Laser-cut paper-based device for the detection of dengue non-structural NS1 protein and specific IgM in human samples.

The incidence of flavivirus infections has increased dramatically in recent decades in tropical and sub-tropical areas worldwide, affecting hundreds of millions of people each year. Dengue viruses are typically transmitted by mosquitoes and can cause a wide range of symptoms from flu-like fever to organ impairment and death. Although conventional diagnostic tests can provide early diagnosis of acute dengue infections, access to these tests is often limited in developing countries. Consequently, there is an urgent need to develop affordable, simple, rapid, and robust diagnostic tools that can be used at 'Point of Care' settings. Early diagnosis is crucial to improve patient management and reduce the risk of complications. In the present study, a novel laser-cut device made of glass-fiber paper was designed and tested for the detection of the dengue Non Structural 1 (NS1) viral protein and specific IgM in blood and plasma. The device, called PAD, was able to detect around 25 ng/mL of NS1 protein in various sample types in 8 minutes, following a few simple steps. The PAD was also able to detect specific IgM in human plasmas in less than 10 minutes. The PAD appears to have all the potential to assist health workers in early diagnosis of dengue fever or other tropical fevers caused by flaviviruses.

An immunoreactive peptide of the FSH involved in autoimmune infertility.

The purpose of this study was to identify autoantigens contained in human ovary extracts. Serum samples from 36 infertile women with anti-ovary antibodies as detected with an ELISA technique were tested in Western blot against human ovary extracts. A reactive protein with a molecular mass matching that of the FSH was detected in 34 cases. These serum samples also reacted strongly in Western blot and ELISA with purified FSH and, in immunofluorescence, with pituitary cells. Using the Pepscan approach, with overlapping peptides matching the amino acid sequence of the human FSH beta-chain, several immunoreactive regions were evidenced. The 78-93 amino acid sequence of the human FSH beta-chain appeared as one of the major epitopes. Synthetic peptides of this region were prepared and demonstrated to react with human serum samples from women with anti-ovary antibodies. These data demonstrate that FSH can be an autoantigen, recognized by autoantibodies associated with infertility.

Differential diagnosis of prostate cancer and benign prostate hyperplasia using two-dimensional electrophoresis.

Prostate specific antigen (PSA) is a protease which is characteristic of the prostate. It is widely used as a serum marker for the early diagnosis of prostate cancer (PCa). Nevertheless, for concentrations between 4 and 10 ng/mL, PSA does not enable PCa to be distinguished from benign diseases, such as benign prostate hyperplasia (BPH). In sera, the use of a ratio between free PSA (PSA uncomplexed with protease inhibitor) and total PSA (free PSA and PSA bound to alpha-1 anti-chymotrypsin) enables the "gray zone" to be reduced, but an important proportion of patients are still wrongly classed. Using two-dimensional electrophoresis, we demonstrated using 52 PCa and 40 BPH well-documented clinical cases that BPH sera show a significantly greater percentage of low-molecular-weight free PSA elements (IwPSA) than PCa sera. In our study, the use of a ratio between IwPSA and standard free PSA enables the correct diagnosis of 100% of PCa and 82.5% of BPH cases as against when 73.1% and 42.5% respectively were correctly diagnozed using the total PSA and the free/total PSA ratio. This important finding may be related to differences in the mechanism secreting PSA from the prostate into the bloodstream. We have shown how a tissue marker may be turned into a powerful tumor marker by events probably unrelated to its expression.

Specificities of multiple sclerosis cerebrospinal fluid and serum antibodies against mimotopes.

In order to characterize antigenic epitopes specifically targeted by the immune response of patients with multiple sclerosis (MS), the antibody specificities of cerebrospinal fluids (CSF) and sera from the same MS patients have been analyzed using a random pentadecapeptide library displayed on phage. The 3 peptides (mimotopes) selected with MS sera were not disease-specific. In contrast, the combination of 4 MS CSF selected mimotopes, allowed the detection of specific antibodies in 21 of 60 MS CSF whereas only 2 of 27 CSF from patients with other neurological diseases equally recognized the 4 mimotopes. Some amino acid similarities were found between two MS CSF selected mimotopes and two envelope regions (319-329 and 433-443, respectively) of MSRV (multiple-sclerosis-associated retrovirus) and the related endogenous retrovirus HERV-W.

Overexpression of the FAD-binding domain of the sulphite reductase flavoprotein component from Escherichia coli and its inhibition by iodonium diphenyl chloride.

