JNK1/2 regulate Bid by direct phosphorylation at Thr59 in response to ALDH1L1.

BH3 interacting-domain death agonist (Bid) is a BH3-only pro-apoptotic member of the Bcl-2 family of proteins. Its function in apoptosis is associated with the proteolytic cleavage to the truncated form tBid, mainly by caspase-8. tBid translocates to mitochondria and assists Bax and Bak in induction of apoptosis. c-Jun N-terminal kinase (JNK)-dependent alternative processing of Bid to jBid was also reported. We have previously shown that the folate stress enzyme 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) activates JNK1 and JNK2 in cancer cells as a pro-apoptotic response. Here we report that in PC-3 prostate cancer cells, JNK1/2 phosphorylate Bid at Thr59 within the caspase cleavage site in response to ALDH1L1. In vitro, all three JNK isoforms, JNK 1-3, phosphorylated Thr59 of Bid with JNK1 being the least active. Thr59 phosphorylation protected Bid from cleavage by caspase-8, resulting in strong accumulation of the full-length protein and its translocation to mitochondria. Interestingly, although we did not observe jBid in response to ALDH1L1 in PC-3 cells, transient expression of Bid mutants lacking the caspase-8 cleavage site resulted in strong accumulation of jBid. Of note, a T59D mutant mimicking constitutive phosphorylation revealed more profound cleavage of Bid to jBid. JNK-driven Bid accumulation had a pro-apoptotic effect in our study: small interfering RNA silencing of either JNK1/2 or Bid prevented Bid phosphorylation and accumulation, and rescued ALDH1L1-expressing cells. As full-length Bid is a weaker apoptogen than tBid, we propose that the phosphorylation of Bid by JNKs, followed by the accumulation of the full-length protein, delays attainment of apoptosis, and allows the cell to evaluate the stress and make a decision regarding the response strategy. This mechanism perhaps can be modified by the alternative cleavage of phospho-T59 Bid to jBid at some conditions.

ALDH1L1 inhibits cell motility via dephosphorylation of cofilin by PP1 and PP2A.

Here we report that ALDH1L1 (FDH, a folate enzyme with tumor suppressor-like properties) inhibits cell motility. The underlying mechanism involves F-actin stabilization, re-distribution of cytoplasmic actin toward strong preponderance of filamentous actin and formation of actin stress fibers. A549 cells expressing FDH showed a much slower recovery of green fluorescent protein-actin fluorescence in a fluorescence recovery after photobleaching assay, as well as an increase in G-actin polymerization and a decrease in F-actin depolymerization rates in pyren-actin fluorescence assays indicating the inhibition of actin dynamics. These effects were associated with robust dephosphorylation of the actin depolymerizing factor cofilin by PP1 and PP2A serine/threonine protein phosphatases, but not the cofilin-specific phosphatases slingshot and chronophin. In fact, the PP1/PP2A inhibitor calyculin prevented cofilin dephosphorylation and restored motility. Inhibition of FDH-induced apoptosis by the Jun N-terminal kinase inhibitor SP600125 or the pan-caspase inhibitor zVAD-fmk did not restore motility or levels of phosphor-cofilin, indicating that the observed effects are independent of FDH function in apoptosis. Interestingly, cofilin small interfering RNA or expression of phosphorylation-deficient S3A cofilin mutant resulted in a decrease of G-actin and the actin stress fiber formation, the effects seen upon FDH expression. In contrast, the expression of S3D mutant, mimicking constitutive phosphorylation, prevented these effects further supporting the cofilin-dependent mechanism. Dephosphorylation of cofilin and inhibition of motility in response to FDH can also be prevented by the increased folate in media. Furthermore, folate depletion itself, in the absence of FDH, resulted in cofilin dephosphorylation and inhibition of motility in several cell lines. Our experiments showed that these effects were folate specific and not a general response to nutrient starvation. Overall, this study shows the presence of distinct intracellular signaling pathways regulating motility in response to folate status and points toward mechanisms involving folates in promoting a malignant phenotype.

