Dimethyl Suberimidate Research Articles

Nucleotide binding and hydrolysis properties of Neurospora crassa cytosolic molecular chaperones, Hsp70 and Hsp80, heat-inducible members of the eukaryotic stress-70 and stress-90 families.

Formation of a hetero-oligomeric complex between Hsp70 and Hsp80 of Neurospora crassa was observed previously by means of chemical crosslinking and enzyme-linked immunosorbent assays (ELISA). The present study documents the effect of nucleotides on the subunit structure of Hsp70 and Hsp80 by crosslinking with bifunctional reagents: glutaraldehyde, dimethyl adipimidate (DMA), and dimethyl suberimidate (DMS). The inter-protomer crosslinking of Hsp80 with DMA and DMS was suppressed by ATP and to a lesser extent by ADP, CTP, and NAD. Crosslinking of purified Hsp70 by glutaraldehyde yielded dimers and higher order oligomers. Binding of ATP, ADP, CTP, and NAD, but not NADH, led to a marked reduction in the yield of oligomers. Similarly, crosslinking by DMA and DMS was suppressed by ADP, ATP, and CTP. Both Hsp70 and Hsp80 exhibited intrinsic ATPase activity. Interestingly, ATP levels exceeding 25 microM resulted in pronounced inhibition of the ATPase activity of Hsp80 and 0.5 mM and 0.25 mM ATP led to a prolonged lag in the reaction. Addition of NAD resulted in the abolition of the lag period. The binding of 2-p-toluidinylnapthalene-6-sulfonate (TNS) to Hsp70 and its displacement by ATP and other nucleotides demonstrated the hydrophobic nature of the nucleotide-binding region.

Modification of beta 2glycoprotein I by glutardialdehyde. Conformational changes and aggregation accompany exposure of the cryptic autoepitope.

Autoantibodies from patients with antiphospholipid syndrome (APS) recognize an epitope on beta 2glycoprotein I (beta 2 GPI) only when native beta 2 GPI is adsorbed on surfaces composed of anionic phospholipids or oxidized polystyrene. beta 2 GPI was modified with the crosslinking agent, glutardialdehyde (GDA), which induced exposure of the anti-beta 2 GPI epitope at GDA:beta 2 GPI mol ratios in the range of 500-2000. A second crosslinking agent, dimethyl-suberimidate (DMS), did not expose the epitope, which may be a consequence of its having less tendency than GDA to form intermolecular links. SDS-PAGE experiments demonstrate that GDA does promote extensive intermolecular crosslinking of beta 2 GPI, and DMS does not. Formaldehyde also reacts with the lysine residues of beta 2 GPI, but does not expose the epitope. The circular dichroism spectra of native and modified beta 2 GPI confirm that GDA induces changes in conformation that are qualitatively different from those caused by formaldehyde. These data provide evidence that binding of lysine residues is not a sufficient condition for exposure of the autoepitope, and also support the likelihood that anti-beta 2 GPI antibodies bind only to aggregates of the protein. Thus, by synthesizing an active holoantigen of beta 2 GPI, conditions were defined that are necessary for binding of human autoantibodies.

Oligomeric state of the tonoplast proteins: Structure of the putative malate transporter in Catharanthus roseus

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.

The accessible surface of the nicotinic acetylcholine receptor. Identification by chemical modification and cross-linking with 14C-dimethyl suberimidate.

To obtain structural information on the nicotinic acetylcholine receptor from Torpedo electric tissue we modified and cross-linked lysine residues with the agonistic bifunctional reagent [14C]dimethyl suberimidate. This reagent labels exposed lysine residues, especially those located near the ligand-binding site, and cross-links lysine residues located not more than 11 A, the length of the cross-linker, apart. Using this method, we identified a cross-link located between betaLys177 and betaLys191 showing that the 13 amino acids in between form a loop with these two residues located at the surface. Cross-linking also occurred between the vicinal lysine residues alphaLys76 and alphaLys77, indicating that these neighbouring lysine residues are not involved in a beta-sheet structure. A total of 21 out of 97 lysine residues present in the receptor were modified by [14C]dimethyl suberimidate. Thus these residues are located on the accessible extramembrane surface. The two lysine residues alphaLys76 and alphaLys179 were predominantly labelled. Because of the agonistic property of [14C]dimethyl suberimidate [Watty, A., Methfessel, C. & Hucho, F. (1997) Proc. Natl Acad. Sci. USA 94, 8202-8209] this might be due to their close proximity to the ligand binding site.

