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.     

Investigation of the quaternary structure of Neurospora pyruvate kinase by cross-linking with bifunctional reagents: the effect of substrates and allosteric ligands.

Pyruvate kinase (EC 2.7.1.40) of Neurospora, a tetramer composed of apparently identical subunits, has been shown to be a dimer of dimers by interprotomeric cross-linking experiments in which bifunctional reagents were used. An analysis of the polyacrylamide gel profiles of the enzyme after cross-linking with glutaraldehyde, dimethyl suberimidate, and dimethyl adipimidate shows that the extent of intersubunit cross-linking is influenced markedly by the ligand bound to the enzyme. Bifunctional cross-linking reagents with a shorter distance between the two functional groups form cross-links effectively in the unliganded enzyme. In the FDP-pyruvate kinase complex, cross-linking was observed over longer distances compared with the unliganded enzyme. It is demonstrated that covalent cross-linkers cah be used as sensitive indicators of conformational changes induced in pyruvate kinase by substrates and allosteric ligands.     

Cross-linking experiments with the adenosine triphosphatase of sarcoplasmic reticulum.

The proteins of sarcoplasmic reticulum were cross-linked by rapid oxidation of thiol groups with I2. About two-thirds of the thiols were oxidized without any significant cross-linking, implying an extensive formation of intramolecular disulphide bonds. When the thiols were completely oxidized at room temperature a series of oligomers containing up to five molecules were observed, as well as large aggregates which were excluded from the gels. Complete oxidation at -10 degrees C left most of the ATPase (adenosine triphosphatase) as monomer. Similar results were obtained when copper-phenanthroline complexes or dimethyl suberimidate were used as cross-linking reagents. We conclude that most of the cross-linked species arise by linking of randomly colliding ATPase molecules which are present in the membrane at very high concentration.     

Effects of amidination and chemical cross-linking on human factor VIII (antihemophilic factor).

The bifunctional reagent dimethyl suberimidate, reacting with primary amino groups of proteins, was used to cross-link highly purified human factor VIII. Reaction products were reduced with beta-mercaptoethanol or treated with Rhizopus arrhizus triglyceride lipase. The proportions of the dissociated subunits and their oligomers were calculated from the relative staining intensities of individual bands following polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. Low concentrations of dimethyl suberimidate (up to 0.5 mM) produced covalently linked dimers which retained full functional (coagulant and von Willebrand factor) activities. Treatment with increasing concentrations of dimethyl suberimidate resulted in an almost simultaneous appearance of both trimeric and tetrameric species, suggesting the existence of specific intra-dimer contacts. A parallel decrease of functional activities was observed at higher concentrations of dimethyl suberimidate. A monofunctional reagent (ethyl acetimidate), reacting similarly with primary amino groups, amidinated factor VIII at rates similar to dimethyl suberimidate. Up to 40% amidinated factor VIII retained full biological activities. We conclude that the most reactive lysine residues are not involved in the active sites responsible for either coagulant or von Willebrand activity.     

Simplified methods for isolation of ubiquitin from erythrocytes. Generation of ubiquitin polymers

1. Ubiquitin has been isolated from bovine erythrocytes by procedures in which the hemoglobin was removed by denaturation with either ethanol-chloroform mixtures or by heating. 2. The proteins soluble to the denaturation step were removed by 3% sodium trichloroacetate (TCA) at pH 2.0-2.5 or by 5% TCA. 3. Ubiquitin was isolated in relatively high yield from the TCA insoluble fraction by use of single ion-exchange chromatographic and gel permeation steps. 4. Ubiquitin shows relatively little cross-linking upon treatment with glutaraldehyde or with dimethyl suberimidate. Heating of the glutaraldehyde treated material in 4 M guanidine, however, leads to marked aggregation. 5. The polymers of ubiquitin react strongly with antibody in an immunoblot assay.     

Citrate synthase from a Gram-positive bacterium. Purification and characterization of the Bacillus megaterium enzyme.

Citrate synthase was purified to homogeneity from a Gram-positive bacterium (Bacillus megaterium) for the first time. The Mr of the native enzyme was determined to be 84 000 (S.E.M. +/- 5000). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in guanidinium chloride revealed a single protein species of Mr 40 300 (S.E.M. +/- 4400), indicating a dimeric enzyme. This dimeric structure was confirmed by cross-linking the native enzyme with dimethyl suberimidate and with glutaraldehyde, followed by electrophoretic analysis. The enzyme follows Michaelis-Menten kinetics with respect to both substrates, acetyl-CoA and oxaloacetate, and is sensitive to non-specific inhibition by a range of adenine nucleotides. In both molecular and catalytic properties the citrate synthase closely resembles the enzyme from eukaryotic sources and contrasts markedly with the larger, hexameric, enzyme from Gram-negative bacteria.     