Cooperation between JNK1 and JNK2 in activation of p53 apoptotic pathway.

FDH (10-formyltetrahydrofolate dehydrogenase) is strongly downregulated in tumors while its elevation suppresses proliferation of cancer cells and induces p53-dependent apoptosis. We have previously shown that FDH induces phosphorylation of p53 at Ser6, which is a required step in the activation of apoptosis. In the present study, we report that FDH-induced p53 phosphorylation is carried out by JNK1 and JNK2 (c-Jun N-terminal kinases) working in concert. We have demonstrated that FDH induces phosphorylation of JNK1 and JNK2, while treatment of FDH-expressing cells with JNK inhibitor SP600125, as well as knockdown of JNK1 or JNK2 by siRNA, prevents phosphorylation of p53 at Ser6 and protects cells from apoptosis. Interestingly, the knockdown of JNK1 abolished phosphorylation of JNK2 in response to FDH, while knockdown of JNK2 did not prevent JNK1 phosphorylation. Pull-down assay with the p53-specific antibody has shown that JNK2, but not JNK1, is physically associated with p53. Our studies revealed a novel mechanism in which phosphorylation of JNK2 is mediated by JNK1 before phosphorylation of p53, and then p53 is directly phosphorylated by JNK2 at Ser6.

On the role of conserved histidine 106 in 10-formyltetrahydrofolate dehydrogenase catalysis: connection between hydrolase and dehydrogenase mechanisms.

The enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate in an NADP(+)-dependent dehydrogenase reaction or an NADP(+)-independent hydrolase reaction. The hydrolase reaction occurs in a 310-amino acid long amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. The amino-terminal domain of FDH shares some sequence identity with several other enzymes utilizing 10-formyl-THF as a substrate. These enzymes have two strictly conserved residues, aspartate and histidine, in the putative catalytic center. We have shown recently that the conserved aspartate is involved in FDH catalysis. In the present work we studied the role of the conserved histidine, His(106), in FDH function. Site-directed mutagenesis experiments showed that replacement of the histidine with alanine, asparagine, aspartate, glutamate, glutamine, or arginine in N(t)-FDH resulted in expression of insoluble proteins. Replacement of the histidine with another positively charged residue, lysine, produced a soluble mutant with no hydrolase activity. The insoluble mutants refolded from inclusion bodies adopted a conformation inherent to the wild-type N(t)-FDH, but they did not exhibit any hydrolase activity. Substitution of alanine for three non-conserved histidines located close to the conserved one did not reveal any significant changes in the hydrolase activity of N(t)-FDH. Expressed full-length FDH with the substitution of lysine for the His(106) completely lost both the hydrolase and dehydrogenase activities. Thus, our study showed that His(106), besides being an important structural residue, is also directly involved in both the hydrolase and dehydrogenase mechanisms of FDH. Modeling of the putative hydrolase catalytic center/folate-binding site suggested that the catalytic residues, aspartate and histidine, are unlikely to be adjacent to the catalytic cysteine in the aldehyde dehydrogenase catalytic center. We hypothesize that 10-formyl-THF dehydrogenase reaction is not an independent reaction but is a combination of hydrolase and aldehyde dehydrogenase reactions.

Aspartate 142 is involved in both hydrolase and dehydrogenase catalytic centers of 10-formyltetrahydrofolate dehydrogenase.