SiR-FP43, the NADPH- and FAD-binding domain of the Escherichia coli sulphite reductase flavoprotein component (SiR-FP), has been overexpressed and characterized. It folds independently, retaining FAD as a cofactor and the catalytic properties associated with the presence of this cofactor. Iodonium diphenyl chloride (IDP) was shown to be a very efficient inhibitor of SiR-FP43 and SiR-FP60, the monomeric form of SiR-FP, containing both FMN and FAD as cofactors (K(i) = 18.5 +/- 5 microM, maximal inactivation rate = 0.053 +/- 0.005 s(-1)). In both cases, inactivation was shown to result from covalent binding of a phenyl group to FAD exclusively, in marked contrast with previous results obtained with cytochrome P450 reductase (CPR), where FMN and a tryptophan were phenylated, but not FAD. However, our kinetic analyses are in agreement with the inhibition mechanism demonstrated with CPR [Tew (1993) Biochemistry 32, 10209-10215]. Nine different FAD phenylated adducts were isolated and, for the first time, two FAD phenylated adducts were identified directly after extraction from a protein. Taken together, our results have shown that flavoprotein inactivation by IDP is not a reliable indicator for a flavin radical intermediate in catalysis.

Two-dimensional electrophoresis of prostate-specific antigen in sera of men with prostate cancer or benign prostate hyperplasia.

Prostate-specific antigen (PSA), the main marker for prostate cancer (PCa), is released from the prostate into the blood stream at nanogram level and may increase in PCa and nonmalignant disease such as benign prostate hyperplasia (BPH). More recently, advantage was taken of PSA's ability to bind to protease inhibitors in serum in order to improve discrimination between PCa and BPH, using the free PSA to total PSA ratio. The understanding of this phenomenon at molecular level, which is still unknown, may promise new improvements in the field of diagnostics. For this purpose, we determined the pattern of PSA forms in PCa and BPH sera, using the high resolving power of two-dimensional electrophoresis (2-DE) in conjunction with the high sensitivity of chemiluminescence detection. Serum PSA differs drastically from seminal PSA: apart from complexed forms, serum PSA shows few cleaved forms. Moreover, 2-DE patterns from PCa are relatively homogeneous, whereas patterns from BPH may in some cases present a higher proportion of cleaved forms and in other cases present slightly more basic spots. We therefore demonstrated, for the first time, that an increase in the free to total PSA ratio in BPH cases may be due to cleaved PSA forms (which are enzymatically inactive and unable to bind inhibitors), or possibly related to basic free PSA, which may represent the zymogen forms.

The epitopes targeted by the rheumatoid arthritis-associated antifilaggrin autoantibodies are posttranslationally generated on various sites of (pro)filaggrin by deimination of arginine residues.

Antifilaggrin autoantibodies (AFA) are a population of IgG autoantibodies associated to rheumatoid arthritis (RA), which includes the so-called "antikeratin" Abs and antiperinuclear factor. AFA are the most specific serological markers of RA. We previously showed that they recognize human epidermal filaggrin and other profilaggrin-related proteins of various epithelial tissues. Here, we report further characterization of the protein Ags and epitopes targeted by AFA. All the Ags that exhibit numerous neutral/ acidic isoelectric variants were immunochemically demonstrated to be deiminated proteins. In vitro deimination of a recombinant human filaggrin by a peptidylarginine deiminase generated AFA epitopes on the protein. Moreover, two of three filaggrin-derived synthetic peptides with a citrulline in the central position were specifically and widely recognized by AFA affinity-purified from a series of RA sera. These results indicate that citrulline residues are constitutive of the AFA epitopes, but only in the context of specific amino acid sequences of filaggrin. In competition experiments, the two peptides abolished the AFA reactivity of RA sera, showing that they present major AFA epitopes. These data should help in the identification of a putative deiminated AFA-inducing or cross-reactive articular autoantigen and provide new insights into the pathogenesis of RA. They could also open the way toward specific immunosuppressive and/or preventive therapy of RA.

HCV core immunodominant region analysis using mouse monoclonal antibodies and human sera: characterization of major epitopes useful for antigen detection.

