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PubMed1999

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.

Adenosine Triphosphatases↗

PubMed1990

Role of membrane proteins in monosodium urate crystal-membrane interactions. II. Effect of pretreatments of erythrocyte membranes with membrane permeable and impermeable protein crosslinking agents.

Intact, human erythrocytes were pretreated with membrane permeable, dimethyl adipimidate (DMA) and dimethyl suberimidate (DMS) and membrane impermeable 3,3' dithiobis (sulfosuccinimidylpropionate) (DTSSP) protein crosslinking agents and incubated with monosodium urate monohydrate (MSUM) crystals. The percent inhibition of lysis values for pretreated cells relative to untreated cells were determined. All 3 agents caused a concentration dependent inhibition of MSUM induced hemolysis that was not due to a decrease in MSUM binding to the pretreated membranes. It was proposed that the inhibition of lysis was due to crosslinking of integral and cytoskeletal membrane proteins, resulting in a reduced mobility of the proteins, inhibition of lateralization of integral proteins into aggregates and decreased "pore" formation in the membrane.

Cell Membrane Permeability↗

PubMed1982

Effect of cross-linking agents on insulin associated responses in adipocytes.

The effect of 10 bifunctional cross-linking agents and four monofunctional analogues was studied on isolated adipocytes. [125I]Insulin binding and degradation, basal and insulin-stimulated glucose oxidation, and 3-O-methyl glucose uptake were measured. Two cross-linkers, which possess succinimide ester residues (disuccinimidyl suberate and dithiobis(succinimidyl propionate)) and react selectively with amino groups, appeared to react relatively specifically with the insulin receptor. Both produced a slight stimulation of basal glucose transport and metabolism, a marked inhibition of insulin-stimulated glucose transport and metabolism, and a marked decrease in insulin binding. Pretreatment of cells with unlabelled insulin partially blocked the effect of disuccinimidyl suberate, and as has been previously shown, disuccinimidyl suberate cross-linked insulin to its receptor. A monofunctional analogue of these compounds was 100-fold less active in altering cellular metabolic activity. Bisimidates, such as dimethyl suberimidate, dimethyl adipimidate, and dimethyl dithiobispropionimidate, also react with free amino groups but are more hydrophilic. These agents produced similar effects on glucose oxidation as the succinimide esters, but had little or no effect on insulin binding. The effects of these agents are not blocked by insulin and they do not cross-link insulin to its receptor. Mixed bifunctional reagents containing either a succinimide ester or an imidate and a group which reacts with thiols produced effects similar to the cross-linkers containing two succinimide groups or bisimidates, respectively. The bifunctional arylating agents difluorodinitrobenzene and bis(fluoronitrophenyl)sulfone produce marked effects on insulin binding and glucose oxidation at micromolar concentrations, but the monofunctional analogue fluorodinitrobenzene is almost equally active suggesting that with these compounds chemical modifications and not cross-linking was important. With neither the mixed bifunctional reagents, nor the arylating agents, did insulin pretreatment alter the effect of cross-linker and none of these agents cross-linked [125I]insulin to its receptor. These data suggest that the insulin receptor possesses a free amino group in a hydrophobic environment in its active site. A reactive amino group in a hydrophilic environment as well as other reactive groups are also present in some component of the insulin receptor-effector complex. Chemical modification or cross-linking of these functional groups results in an inhibition or mimicking of insulin action. Further study will be required to identify the exact locus of these sites.

Adipose Tissue↗

PubMed1979

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.

Allosteric Regulation↗

PubMed1982

Cross-linking of synaptic vesicle proteins. Effect of ATP.