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.     

Cross-linking of Salmonella isopropylmalate synthesis with dimethyl suberimidate: evidence for antagonistic effects of leucine and acetyl-CoA on the guaternary structure

Reaction of α-isopropylmalate synthase from Salmonella typhimurium with the bifunctional cross-linking reagent dimethyl suberimidate and subsequent sodium dodecyl sulfate gel electrophoresis results in the appearance of four protein species with molecular weights of about 50,000, 100,000, 150,000, and 200,000. In the absence of ligands, the pattern consists predominantly of tetramers and dimers (“native” pattern). This pattern is changed to one of dimers and monomers in the presence of inhibitory concentrations of leucine. Acetyl-CoA, known to kinetically compete with leucine, restores the “native” pattern. On the basis of previous findings, the results are discussed in terms of “frozen” equilibria.     

Dimethyl suberimidate cross-linking of oligo(dT) to DNA-binding proteins

Dimethyl suberimidate is a bifunctional reagent that is used for cross-linking the protein components of oligomeric macromolecules. In this report, dimethyl suberimidate is shown to specifically cross-link oligo(dT) of varying lengths to the DNA-binding subunits of a multimeric helicase-primase encoded by herpes simplex virus type 1. This result indicates that dimethyl suberimidate and other imidoester cross-linking reagents may be useful for characterizing the interaction of oligo(dT) with proteins that bind single-stranded DNA.     

Macromolecular association of ADP-ribosyltransferase and its correlation with enzymic activity.

The macromolecular self-association of ADP-ribosyltransferase protein in solution was studied by several experimental techniques: quantitative gel filtration, electrophoretic analyses in non-denaturing gels, and cross-linking the enzyme protein with glutaraldehyde, dimethyl pimelimidate, dimethyl suberimidate, dimethyl 3,3'-dithiobisproprionimidate and tetranitromethane. The self-association of the polypeptide components obtained by plasmin digestion was also determined by using the above cross-linking agents. Monomers and cross-linked dimers of the enzyme protein, possessing enzymic activity, were separated in non-denaturing gels by electrophoresis. The basic polypeptide fragments, exhibiting molecular masses of 29 kDa and 36 kDa, self-associated, whereas the polypeptides with molecular masses of 56 kDa and 42 kDa associated only to a negligible extent, indicating that the peptide regions that also bind DNA and histones are probable sites of self-association in the intact enzyme molecule. Macromolecular association of the enzyme was indicated by a protein-concentration-dependent red-shift in protein fluorescence. The specific enzymic activity of the isolated ADP-ribosyltransferase depended on the concentration of the enzyme protein, and at 2.00 microM concentration the enzyme was self-inhibitory. Dilution of the enzyme protein to 30-40 nM resulted in a large increase in its specific activity. Further dilution to 1-3 nM coincided with a marked decrease of specific activity. Direct enzymic assays of electrophoretically separated monomers and cross-linked dimers demonstrated that the dimer appears to be the active molecular species that catalyses poly(ADP-ribose) synthesis. The NAD+ glycohydrolase activity of the enzyme was also dependent on protein concentration and was highest at 1-3 nM enzyme concentration, when polymerase activity was minimal, indicating that the monomeric enzyme behaved as a glycohydrolase, whereas poly(ADP-ribosyl)ation of enzyme molecules was maximal when the enzyme tends to be self-associated to the dimeric form.     

Physicochemical and immunological homogeneity of spinin, the subunit-protein of bacterial spinae.

Bacterial spinae from marine bacterium D71 are multi-subunit structures of a single protein. This protein, called spinin, is homogeneous by immunodiffusion and immunoelectrophoresis, amino acid composition, polyacrylamide gel electrophoresis with a number of buffer systems, sedimentation velocity and diffusion boundary analysis. Sedimentation equilibrium gives Mr = 19,000, while phosphate polyacryl-amide gel electrophoresis in presence of dodecyl sulfate gives Mr = 32,000. The lower Mr estimate for spinin is supported by sedimentation equilibrium in 6 M guanidine . HCl, and covalent cross-linking with dimethyl suberimidate or glutaraldehyde. The higher Mr value probably arises from an anomalous spinin-dodecyl sulfate interaction. Isoelectric focusing in polyacrylamide gel gives pI = 3.45; however, the focusing pattern also contains three distinct bands that may arise from hydrolysis of the spinin protomer during anodic migration. This study presents the first extensive physicochemical characterization of spinin and provides the basis for investigating the subunit assembly of spinae.     

