Tetrameric structure of the nonactivated glucocorticoid receptor in cell extracts and intact cells

Mouse lymphoma cells contain a nonactivated glucocorticoid receptor of Mr approximately 330,000 which is heteromeric in nature and is unable to bind to DNA. Following affinity labeling of the steroid-binding subunit and subsequent cross-linking with dimethyl suberimidate at various times either in cell extracts or in intact cells, a series of labeled bands was detected in SDS gels. From the molecular masses of completely and partially cross-linked complexes we conclude that the large nonactivated receptor is a tetramer composed of two 90 kDa subunits, one 50 kDa polypeptide and one steroid-binding subunit.     

F0F1-ATPase of plant mitochondria: isolation and polypeptide composition

A simple and high yield purification procedure for the isolation of F0F1-ATPase from spinach leaf mitochondria has been developed. This is the first report concerning purification and composition of the plant mitochondrial F0F1-ATPase. The enzyme is selectively extracted from inner membrane vesicles with the zwitterionic detergent, 3-[(3-cholamidopropyl) dimethyl ammonio]-1- propane sulfonate (CHAPS). The purified enzyme exhibits a high oligomycin-sensitive ATPase activity (3,6 mumol.min-1.mg-1). SDS-PAGE of the purified F0F1-ATPase complex reveals protein bands of molecular masses of 54 kDa (F1 alpha,beta), 33 kDa (F1 gamma), 28 kDa, 23 kDa, 21 kDa (F1 delta), 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa (F1 epsilon) and 8.5 kDa. All polypeptides migrate as one complex in a polyacrylamide gradient gel under non-denaturing conditions in the presence of 0.1% Triton X-100. Five polypeptides could be identified as subunits of F1. Polypeptides of molecular masses 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa constitute the F0 part of the complex. Our results show that polypeptide composition of the plant mitochondrial F0 differs from other eukaryotic F0 of yeast, mammals and chloroplasts.     

Isolation and purification of poly(ADP-ribose) glycohydrolase from pig thymus

Poly(ADP-ribose) glycohydrolase has been purified about 12 300-fold from pig thymus with a recovery of 8.5%. The specific activity of the purified enzyme is 13.8 mumol min -1 mg protein -1. The molecular weight was estimated to be 59 000 by gel filtration through Sephadex G-100 in a non-denaturing solvent. Analysis of the final preparation by sodium dodecyl sulphate gel electrophoresis reveals two protein bands of molecular weight, 61 500 and 67 500. The Km value for poly(ADP-ribose) is estimated to be 1.8 microM monomer units. The enzyme preparation is free from phosphodiesterase, NADase and ADP-ribosyltransferase activities. The purified enzyme is inhibited by cyclic AMP, ADP-ribose, naphthylamine, histones H1, H2A, H2B, H3, polylysine, polyarginine, polyornithine and protamine. The inhibition by histone is relieved by an equal mass of DNA. Single-stranded DNA, poly(A), poly(I) and polyvinyl sulphate were inhibitory, but double-stranded DNA was not inhibitory.     

Chlorophyll catabolism and gene expression in the peel of ripening banana fruits

Vacuolar malate transport in Catharanthus roseus is probably mediated by a 37 kDa intrinsic tonoplast protein identified with a photolyzable malate analog. Antibodies raised against the protein inhibit malate uptake in isolated vacuoles. We report here the native molecular mass and the oligomeric state of the putative malate transporter which were determined from two-dimensional native electrophoresis. In its first dimension, the electrophoresis used a charge shift method developed for isolating native membrane protein complexes from purified tonoplast vesicles. In combination with a second dimension of sodium dodecylsulfate electrophoresis, it enables the determination of the oligomeric state and subunit composition of non-dissociated complexes. In such analyses, most of the tonoplast proteins of Catharanthus roseus appear to have a complex structure. In native gels (first dimension), both the photoprobe and the antibodies recognized a 160 kDa protein. The photolabelling characteristics correlate well with the main features of malate transport activity. The 160 kDa protein, when analyzed in the second dimension, contained the 37 kDa polypeptide as a subunit. In addition, cross-linking with dimethyl suberimidate (DMS) in the intact tonoplast vesicles resulted in the disappearance of the 37 kDa monomer protein band with concomitant appearance of additional bands of molecular masses higher than the monomer, i.e. 73 and 160 kDa. These results, taken together, suggest that the putative malate transporter exists in the tonoplast as a tetramer.     

