[Reactions with imidoesters. 13. Preparation of fluorescent nitriles and imidoesters. Possible application as indicators in peptide chemistry].
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Chemical Phenomena↗
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Imidoester inhibition of lymphocyte DNA synthesis.
Imidoesters amidinate free amino groups and produce inter- and intramolecular covalent bonds. To determine whether imidoesters influenced lymphocyte transformation, human peripheral blood or calf lymph node lymphocytes were cultured with dimethyladipimate (DMA), a bifunctional (cross-linking) imidoester, or methyl acetimidate (MAC), a monofunctional (noncross-linking) imidoester. Both DMA and MAC decreased the rate of endogenous DNA synthesis in a dose-dependent fashion. In further work, lymphocytes were treated with Phaseolus vulgaris phytohemagglutinin, concanavalin A, or periodate. DMA (1 mM) decreased DNA synthesis in P. vulgaris phytohemagglutinin-stimulated human cells by 65%, Concanavalin A-stimulated cells by 98.2%, and periodate-stimulated cells by 85%. Similar results were obtained with 1 mM MAC. Inhibition by DMA was slightly greater than was the inhibition by MAC. Decreased DNA synthesis resulted if DMA was added to P. vulgaris phytohemagglutinin-stimulated human lymphocytes at initiation of culture (72%) or after 16 hr (75%); inhibition was less when DMA was added after 24 hr (43%) and was not apparent if added after 48 hr. Therefore, both monofunctional and bifunctional imidoesters inhibit endogenous and stimulated DNA synthesis in human and calf lymphocytes.
Cells, Cultured↗
Quaternary structure of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus studied by a new reversible cross-linking procedure with bis(imidoesters).
The bis(amidine) cross-links formed between protein subunits by treating them with bis(imidoesters) were found to be rapidly broken by exposing the cross-linked proteins to methylamine buffers containing the aprotic solvent acetonitrile. This cleavage step could be introduced between the two dimensions of a diagonal gel electrophoretic separation of cross-linked proteins to facilitate identification of the contributors to a cross-linked species. Tests with the tetrameric enzyme glyceraldehyde-3-phosphate dehydrogenase demonstrated the simplicity and effectiveness of the technique. When the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus was treated with a range of bis(imidoesters), from dimethyl succinimidate to dimethyl suberimidate, the most informative set of products was obtained with dimethyl glutarimidate. The longer bis(imidoesters) caused too extensive cross-linking of the enzyme subunits, although the beta chain of the pyruvate decarboxylase component always appeared to be the most resistant. Almost all the cross-linked species up to pentamers of the lipoate acetyltransferase polypeptide chain (apparent Mr approximately 280 000) were identified by means of the diagonal gel electrophoretic procedure after cleavage of the cross-links. The introduction of the methylamine cleavage step enables the bis(imidoester) for such experiments to be selected purely for the efficacity of its cross-linking reaction with the protein and dispenses with the need to incorporate a specially cleavable bond in the reagent.
Chemical Phenomena↗
Stabilization of vasopressin-induced membrane events by bifunctional imidoesters.
Vasopressin increases the water permeability of the luminal membrane of the toad bladder epithelial cell. This change in permeability correlates with the occurrence in luminal membranes of intramembrane particle aggregates, which may be the sites for transmembrane water flow. Withdrawal of vasopressin is ordinarily associated with a rapid reduction of water flow to baseline values and a simultaneous disappearance of the particle aggregates. The bifunctional imidoesters dithiobispropionimidate (DTBP) and dimethylsuberimidate (DMS), which cross-link amino groups in membrane proteins and lipids, slow the return of water flow to baseline after vasopressin withdrawal. Cross-linking is maximal at pH 10, and is reduced as pH is lowered. Freeze-fracture studies show persistence of luminal membrane particle aggregates in cross-linked bladders and a reduction in their frequency as water flow diminishes. Fusion of aggregate-containing cytoplasmic tubular membrane structures with the luminal membrane is also maintained by the imidoesters. Reductive cleavage of the central S-S bond of DTBP by beta-mercaptoethanol reverses cross-linking, permitting resumption of the rapid disappearance of the vasopressin effect. Bladders that have undergone DTBP cross-linking and beta-mercaptoethanol reduction respond to a second stimulation by vasopressin. Thus, the imidoesters provide a physiologic and reversible means of stabilizing normally rapid membrane events.
Animals↗
The cross-linking of tubulin with imidoesters.
Tubulin was reacted with a monofunctional imidoester, ethyl acetimidate, and three bifunctional imidoesters ranging in extension from 5 to 10 A. Extensive cross-linking was found to occur with the three bifunctional reagents resulting in the formation of dimers, trimers, tetramers, pentamers, and hexamers. In addition, a similar cross-linking patterns was observed with the monofunctional reagent. This type of cross-linking is rarely seen when proteins are reacted with imidoesters. Given that the cross-linking span of ethyl acetimidate is only 3 A it is reasonable to infer that there are nucleophilic groups in close proximity within the tubulin dimer. Amidinated tubulin is still capable of assembling into microtubules.
