Cross-linking of minor subunits in the adenosine triphosphatase of rat liver mitochondria.

The subunits of the purified ATPase of rat liver mitochondria were cross-linked with the cleavable disulfide crosslinking reagent dithiobis(succinimidyl propionate). Besides cross-linking of the major (alpha,beta) subunits interactions between beta and gamma and between gamma and epsilon subunits were observed.     

Cross-linking of the proteins in the outer membrane of Escherichia coli.

1. The organization of the proteins in the outer membrane of Escherichia coli was examined by the use of cross-linking agents and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of protein A-peptidoglycan complexes with dithiobis(succinimidyl propionate) or glutaraldehyde produced the dimer, trimer, and higher oligomers of protein A. Both forms of this protein, proteins A1 and A2, produced similar cross-linking products. No cross-linking of protein A to the peptidoglycan was detected. 2. The proteins of the isolated outer membrane varied in their ease of cross-linking. The heat-modifiable protein, protein B, was readily cross-linked to give high molecular weight oligomers, while protein A formed mainly the dimer and trimer under the same conditions. The pronase resistant fragment, protein Bp, derived from protein B was not readily cross-linked. No linkage of protein A to protein B was detected. 3. Cross-linking of cell wall preparations, consisting of the outer membrane and peptidoglycan, showed that protein B and the free form of the lipoprotein, protein F, could be linked to the peptidoglycan. A dimer of protein F, and protein F linked to protein B, were detected. 4. These results suggest that specific protein-protein interactions occur in the outer membrane.     

The Fo subunits of the Escherichia coli F1Fo-ATP synthase are sufficient to form a functional proton pore

The assembly of the Fo sector of the Escherichia coli ATP synthase has been studied using both structural and functional criteria for assembly. Cross-linking E. coli minicell membranes containing only the Fo subunits a, b, and c with dithiobis(succinimidyl propionate) (DSP) produces b2 and c2 dimers that are generated by cross-linking membranes containing the assembled holoenzyme. Five plasmids carrying the genes specifying the Fo polypeptides in a bacterial strain lacking all of the unc (ATP synthase) genes show a good correlation between Fo function and the amount of the membrane-bound Fo polypeptides. In this report we revise a conclusion reached previously (Klionsky, D.J., Brusilow, W.S.A., and Simoni, R.D. (1983) J. Biol. Chem. 258, 10136-10143) and present evidence that the Fo subunits alone are sufficient to assemble a functional proton pore.     

Murine mammary tumor virus structural protein interactions: formation of oligomeric complexes with cleavable cross-linking agents

Murine mammary tumor virus protein interactions in the intact virion structure were studied with the use of the cleavable cross-linking reagents dithiobis(succinimidyl propionate) and methyl 4-mercaptobutyrimidate hydrochloride. Cross-linked oligomeric complexes of murine mammary tumor virus proteins were analyzed by two-dimensional gel electrophoresis. Among the complexes most consistently formed were a heterodimer of the two glycoproteins gp36 and gp52, the homodimer of gp36, and the homotrimer of gp52. A very prominent oligomer formed at higher concentrations of dithiobis(succinimidyl propionate) was a complex of about 230,000 molecular weight, made up of three molecules each of gp36 and gp52. A number of lines of evidence, including electron microscopic analysis, suggest that the 230,000-molecular-weight complex actually represents the murine mammary tumor virus spike structure. Of the murine mammary tumor virus core proteins, p14 forms homooligomers most readily. Upon cross-linking with methyl 4-mercaptobutyrimidate hydrochloride a small amount of what seems to be a heterodimer made up of the N-terminal gag protein p10 and the hydrophobic membrane glycoprotein gp36 can be observed.     