The effect of chemical crosslinking of invertase with dimethyl suberimidate on its pH stability

The effect of chemical crosslinking of invertase with a homo-bifunctional bisimidoester on its pH stability was studied. Dimethyl suberimidate (DMS) was used as the crosslinker and pH inactivation was investigated in the pH range of 2.5–10. The inactivation mechanisms of both native and DMS crosslinked invertases were observed to follow first-order kinetics during prolonged incubation. Although DMS crosslinking of invertase increased the pH stability of the enzyme over the complete pH range, it was especially effective over the acidic range. The half-lives of invertase increased almost twofold, on average, by crosslinking at the neutral to the acidic range. The effect of crosslinking was especially pronounced at pH 5 since both the half-life of the native invertase and the stabilization factor were very high. Higher activation free energies of inactivation were obtained for DMS crosslinked invertases over the whole pH range which also indicates the stabilization of invertase by crosslinking.

Short Communication: Immobilization of urease from pigeonpea (Cajanus cajan L.) on flannel cloth using polyethyleneimine

Urease of pigeonpea has been immobilized on polyethyleneimine-activated cotton cloth followed by cross-linking with dimethyl suberimidate. Optimum immobilization (56%) was obtained at a protein loading of 1.2mg/5×5cm2 cloth piece. The immobilized enzyme stored in 0.1M Tris/acetate buffer, pH6.5, at 4°C had a t1/2 of 70 days. There was practically no leaching of the enzyme from the immobilization matrix in 15 days. The immobilized enzyme was used 7 times at an interval of 24h between each use with 75% residual activity at the end of the period. Blood urea analysis was carried out with immobilized urease for some clinical samples.

Cross-linked trimers of bovine ribonuclease A: activity on double-stranded RNA and antitumor action

Trimers of bovine pancreatic RNase A were obtained by cross-linking native RNase A with dimethyl suberimidate. They degrade double-stranded RNA more efficiently than dimers and monomers of RNase A, and display significant cytotoxic and/or cytostatic actions against C4-I cells (a human cell line derived from squamous carcinoma of the uterus cervix). On the same cell line cross-linked dimers of RNase A appear to be ineffective.

Crosslinking density and resorption of dimethyl suberimidate-treated collagen.

Collagen was purified from bovine Achilles tendon and crosslinked with dimethyl suberimidate (DMS) and glutaraldehyde (GTA). Under optimal conditions, the shrinkage temperature (Ts) was raised to 74 degrees C for collagen crosslinked with DMS and to 80 degrees C for those crosslinked with GTA. Crosslinking density measurements were done on the hydrothermally denatured collagen by the method based on the Flory-Rehner equation. GTA treatment was found to introduce more number of crosslinks than DMS. The maximum tension attained during heating (after shrinkage has occurred) was greater for GTA-treated collagen than for DMS and control. The control collagen membranes broke during heating (at 73 degrees C), while for the crosslinked membranes the tension kept on increasing up to 100 degrees C. The crosslinking density correlated well with the data determined from the in vitro and in vivo degradation studies. Uncrosslinked and DMS crosslinked collagen membranes were more susceptible to degradation by enzymes in vitro, while GTA-treated collagen was highly resistant to degradation. The biocompatibility of the collagen membranes was studied by subcutaneous implantation in rats. Uncrosslinked collagen membranes degraded within 14 days with the formation of granulation tissue. DMS crosslinked membranes degraded within 21 days and the area was replaced by numerous fibroblasts and newly formed collagen. No calcification was observed. For GTA-treated membranes, necrosis was observed after 7 days implantation and by 14 days the membrane had started to calcify.