Effects of chaotropic electrolytes on the structure and electronic excitation coupling of glutaraldehyde- and diimido ester-cross-linked phycobilisomes

The tendency of isolated intact phycobilisomes to disperse into multimeric phycobiliprotein subunits in dilute phosphate buffer is diminished, or even eliminated, by means of covalent polypeptide cross-linking with glutaraldehyde, or dimethyl suberimidate. Employing sensitized fluorescence spectrophotometry in order to detect transitions of the type phycobilisome → phycobiliprotein multimers, and second-derivative absorption spectrophotometry to detect transitions of the type phycobiliprotein multimers → monomers, we investigated the molecular basis of phycobilisome stabilization by these two cross-linkers. A network of intrahexamer and interhexamer covalent cross-links prevents the dissociation of glutaraldehyde-treated phycobilisomes into monomers, even under the strong chaotropic effect of KNO₃ and KSCN solutions, but fails to protect against polypeptide unfolding in concentrated urea solution. Dimethyl suberimidate, on the other hand, introduces only intramonomer cross-links, and thus it does not prevent dissociation to monomers in the presence of KNO₃ and KSCN. Increased hydrophobicity of phycobiliprotein subunits, as a result of the alkylation of ?NH₂ by the diimido ester, or the dialdehyde, is an additional structure-stabilizing factor.     

Stabilization of restriction endonuclease Bam HI by cross-linking reagents

Bacillus amyloliquefaciens H produces a restriction endonuclease enzyme BamHl which is heat labile even at low temperatures. Studies were conducted to enhance thermal stability of BamHl using cross-linking reagents, namely, glutaraldehyde, dimethyl adipimidate (DMA), dimethyl suberimidate (DMS), and dimethyl 3,3'-dithiobispropionimidate (DTBP). Reaction with glutaraldehyde did not result in a preparation with enhanced thermal stability. However, the DMA-, DMS-, and DTBP-cross-linked preparations of BamHI exhibited significant improvement in thermal stability. Studies on thermal denaturation of the cross-linked enzyme preparations revealed that these do not follow a true first-order kinetics A possible deactivation scheme has been proposed in which the enzyme has been envisaged to go through a fully active but more susceptible transient state which, on prolonged heat exposure, exhibits a first-order decay kinetics. At 35 degrees C, which is close to the optimum reaction temperature of 37 degrees C for BamHl activity, the half-line of DMA-, DMS-, and DTBP-cross-linked preparations were 4.0, 5.25, and 5.5 h, respectively, whereas the native enzyme exhibited a half-line of 1.2 h only. The apparent values of deactivation rate constants for native, DMA-, DMS-, and DTBP-cross-linked BamHl were 1.13, 0.39, 0.29, and 0.26 h(-1), respectively, at the same temperature, and the apparent values of activation energies for denaturation of native, DMA-, DMS-, and DTBP-cross-linked BamHl were 2.63, 5.24, 6.55, and 9.2 kcal/mol, respectively. The DTBP-cross-linked Bam HI was, therefore, the best heat-stable preparation among those tested. The unusually low values of activation energies for denaturation of Bam Hl represent their highly thermolabile nature compared to other commonly encountered enzymes such as trypsin, having activation energies of more than 40 kcal/mol for their denaturation.     

State of aggregation of the (Ca2+ + Mg2+)-ATPase studied using chemical cross-linking

We have studied cross-linking of the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum and in reconstituted systems, using glutaraldehyde, cupric-1,10-phenanthroline and 3,3'-dithiobis (sulphosuccinimidylpropionate). All reagents produce extensive cross-linking, forming aggregates too large to enter polyacrylamide gels. Only traces of cross-linked dimeric ATPase species are formed. Saturation transfer electron spin resonance spectra of spin-labelled sarcoplasmic reticulum cross-linked with glutaraldehyde are also consistent with the formation of extensively cross-linked aggregates in the membrane. The results are interpreted in terms of dynamic clusters of ATPase molecules in the membrane, probably in the form of rows of ATPase molecules.     

Cross-linking with diimidates of glutamine synthetase from Bacillus stearothermophilus.

Glutamine synthetase [EC 6.3.2.1] from Bacillus stearothermophilus was modified with diethyl malonimidate (DEM), dimethyl adipimidate (DMA), and dimethyl suberimidate (DMS). DMA modified most epsilon-amino groups. On modification with DMA, formation of 3 to 4 cross-links/subunit resulted in a large increase in thermostability. The activity, allosteric properties and fluorescence spectrum of the enzyme were not changed on cross-linking. The SDS-polyacrylamide gel electrophoretic profiles of DEM-, DMA-, and DMS-modified enzymes suggested that the interaction berween six subunits in each of the two hexagonal rings of the protein are heterologous and are different from those between the piled subunits on different rings.     