Monoclonal antibodies (MAbs) were generated by immunizing mice with a truncated recombinant protein corresponding to the immunodominant region (residues 1-120) of hepatitis C virus (HCV) nucleocapsid protein. The specific recognition by either human sera or mouse monoclonal antibodies of overlapping peptides spanning the core region 1-120 as well as the comparison with epitopes described earlier allowed the fine mapping of HCV core. Within the region 1-120, the major antigenic domain could be restricted to the first 45 amino acids. Indeed, the peptide S42G (residues 2-45) allowed the detection of an anti-HCV core response by all anticore-positive human sera examined. According to their epitope localization, three groups of mouse MABs could be evidenced that were directed against different regions of core. Group II MAbs recognized a strictly linear epitope (QDVKF, residues 20-24), whereas group I MABs were directed against a conformational epitope mainly located at the amino acid residues (QIVGG, 29-33). The epitope of group III MABs was also conformational (PRGRRQPI, residues 58-65). These three epitopes appeared close but different from the three major human epitopes RKTKRNTN, VYLLPR, and GRTWAQPGYPWPLY (residues 7-17, 34-39, and 73-86, respectively). Group II MAB 7G12A8 and group I MAB 19D9D6 were used in a sandwich ELISA for the capture and the detection, respectively, of viral core antigen in sera of patients with chronic HCV infection. After treatment of sera with triton x 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected.

Solid-phase synthesis and on-resin cyclization of a disulfide bond peptide and lactam analogues corresponding to the major antigenic site of HIV gp41 protein.

A cyclic peptide that spans the major antigenic determinant of the human immunodeficiency virus (HIV) glycoprotein 41 (gp41) has been synthesized according to various strategies. For immunodiagnostic applications, biotin was added at the N-terminus of the peptide and aminohexanoic acid was used as a spacer. Polymer-supported oxidations were carried out in a variety of ways with thallium (III) trifluoroacetate. The biotinylcyclic peptide was released from the support using trimethylsilyl trifluoromethane sulfonate and various scavengers. The efficacy of these different cyclization and cleavage procedures was compared. Side reactions were studied, and a simple and efficient procedure was set up to monitor peptide cyclization by mass spectrometry. In a second series of syntheses the disulfide bridge was replaced by an amide bond. For this purpose, an aspartic acid derivative and a diaminopropionic acid were introduced during the synthesis in place of the two cysteine residues in the parent sequence. On-resin cyclization was performed and led to a major side-product identified as a piperidide. This undesired base-mediated side reaction was prevented when, instead of piperidine, 1,8-diazabicyclo-[5.4.0]undec-7-ene was used for fluorenylmethyl ester deprotection. Reactivity of these peptides with different patients' sera and with a monoclonal antibody directed against the whole gp41 was tested using an enzyme-linked immunosorbent assay.

Synthetic peptide ELISAs for detection of and discrimination between group M and group O HIV type 1 infection.

We developed and evaluated two peptide-based immunoassays to confirm and discriminate between group M and group O HIV-1 infection. These assays are based on in vitro competition for antibody binding between M and O peptides. The first EIA is based on competition between group M and group O gp41 immunodominant domains and the second on competition between group O and group M V3 regions of gp120. Two panels of sera were used: the first consisted of 109 sera collected from 27 group O- and 92 group M-infected patients in whom the HIV isolates had been genotyped by sequencing or heteroduplex mobility assay. In this panel, the combination of the two assays correctly discriminated 106 samples (100% group O and 96.7% group M samples). The second panel, used for the field evaluation of the two assays, consisted of 157 samples from HIV-1-infected Cameroonian patients, 33 strains having been genotyped. The combination of the two techniques in a serogrouping algorithm discriminated 147 of these samples, 74 being HIV-1 group O and 73 group M. These results always correlated with genotyping results. The 10 sera that were not successfully classified by these assays were from early seroconverters. Altogether, the two assays clearly differentiated 263 of 276 (94.9%) samples in the two panels. On the basis of the genotyping results, the positive predictive value for group discrimination in the two panels was 100% for both GSEIA assays. Our peptide-blocking group-specific EIAs for differentiation and confirmation of HIV-1 group M and group O infection are complementary tools for epidemiological studies and surveillance of HIV-1 group O strain trafficking.

Antigenic variability of an immunodominant region (239-261) of the surface glycoprotein of HTLV-I.