Fixation of allosteric states of the nicotinic acetylcholine receptor by chemical cross-linking.

Receptor activity can be described in terms of ligand-induced transitions between functional states. The nicotinic acetylcholine receptor (nAChR), a prototypic ligand-gated ion channel, is an "unconventional allosteric protein" which exists in at least three interconvertible conformations, referred to as resting (low agonist affinity, closed channel), activated (open channel), and desensitized (high agonist affinity, closed channel). Here we show that 3,3'-dimethyl suberimidate (DMS) is an agonistic bifunctional cross-linking reagent, which irreversibly "freezes" the nAChR in a high agonist affinity/closed-channel state. The monofunctional homologue methyl acetoimidate, which is also a weak cholinergic agonist, has no such irreversible effect. Glutardialdehyde, a cross-linker that is not a cholinergic effector, fixes the receptor in a low-affinity state in the absence of carbamoylcholine, but, like DMS, in a high-affinity state in its presence. Covalent cross-linking thus allows us to arrest the nAChR in defined conformational states.

Annexin XII forms calcium-dependent multimers in solution and on phospholipid bilayers: a chemical cross-linking study.

The annexins are a family of proteins that bind in a Ca2+-dependent manner to phospholipids that are preferentially located on the intracellular face of plasma membranes. Recent X-ray studies of hydra annexin XII showed that it crystallized as a homohexamer with an intermolecular Ca2+ binding site separate from the type II Ca2+-dependent phospholipid binding site. On the basis of this hexamer structure, a novel mechanism was proposed to explain how annexins interact with membranes. The first step toward evaluating this proposal is to determine whether the annexin XII hexamer exists when the protein is not in a crystalline form. We now report that annexin XII in solution can be cross-linked with dimethyl suberimidate into multimers with apparent Mr's corresponding to trimers and hexamers as determined by SDS--polyacrylamide gel electrophoresis--the trimer band may correspond to incompletely cross-linked hexamers. Multimer formation was dependent on Ca2+ and was enhanced when the protein first was bound to phospholipid vesicles. To evaluate the role of the intermolecular Ca2+ site in annexin XII hexamer formation, one of the residues used to coordinate Ca2+, glutamate 105, was replaced with lysine (E105K). In solution, the E105K mutation inhibited hexamer formation in the presence of moderate (3 mM) but not high (25 mM) Ca2+. No inhibition of E105K annexin XII hexamer formation was observed in the presence of phospholipid, thereby suggesting that either (i) other interactions are capable of stabilizing the hexamer when bound to bilayers or (ii) only trimers form on bilayers and the observed hexamer bands were due to cross-linking of closely packed trimers. In summary, this study shows for the first time that annexin XII can form hexamers in solution and implicates the intermolecular Ca2+ site in hexamer formation. This study also shows that multimers form on bilayers but does not clearly establish whether the multimers are trimers or hexamers.

The quaternary geometry of transcription termination factor rho: assignment by chemical cross-linking

Transcription termination factor rho from Escherichia coli is a ring-shaped homohexamer of 419 amino acid subunits and catalyzes an ATP-dependent release of nascent RNA transcripts. Previous chemical cross-linking studies suggested that the rho hexamer might have D 3 symmetry with three isologous dimers as protomers. However, our recent mutational analysis of rho alongside its putative structural homology to F 1-ATPase rather argued for C 6 symmetry. To resolve this discrepancy, we have re-investigated the pattern of cross-linking of rho using various cross-linkers with different functional groups and spacer lengths. Upon reaction with dimethyl suberimidate followed by SDS-polyacrylamide gel electrophoresis, rho protein generated a series of cross-linked oligomers up to hexamers, of which dimers migrated as distinct doublet bands of approximately equal intensities. However, the lower band became much stronger than the upper one with dimethyl adipimidate and difluorodinitrobenzene, and vice versa with disuccinimidyl glutarate, disuccinimidyl suberate and disulfosuccinimidyl tartarate. Furthermore, the trimeric products also produced doublet bands, whose relative intensities were again variable with cross-linkers, but in an inverse correlation with those of the dimer bands. These results combined with theoretical considerations support a C 6 symmetry model in which cross-linking is assumed to occur stochastically at one of two alternative sites within each subunit interface with variable relative frequencies depending on cross-linkers. The D 3 symmetry is excluded, for the putative trimeric subspecies should always retain mutually equal intensities in that case. Detailed inspections of the cross-linking kinetics further revealed a moderate characteristic of C 3 symmetry for the rho hexamer such that the collective as well as relative rates of cross-linking at the two available sites could fluctuate between alternating interfaces. The final model designated as C 3/6 is also compatible with other functional and structural properties known for rho.