The subunit structure of bovine heart mitochondrial transhydrogenase

Reaction of purified bovine heart transhydrogenase with bifunctional cross-linking reagents dimethyl adipimidate, dimethyl pimelimidate, dimethyl suberimidate, and dithiobis(succinimidyl propionate) results in the appearance of a dimer band on sodium dodecyl sulfate polyacrylamide gels with no higher oligomers formed. Treatment of the enzyme with 6 M urea led to inactivation and prevented cross-linking by dimethyl suberimidate. Transhydrogenase reconstituted into phosphatidylcholine proteoliposomes also yielded a dimer band on cross-linking. These data indicate that soluble and functionally reconstituted transhydrogenase possesses a dimeric structure.     

Identification of the calmodulin-binding components in bovine lens plasma membranes

Lens membranes, purified from calf lenses, have been labeled by covalent cross-linking to membrane-bound 125I-calmodulin with dithiobis(succinimidyl propionate). Electrophoretic analysis in sodium dodecyl sulfate demonstrated two major 125I-containing products of Mr = 49 000 and 36 000. That the formation of these two components was specifically inhibited by unlabeled calmodulin, or calmodulin antagonists, would indicate that the formation of these components was calmodulin-specific. The size of these two 125I-labeled components was unchanged over a range of 125I-calmodulin or dithiobis(succinimidyl propionate) concentrations indicating that they represent 1:1 complexes between 125I-calmodulin (Mr = 17 000) and Mr-32 000 and Mr-19 000 lens membrane components respectively. Although formation of both cross-linked components exhibited an absolute dependence on Mg2+, the autoradiographic intensity of these components was enhanced when Ca2+ was included with Mg2+ during the cross-linking reaction. Labeling was maximal in 10 mM MgCl2 and approximately 1 microM Ca2+. Treatment of lens membranes with chymotrypsin resulted in the cleavage of MP26 (the major lens membrane protein), with the appearance of a major proteolytic fragment of Mr = 22 000. This proteolysis was not associated with any significant change in either the size or amount of the 125I-calmodulin-labeled membrane components. These results suggest that calmodulin interacts with two membrane proteins, but not significantly with MP26, in the intact lens cell membrane. Our results indicate the need to maintain caution in interpreting direct calcium plus calmodulin effects on MP26 and lens cell junctions.     

Neighboring proteins in rat liver 60 S ribosomal subunits disulfide-linked by hydrogen peroxide oxidation or cross-linked with dithiobis(succinimidyl propionate)

Neighboring proteins in rat liver 60 S ribosomal subunits were investigated by two kinds of cross-linking techniques: treatment of 60 S subunits with 1) hydrogen peroxide, which promotes the formation of protein-protein disulfide linkages and 2) a disulfide-bridged bifunctional reagent dithiobis(succinimidyl propionate). The cross-linked protein complexes formed were separated by two-dimensional polyacrylamide gel electrophoresis in a basic-sodium dodecyl sulfate gel system under nonreducing conditions. Each complex in the gel was labeled with 125I and extracted under reducing conditions. The protein components of the complex were analyzed by two kinds of two-dimensional polyacrylamide gel electrophoresis, followed by autoradiography. Closely neighboring pairs disulfide-linked by hydrogen peroxide were identified as L4-L6, L4-L29, L6-L29, L18a-L29, and L29-L32; more distant pairs cross-linked with dithiobis(succinimidyl propionate) were identified as L3-L5, L3-L24, L3-L37a, L4-L14, L4-L18a, L5-L10, L5-L11, L7/L7a-L27, L7/L7a-L36, L13-L35, and L13a-L14.     

Pharmacology and molecular identification of secretin receptors in rat gastric glands

The structure of the secretin receptor in purified plasma membranes isolated from the antral and fundic parts of the rat gastric mucosa was probed, using the cross linking reagent dithiobis succinimidyl propionate (DSP) and HPLC-purified [125I] secretin. [125I] secretin binding sites were preferentially located in rat antrum and displayed the pharmacological properties expected for specific secretin receptors: secretin greater than helodermin greater than rhGRF greater than rPHI. SDS gel electrophoresis of the solubilized receptor allowed identification of two radiolabeled peptides of 62 and 33 KDa connected by disulfide bonds. According to the sensitivity of the 62 KDa component to low doses of secretin and to GTP, it constitutes the membrane domain involved in the physiological regulation of adenylate cyclase by secretin in rat gastric glands.     