One interferon gamma receptor binds one interferon gamma dimer

We investigated the stoichiometry of the interferon gamma and interferon gamma receptor interaction, using recombinant interferon gamma and recombinant soluble interferon gamma receptor, applying chemical cross-linking and chromatographic techniques, and analyzing the resulting products in denaturing polyacrylamide gels. Interferon gamma cross-linked to itself produced a major band of an apparent molecular mass of 34 kDa, which suggests that it exists as a dimer in physiological buffer and which agrees with published data. Soluble interferon gamma receptor cross-linked to itself produced mainly a 28-kDa band, suggesting that the interferon gamma receptor exists as a monomer. Interferon gamma cross-linked to the soluble interferon gamma receptor resulted in the formation of two main products of apparent molecular masses of 60 and 44 kDa. The predominant 60-kDa band resulted from the cross-linking of one interferon gamma dimer (34 kDa) to one interferon gamma receptor molecule (27 kDa). The 44-kDa band was formed by the cross-linking of one interferon gamma molecule to one interferon gamma receptor. Kinetic studies showed that the cross-linking of interferon gamma dimer to the soluble receptor proceeds through the intermediate formed by cross-linking one molecule of the interferon gamma dimer to the receptor. Reducing and dissociating agents inhibited complex formation. When chromatographed on Sephadex G-100, interferon gamma was eluted as a protein of 34-kDa molecular mass, the soluble interferon gamma receptor as a protein of 40 kDa, and their mixture was eluted in one peak corresponding to an apparent molecular mass of 73 kDa. Sodium dodecyl sulfate-polyacrylamide gel analysis of the eluted mixture showed the presence of both interferon gamma and interferon gamma receptor at a ratio of 2:1. The found results suggest that the interferon gamma receptor binds interferon gamma as a dimer.     

Receptors for Sperm-activating Peptides, SAP-I and SAP-IIB, on Spermatozoa of Sea Urchins, Hemicentrotus pulcherrimus and Glyptocidaris crenularis

Sperm-activating peptide analogues, GYGG-SAP-I (Gly-Tyr-Gly-Gly-Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and GYGG-SAP-IIB (Gly-Tyr-Gly-Gly-Lys-Leu-Cys-Pro-Gly-Gly-Asn-Cys-Val) which exibit almost the same respiration-stimulating activity as the respective original peptide were chemically synthesized, radiolabeled with Na¹²⁵1, and used for experiments of binding and cross-linking to Hemicentrotus pulcherrimus or Glyptocidaris crenularis spermatozoa. In competitive binding, SAP-I caused 50% decrease in ¹²⁵l-GYGG-SAP-l binding to intact spermatozoa, sperm heads and sperm tails of H. pulcherrimus at the concentrations of 282 nM, 3 nM and 141 nM, respectively. The incubations of H. pulcherrimus sperm tails with ¹²⁵l-GYGG-SAP-I and the chemical cross-linking reagent, disuccinimidyl suberate, resulted in the appearance of two radiolabeled bands with apparent molecular masses of 71 kDa and 63 kDa (determined by sodium dodecyl sulfate-gel electrophoresis under reducing conditions). ¹²⁵l-GYGG-SAP-IIB bound almost equally to sperm heads and sperm tails of G. crenularis , and was cross-linked to 172 kDa, 62 kDa and 58 kDa proteins in sperm heads, and 157 kDa and 62 kDa proteins in sperm tails with disuccinimidyl suberate.     