The enzyme 10-formyltetrahydrofolate dehydrogenase (FDH) catalyzes conversion of 10-formyltetrahydrofolate to tetrahydrofolate in either a dehydrogenase or hydrolase reaction. The hydrolase reaction occurs in a 310-residue amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. N(t)-FDH shares some sequence identity with several 10-formyltetrahydrofolate-utilizing enzymes. All these enzymes have a strictly conserved aspartate, which is Asp(142) in the case of N(t)-FDH. Replacement of the aspartate with alanine, asparagine, glutamate, or glutamine in N(t)-FDH resulted in complete loss of hydrolase activity. All the mutants, however, were able to bind folate, although with lower affinity than wild-type N(t)-FDH. Six other aspartate residues located near the conserved Asp(142) were substituted with an alanine, and these substitutions did not result in any significant changes in the hydrolase activity. The expressed D142A mutant of the full-length enzyme completely lost both hydrolase and dehydrogenase activities. This study shows that Asp(142) is an essential residue in the enzyme mechanism for both the hydrolase and dehydrogenase reactions of FDH, suggesting that either the two catalytic centers of FDH are overlapped or the dehydrogenase reaction occurs within the hydrolase catalytic center.

Overexpression of functional hydrolase domain of rat liver 10-formyltetrahydrofolate dehydrogenase in Escherichia coli.

Rat liver 10-formyltetrahydrofolate dehydrogenase (FDH) is a tetrameric enzyme composed of four identical 902-amino-acid-residue (99 kDa) monomers. We expressed the enzyme and its 310-amino-acid-residue amino-terminal domain, which is 10-formyltetrahydrofolate hydrolase, in Escherichia coli BL21 (DE3) cells using the pRSET expression vector. We removed the entire translated region of the vector including the polyhistidyl tag and the recombinant proteins were expressed, not as a fusion constructs, but as unmodified sequences. The expressed full-length enzyme was found to be an insoluble protein and was not purified and characterized, while the amino-terminal domain was expressed as a soluble protein possessing hydrolase activity. The recombinant amino-terminal domain was purified in one step on a DEAE MemSep 1000 HP Ion-Exchange Membrane Chromatography Cartridge (Millipore) using a ConSep LC100 chromatographic system (Millipore). The chromatography gave a homogenous and active preparation of the recombinant protein with a yield of about 2 mg per 100 ml of bacterial culture. Kinetic parameters of the hydrolase reaction displayed by the amino-terminal domain expressed in E. coli were similar to those of the recombinant full-length enzyme and its amino-terminal domain previously expressed in insect cells. The purified recombinant enzyme remained active for at least 4 weeks at 4 degreesC. These results show that the hydrolase amino-terminal domain of FDH can be overexpressed as a functional enzyme in E. coli cells and purified in one step by a simple chromatographic procedure.

Expression, purification, and properties of the aldehyde dehydrogenase homologous carboxyl-terminal domain of rat 10-formyltetrahydrofolate dehydrogenase.

The liver cytosolic enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH) (EC 1.5.1.6) catalyzes two reactions: the NADP+-dependent oxidation of 10-formyltetrahydrofolate to tetrahydrofolate and CO2 and the NADP+-independent hydrolysis of 10-formyltetrahydrofolate to tetrahydrofolate and formate. The COOH-terminal domain of the enzyme (residues 420-902) is about 48% identical to a family of NAD-dependent aldehyde dehydrogenases (EC 1.2.1.3), and FDH possesses aldehyde dehydrogenase activity. We expressed the COOH-terminal domain (residues 420-902) of FDH in insect cells using a baculovirus expression system. The recombinant protein was released from insect cells to the culture medium and was purified from the medium by a two-step procedure: precipitation with 35% saturated ammonium sulfate followed by chromatography on hydroxyapatite. The purified COOH-terminal domain displayed aldehyde dehydrogenase activity similar to that of native FDH but had neither dehydrogenase nor hydrolase activity toward folate substrates. Aldehyde dehydrogenase activity of the COOH-terminal domain and FDH was independent of the presence of 2-mercaptoethanol while 10-FDDF dehydrogenase activity of FDH occurred only in the presence of 2-mercaptoethanol. The COOH-terminal domain existed as a tetramer showing that the sites for oligomerization of subunits in native FDH resides in this domain. Using titration of tryptophan fluorescence, it was found that the COOH-terminal domain bound NADP+ to the same extent as FDH (Kd 0.2 and 0.3 microM, respectively) but did not bind folate. Both FDH and its COOH-terminal domain also bound NAD+ (Kd 11 and 16 microM, respectively) as measured by fluorescence titration. Both proteins were able to catalyze the aldehyde dehydrogenase reaction utilizing NADP+ or NAD+, but the Km for NAD+ was three orders higher than that for NADP+ (2 mM and 1.5-2.0 microM, respectively). The concentration of NAD+ required for the reaction was high compared with the physiological level of NAD+, suggesting that the reaction does not occur in vivo. NAD+ at physiological concentrations stimulated the aldehyde dehydrogenase reaction performed by FDH or its COOH-terminal domain using NADP+.