The effects of deoxycholate and trypsin on the cross-linking of rabbit skeletal muscle sarcoplasmic reticulum proteins.

Dithiobis (succinimidyl propionate) has been used to cross-link sarcoplasmic reticulum microsome proteins. Although the 100,000 dalton calcium stimulated ATPase and the 60,000 dalton calcium-binding protein calsequestrin were readily cross-linked to form homopolymers, no heteropolymer formation between these two proteins were detected. The 90,000 dalton protein A1 which is always observed in our preparations appeared to preferrentially form dimers on cross-linking. When calsequestrin was solubilized using 0.1 mg deoxycholate/mg protein, this protein was not cross-linked even at dithiobis(succinimidyl propionate) concentrations ten times those used to cross-link this protein in the intact membrane. In a similar manner the deoxycholate-solubilized ATPase (0.5 mg deoxycholate/mg protein) was not cross-linked by dithiobis (succinimidyl propionate). These results suggest that the state of aggregation of the sarcoplasmic reticulum proteins may be modified when solubilized in detergents such as deoxycholate. When the 100,000 dalton ATPase polypeptide was cleaved with trypsin to two fragments with molecular weights of approximately 55,000, these could be readily cross-linked. The fragments were capable of forming polymers with either other 55,000 dalton fragments or with the 100,000 dalton ATPase. The 29,000 and 22,000 dalton fragments, produced by further tryptic cleavage of the 55,000 dalton fragments, were not cross-linked at dithiobis (succinimidyl propionate) concentrations which readily cross-linked the 55,000 dalton fragments. Thus tryptic cleavage of the ATPase to fragments smaller than 55,000 dalton altered associations made by the ATPase in the membrane.     

Interactions among the three adenovirus core proteins.

Interactions among the three adenovirus core polypeptides V, VII, and mu were examined, using the reversible chemical cross-linker dithiobis(succinimidyl propionate) and two-dimensional polyacrylamide gel electrophoresis. Cross-linked species obtained from gradient-purified adenovirus type 2 cores were well represented among the cross-linked products of pentonless virions and crude core preparations. The more efficiently formed cross-linked core species were also identified with the arginine-specific cross-linker, p-azidophenyl glyoxal. In addition to dimers of polypeptides V and VII, efficient cross-linking of V to VII, V to mu, and VII to V to mu was detected in adenovirus cores. Notably absent were cross-linked species corresponding to higher multimers of polypeptide VII. A major core-capsid interaction appeared to be via the association of polypeptide V with a dimer of polypeptide VI.     

Highly selective extraction of spiralin from the Spiroplasma citri cell membrane with alkyl-N-sulfobetaines

The extraction of proteins from the membrane of the mollicute (mycoplasma) Spiroplasma citri by sodium N-dodecyl-N,N-dimethyl-3-amino-1-propane sulfonate (SB12) and sodium N-tetradecyl-N,N-dimethyl-3-amino-1-propane sulfonate (SB14) was studied with electrophoretic methods. The membranes were prepared by osmotic lysis of the cells and depleted of the bulk of extrinsic proteins. It was possible to extract up to 35 and 45% of membrane proteins with SB12 and SB14, respectively. Maximal yield was obtained in both cases with detergent concentrations greater than or equal to 5 mumoles/mg of membrane protein. Spiralin, the major protein in the S. citri membrane, was highly selectively solubilized without the loss of antigenicity, with a yield of about 90% with SB12 and close to 100% with SB14, for a detergent concentration greater than or equal to 0.2 M. The degree of selectivity in favour of spiralin was higher with SB12 (purity approximately equal to 70%) than with SB14 (purity approximately equal to 50%). Treatment of the S. citri membrane with high concentrations of SB12 is a simple and fast procedure for partial purification of spiralin. This example shows that, in some cases, it should be possible to modulate the selectivity of the extraction of membrane proteins simply by varying the relative concentration of detergent.     