Investigation of the properties of bovine heart creatine kinase cross-linked with dimethyl suberimidate

Dimeric bovine heart creatine kinase (EC 2.7.3.2, ATP: creatine N-phosphotransferase) has been cross-linked with the bifunctional reagent dimethyl suberimidate at several concentrations to yield modified enzyme with enhanced stability towards heat denaturation. The degree of thermal stability is dependent on the degree of cross-linking with optimal stabilization occurring when approx. half of all the available amino groups are covalently attached to dimethyl suberimidate. Accelerated storage studies were performed and the results used to predict the storage time of the native and modified enzyme at lower temperatures. The cross-linked derivative was predicted to have a longer shelf-life at 4 degrees C than the native enzyme. Modification caused a reduction in the specific activity of the enzyme. The pH profile was altered following cross-linking, but the Michaelis constants were not changed. The modified enzyme exhibited a marked resistance to the action of some denaturing agents.     

Augmentation of alpha-actinin-induced gelation of actin by talin.

Interactions among the three major constituents of focal adhesions, talin, actin, and alpha-actinin, were studied. No evidence was obtained for the direct interaction between talin and alpha-actinin. Both talin and alpha-actinin increased the rate and extent of polymerization of actin, and their effects were additive. Whereas talin alone exhibited very little actin-gelating activity, it potentiated markedly the gelation in the presence of alpha-actinin and lowered the concentration of alpha-actinin necessary for the gel formation. Its gelation-potentiating activity on prepolymerized actin was much smaller than observed on G-actin. Treatment of talin with a cross-linking reagent, 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide or dimethyl suberimidate, resulted in the formation of its oligomeric polypeptides. The complexes of talin and G-actin were also demonstrated with the cross-linking reagents and fluorescence-labeled actin. These results indicate that talin is able to cross-link some limited regions of actin filaments.     

Polydispersity of Serratia marcescens nuclease at optimal pH values]

Treatment with dimethyl suberimidate, a cross-linking bifunctional agent, showed that Sm1 and Sm2 nucleases of Serratia marcescens B10M1 are polydisperse in solution and consist of monomers and dimers at the level of pH optimal for the enzyme activity. The data suggest that nucleases from the strain B10M1 and any other strain are polydisperse at pH optimum if their amino acid sequences are identical.     

Characterization of the gamma subunits of the 7S nerve growth factor complex.

The gamma subunits of the 7S nerve growth factor complex (7S NGF) display arginine esteropeptidase activity. By varying the conditions of electrophoresis in acrylamide gel, it has been demonstrated that the gamma-subunit fraction of 7S NGF contains five different proteins, in contrast to the three (gamma1, gamma2, and gamma3) originally described (Smith, A.P., Varon, S. and Shooter, E.M. (1968), Biochemistry 7, 3259-3268); the gamma1 and gamma2 subunits, previously thought to be single species, can each be resolved into two components. The two components of the gamma1 subunit have the same isoelectric point, as do the two components of the gamma2 subunit. The distribution of protein among the two components of each of the gamma1 and gamma2 subunits varied from preparation to preparation. Moreover, a shift in the distribution for the gamma1 subunit was accompanied by a parallel shift for the gamma2 subunit. All of the different gamma proteins have the same molecular weight. On the basis of the molecular weights of the peptide chains of the gamma subunits and of the species which are formed by cross-linking with dimethyl suberimidate, it was concluded, that both the gamma1 and gamma2 subunits contain one species with two peptide chains and another with three peptide chains, while the gamma3 subunit is a single species with three peptide chains. The results also suggest that two of the chains in the three-chain species are derived, by proteolytic cleavage, from the larger chain in the two-chain species.     

Subunit topography of RNA polymerase from Escherichia coli. A cross-linking study with bifunctional reagents.

The quaternary structures of Escherichia coli DNA-dependent RNA polymerase holenzyme (alpha 2 beta beta' sigma) and core enzyme (alpha 2 beta beta') have been investigated by chemical cross-linking with a cleavable bifunctional reagent, methyl 4-mercaptobutyrimidate, and noncleavable reagents, dimethyl suberimidate and N,N'-(1,4-phenylene)bismaleimide. A model of the subunit organization deduced from cross-linked subunit neighbors identified by dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the large beta and beta' subunits constitute the backbone of both core and holoenzyme, while sigma and two alpha subunits interact with this structure along the contact domain of beta and beta' subunits. In holoenzyme, sigma subunit is in the vicinity of at least one alpha subunit. The two alpha subunits are close to each other in holoenzyme, core enzyme, and the isolated alpha 2 beta complex. Cross-linking of the "premature" core and holoenzyme intermediates in the in vitro reconstitution of active enzyme from isolated subunits suggests that these species are composed of subunit complexes of molecular weight lower than that of native core and holoenzyme, respectively. The structural information obtained for RNA polymerase and its subcomplexes has important implications for the enzyme-promoter recognition as well as the mechanism of subunit assembly of the enzyme.