Purification and properties of dinitrogenase reductase ADP-ribosyltransferase from the photosynthetic bacterium Rhodospirillum rubrum

The enzyme that catalyzes the ADP-ribosylation and concomitant inactivation of dinitrogenase reductase in Rhodospirillum rubrum has been purified greater than 19,000-fold to near homogeneity. We propose dinitrogenase reductase ADP-ribosyltransferase (DRAT) as the working name for the enzyme. DRAT activity is stabilized by NaCl and ADP. The enzyme is a monomer with a molecular mass of 30 kDa and is a different polypeptide than dinitrogenase reductase activating glycohydrolase. NAD (Km = 2 mM), etheno-NAD, nicotinamide hypoxanthine dinucleotide, and nicotinamide guanine dinucleotide will serve as donor molecules in DRAT-catalyzed ADP-ribosylation reaction, and dinitrogenase reductases from R. rubrum, Azotobacter vinelandii, Klebsiella pneumoniae, and Clostridium pasteurianium will serve as acceptors. No other proteins or small molecules, including water, have been found to be effective as acceptors. Nicotinamide is released stoichiometrically with formation of the ADP-ribosylated product. DRAT is inhibited by NaCl and has maximal activity at a pH of 7.0.     

Glycosylation of procathepsin L does not account for species molecular-mass differences and is not required for proteolytic activity.

Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function.     

Covalent cross-linking of the bovine somatotropin dimer. Effects on growth-promoting, receptor-binding and immunological activities and preliminary characterization of the self-association.

Bovine somatotropin, at pH 8.5 in 0.02 M-Bicine [NN-bis-(2-hydroxyethyl)glycine]/0.09M-NaCl, showed by frontal analysis the characteristics of a rapid monomer-dimer equilibrium whose dissociation constant was estimated to be 6.6 X 10(-6)M. Reaction of the hormone with dimethyl suberimidate lead to covalent cross-linking of the dimeric species. Under the conditions chosen (0.4 mg of bifunctional imidate and 1 mg of protein/ml at room temperature for 1 h) the cross-linked dimers accounted for 26% of the total protein, and these were isolated by molecular sieving in 0.29M-NH3/0.12M-NaCl. Covalent stabilization greatly diminished the growth-promoting activity and the ability to interact with somatogenic sites in both rat liver in vivo and rabbit liver microsomal fractions. Evidence indicating a non-critical role for amino groups involved in the covalent cross-linking was provided by a nearly equivalent derivative obtained after reaction with 3,3'-dithiobispropionimidate, which had substantial hormonal activity upon cleavage of the disulphide links. Conversely, immunological reactivity as demonstrated by radioimmunoassay was not affected by cross-linking. Details of the least-squares procedure employed to evaluate the self-association equilibrium constant has been deposited as Supplement SUP 50115 (7 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem.J. (1981) 193,5.     

Purification of aspartate transcarbamoylase from Pseudomonas syringae

Abstract The aspartate transcarbamoylase (ATCase) from Pseudomonas syringae has been purified. The purified enzyme was shown by SDS-PAGE to give two bands. Unambiguous results from N-terminal sequencing suggested that each band represented a homogeneous polypeptide. The M _r (relative molecular mass) of the polypeptides was estimated to be 47 kDa and 34 kDa. The M _r of the holoenzyme determined by gel filtration and electrophoretic migration in polyacrylamide gradient gels under non-denaturing conditions was estimated at approximately 490 kDa. These findings suggest a subunit structure different from any previously described for a bacterial ATCase.     

Covalent complexes formed between plasma gelsolin and actin with a zero-length cross-linking compound

Actin and plasma gelsolin were covalently cross-linked with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Two major intermolecularly linked products were identified on polyacrylamide gels. By use of 14C-labeled actin and 125I-labeled gelsolin, these were shown to be the 1:1 and 2:1 complexes of actin with gelsolin, respectively. The higher molecular weight complex predominated under all conditions tested including the presence and absence of Ca2+. In titration experiments in which actin at different concentrations was reacted with a fixed concentration of gelsolin, end points were obtained for the formation of both cross-linked species at about two actins per gelsolin, implying that a 2:1 noncovalent complex is cross-linked. In 0.1 mM Ca2+, the extent of cross-linking was independent of protein concentration down to 50 nM gelsolin. At low Ca2+ concentrations (less than 10(-8)M), the extent of cross-linking was very much reduced at micromolar gelsolin and fell to zero at about 100 nM gelsolin. The binding of actin to gelsolin to give a cross-linkable complex is therefore very strong at 0.1 mM Ca2+ but much weaker at low Ca2+ concentrations.     