Identification of cross-linked cytochrome P-450 in rat liver microsomes by enzyme-immunoassay

The topography of microsomal proteins was studied by 2-dimensional gelelectrophoresis. The second dimension was run in the presence of 2-mercaptoethanol, thus allowing detection of proteins previously cross-linked by disulfide bonds as off-diagonal spots. With hepatic microsomes from phenobarbital pretreated rats, several off-diagonal spots were seen. The most intense spot, with a molecular weight of 52,000, was derived from a dimer of this protein. It was identified as cytochrome P-450 (P-450) by a double antibody enzyme-immunoassay. The dimer is probably formed by oxidation of sulfhydryl groups of P-450 molecules during the preparation of microsomes. P-450 can also be cross-linked to form 105,000, 167,000, and 240,000 dal oligomers by treating microsomes with dithiobis(succinimidyl propionate) at 0°C. Cross-linking of P-450 to other proteins was also observed with one-dimensional gel-electrophoresis. The results suggest that the cross-linked proteins are close neighbors of P-450 in the membrane.     

Chemical cross-linking studies of the light-harvesting pigment-protein complex B800-850 of Rhodopseudomonas capsulata

The spatial relationship of the three polypeptides contained in the B800-850 light-harvesting complex of Rhodopseudomonas capsulata has been studied with chemical cross-linking of crude membrane preparations of the phototrophic negative mutant strain Y5. Samples were cross-linked with the cleavable reagent dithiobis (succinimidyl propionate) (1.1 nm chain length) and analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Membranes labelled with 14C-amino acids were used to determine the compositional stoichiometry of cross-linked products. It was found that the two polypeptides with an apparent Mr of 8000 and 10 000, respectively, that are associated with the pigments bacteriochlorophyll a and carotenoid form homooligomers as well as heterooligomers. The data support the idea that these polypeptides are closely arranged in clusters probably containing at least four of each species. The third subunit with an Mr of 14 000, which is not associated with pigments, was found to be most susceptible to cross-linking and formed homooligomers but no heterooligomers with the other two subunits, and is thus likely to be loosely attached to these clusters. Comparative studies with the phototrophic positive wild type strain indicated that the results found with the phototrophic negative mutant strain Y5 reflect the organization of the B800-850 complex in the membrane of Rhodopseudomonas capsulata. Studies with the isolated B800-850 complex revealed that the sterical arrangement of the three constituent polypeptides in dodecyl dimethylamine-N-oxide containing solutions must be very similar to that in the membrane.     

Investigation of the spatial relationships between photosystem 2 polypeptides by reversible crosslinking and diagonal electrophoresis

Nearest neighbour relationships within the LHC2-PS2 complex were investigated by using the reversible crosslinking agent dithiobis(succinimidyl propionate) (DSP). This was accomplished by treating PS2-enriched membranes, prepared from chloroplasts of Pisum sativum, with the crosslinker followed by diagonal electrophoresis of the solubilised polypeptides.Analysis of the off-diagonal spot patterns produced by crosslinker cleavage and second dimension electrophoresis was made on the basis of: staining with Coomassie blue or silver, labelling with [³⁵S]-methionine, and sensitivity to 1 M NaCl washing. It was concluded that LHC2 polypeptides crosslinked with several components of the PS2 complex and that the extrinsic polypeptides associated with water oxidation, having approximate molecular weights of 16 and 23 kDa, crosslink to form homodimers. The latter finding suggests that there may be more than one copy of each of these polypeptides per PS2 complex.Abbreviations DMSO dimethylsulphoxide - DSP dithiobis(succinimidylpropionate) - DCPIP dichlorophenolindophenol - SDS sodium dodecylsulphate - PS2 photosystem 2 - LHC2 light harvesting chlorophyll a/b complex associated with photosystem 2 - MES 2[N-morpholino] ethanesulphonic acid     

Isolation of the prohead core of bacteriophage T4 after cross-linking and determination of protein composition