Synaptic vesicles isolated from bovine brain were subjected to cross-linking with the bifunctional amino group reagent dimethyl adipimidate. The resulting proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The reagent (10 mM) caused partial or complete disappearance from the SDS gel of most of the major polypeptides of the vesicles and the formation of new polymeric species with molecular weights greater than 500000. Using lower concentrations of the adipimidate (2-5 mM) a more selective cross-linking occurred, with the disappearance of a group of protein bands having apparent molecular weight values of 60000-68000, 40000-41000 and 25000-30000. The extent of cross-linking was independent of vesicle concentration in the range 0.3-3.0 mg protein per ml. Addition of ATP or AMP to the cross-linking reaction mixture resulted in a marked reduction in cross-linking of all of the major vesicle polypeptides (apparent molecular weight values 160000, 77000, 55000, 42000, 32000, 28000 and 26000). Several proteins were less affected by ATP or AMP; these were mostly the same vesicle proteins as those which had become cross-linked with low concentrations (2 mM) of dimethyl adipimidate. The ATP effect was markedly reduced if the vesicles were pretreated prior to the cross-linking reaction with alkaline buffer (pH 8.5) in either the presence or the absence of ATP. In the presence of 32P-labeled ATP, several of the vesicle protein bands became phosphorylated, but the extent of their cross-linking did not depend upon the state of phosphorylation of the major phoshorylated proteins. The results are consistent with the presence of aggregated protein complexes and of stabilized arrays of the major proteins within the vesicle membrane.

Adenosine Monophosphate↗

PubMedDate not supplied

A crosslinked preparation of E. coli beta-D-galactosidase.

beta-D-Galactosidase from E. Coli was crosslinked using glutaraldehyde and two bisimidoesters. With glutaraldehyde and dimethyl adipimidate (DMA), it is possible to obtain preparations having higher activity than the native enzyme. Glutaraldehyde and DMA gave preparations showing enhanced thermal stability. The preparation crosslinked with DMA, when used for continuous hydrolysis of lactose in milk, was found to be significantly better than the native enzyme.

Cross-Linking Reagents↗

PubMed1979

Topographical studies on poliovirus capsid proteins by chemical modification and cross-linking with bifunctional reagents.

Poliovirus capsid proteins comprise 15.1 lysines in VP1, 5.6 lysines in VP2, 11.7 lysines in VP3 and 5.5 lysines in VP4. Treatment with monofunctional reagent N-succinimidyl 2,3-3H-proprionate leads to the modification of 3.4 lysines in VP1, 0.6 lysines in VP2, 2.0 lysines in VP3 and 0.03 lysines in VP4. Chemical modification with the monofunctional reagent N-succinimidyl 3-(4-hydroxy,5-125I-iodophenyl)propionate results in a predominant labelling of VP1 and VP3, whereas VP2 is less accessible and VP4 is not modified. Cross-linking of poliovirus with bifunctional imidoesters, dimethyl suberimidate (DMS, 1.1 nm) and dimethyl adipimidate (DMA, 0.8 nm) leads to a new protein complex of mol. wt. which corresponds to the sum of VP1 and VP3. By cleavage with ammonia and electrophoresis on polyacrylamide gels in SDS, the proteins are identified as VP1 and VP3. This result gives evidence for a direct neighbourhood of VP1 and VP3 in the virus capsid. Treatment of the virus with the mono- and bifunctional reagents has no influence on the stability of the particle. The infectivity is reduced only by the bifunctional reagent.

Chemical Phenomena↗

PubMed1982

The influence of calcium ions on the conformation of fibrinogen fragment D: the use of chemical cross-linking agents.

Fibrinogen fragment D prepared in the presence of calcium ions (fragment D[Ca++]) shows qualitatively similar cross-linking patterns with dimethyl suberimidate, dimethyl adipimidate and tetranitromethane. Fragment D prepared in the presence of EDTA (fragment D[EDTA]) gives a consistently different pattern with these reagents. In the case of fragment D[EDTA] there is much more intermolecular cross-linking suggesting that the loss of the C-terminus of the gamma-chain remnant results in fragment D adopting a more open conformation. Neither the addition of 2M urea nor EDTA to fragment D[Ca++] alters its cross-linking pattern suggesting that the proposed conformational change follows cleavage of a plasmin susceptible bond which is normally protected by the presence of calcium ions.