Catalytic properties of a calmodulin-regulated transglutaminase from human platelet and chicken gizzard

The kinetic parameters and some enzymatic characteristics of human platelet and chicken gizzard transglutaminases were determined. Activity of the transglutaminases was regulated by calmodulin. These enzymes co-isolated with alpha-actinin and were dissociated from alpha-actinin by gel filtration and absorption onto a calmodulin affinity column. Silver-stained polyacrylamide gels showed that the protein peak eluted by EGTA from this column contained polypeptides of Mr approximately 58,000 and 63,000. The transglutaminases required Ca2+ for incorporation of monodansylcadaverine into casein and actin substrates. Activity was enhanced 3-fold by calmodulin with a biphasic effect, showing stimulation at 10-200 nM and inhibition at concentrations higher than 300 nM. In the presence of 200 nM calmodulin, half-maximal transglutaminase stimulation was obtained with 2.5 microM free [Ca2+]. Chlorpromazine inhibited calmodulin enhancement of the transglutaminases. Activity of the transglutaminases was independent of proteolytic activation, since inhibitors for Ca2+-dependent proteases failed to inhibit filamin cross-linking. For comparison, factor XIIa, a plasma and platelet transglutaminase, required both Ca2+ and thrombin for activation and was insensitive to calmodulin. The cross-linking pattern of fibrin, fibrin monomers, and fibrinogen by the calmodulin-regulated transglutaminases showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, disappearance of fibrinogen alpha-chains with no decrease of beta- and gamma-chains or formation of gamma-gamma dimers. By autoradiography, cross-linked products of 125I-fibrinogen revealed heavily labeled high molecular weight polymers and polypeptides of Mr 98,000, 116,000, and 148,000; the latter appeared to be a transient species. However, when fibrin, fibrin monomers, and fibrinogen were used as factor XIIIa substrates, gamma-gamma dimers and alpha-polymers were formed. Formation of gamma-gamma dimers was slower with fibrinogen than with fibrin. Iodoacetamide blocked activity of factor XIIIa but not of the calmodulin-regulated transglutaminases.     

Gene 5 protein of bacteriophage fd: A dimer which interacts Co-operatively with DNA

Isolated gene 5 protein from bacteriophage fd-infected Escherichia coli has been shown by sedimentation equilibrium to exist primarily as a dimer under non-denaturing conditions. The dimer was stable under conditions of high ionic strength, extremes in pH, dilution to 0.075 mg/ml, and increased temperature. Gene 5 protein did not undergo the indefinite self-association observed with gene 32 protein.Three lines of evidence for co-operative binding of gene 5 protein to DNA were developed. First, the interaction between gene 5 protein and phage T4 DNA was examined using a nitrocellulose filter assay. Scatchard plots of the binding data indicated that the interaction was co-operative. Similar results were obtained with gene 32 protein. Second, the co-operative binding of both proteins to DNA was shown by the sensitivity of the protein-DNA interaction to increasing ionic strength at various ratios of protein to DNA. Finally, by using the cross-linking agent, dimethyl suberixmidate, oligomeric structures containing at least seven monomers were found when the DNA was less than saturated.The possibility that gene 5 protein dimers undergo indefinite self-association in the presence of oligonucleotides was examined by sedimentation equilibrium. With oligo[d(pT)₄], the protein dimer was complexed with this oligonucleotide but no self-association was observed. With oligo[d(pT)₈], gene 5 protein formed tetramers, but no significant indefinite association was noted. These results do not suggest a DNA-induced conformational change, which results in indefinite association. A model for the co-operative binding of gene 5 protein to DNA is presented.     