The naked core of bacteriophage T4 was isolated ex vivo after cross-linking with either glutaraldehyde or dithiobis(succinimidyl propionate). The isolated particles appeared to be morphologically identical to the cores found in thin sections, to those demonstrated in in situ lysis preparations, and to core structures assembled in vitro. Treatment with glutaraldehyde provided core particles which were morphologically well preserved, whereas dithiobis(succinimidyl propionate)-induced cross-linking was reversible and allowed analysis of the protein composition of the isolated particles. The identity of the reversibly cross-linked particles with those obtained after irreversible cross-linking was suggested by their morphology and their similar sedimentation behavior. Immunolabeling confirmed the structural presence of the main core protein in both structures. Gel electrophoresis of reversibly cross-linked cores revealed the essential head proteins gp22, gp67, and gp21, the three internal proteins IPI, IPII, and IPIII, and a 17K protein.     

Chemical cross-linking of proteins of Semliki Forest virus: virus particles and plasma membranes from BHK-21 cells treated with colchicine or dibucaine.

Chemical cross-linking of the proteins of Semliki Forest virus has been performed in virus particles and in baby hamster kidney-21 (BHK-21) cells infected with Semliki Forest virus. Most of the studies were done with the reversible cross-linkers dimethyl 3,3'-thiobis(propionimidate) and dithiobis(succinimidyl propionate). The identity of the cross-linked species was determined by two-dimensional electrophoresis. The results with virus particles showed extensive cross-linking of the nucleocapsid proteins and the formation of dimers of the two large envelope glycoproteins (E1 and E2). Similar patterns for the cross-linked virus proteins were observed in plasma membranes isolated from BHK-21 cells infected with Semliki Forest virus. No cross-linking of the third envelope glycoprotein (E3) was observed. Also, there was no evidence for significant cross-linking between host and virus proteins. The addition of colchicine, a drug that disrupts microtubules, to infected BHK-21 cells had no effect on the cross-linking of virus proteins in the plasma membrane. In contrast, dibucaine, a local anesthetic, greatly inhibited the formation of envelope dimers (E1-E2) in plasma membranes, but not in virus particles. The implication of these results for the involvement of the cytoskeletal system in the morphogenesis of Semliki Forest virus is discussed.     

Reversible effects of cross-linking on the regulatory cooperativity of Acinetobacter citrate synthase

Citrate synthase was purified from Acinetobacter calcoaceticus and treated with the cleavable cross-linking reagent dithiobis(succinimidyl propionate). Cross-linking of the enzyme resulted in the abolition of the sigmoidal responses to inhibition by NADH and re-activation by AMP displayed by the native enzyme. Inhibition and re-activation were still observed but without any cooperativity. Cleavage of the disulphide bonds in the cross-links by treatment with dithiothreitol restored the sigmoidal characteristics of both inhibition and re-activation.     

Characterization of Serologically Dominant Outer Membrane Proteins of Neisseria gonorrhoeae

The proteins of the outer membrane of Neisseria gonorrhoeae play an important role in the serotyping system defined by K. H. Johnston et al. (J. Exp. Med. 143:741–758, 1976). This study attempted to delineate the molecular arrangement of the major proteins of the outer membrane of the gonococcus by using three approaches. First, natural protein-protein relationships were demonstrated by symmetrical, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Second, proteins exposed on the surface of outer membrane vesicles were cross-linked by using the bifunctional reagents dimethyl-3,3′-dithiobispropionimidate and dithiobis[succinimidyl propionate]. Third, specific antigen-antibody interactions on the surface of membrane vesicles were analyzed by radioautographic techniques. The major proteins of the outer membrane of the gonococcus were defined, and a nomenclature was devised to take into account the effects of heat and reducing agents on the resolution of these proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results of cross-linking experiments strongly suggest that two of the major proteins of the gonococcal outer membrane (proteins 1 and 3) form a hydrophobically associated trimeric unit in situ which can be stabilized by selective cross-linking reagents. Results substantiated that these proteins are responsible for imparting serotypic specificity.     

