system with g = 2.012(7), −2J = 34.0(8) cm⁻¹. Crystal data for 1a: C₂₇H₅₁N₆O₉F₉Cu₂Sr/THF, monoclinic space group P2₁/m, A = 10.708(6), B = 15.20(1), C = 15.404(7) Å, β = 107.94(4)°, V = 2386(2) ų, Z = 2; for 1b: C₂₇H₆₀N₆O₉Cu₂Sr/THF, orthorhombic space group Pbcn, A = 19.164(9), B = 26.829(8), C = 17.240(9) Å, V = 8864(5) ų, Z = 8; for 2: C₂₂H₄₈O₁₁N₄Bi₂, monoclinic space group P2₁/c, A = 17.614(9), B = 10.741(3), C = 18.910(7) Å, β = 109.99(3)°, V = 3362(2) ų, Z = 4.     

Identification of tubulin isoforms in different tissues of Ascaris suum using anti-tubulin monoclonal antibodies.

Three monoclonal antibodies specific to - and β-tubulin were used to examine the expression of tubulin isofoms in the intestine, reproductive tract and body wall muscle of A. suum. The tubulins were found to be different in their isoelectric points, number of isoforms and peptide maps with Western blot analysis of one-dimensional polyacrylamide gel confirming the presence of -, β₁- and β₂- tubulin. Commercial cross-reactive anti- and anti-β MAbs 356 and 357 recognized tubulin from A. suum tissues as well as from pig brain, whereas anti-A. suum β-tubulin specific MAb P3D6 recognized tubulin from the A. suum tissues only. Two-dimensional gel analysis showed different isoform patterns in different A. suum tissues with anti-A. suum β-tubulin MAb P3D6 and cross-reactive β-tubulin MAb 357 recognizing 2–4 β- tubulin isoforms and anti--tubulin MAb 356 recognizing 1–6 -tubulin isoforms. Different peptide maps of tubulin were observed in the three tissues, when subjected to limited proteolysis followed by SDS-PAGE. The data indicate that different tubulins are found in different tissues of adult A. suum.     

The coordination chemistry of cationic main group clusters: syntheses and structures of [Fe3(Se2)2(CO)10] and [Fe4(Se2)3(CO)12]

The reactions of the polysulfur and selenium cationic clusters S₈²⁺ and Se₈²⁺ with various iron carbonyls were investigated. Several new chalcogen containing iron carbonyl cluster cations were isolated, depending on the nature of the counteranion. In the presence of SbF₆⁻ as a counterion, the cluster [Fe₃(E₂)₂(CO)₁₀] [SbF₆]₂·SO₂ (E = S, Se) could be isolated from the reaction of E₈²⁺ and excess iron carbonyl. The cluster is a picnic-basket shaped molecule of two iron centers linked by two Se₂ groups, with the whole fragment capped by an Fe(CO)₄ group. Crystallographic data for C₁₀O₁₂Fe₃Se₄Sb₂F₁₂S (I): space group monoclinic P2₁/c, A = 11.810(9), b = 24.023(6), c = 10.853(7) Å, β = 107.15(5)°, V = 2942(3) ų, Z = 4, R = 0.0426, R_w = 0.0503. When Sb₂F₁₁⁻ is present as the counterion, or Se₄[Sb₂F₁₁]₂ is used as the cluster cation source, a different cluster can be isolated, which has the formula [Fe₄(Se₂)₃(CO)₁₂] [SbF₆]₂·3SO₂. The dication contains two Fe₂Se₂ fragments bridged by an Se₂ group. Crystallographic data for C₁₂O₁₈Fe₄Se₆Sb₂F₁₂S₃ (III): space group triclinic , b = 18.400(9), C = 10.253(4) Å, = 93.10(4), β = 103.74(3), γ = 93.98(3)°, V = 1995(1) ų, Z = 2, R = 0.0328, R_w = 0.0325. The CO stretches in the IR spectrum all show a large shift to higher wavenumbers, suggesting almost no τ backbonding from the metals. This also correlates with the observed bond distances. All the compounds are extremely sensitive to air and water, and readily lose SO₂ when removed from the solvent. Thus all the crystals were handled at −100°C. The clusters seem to be either insoluble or unstable in all solvents investigated.     