Amino Acids↗
Preparation and reactions of an iodinated imidoester reagent with actin and alpha-actinin.
The chemical iodination of an imidoester (methyl-p-hydroxybenzimidate, Wood et al. (1975) Anal. Biochem. 68, 339) and subsequent coupling of iodinated imidoester (IIE) to protein is an indirect method of iodinating proteins that is specific for the epsilon amino group of lysine residues and maintains the positive charge on the amino group at physiological pH. Purification of the IIE from chloramine-T and free iodine by benzene extraction eliminates the need for isoelectric precipitation and produces a more time- and cost-efficient IIE preparation and purification protocol. The separation of free from protein-bound label by chromatography, using centrifugal elution, provides a separation method that is rapid and efficient, without the generation of large volumes of radioactive wastes characteristic of conventional chromatographic and dialysis methods. To optimize the parameters of labeling protein with IIE, a systematic assessment of the effects of pH, reactant concentrations, and reaction time was made using purified cardiac actin and gizzard alpha-actinin. The parameters were defined to achieve an average labeling ratio of one IIE per protein polypeptide. The data demonstrate that both proteins appear to be labeled at the same rate and define several determining factors that limit the rate and extent of IIE incorporation into protein.
Actinin↗
Effect of chemical modification by (di)imidoesters on cells and cell envelope components of Escherichia coli and Salmonella typhimurium.
Cells and cell envelope components of Escherichia coli and Salmonella typhimurium were treated with mono- and bifunctional imidates like methylbutyrimidate, methyl-4-mercaptobutyrimidate and dimethylsuberimidate, and after determination of the appropriate conditions, the effects on structure and function were investigated. Within 10 min after treatment of the bacteria with 5 mM of the (di)imidoesters, active transport, DNA-, RNA-, protein- and lipid-synthesis were severely inhibited. After crosslinking, cell envelope components, e.g. phospholipids, lipopolysaccharide, and periplasmic proteins, became partly inextractable. Crosslinked cells showed increased resistance to ultrasonic treatment and osmotic or detergent induced lysis. By the reaction of (di)imidoesters with phospholipids containing no amino groups, it could be demonstrated that non-amidine products were also formed. In addition, freeze-fracturing and freeze-etching demonstrated distinct crosslinked areas in the cell envelope of E. coli B.
Bacterial Proteins↗
Cross-linking of membrane phospholipid and protein using putative monofunctional imidoesters.
The reaction of methyl acetimidate or isethionyl acetimidate with mitoplasts at pH 8.5 yields two derivatives of phosphatidylethanolamine. These derivatives are shown to be the mono-amidine derivative and the bis-derivative of phosphatidylethanolamine. The bis-derivative represents one phosphatidylethanolamine cross-linked to another phosphatidylethanolamine. Similar derivatives are formed by the reaction of dipalmitoyl phosphatidylethanolamine with these imidoesters in organic solution with the exception that much more monoderivative is produced. Methyl picolinimidate reacts with phosphatidylethanolamine of mitoplasts to form primarily the mono-derivative. The bis-derivative was not detected. The reaction of bovine rod outer segment discs with methyl acetimidate causes cross-linking of 30% of the membrane rhodospin as dimers. Putative monofunctional imidoesters cause considerable cross-linking of both phospholipids and proteins in cell membranes. Cross-linking can be minimized at pH 9.0.
Animals↗
Activation of liver alcohol dehydrogenases by imidoesters generated in solution.
Various omega-halogenated carboxy acids and amides were evaluated as potential active-site-directed reagents for alcohol dehydrogenase. 2-Bromoacetamide and bromoacetic and 3-bromopropionic acids inactivated the enzyme; AMP, NAD+, and NADH markedly decreased the rate of inactivation. Some omega-halogenated carboxyamides, X(CH2)nCONH2, increased the activity of the enzyme with the rate and extent of activation depending on the number of methylene units (n) in the order 3 greater than 4 greater than 2 and on X in the order Br greater than Cl. 4-Chlorobutyramide (0.1 M) activated the horse liver enzyme 20-fold in 24 hr at pH 8.0 and 25 degrees. The activation was not prevented by AMP or 2,2-bipyridine, but was by NADH. The kinetic constants and turnover numbers for human and horse liver alcohol dehydrogenases treated with chlorobutyramide were increased markedly compared to those for native enzymes. Alcohol dehydrogenase treated with chlorobutyramide was not further activated by methyl picolinimidate, an imidoester which activates native enzyme by modifying amino groups in the active sites. Chlorobutyramide does not appear to react directly with the enzyme but cyclizes in the reaction medium to form an intermediate imidoester, 2-iminotetrahydrofuran, which reacts with most of the amino groups of the enzyme.
Adenosine Monophosphate↗
Effects of chemical modification of amino groups by two different imidoesters on voltage-clamped nerve fibres of the frog.