Oxygenation capacity of hypotonized and crosslinked rat erythrocytes

Rat erythrocytes subjected to hypotonic-isotonic dialysis, or crosslinking with bifunctional reagents (glutaraldehyde and dimethyl suberimidate hydrochloride) show a high percentage of methemoglobin and decreased oxyhemoglobin content which implies a low oxygen carrying capacity. Such modified cells maintain reversible oxygen binding properties although, they present a high hemoglobin oxygen affinity (low P 50) and a diminished cooperativity in binding oxygen to hemoglobin (low n). These results suggest a reduced capacity of liberating oxygen to tissues under low PO 2. Changes produced in erythrocytes can not be restored even in the presence of energy (ATP), reduced glutathione and 2,3-bisphosphoglyceric acid during the dialysis process or after crosslinking/permeabilizing treatment.

D-glucose metabolism in dimethyl suberimidate-treated tumoral pancreatic islet cells.

After protein cross-linking by dimethyl suberimidate, tumoral insulin-producing cells of the RINm5F line were either exposed to digitonin for measurement of hexokinase activity in the resulting cell pellet and supernatant, or incubated in the presence of D-[5-3H]glucose, D-[U-14C]glucose or L-[U-14C]glutamine to assess the metabolism of these nutrients. After digitonin treatment, the activity of hexokinase recovered in the cell pellet was about 40% higher in cross-linked than control RINm5F cells. Although failing to affect the metabolism of L-[U-14C]glutamine, and severely decreasing the oxidation of D-[U-14C]glucose, the cross-linking of proteins accentuated the increase in D-[5-3H]glucose utilization and D-[U-14C]glucose conversion to acidic metabolites resulting from a rise in hexose concentration from 2.8 to 16.7 mM. The latter change represents a mirror image of that previously found in cross-linked pancreatic islets. Taking into account the vastly different participation of glucokinase to hexose phosphorylation in RINm5F cells and normal islet cells, the present findings further support, therefore, the regulatory role of protein-to-protein interaction in the control of glucokinase catalytic activity in these fuel-sensing cells.

GTP Cyclohydrolase I Feedback Regulatory Protein Is a Pentamer of Identical Subunits: PURIFICATION, cDNA CLONING, AND BACTERIAL EXPRESSION

GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by tetrahydrobiopterin and also mediates the stimulatory effect of phenylalanine on the enzyme activity. To characterize the molecular structure of GFRP, we have purified it from rat liver using an efficient step of affinity chromatography and isolated cDNA clones, based on partial amino acid sequences of peptides derived from purified GFRP. Comparison between the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of purified GFRP showed that the mature form of GFRP consists of 83 amino acid residues with a calculated Mr of 9,542. The isolated GFRP cDNA was expressed in Escherichia coli as a fusion protein with six consecutive histidine residues at its N terminus. The fusion protein was affinity-purified and digested with thrombin to remove the histidine tag. The resulting recombinant GFRP showed kinetic properties similar to those of GFRP purified from rat liver. Cross-linking experiments using dimethyl suberimidate indicated that GFRP was a pentamer of 52 kDa. Sedimentation equilibrium measurements confirmed the pentameric structure of GFRP by giving an average Mr of 49,734, which is 5 times the calculated molecular weight of the recombinant GFRP polypeptide. Based on the pentameric structure of GFRP, we have proposed a model for the quaternary structure of GFRP and GTP cyclohydrolase I complexes.