In the 239-261 region of the surface glycoprotein of HTLV-I, we delineated five epitopes recognized by antibodies present in sera from HTLV-I infected patients. Three epitopes are located between the amino acids 252 and 261, one is of a linear type and the two others of a conformational type. One epitope is comprised between amino acids 244 and 249. The last epitope described lies between the amino acids 244 and 257 and strictly requires the presence of a serine at position 250 for its recognition. When patients are infected by viral isolates presenting a substitution of the serine at position 250 by a proline, no antibodies recognizing the 244-257 region could be found. Altogether, our data demonstrate that the immunodominant 239-261 region is complex and is subject to antigenic variability.

Immunogenicity of variable regions of the surface envelope glycoprotein of HTLV type I and identification of new major epitopes in the 239-261 region.

The reactivity of sera of 96 individuals infected with human T-cell leukemia virus type I (HTLV-I) was tested against various synthetic peptides corresponding to the gp46 immunodominant antigenic domains: residues 86-107, 175-199, and 239-261. The frequency of reactive sera was higher for 175-199 (93%) than for 239-261 (78%) or 86-107 (24%) with some variations in geographical regions and in diseases. The region 239-261 was extensively analyzed and five (linear or conformational) epitopes were found. The reactivity of sera toward functional or immunodominant domains may depend on the sequence of the infecting virus, and the role of three frequent substitutions (asparagine by tyrosine, proline by serine, and serine by proline or leucine at positions 93, 192, and 250 respectively) was established. Finally, the role of the genetic background of the host may condition the humoral immune response as individuals infected by HTLV-Is harboring the same predicted gp46 peptide sequence may recognize one, several, or all regions examined.

Characterization of monoclonal antibodies directed against the gp46 of human T-cell leukemia virus type I.

Essential HTLV-I biological functions depend on the structural motives of the surface glycoprotein (gp46). Monoclonal antibodies (mAbs) have been generated in order to identify functional regions of gp46. We obtained three monoclonal antibodies (3F3F10, 4F5F6 and 7G5D8) by immunizing Balb/c mice with beta-propiolactone inactivated HTLV-I producing cells and partially purified gp46. The mAbs are of the IgG 1 subclass. They have been characterized by western blot analysis, reactivity with HTLV-I and HTLV-II producing cells and ELISA binding assays using synthetic peptides. The immunoblot analysis performed with sheets prepared with the virus released by HUT 102 and 2060 cells (an HTLV-I virus producing cell line established in our laboratory) indicate that the three mAbs recognize a 46 kDa product as did the anti -gp46 mAb 0.5 alpha (18). Reactivity of the three mAbs with various cell lines was examined by indirect immunofluorescence assay. The mAb 7G5D8 stained strongly the membrane of all HTLV-I producing cells (MT2, C91/PL, HUT102 and cells of seven lines established in our laboratory and by A. Gessain); uninfected lymphoid cells (HSB-2, MOLT 4, CEM and PHA activated lymphocytes from normal donors) were negative. Interestingly cells of a HTLV-II producing line (344 MO) were positive. The mAbs 3F3F10 and 4F5F6 reacted with the same cells as did 7G5D8 but the fluorescence intensity was much lower than that observed with this later. A long synthetic peptide corresponding to the immunodominant region of the gp46 defined by the amino acids 175-199 and 10-mer peptides overlapping this region were used in an approach to identify the recognized epitope(s). The long 175-199 peptide was recognized by the three mAbs. 3F3F10 and 4F5F6 recognized none of the 10-mer peptides whereas 7G5D8 bound to 186-195 and 182-191 peptides. In addition 7G5D8 did not inhibit either syncytia formation or virus infection. In view of the data concerning the previously described mAbs 0.5 alpha, LAT 27 (5) and KE36-11 (6), our results suggest that the epitope recognized by 7G5D8 is different from those recognized by the former ones. As the 183-191 sequence corresponds to a region in which HTLV-I and HTLV-II harbour six common amino acids and two similar ones, this is consistent with the observation that 7G5D8 stained the HTLV-II producing cells 344 MO as well as all HTLV-I producing ones. Altogether our data support the hypothesis whereby this epitope recognized by 7G5D8 is contained within a sequence defined by amino acids 183-191.

Recognition by human sera of a variable region of the surface glycoprotein of HTLV-I.