Domain structure of rat 10-formyltetrahydrofolate dehydrogenase. Resolution of the amino-terminal domain as 10-formyltetrahydrofolate hydrolase.

We expressed the NH2-terminal domain of the multidomain, multifunctional enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), using a baculovirus expression system in insect cells. Expression of the 203-amino acid NH2-terminal domain (residues 1-203), which is 24-30% identical to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.2), resulted in the appearance of insoluble recombinant protein apparently due to incorrect folding. The longer NH2-terminal recombinant protein (residues 1-310), which shares 32% identity with Escherichia coli L-methionyl-tRNA formyltransferase (EC 2.1.2.9), was expressed as a soluble protein. During expression, this protein was released from cells to the culture medium and was purified from the culture medium by 5-formyltetrahydrofolate-Sepharose affinity chromatography followed by chromatography on a Mono-Q column. We found that the purified NH2-terminal domain bears a folate binding site, possesses 10-formyltetrahydrofolate hydrolase activity, and exists as a monomer. Titration of tryptophan fluorescence showed that native FDH bound both the substrate of the reaction, 10-formyl-5, 8-dideazafolate, and the product of the reaction, 5,8-dideazafolate, with the same affinities as its NH2-terminal domain did and that both proteins bound the substrate with a 50-fold higher affinity than the product. Neither the NH2-terminal domain nor its mixture with the previously purified COOH-terminal domain had 10-formyltetrahydrofolate dehydrogenase activity. Formation of complexes between the COOH- and NH2-terminal domains also was not observed. We conclude that the 10-formyltetrahydrofolate dehydrogenase activity of FDH is a result of the action of the aldehyde dehydrogenase catalytic center residing in the COOH-terminal domain on the substrate bound in the NH2-terminal domain and that the intermediate domain is necessary to bring the two functional domains together in the correct orientation.

Baculovirus expression and purification of rat 10-formyltetrahydrofolate dehydrogenase.

The liver cytosolic enzyme, 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) (EC 1.5.1.6) catalyzes two reactions: the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate to tetrahydrofolate and CO2 and the NADP(+)-independent hydrolysis of 10-formyltetrahydrofolate to tetrahydrofolate and formate. It exists as a tetramer of 99-kDa subunits in rat liver. We expressed rat liver cDNA encoding 10-FTHFDH in insect cells using the pVL 1393 expression vector and MaxBac expression kit. Despite the absence of a leader peptide the recombinant 10-FTHFDH was released from the cells to the culture medium during production in both Sf9 and High five cell lines. The enzyme released into the medium was no less than 80% of the total recombinant 10-FTHFDH. Both enzyme pools, from the medium and from cell extracts, displayed high activity. The maximum expression of 10-FTHFDH was observed 72 h postinfection in High five cells and 96 h postinfection in Sf9 cells. High five cells revealed four times higher expression of the recombinant enzyme per milligram of total cell protein than Sf9 cells. Passage of the cell-free culture medium over an affinity column of 5-formyltetrahydrofolate-Sepharose provided 10-FTHFDH that was more than 95% pure. Additional purification on a Mono Q column resulted in an homogenous preparation of the enzyme. Purified recombinant 10-FTHFDH displayed both dehydrogenase and hydrolase activities, similar to those of the rat liver enzyme, and the recombinant enzyme remained active at least 12 months when stored appropriately. These results show that 10-FTHFDH can be overexpressed as a functional enzyme in baculovirus-infected insect cells and purified in two steps by simple chromatographic procedures.