Activity and stability of native and modified alanine aminotransferase in cosolvent systems and denaturants

Alanine aminotransferase (ALT) is used in clinical diagnostics, amino acid synthesis and in biosensors. Here we describe the stabilization of soluble porcine ALT by chemical modification with mono- and bis-imidates. The apparent transition temperatures (‘ T m ’, the temperature where 50% of initial activity was lost in 10 min) for native and DMS-modified ALT were 46 and 56 °C respectively. The effects of water-miscible organic solvents (methanol, dimethylformamide, dimethylsulphoxide and 1,4-dioxane) on the activity/stability of native and modified forms were determined. In all systems studied, an abrupt decrease in ALT catalytic activity was observed on reaching a certain threshold concentration of the organic solvent. The modified derivatives were more organotolerant than native enzyme. Comparison of the apparent V max and K m for 2-oxoglutarate as substrate, determined in 10% ( v v ) organic solvent, with the results of thermal inactivation studies showed that the solvents have different effects on ALT's catalytic parameters and on its conformational stability. At 35 °C with no organic solvent the dimethylsuberimidate (DMS)-modified derivative's half-life was 16 times greater than that for native enzyme; in 30% ( v v ) solvent at 35 °C, the DMS-modified ALT's half-life was up to 4.6 times greater than native enzyme's. DMS-modified ALT was also more stable in urea and guanidine HCl, and its refolding was more noticeable, than that of native enzyme.

A study of the oligomeric state of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-preferring glutamate receptors in the synaptic junctions of porcine brain.

The number of the subunits in an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring L-glutamate receptor in the synaptic junctions of porcine brain was investigated in this study. Upon incubation of the synaptic junctions with three cross-linking regents, dimethyl adipimidate (DMA), dimethyl suberimidate (DMS) and N-succinimidyl-(4-azidophenyl)-1,3'-dithiopropionate (SADP), AMPA receptor subunits in higher-molecular-mass aggregates were detected by immunoblotting. These aggregates migrated as proteins of approx. 200, 300 and 400 kDa. The number and identity of the subunits in a solubilized AMPA receptor were also investigated here. Two samples, W1 and W2, enriched in AMPA receptors were prepared from synaptic junctions by a combination of detergent-solubilization, anion-exchange chromatography and wheatgerm agglutinin affinity chromatography. Hydrodynamic behaviour analyses revealed that the majority of the AMPA receptors in either one of these samples were asymmetrical detergent-surrounded particles with a protein mass around 350 kDa. SDS/PAGE analysis revealed that the majority of AMPA receptors in the W1 sample were comprised of dimers of 106 kDa subunits which were covalently linked by disulphide bonds. Cross-linking these receptors with SADP yielded a new band of approx. 400 kDa. The results obtained here, either from the studies of AMPA receptors embedding in synaptic junctions or from those of detergent-solubilized and partially purified receptors, suggest that AMPA receptors contain a basic core structure comprising of four 106 kDa subunits.