Calcium↗

PubMed1997

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 D3 symmetry with three isologous dimers as protomers. However, our recent mutational analysis of rho alongside its putative structural homology to F1-ATPase rather argued for C6 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 C6 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 D3 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 C3 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 C3/6 is also compatible with other functional and structural properties known for rho.

Cross-Linking Reagents↗

PubMed2006

Effect of intracellular magnesium and oxygen tension on K+-Cl- cotransport in normal and sickle human red cells.

In red cells from normal individuals (HbA cells), the K+-Cl- cotransporter (KCC) is inactivated by low O2 tension whilst in those from sickle cell patients (HbS cells), it remains fully active. Changes in free intracellular [Mg2+] have been proposed as a mechanism. In HbA cells, KCC activity was stimulated by Mg2+ depletion and inhibited by Mg2+ loading but the effect of O2 was independent of Mg2+. At all [Mg2+]is, the transporter was stimulated in oxygenated cells, minimally active in deoxygenated ones. By contrast, the stimulatory effects of O2 was abolished by inhibitors of protein (de)phosphorylation. HbS cells had elevated KCC activity, which was of similar magnitude in oxygenated and deoxygenated cells, regardless of Mg2+ clamping. In deoxygenated cells, the antisickling agent dimethyl adipimidate inhibited sickling, Psickle and KCC. Results indicate a role for protein phosphorylation in O2 dependence of KCC, with different activities of the relevant enzymes in HbA and HbS cells, probably dependent on Hb.

Anemia, Sickle Cell↗

PubMed1980

Purification and some properties of 6-phosphoglucose isomerase from Bacillus caldotenax.

6-Phosphoglucose isomerase [EC 5.3.1.9] was purified from Bacillus caldotenax. The isomerase shared many common properties with the isomerase from B. stearothermophilus, i.e, pH and temperature optima, thermostability, competitive inhibition by 6-phosphogluconate and Pi, and amino acid composition. The enzyme activity of the former, however, was lower than that of the latter. The molecular weight of the B. caldotenax isomerase was estimated to be 202,000-204,000 by gel filtration and electrophoresis of the enzyme cross-linked with dimethyl adipimidate (DMA). The enzyme was shown to consist of four subunits of equal molecular weight (50,600), and the four subunits were concluded to be identical based on the results of dansylation and cyanogen bromide cleavage of the enzyme. The interaction between the subunits were shown to be isologous by cross-linking with DMA.

Amino Acids↗

PubMed1987

Concanavalin A binding to human erythrocytes leads to alterations in properties of the membrane skeleton.

Three properties related to the erythrocyte membrane skeleton are found to be altered after the binding of concanavalin A (Con A) to erythrocytes or their isolated membranes. Con A binding to normal erythrocytes imparts resistance to heat (49 degrees C)-induced fragmentation of the cells. The fragmentation, due to denaturation of spectrin at 49 degrees C, is prevented by Con A in a dose-dependent manner, but levels off at concentrations of Con A in excess of 100 micrograms/ml. The binding of Con A to ghosts isolated from normal, trypsin- or Pronase-treated cells prevents (completely or substantially) the elution of the skeletal protein complex when the membranes are extracted under low-ionic-strength conditions in the cold. The Con A-agglutinated membranes of trypsin- and Pronase-treated, but not normal, cells show cross-linking of skeletal proteins and band 3 with dimethyl adipimidate, a 0.86 nm (8.6 A)-span bifunctional reagent. The extent of cross-linking is greater in the Pronase-treated membrane than in the less-agglutinable trypsin-treated membranes. The results show that, after Con A has bound, rearrangements occur in the membrane that alter properties of the skeletal proteins. Additionally, redistribution of the skeletal proteins and the Con A receptor occurs in the lectin-agglutinated membranes.