Organization of the transcortin-binding domain on placental plasma membranes.

Complex formation between transcortin (corticosteroid-binding globulin) and 20 kDa sialoglycoprotein from human syncytiotrophoblast plasma membranes (presumably a transcortin-recognizing subunit of the transcortin membrane receptor) was studied using FPLC and cross-linking with bifunctional reagents. The action of 1,5-difluoro-2,4-dinitrobenzene (DFDNB) on a solution of the purified 20 kDa sialoglycoprotein and transcortin resulted in formation of covalently linked complexes of 95 kDa and 140 kDa consisting of one transcortin molecule and either two or four molecules of the membrane sialoglycoprotein (the molecular mass of transcortin is 55 kDa). Additionally, cross-linking resulted in the appearance of a 43 kDa species which is the cross-linked dimer of the membrane protein. The dimer was also observed during chromatography on a Superose 12 column in the absence of DFDNB treatment. Treatment of intact syncytiotrophoblast membranes with DFDNB resulted in isolation of the transcortin binding protein dimer as the major portion of total pool of the protein. Formation of the transcortin complexes with two and four molecules of the membrane protein was also observed when the membranes were incubated with 125I-labeled transcortin and treated with DFDNB, but formation of the latter complexes predominated. The results obtained suggest that the recognizing and binding domain for transcortin in placental membranes is organized as dimers consisting of non-covalently linked sialoglycoprotein monomers of a 20 kDa each and that transcortin has two sites for interaction with this dimer. Apparently, binding of two dimers results in the formation of the functional form of the transcortin-receptor complex. The possible biological role of such a complex is discussed.

Recombinant 10-formyltetrahydrofolate dehydrogenase catalyses both dehydrogenase and hydrolase reactions utilizing the synthetic substrate 10-formyl-5,8-dideazafolate.

10-Formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) is a bifunctional enzyme, displaying both NADP(+)-dependent dehydrogenase activity for the formation of tetrahydrofolate and CO2, and NADP(+)-independent hydrolase activity for the formation of tetrahydrofolate and formate. A previous report [Case, Kaisaki and Steele (1988) J. Biol. Chem. 263, 1024-1027] claimed that dehydrogenase and hydrolase activities were products of separate cytosolic and mitochondrial forms of this enzyme. Here we report that recombinant 10-formyltetrahydrofolate dehydrogenase carries out both enzymic reactions, proving that a product of a single gene, i.e. one protein, not two, has both activities. The stable synthetic analogue 10-formyl-5,8-dideazafolate can substitute for the labile natural substrate, 10-formyltetrahydrofolate, in both reactions. This was shown with both native and recombinant rat liver enzyme. The Km values for 10-formyl-5,8-dideazafolate were half of those for 10-formyltetrahydrofolate in both the dehydrogenase and hydrolytic reactions. The Vmax, values were similar for both substrates. Both dehydrogenase and hydrolase reactions were dependent on the presence of 2-mercaptoethanol. The pH optima were 7.8 and 5.6 for the dehydrogenase and hydrolase reactions respectively, consistent with the presence of two active sites in the enzyme.

Cysteine 707 is involved in the dehydrogenase activity site of rat 10-formyltetrahydrofolate dehydrogenase.