Cleavage Specificity of Enterococcus faecalis EnpA (EF1473), a Peptidoglycan Endopeptidase Related to the LytM/Lysostaphin Family of Metallopeptidases

Enterococcus faecalis EnpA (EF1473) is a 1721-residue predicted protein encoded by prophage 03 that displays similarity to the staphylolytic glycyl-glycyl endopeptidases lysostaphin and LytM. We purified a catalytically active fragment of the protein, EnpA_C, comprising residues 1374-1505 and showed that the recombinant polypeptide efficiently cleaved cross-linked muropeptides generated by muramidases, but was poorly active in intact sacculi. Analysis of the products of digestion of purified dimers by mass spectrometry indicated that EnpA_C cleaves the d-Ala-l-Ala bond formed by the d,d-transpeptidase activity of penicillin-binding proteins in the last cross-linking step of peptidoglycan synthesis. Synthetic d was identified as the minimum substrate of EnpA_C indicating that interaction of the enzyme with the donor peptide stem of cross-linked dimers is sufficient for its activity. Peptidoglycan was purified from various bacterial species and digested with mutanolysin and EnpA_C to assess enzyme specificity. EnpA_C did not cleave direct cross-links, but tolerated extensive variation in cross-bridges with respect to both their length (one to five residues) and their amino acid sequence. Recognition of the donor stem of cross-linked dimers could account for the substrate specificity of EnpA_C, which is significantly broader in comparison to endopeptidases belonging to the lysostaphin family.     

Comparative analysis of proteins from the fibrous sheath and outer dense fibers of rat spermatozoa

The protein composition of the fibrous sheath (FS) and the outer dense fibers (ODF), two cytoskeletal components of the tail of spermatozoa, was compared by using polyacrylamide gel electrophoresis and immunochemistry applied to Western blots and to spermatozoa. Isolated FS and ODF, the purity of which were verified by electron microscopy (EM), were denatured and either run on sodium dodecyl sulfate-polyacrylamide gels or used to raise antibodies. The gels revealed at least 18 and 14 polypeptide bands for the FS and ODF, respectively. The major bands of the FS had molecular masses of 75, 27.5, and 14.4 kDa, whereas the major bands of the ODF-connecting piece had molecular masses of 32-26, 20, 14.4, 84, and 80 kDa. Several prominent FS and ODF bands were found to comigrate on gels, and the 14.4 kDa polypeptides had similar electrophoretic properties. Anti-FS serum reacted with the majority of Western blot-transferred FS polypeptides, but also cross-reacted strongly with a major 14.4 kDa ODF polypeptide and with less affinity to other major ODF polypeptides. Anti-ODF serum reacted with the majority of ODF polypeptides, but also cross-reacted strongly with a major 14.4 kDa FS polypeptide, and with less affinity to several other FS polypeptides including the 75 kDa band. Antibodies affinity-purified from the 14.4 kDa FS polypeptide only cross-reacted with the 14.4 kDa ODF polypeptide, whereas antibodies purified from the 14.4 kDa ODF polypeptide cross-reacted with 14.4, 27.5, 57, and 63 kDa FS polypeptides. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. This study demonstrates that the FS and ODF, both composed of many polypeptides, several having similar molecular weights, are related cytoskeletal structures as they have epitopes in common, and both contain 14.4 kDa polypeptides with common antigenic and electrophoretic properties.     

Target size analysis of prostaglandin endoperoxide synthase. Radiation inactivation of both cyclooxygenase and peroxidase correlated with the monomer of 72 kDa.

To determine the size of the functional catalytic unit of prostaglandin endoperoxide (prostaglandin H) synthase, radiation inactivation experiments were performed. Both microsomes from ovine seminal vesicles and purified enzyme were irradiated with 10 MeV electrons. The enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, showed mono-exponential inactivation curves dependent on radiation dose, indicating molecular masses of approximately 72 kDa. The enzyme in microsomes, in its native environment, as well as in its purified state after solubilisation with nonionic detergent showed identical molecular masses. The results clearly demonstrate that the monomer of the enzyme with an apparent molecular mass of 72 kDa (SDS/PAGE) is the functional unit for catalysis of both activities. Hence the two active sites of cyclooxygenase and peroxidase reside on the same polypeptide chain.     