Arrangement of the subunits of the nicotinic acetylcholine receptor of Torpedo californica as determined by alpha-neurotoxin cross-linking

[3H]Methyl-alpha-neurotoxin prereacted with dithiobis(succinimidyl propionate) (DTSP) can be covalently linked to each of the subunits of the nicotinic acetylcholine receptor in membranes from the electric tissue of Torpedo californica. Pronounced changes in the cross-linking pattern are observed upon prior incubation with receptor specific ligands and upon reduction and/or alkylation of the receptor. d-Tubocurarine has been shown to bind to two different sites in receptor-rich membranes. These sites are present in equal numbers but have different affinities [Neubig, R. R., & Cohen, J. B. (1979) Biochemistry 18, 5464-5475; Sine, S., & Taylor, P. (1981) J. Biol. Chem. 256, 6692-6699]. Using d-tubocurarine inhibition of [3H]-methyl-alpha-neurotoxin binding, we demonstrate two inhibitory constants for d-tubocurarine of 67 +/- 21 nM and 4.9 +/- 1.7 microM in unreduced membranes. We utilize the large difference in Ki's to preferentially block toxin cross-linking at the high affinity site for d-tubocurarine. Low concentrations of this competitive antagonist selectively block the cross-linking of toxin to the beta and gamma subunits of the receptor, suggesting that these subunits are located close to the toxin binding site which is also the high-affinity binding site for d-tubocurarine. Reduction of disulfide bonds alters the affinity of the receptor for alpha-neurotoxin. Alterations are also seen in the cross-linking pattern of DTSP-activated [3H]methyl-alpha-neurotoxin to reduced and alkylated membranes in the presence of tubocurarine. The constants for d-tubocurarine inhibition of [3H]methyl-alpha-neurotoxin binding to reduced and alkylated membranes are 172 +/- 52 nM and 2.4 +/- 0.4 microM. The effects of bromoacetylcholine, carbamoylcholine, gallamine, and procaine on the cross-linking pattern are also examined. Our observations are consistent with an arrangement of the subunits in the membrane of alpha beta alpha gamma delta.     

The molecular morphology of bovine heart mitochondrial NADH-ubiquinone reductase. Cross-linking with dithiobis(succinimidyl propionate)

The molecular morphology of NADH-ubiquinone reductase (complex I) was investigated by cross-linking with the cleavable bifunctional reagent, dithiobis(succinimidyl propionate). Cross-linking inhibits the following activities of the complex--NADH----3-acetylpyridine adenine dinucleotide (oxidized), NADH----2,6-dichloroindophenol, NADH----ferricyanide, and NADH----menadione--to different degrees with the greatest inhibition occurring with either ferricyanide or 3-acetylpyridine adenine dinucleotide as electron acceptor. Addition of 150 microM NADH affords partial protection from inhibition. Cross-linking quenches the FMN fluorescence of complex I (288 nm excitation/515 nm emission), and addition of 150 microM NADH greatly reduces the quenching. Treatment of complex I (1 mg/ml) for 2 min with dithiobis(succinimidyl propionate) (0.2 mg/ml) at 4 degrees C revealed a cross-linked product consisting of the following seven subunits: 75-80, 53-57, 42, 33-35, 24-27, 17-18, and 12.5-15.5 kDa. Five minutes of treatment cross-linked the unidentified polypeptides of 69 and 51 kDa to six of the seven complex I subunits, but the 12.5-15.5-kDa subunit may be missing from this cross-linked product, while 15 min of treatment cross-linked additional unidentified polypeptides of 177, 107, 72, and 63 kDa. Since longer times of cross-linking result in a larger number of unidentifiable polypeptide spots, the shorter cross-linking time results are taken as a more accurate picture of the native enzyme conformation. This would indicate that within complex I the following subunits are within 12 A of each other at one or more points in space: 75-80, 53-57, 42-45, 33-35, 24-27, 17-18, and, perhaps, 12.5-15.5 kDa. These subunits represent portions of all three fractions of the enzyme, i.e. flavoprotein, iron-protein, and insoluble or hydrophobic fractions.