Chromatography, Affinity↗

PubMed1978

Membrane effects of imidoesters in hereditary stomatocytosis.

The marked increase in cation (Na+, K+) permeability that results in swollen, cup-shaped red cells in the hereditary stomatocytosis syndrome can be corrected in vitro with a bifunctional crosslinking reagent, dimethyl adipimidate (DMA). 45Ca influx in intact RBC, 45Ca efflux in red ghosts, and 45Ca retention in red ghosts are normal and not influenced by DMA. Endocytosis in resealed red ghosts is strikingly impaired but becomes normal if cells are first treated with 2 mM DMA. Protein kinase mediated phosphorylation of membrane proteins by AT32P--only 20--40% of normal control values in both shortterm (5 min) and more extended (60 min) incubations--is not improved by DMA. After reaction of 14C-DMA with stomatocytes, radiolabel is found associated with phosphatidyl serine and phosphatidyl ethanolamine and is also widely distributed among membrane proteins. Cation permeability of stomatocytes in corrected at DMA concentrations (1 mM) that result in barely detectable crosslinking of aminophospholipids or proteins, suggesting that either crosslinking of a minor component present in only small quantities or intramolecular (rather than intermolecular) crosslinking is responsible for the permeability effects. DMA, whose maximal crosslinking dimension is 7.3--9 A, is the most effective bifunctional imidoester of those tested. Shorter (dimethyl malonimidate) or longer (dimethylsuberimidate) reagents are either less effective than DMA or totally without effect.

Anemia, Hemolytic↗

PubMed1988

Actin-binding and dimerization domains of HeLa cell filamin.

HeLa cell filamin is a linear, bivalent, homodimer of high molecular weight subunits (Mr 250,000 that may cross-link actin filaments in vivo into supramolecular structures such as networks and bundles. We used millimolar Ca protease from chicken breast muscle to cleave the subunit into smaller fragments that we mapped with respect to the overall structure of the dimer. The enzyme cleaves HeLa filamin into a larger (Mr 192,000) and a smaller (Mr 104,000) fragment; the smaller fragment is the precursor of a still smaller (Mr 92,000) fragment. Only the larger fragment bound to actin in a cosedimentation test, suggesting that it contains the actin-binding region of the subunit. Digestion of HeLa filamin that had been cross-linked with dimethyl adipimidate produced a good yield of the Mr 192,000 fragment but a poor yield of the Mr 104,000/92,000 fragments. Since native filamins are head-to-head dimers, it was expected that cross-linking would proceed most readily at the dimerization site and, therefore, it appears that the Mr 192,000 fragment is cleaved from cross-linked filamin because it is distal to the dimerization region, while the Mr 104,000/92,000 fragments are absent because they lie at the dimerization region and were cross-linked to a form that was not identifiable by sodium dodecyl sulfate electrophoresis.

Actins↗

PubMed1990

Abnormalities of the erythrocyte membrane in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a hematologic malignancy characterized by excessive growth of myeloid cells and their progenitors. The proportion of spectrin dimers compared to tetramers extracted from membranes at 4 degrees C, under low ionic strength conditions, increased in CML erythrocytes. These also displayed abnormal thermal sensitivity (between 45 and 46 instead of 49 degrees C). Crosslinking with the bifunctional reagent, dimethyl adipimidate (8.6 A) showed significant organizational modification of not only spectrin, but other cytoskeletal components such as ankyrin, bands 4.2 and 5. Enhanced concanavalin A (Con-A) agglutinability of CML erythrocytes also suggests altered topographic distribution of a functionally important membrane protein, band 3. The anion transport activities of erythrocytes from patients with CML and normal donors were comparable. In CML erythrocytes, significant reduction in the number of ankyrin-binding sites, present in the cytoplasmic domain of band 3, may lead to partial loss of cytoskeletal anchorage to the bilayer and account for their increased Con-A agglutinability and heat sensitivity and may lead to their premature removal from the circulation.