The enzyme, 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) (EC 1.5.1.6) catalyzes both the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate to tetrahydrofolate and CO2 and the NADP(+)-independent hydrolysis of 10-formyltetrahydrofolate to tetrahydrofolate and formate. The COOH-terminal domain of the 10-FTHFDH (residues 417-902) shows a 46% identity with a series of NAD(+)-dependent aldehyde dehydrogenases (EC 1.2.1.3). All known members of the aldehyde dehydrogenase family and 10-FTHFDH have a strictly conserved cysteine (Cys-707 for 10-FTHFDH), which has been predicted to be at the active site of these enzymes. Rat liver 10-FTHFDH was expressed in a baculovirus system, and site-directed mutagenesis has been used to study the role of cysteine 707 in the activity of 10-FTHFDH. 10-FTHFDH with alanine substituted for cysteine at position 707 had no dehydrogenase activity, while hydrolase activity and binding of NADP+ were unchanged. Light scattering analysis revealed that wild type and mutant 10-FTHFDH exist as tetramers. We conclude that cysteine 707 is directly involved in the active site of 10-FTHFDH responsible for dehydrogenase activity, and there is a separate site for the hydrolase activity.

Interaction of sex hormone-binding globulin with plasma membranes from the rat epididymis and other tissues.

The binding of human sex hormone-binding globulin (hSHBG) to plasma membranes prepared from the adult rat epididymis and other potential target and non-target tissues was examined. Specific binding sites were detected in the epididymis, testis, prostate, skeletal muscle and liver. The first three organs exhibited a higher (KD approx. 0.1 nM; Bmax approx. 0.05-0.10 pmol/mg membrane protein, Site I) and a lower (KD approx. 5 nM; Bmax approx. 1.0-2.5 pmol/mg membrane protein, Site II) affinity binding site. Only Site I was detected in muscle membranes and only Site II was detected in membranes isolated from liver. Specific binding was not detectable in either spleen or brain. Regional distribution of hSHBG binding sites occurred in the epididymis. Both Site I and Site II were present in the proximal caput and distal cauda. The distal caput and proximal cauda contained only Site II; no specific binding was detected in the corpus. Binding of hSHBG to epididymal membranes was time- and temperature-dependent. The presence of Ca2+ did not affect binding. Non-liganded [125I]-labeled hSHBG can bind to both sites in epididymal membranes. The affinity of hSHBG for Site I increased 2-fold when it was complexed with 5 alpha-dihydrotestosterone, testosterone or estradiol. The hSHBG-androgen complex had little effect on Site II versus steroid-free SHBG. However, the affinity of the hSHBG-estradiol complex for these sites was increased 10-fold. Cortisol, which has a low affinity for hSHBG, did not influence its binding to either the higher or lower affinity membrane sites.

Testosterone destroys the transcortin-receptor complex.

Dissociation of the complex of transcortin receptor with immobilized transcortin in the presence of 10(-5) M testosterone has been shown with the use of affinity chromatography on transcortin-Sepharose. The specificity of this effect is confirmed by its abrogation in the presence of cortisol. The testosterone effect has been used for the elution of transcortin receptor from affinity column. The receptor retained transcortin-binding capacity after the elution and removal of testosterone. Characteristics of the receptor obtained by testosterone elution were identical with those of the transcortin eluted preparation.

A transcortin-binding protein in the plasma membrane of human syncytiotrophoblast.

Using affinity chromatography on immobilized transcortin of 125I-labeled, cholate-solubilized plasma membranes of human syncytiotrophoblast, a transcortin-binding protein with a minimal Mr of about 20 kDa has been isolated. It was found to be a sialoglycoprotein with an isoelectric point at pH 4.4 (about 5.0 after the treatment with neuraminidase). We assume that this protein is a component of membrane recognition system for transcortin-steroid complexes.

On the functional form of transcortin-recognizing subunit of transcortin membrane receptor.