Mutation of fungal endoglucanases into glycosynthases and characterization of their acceptor substrate specificity

Humicola insolens mutant Cel7B E197A is a powerful endo-glycosynthase displaying an acceptor substrate specificity restricted to β-d-glucosyl, β-d-xylosyl, β-d-mannosyl and β-d-glucosaminyl in +1 subsite. Our aim was to extend this substrate specificity to β-d-N-acetylglucosaminyl, in order to get access to a wider array of oligosaccharidic structures obtained through glycosynthase assisted synthesis. In a first approach a trisaccharide bearing a β-d-N-acetylglucosaminyl residue was docked at the +1 subsite of H. insolens Cel7B, indicating that the mutation of only one residue, His209, could lead to the expected wider acceptor specificity. Three H. insolens Cel7B glycosynthase mutants (H209A, H209G and H209A/A211T) were produced and expressed in Aspergillus oryzae. In parallel, sequence alignment investigations showed that several cellulases from family GH7 display an alanine residue instead of histidine at position 209. Amongst them, Trichoderma reesei Cel7B, an endoglucanase sharing the highest degree of sequence identity with Humicola Cel7B, was found to naturally accept a β-d-N-acetylglucosaminyl residue at +1 subsite. The T. reesei Cel7B mutant nucleophile E196A was produced and expressed in Saccharomyces cerevisiae, and its activity as glycosynthase, together with the H. insolens glycosynthase mutants, was evaluated toward various glycosidic acceptors.     

The synthesis, fluctionality and structural characterization of ReMo3H4(CO)12

The reaction of ReH₉²⁻ with Mo(diglyme)(CO)₃ leads to the formation of the mixed metal cluster trianion, ReMo₃H₄(CO)₁₂³⁻. This species has been characterized analytically, spectroscopically and through X-ray diffraction analysis. A pseudo-tetrahedral arrangement of M(CO)₃ fragments is adopted, such that each set of three carbonyl ligands eclipses the adjacent three tetrahedral edges, an apparent result of the location of the hydride ligands on the tetrahedral faces. Variable temperature NMR studies revealed a fluctional process for some of the carbonyl ligands, but not for the hydrides. Crystal data for [Me₄N]₃[ReMo₃H₄(CO)₁₂]·THF; space group P2₁/n, a = 12.157(2), B = 21.480(4), C = 15.964(3) Å, β = 98.26(1)°, Z = 4, R = 0.067 and R_w = 0.076.     

Over-expression of human type I (placental) 3β-hydroxy-5-ene-steroid dehydrogenase/isomerase in insect cells infected with recombinant baculovirus

Human type I placental 3β-hydroxy-5-ene-steroid dehydrogenase/steroid 5→4-ene-isomerase (3β-HSD/isomerase) synthesizes androstenedione from fetal dehydroepiandrosterone and progesterone from pregnenolone. The full length cDNA that encodes type I 3β-HSD/isomerase was inserted into the baculovirus, Autographa californica multiple nucleocapsid polyhedrosis virus, and expressed in Spodoptera fungiperda (Sf-9) insect cells. Western blots showed that the baculovirus-infected Sf-9 cells produced an immunoreactive protein that co-migrated with purified placental 3β-HSD/isomerase. Ultracentrifugation localized the expressed enzyme activities in all the membrane-associated organelles of the Sf-9 cell (nuclear, mitochondrial and microsomal). Kinetic studies showed that the expressed enzyme has 3β-HSD and isomerase activities. The Michaelis-Menton constant is very similar for the 3β-HSD substrate, 5-androstan-3β-o1-17-one, in the Sf-9 cell homogenate (K_m = 17.9 μM) and placental microsomes (K_m = 16.7 μM). The 3β-HSD activity (V_max = 14.5 nmol/min/mg) is 1.6-fold higher in the Sf-9 cell homogenate compared to placental microsomes (V_max = 9.1 nmol/min/mg). The K_m values are almost identical for the isomerase substrate, 5-androstene-3,17-dione, in the Sf-9 cell homogenate (K_m = 14.7 μM) and placental microsomes (K_m = 14.4 μM). The specific isomerase activity is 1.5-fold higher in the Sf-9 cells (V_max = 25.7 nmol/min/mg) relative to placenta (V_max = 17.2 nmol/min/mg). These studies show that our recombinant baculovirus system over-expresses fully active enzyme that is kinetically identical to native 3β-HSD/isomerase in human placenta.