Proteomic and biochemical evidence links the callose synthase in Nicotiana alata pollen tubes to the product of the NaGSL1 gene

The NaGSL1 gene has been proposed to encode the callose synthase (CalS) enzyme from Nicotiana alata pollen tubes based on its similarity to fungal 1,3-beta-glucan synthases and its high expression in pollen and pollen tubes. We have used a biochemical approach to link the NaGSL1 protein with CalS enzymic activity. The CalS enzyme from N. alata pollen tubes was enriched over 100-fold using membrane fractionation and product entrapment. A 220 kDa polypeptide, the correct molecular weight to be NaGSL1, was specifically detected by anti-GSL antibodies, was specifically enriched with CalS activity, and was the most abundant polypeptide in the CalS-enriched fraction. This polypeptide was positively identified as NaGSL1 using both MALDI-TOF MS and LC-ESI-MS/MS analysis of tryptic peptides. Other low-abundance polypeptides in the CalS-enriched fractions were identified by MALDI-TOF MS as deriving from a 103 kDa plasma membrane H+-ATPase and a 60 kDa beta-subunit of mitochondrial ATPase, both of which were deduced to be contaminants in the product-entrapped material. These analyses thus suggest that NaGSL1 is required for CalS activity, although other smaller (<30 kDa) or low-abundance proteins could also be involved.     

The formation of a disulfide cross-link between the two subunits demonstrates the dimeric structure of the mitochondrial oxoglutarate carrier

Isolated oxoglutarate carrier (OGC) can be cross-linked to dimers by disulfide-forming reagents such as Cu²⁺-phenanthroline and diamide. Acetone and other solvents increase the extent of Cu²⁺-phenanthroline-induced cross-linking of OGC. Cross-linked OGC re-incorporated in photeoliposomes fully retains the oxoglutarate transport activity. The amount of cross-linked OGC calculated by densitometry of scanned gels depends on the method of staining, since cross-linked OGC exhibits a higher sensitivity to Coomassie brilliant blue as compared to silver nitrate. Under optimal conditions the formation of cross-linked OGC dimer (stained with Coomassie brilliant blue) amounts to 75% of the total protein. Approximately the same cross-linking efficiency was evaluated from Western blots. Cross-linking of OGC is prevented by SH reagents and reversed by SH-reducing reagents, which shows that it is mediated by disulfide bridge(s). The formation of SS bridge(s) requires the native state of the protein, since it is suppressed by SDS and by heating. Furthermore, the extent of cross-linking is independent of OGC concentration indicating that disulfide bridge(s) must be formed between the two subunits of native dimers. The number and localization of disulfide bridge(s) in the cross-linked OGC were examined by peptide fragmentation and subsequent cleavage of disulfide bond(s) by β-mercaptoethanol. Our experimental results show that cross-linking of OGC is accomplished by a single disulfide bond between the cysteines 184 of the two subunits and suggest that these residues in the putative transmembrane helix four are fairly close to the twofold axis of the native dimer structure.     

Transmembrane organization of mitochondrial NADH dehydrogenase as revealed by radiochemical labelling and cross-linking.

The organization of bovine heart NADH dehydrogenase in the mitochondrial inner membrane was investigated by chemical cross-linking and radiolabelling with [125I]iododiazobenzenesulphonate (IDABS). Mitochondria or submitochondrial particles were cross-linked with disulphosuccinimidyl tartrate and dimethyl suberimidate, and dimeric products containing subunits of the NADH dehydrogenase were analysed by Western blotting with subunit-specific antisera. Cross-linking of mitochondria gave rise to (49 + 30) kDa and (49 + 19) kDa dimers and an additional dimer containing the 30 kDa subunit. Cross-linking of submitochondrial particles gave rise to (75 + 51) kDa, (75 + 30) kDa and (49 + 13) kDa dimers and a further dimer containing the 30 kDa subunit. We conclude that the 49 kDa and 30 kDa subunits are transmembranous, the 19 kDa subunit is exposed on the cytoplasmic face of the membrane, whereas the 75, 51 and 13 kDa subunits are exposed on the matrix face of the membrane. Reaction of the isolated enzyme with IDABS results in labelling of 75, 49, 42, 33, 30, 13 and 10 kDa subunits. From experiments in which mitochondria or submitochondrial particles were first labelled and NADH dehydrogenase then isolated by immunoprecipitation, it was found that labelling of the 49 kDa subunit occurs predominantly from the cytoplasmic side of the membrane. On the other hand, labelling of the 75, 13 and 10 kDa subunits occurs predominantly from the matrix side of the membrane, whereas the 30 and 33 kDa subunits are heavily labelled from either side. These findings are consistent with those obtained from cross-linking.