Complex formation between transcortin and the 20 kDa sialoglycoprotein from the plasma membrane of human decidual endometrium (presumably a transcortin-recognizing subunit of transcortin membrane receptor) was studied using cross-linking reagents. The action of 1,5-difluoro-2,4-dinitrobenzene (DFDNB) on a solution of 125I-labelled 20 kDa sialoglycoprotein and unlabelled transcortin resulted in the formation of two 125I-containing containing species that corresponded to covalently linked complexes of one transcortin molecule and either 2 or 4 molecules of the labeled membrane sialoglycoprotein. Only the latter complex was observed when the endometrium membranes were incubated with [125I]transcortin and treated with DFDNB. This suggests that the functional form of transcortin-recognizing subunit of the membrane receptor is a tetramer.

Study of the transcortin binding to human endometrium plasma membrane.

Transcortin complexed with progesterone was shown to bind specifically to the plasma membrane of human decidual endometrium. The binding reaction was characterized by a high affinity (an apparent Kd value was (1.0 +/- 0.2).10(-10) mol/l) and high selectivity: such human serum proteins as albumin, orosomucoid, transferrin, thyroxine-binding globulin and sex hormone-binding globulin did not compete with transcortin for the membrane binding sites. Transcortin binding to the membrane was steroid-dependent: transcortin-cortisol complex bound to the membranes substantially more weakly than transcortin-progesterone, and specific binding of transcortin devoid of steroid was not detected. Using a radioimmunoassay, we have measured the concentration of endogenous transcortin in highly purified membrane preparations solubilized with sodium cholate. It was found that an extensive washing of decidual strips with a physiological buffer prior to the membrane isolation resulted in a decrease of the endogenous transcortin level along with an increase of the specific membrane binding of exogenous 125I-labeled transcortin. Affinity chromatography on immobilized transcortin was used to isolate transcortin-binding components from 125I-labeled, cholate-solubilized plasma membrane of decidual endometrium. Along with lipid components, the structure of which was not investigated, a 125I-labeled transcortin-binding sialoglycoprotein with a minimal Mr of 20.0 +/- 1.5 kDa and a pI of approx. 3.3 was detected. In the presence of transcortin, this sialoglycoprotein could be precipitated with a monospecific antitranscortin antiserum. Using hydroxylapatite as a separating agent, the interaction of transcortin and the membrane sialoglycoprotein in model systems containing the two proteins and various steroid hormones was studied. It was found that the membrane sialoglycoprotein displayed a higher affinity for transcortin-progesterone than for transcortin-cortisol (the Kd values were, respectively, 2.10(-11) and 7.10(-11) mol/l) and it did not bind transcortin complexed with testosterone.

[Characteristics of a transcortin-binding component of the plasma membrane of cells of the human decidual endometrium]. / Kharakteristika transkortin-sviazyvaiushchego komponenta plazmaticheskoi membrany kletok detsidual'nogo éndometriia cheloveka.

A sialoglycoprotein was isolated by affinity chromatography on immobilized transcortin from plasma membranes of human decidual endometrium cells, whose components were labeled with 125I and solubilized with sodium cholate. The apparent molecular mass of the monomer is 20.0 +/- 1.5 kDa, pI is at pH 3.3. The sialoglycoprotein specifically binds transcortin complexed to progesterone with Kd approximately 10(-10) M.

[Interaction of the transcortin-progesterone complex with plasma membranes of human decidual epithelium]. / Vzaimodeistvie kompleksa transkortin-progesteron s plazmaticheskimi membranami kletok detsidual'nogo éndometriia cheloveka.

It was demonstrated that transcortin complexed with progesterone specifically binds to plasma membranes of human decidual endometrium, a progesterone target tissue. This interaction is characterized by a high affinity [Kd = (1.0 +/- 0.2).10(-10) mol/l] and selectivity. Such human serum proteins as albumin, orosomucoid, transferrin, thyroxine- and sex steroid-binding globulins do not compete with transcortin for the binding sites on the membranes. The concentration of endogenous transcortin in sodium cholate-solubilized endometrium cell membranes was determined by the radioimmunoassay method.