The region comprised between the amino acids 175 and 199 of the HTLV-I envelope surface glycoprotein is one of the immunodominant domains of this molecule. In this region, which is well recognized by sera from HTLV-I infected patients, a substitution of the proline at position 192 by a serine has been described in some isolates. Because this mutation could modify the secondary structure of the glycoprotein molecule, we studied the inference of the presence of proline or serine on the recognition of the region 175-199 by human sera. For this, three peptides have been synthetized (a 25-mer 175-199 corresponding to the sequence of the ATK prototype, and two internal 10-mer 190-Pro-199 and 190-Ser-199 having a proline or a serine at position 192) and tested by immunosorbent assay. While most sera reacted with 190-Pro-199 and with 190-Ser-199 synthetic peptides, a differential recognition was observed according to the pathology associated to HTLV-I infection. Moreover sera corresponding to patients infected with a virus harboring a serine at position 192 were found to recognize only the 10-mer with a serine. These data indicates that HTLV-I is subject to antigenic variability.

Prevalence and persistence of antibody titers to recombinant HIV-1 core and matrix proteins in HIV-1 infection.

Numerous studies have established the correlation between antibodies to the core protein p24 of HIV-1 and the progression of the acquired immunodeficiency syndrome. In this study, we analyzed the immune response to two recombinant gag proteins, p24 and p17, in order to evaluate their diagnostic or prognostic significance. Immune response to the immunodominant domain of the transmembrane glycoprotein gp41 was used as a reference. Sera collected from individuals from France and Burundi (Central Africa) at various CDC stages of HIV-1 infection were tested using three sandwich enzyme-linked immunoassays developed with a synthetic peptide corresponding to the immunodominant domain of gp41, SP gp41, or recombinant p24 and p17 cloned and expressed in Escherichia coli. These assays allowed detection of titer antibodies to the three cited antigens. Antibodies to SP gp41 were detected in every HIV-1-positive patient from France and Burundi, generally at a high and stable level. Results obtained with p24 confirmed the value of antibodies to p24 as a prognostic marker only in European and North American populations, since the African population had very high levels of these antibodies even at an advanced stage of the disease. They also confirmed that initial antibody response to p24 is more predictive of outcome than antibody titer change over time. Although antibodies to p17 decline during progression to AIDS, they are frequently absent in French patients at early, asymptomatic stages and therefore could not be used as a prognostic marker. In contrast, antibodies to p17 are significantly less common in African patients with AIDS when compared with symptomless HIV-1-infected African individuals.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of cyclins in the maturation of Patella vulgata oocytes.

We have cloned and sequenced the cDNAs encoding Patella vulgata cyclins A and B. The cDNA clones contain an open reading frame of 426 and 408 amino acids respectively, which present similarity with cyclins from other species. Cyclin A and B RNAs are present as polyadenylated and non-polyadenylated RNA in prophase oocytes and are completely polyadenylated in metaphase I. During the first cleavages after fertilization the level of cyclin A and B mRNAs is high and drops when the free swimming stage is reached. Using p13suc1-Sepharose bead precipitation we demonstrate that cyclin synthesis is triggered during maturation and that inhibition of protein synthesis makes the cyclins disappear rapidly from the metaphase I oocytes, which shift to interphase condition. By microinjecting antisense oligonucleotides into metaphase I oocytes, we demonstrate that in vivo ablation of cyclin A and B messengers together gives the same result, whereas microinjection of only one oligonucleotide does not show any effect.

Orientation of the N-terminal region of the membrane-bound ADP/ATP carrier protein explored by antipeptide antibodies and an arginine-specific endoprotease. Evidence that the accessibility of the N-terminal residues depends on the conformational state of the carrier.

Two peptides corresponding to the amino acid sequences 1-11 (N-terminal peptide) and 288-297 (C-terminal peptide) of beef heart ADP/ATP carrier have been synthesized. After coupling to ovalbumin, they were injected into rabbits to raise polyclonal antibodies. The specificities of the generated antibodies were tested by enzyme-linked immunosorbent assay (ELISA) and (or) Western blot. Anti-N-terminal antibodies and anti-C-terminal antibodies reacted specifically with the corresponding peptide. However, only anti-N-terminal antibodies reacted with the isolated ADP/ATP carrier; they also reacted with the membrane-bound carrier in freeze-thawed mitochondria and mitoplasts, indicating that the first 10 amino acid residues of the membrane-bound carrier in mitochondria face the cytosol. On the basis that the ADP/ATP carrier can adopt two conformations, one trapped by carboxyatractyloside (CATR conformation) and the other by bongkrekic acid (BA conformation), the reactivity of the anti-N-terminal antibodies to the ADP/ATP carrier in mitoplasts or freeze-thawed mitochondria was tested for each conformation of the carrier. Only in the CATR conformation was the N-terminal region of the membrane-bound carrier reactive to the N-terminal antibodies; the contrasting weak reactivity of the carrier in the BA conformation suggested that the transition from the CATR conformation to the BA conformation results in a restricted conformation of the peptide chain corresponding to the first 10 amino acid residues or its partial burying in the lipid bilayer. These immunological data were complemented by enzymatic data pertaining to proteolysis of the membrane-bound ADP/ATP carrier by an arginine-specific endoprotease.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping of the nucleotide-binding sites in the ADP/ATP carrier of beef heart mitochondria by photolabeling with 2-azido[alpha-32P]adenosine diphosphate.