Subunit Structure of Rod cGMP-Phosphodiesterase

The rod cGMP phosphodiesterase (PDE) is the G-protein-activated effector enzyme that regulates the level of cGMP in vertebrate photoreceptor cells. Rod cGMP PDE is generally viewed as a heterotrimeric protein composed of catalytic alpha and beta subunits ( approximately90 kDa each) and two copies of the inhibitory subunit gamma ( approximately 10 kDa). However, the possibility that rod PDE could exist as distinct isoforms, such as alphaalphagamma2 and betabetagamma2 has not been ruled out. We have studied this question using cross-linking of PDE subunits with maleimidobenzoyl-N-hydroxysuccinimide ester and para-phenyldimaleimide. The cross-linking resulted in major products with molecular mass of 100 and 150 kDa, a doublet at approximately 180-190 kDa, and a doublet at approximately 210-220 kDa. Cross-linked products were analyzed using polyclonal-specific anti-PDEalphabeta, anti-PDEalpha, anti-PDEbeta, or anti-PDEgamma antibodies. The anti-PDEalpha and anti-PDEalphabeta antibodies recognized all the cross-linked products, whereas anti-PDEbeta and anti-PDEgamma antibodies did not interact with the 150-kDa band, indicating that the composition of this band is most likely alphaalpha. Similar analysis of cross-linked products of trypsin-treated PDE preparations revealed bands that are likely formed by PDEbeta subunit. The molecular size of holo-PDE and trypsin-activated PDE were studied using analytical ultracentrifugation in order to determine if oligomerization of PDE could account for the cross-linking of identical PDE subunits. The sedimentation analysis of both holo-PDE and ta-PDE revealed homogeneous samples with molecular masses of approximately220 and approximately150 kDa, respectively. These results indicate that PDE is likely a mixture of the major species alphabetagamma2, minor species alphaalphagamma2, and possibly betabetagamma2. Our data are consistent with the detection of low PDE activity in the rd mouse, which lacks any functional PDEbeta subunit.

Hexokinase and glucokinase activity in cross-linked and permeabilized pancreatic islets

The activities of hexokinase and glucokinase were measured in cross-linked and permeabilized rat pancreatic islets. After exposure to dimethyl suberimidate (20 mM) and digitonin (0.4 mM), the activity of hexokinase represented about half of that found in homogenates of freshly isolated islets. The K m of hexokinase for d-glucose and the K i for its inhibition by d-glucose-6-phosphate were similar, however, in the cross-linked and permeabilized islets and in homogenates of freshly isolated islets. Glucokinase activity also was documented in the cross-linked and permeabilized islets, it being less sensitive than hexokinase activity to inhibition by d-glucose-6-phosphate. At a high concentration of d-glucose (16.7 mM), the phosphorylation of the hexose failed to be increased by d-fructose-1-phosphate, whether in the absence or presence of d-glucose-6-phosphate. These findings indicate that the intrinsic properties of hexokinase isoenzymes are preserved in cross-linked islets, but suggest that the cross-linking of proteins prevents the activation of glucokinase by its regulatory protein.

The membrane-bound rat serotonin transporter, SERT1, is an oligomeric protein

The synaptic actions of the neurotransmitter serotonin are terminated by a selective re-uptake system located in the axonal membrane. To gain information about the quaternary structure of this membrane protein, we transiently expressed the recombinant rat serotonin transporter, SERT1, in human embryonic kidney 293 cells. Treatment with sulfhydryl oxidizing agents and the homobifunctional cross-linker dimethyl suberimidate (DMS) generated adducts of 130–180 kDa and 220–270 kDa, respectively. These data indicate an oligomeric structure of SERT1.

D-Glucose Metabolism in Cross-Linked Pancreatic Islets

D-glucose metabolism was investigated in pancreatic islets that underwent cross-linking of intracellular proteins by dimethyl suberimidate. Both the utilization of D-[5-3H]glucose and oxidation of D-[U-14C]glucose were much more severely affected at high (16.7 mM) than at low (2.8 mM) concentration of the hexose, when comparing cross-linked to control islets. The preferential stimulation of D-[U-14C]glucose oxidation relative to D-[5-3H]glucose utilization, resulting from an increase in hexose concentration, was not abolished, however, in cross-linked islets. Likewise, the activation of phosphofructokinase in glucose-stimulated islets did not appear to be impaired in cross-linked islets, the islet content in tritiated acidic metabolites generated from D-[5-3H]glucose failing to be increased in cross-linked islets. It is proposed, therefore, that the impaired responsiveness of cross-linked islets to a rise in D-glucose concentration might be attributable to an altered regulation of glucokinase, as possibly resulting from a missing interaction of the enzyme with GLUT-2.