Agglutination↗

PubMed1998

Activation of bovine tracheal chloride channels by amino group-specific reagents.

1. The predominant Cl- channel in bovine tracheal epithelial cells has a conductance of approximately 71 pS and accounts for more than 80 % of the total chloride conductance. We examined the effects of protein-modifying reagents on channel function and found that amino groups are critically involved in gating. 2. Patch clamp studies showed that lysine-specific reagents, such as dimethyl adipimidate (DMA), significantly increased the channel open probability, but not its conductance. This suggests that modified residues are involved in the gating mechanism, but are distant from the channel permeation pathway. 3. Kinetic analysis of channel activity showed that histograms of open and closed durations could be well fitted by double exponential distributions, suggesting that the channel has at least two open and two closed states. DMA did not change the number of open or closed states, but increased channel mean open time. 4. Since membrane impermeant reagents were effective only from the extracellular side, we conclude that lysine residues in the extracellular domain of the channel are critically involved in gating. These residues may present an important target for site-directed mutagenesis and pharmacological activation of Cl- channels in epithelial cells.

Animals↗

PubMed1995

Production of specific antibodies and development of a non-isotopic immunoassay for carbamazepine by the carbonyl metallo-immunoassay (CMIA) method.

As part of our ongoing work to extend the range of applications of the non-isotopic carbonyl metalloimmunoassay (CMIA), previously developed in our laboratory, we describe here the first CMIA study of carbamazepine. The CMIA method uses a metal carbonyl complex as a non-isotopic tracer, and in this case we chose to employ the dicobalt hexacarbonyl moiety (Co2(CO)6) attached to an alkyne. Two organometallic tracers, 3 and 7, were synthesized, differentiated by the nature and length of the spacer arm of the Co2(CO)6 moiety. Two different coupling methods were subsequently used to synthesize the immunogens 1 and 2, the first one used a carbodiimide, while the second, employed dimethyl adipimidate as coupling agent. Titer values of the antisera obtained by injection of these immunogens into rabbits, were determined by CMIA, using one of the organometallic complexes, 3 or 7, as tracer. Both antisera had higher titer values with the long-chain tracer, 7, than with the short-chain tracer, 3. However these titer values were very different: low for antiserum 1 and high for antiserum 2. The cross-reactivity of antiserum 2 with other antiepileptic drugs was negligible. For competition curves, there was good sensitivity with the antibody 2/3 pairing, while a broad assay range was obtained with antibody 2/7 pairing. These results demonstrate the viability of CMIA as an immunoassay method for carbamazepine, and open the way to development of a simultaneous multiassay by CMIA of the principal antiepileptic drugs.

Animals↗

PubMed1987

Cross-linking of poliovirus with bifunctional reagents: biochemical and immunological identification of protein neighbourhoods.

Poliovirus was treated with the homobifunctional reagents 1,5-bis (succinimidooxycarbonyloxy)pentane (BSOCOP) and dimethyl adipimidate, both reacting with epsilon-amino-groups of lysines and N-terminal amino acids, respectively. Cross-links between separate virus particles did not occur and their physical stability remained unaltered as determined by sucrose gradient centrifugation. Using BSOCOP intermolecular protein complexes of different sizes, namely 59K, 71K and 92K, were obtained. Their compositions were identified after cleavage by the relative mobilities of the proteins in SDS-PAGE and particularly by immunoblot analysis using protein-specific polyclonal antisera. The three major capsid proteins were involved in cross-linking. The 59K complex consisted of VP1-VP3, the 71K complex of VP1-VP2 and the 92K complex of VP2-VP1-VP3, reflecting neighbourhoods of the respective proteins in the icosahedral virus capsid. It is suggested that cross-linking took place between proteins in a protomer rather than between proteins of adjacent protomers, trimers or pentamers.

Antigens, Viral↗