2-Azido[alpha-32P]adenosine diphosphate (2-azido[alpha-32P]ADP) has been used to photolabel the ADP/ATP carrier in beef heart mitochondria. In reversible binding assays carried out in the dark, this photoprobe was found to inhibit ADP/ATP transport in beef heart mitochondria and to bind to two types of specific sites of the ADP/ATP carrier characterized by high-affinity binding (Kd = 20 microM) and low-affinity binding (Kd = 400 microM). In contrast, it was unable to bind to specific carrier sites in inverted submitochondrial particles. Upon photoirradiation of beef heart mitochondria in the presence of 2-azido[alpha-32P]ADP, the ADP/ATP carrier was covalently labeled. After purification, the photolabeled carrier protein was cleaved chemically by acidolysis or cyanogen bromide and enzymatically with the Staphylococcus aureus V8 protease. In the ADP/ATP carrier protein, which is 297 amino acid residues in length, two discrete regions extending from Phe-153 to Met-200 and from Tyr-250 to Met-281 were labeled by 2-azido[alpha-32P]ADP. The peptide fragments corresponding to these regions were sequenced, and the labeled amino acids were identified. As 2-azido-ADP is not transported into mitochondria and competes against transport of externally added ADP, it is concluded that the two regions of the carrier which are photolabeled are facing the cytosol. Whether the two photolabeled regions are located in a single peptide chain of the carrier or in different peptide chains of an oligomeric structure is discussed.

Photoaffinity labeling of mitochondrial adenosinetriphosphatase by 2-azidoadenosine 5'-[alpha-32P]diphosphate.

2-Azidoadenosine 5'-diphosphate (2-azido-ADP) labeled with 32P in the alpha-position was prepared and used to photolabel the nucleotide binding sites of beef heart mitochondrial F1-ATPase. The native F1 prepared by the procedure of Knowles and Penefsky [Knowles, A. F., & Penefsky, H. S. (1972) J. Biol. Chem. 247, 6617-6623] contained an average of 2.9 mol of tightly bound ADP plus ATP per mole of enzyme. Short-term incubation of F1 with micromolar concentrations of [alpha-32P]-2-azido-ADP in the dark in a Mg2+-supplemented medium resulted in the rapid supplementary binding of 3 mol of label/mol of F1, consistent with the presence of six nucleotide binding sites per F1. The Kd relative to the reversible binding of [alpha-32P]-2-azido-ADP to mitochondrial F1 in the dark was 5 microM in the presence of MgCl2 and 30 microM in the presence of ethylenediaminetetraacetic acid. A linear relationship between the percentage of inactivation of F1 and the extent of covalent photolabeling by [alpha-32P]-2-azido-ADP was observed for percentages of inactivation up to 90%, extrapolating to 2 mol of covalently bound [alpha-32P]-2-azido-ADP/mol of F1. Under these conditions, only the beta subunit was photolabeled. Covalent binding of one photolabel per beta subunit was ascertained by electrophoretic separation of labeled and unlabeled beta subunits based on charge differences and by mapping studies showing one major radioactive peptide segment per photolabeled beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[alpha-32P]ADP.

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[alpha-32P]ADP in the dark with a Kd value of congruent to 8 microM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[alpha-32P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ATP transport. Upon photoirradiation of inside-out submitochondrial particles with 2-azido-[alpha-32P]ADP, both the ADP/ATP carrier and the beta subunit of the membrane-bound F1-ATPase are covalently labeled. The binding specificity of 2-azido-[alpha-32P]ADP for the beta subunit of F1-ATPase is ascertained by prevention of photolabeling of isolated F1 by preincubation with an excess of ADP.