Voltage clamped single nerve fibres of the frog Rana esculenta were treated with the amino groups specific reagents ethyl acetimidate and isethionyl acetimidate. Ethyl acetimidate is lipid soluble, relatively non-polar and can penetrate a membrane. Isethionyl acetimidate is lipid-insoluble, polar and membrane-impermeant. Treatment with ethyl acetimidate caused an irreversible reduction of Na currents and a shift of the voltage dependence of the steady-state sodium inactivation, h infinity (E), in the hyperpolarizing direction. The voltage dependence of sodium activation was much less affected and a small shift into the depolarizing direction was observed. By contrast, the non-permeant reagent did not reduce the sodium currents and the shifts of the h infinity (E) curve were smaller than the shifts caused by ethyl acetimidate. Furthermore, a small shift of the voltage-dependence of activation in the hyperpolarizing direction was observed. As the modification procedure with imidoesters does not cause a change of net charge, the results cannot be explained by an effect of surface charge modification; rather, they seem to be due to a direct effect of amino group modification on the voltage dependence of inactivation.
Alkanesulfonates↗
The effect of imidoesters, fluorodinitrobenzene and trinitrobenzenesulfonate on ion transport in human erythrocytes.
Several amino-reactive chemical probes which differ in hydrophobicity and charge and in their ability to penetrate the red cell membrane were tested for their ability to modify K+ leak and inorganic phosphate (Pi) leak in intact human red cells. Methyl picolinimidate (MP), ethyl acetimidate (EA), methyl acetimidate (MA) are hydrophilic penetrating probes whereas isethionylacetimidate (IA) is a hydrophilic non-penetrating probe. The order of their effectiveness in inhibiting Pi leak was found to be MP > EA > MA > IA. This order is in decreasing hydrophobicity and suggests that some penetration into the bilayer or into hydrophoblic domains of the anion transport protein is required to modify an amino group required for Pi permeability through the membrane. These imidoesters have little or no effect on K+ leak in the red cell. Trinitrobenzenesulfonate (TNBS) a relatively non-penetrating hydrophobic anionic probe and fluorodinitrobenzene (FDNB) a penetrating hydrophobic neutral probe have markedly different effects on K+ and Pi leak. TNBS has little effect on K+ leak but markedly inhibits Pi leak. The effect of TNBS on Pi leak is not blocked by prior treatment with IA suggesting that these probes sense different populations of amino groups in the membrane. FDNB nearly completely blocks Pi leak and markedly increases K+ leak. The results with TNBS and FDNB indicate an asymmetric arrangement of amino groups on the red cell membrane. Certain amino groups on the outer surface of the membrane regulate Pi permeability whereas certain amino groups on the inner surface of the membrane regulate K+ permeability. The data also suggest that these amino groups are in a hydrophobic domain.
Biological Transport↗
Covalent modification of rhodopsin with imidoesters: evidence for transmembrane arragnement of rhodopsin in rod outer segment disk membranes.
The transmembrane disposition of the visual pigment rhodopsin was studied by the covalent labeling of protein amino groups with membrane-permeable and -impermeable imidoesters. A new, highly reactive permeable reagent, 2-(methylsulfonyl)ethyl acetimidate (SAI) was developed for this purpose. The permeabilities of both this compound and the "impermeable" reagent isethionyl acetimidate (IAI) across the rod outer segment disk membrane were directly measured. Our results indicate that rhodopsin contains three classes of amino groups. One class (35--55% of the total) reacts rapidly with the membrane-impermeable reagent and is presumably exposed on the outside surface of the membrane. A second class (35--55% of the total) is located on the internal surface of the disk since its rate of reaction is dependent on the relative permeabilities of the labeling reagents. The remaining 10% of the rhodopsin amino groups are inaccessible to either type of imidate and are largely accounted for by the single lysine residue which specifically binds the chromophore retinal. These findings, taken together with evidence from freeze--fracture electron microscopy, imply that rhodopsin is a transmembrane protein.
Animals↗
Mitochondrial membrane-bound hexokinase of ascites tumor cells. Functional implications of lysine residues studied by modification with imidoesters.
Conformational changes of hexokinase from ascites tumor cells have been studied by chemical modification of lysine residues with imidoesters with the following results: 1) The membrane-bound enzyme, in contrast to the soluble enzyme, is not inactivated by treatment with dimethyl suberimidate, which suggests (a) lysine residue(s) essential for the activity that is protected in the membrane-bound enzyme. 2) Three different conformations have been detected in the membrane-bound enzyme. Two of these are induced by glucose and glucose 6-phosphate, respectively. 3) Treatment of the membrane-bound enzyme with dimethyl suberimidate affects its sensitivity to the inhibition by glucose 6-phosphate, but not its activity or degree of maximal inhibition. This suggests that lysine(s) is related to the binding of glucose 6-phosphate to its allosteric regulatory site. 4) In intact tumor cells, most, if not all, of the hexokinase activity seems to be in a membrane-bound form.
Allosteric Regulation↗
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↗
Aggregation of hemoglobin S modified by bifunctional imidoesters.
Sickle hemoglobin (Hb S) was cross-linked by two types of bifunctional imidoesters, dimethyladipimidate (DMA) and dimethyl-3,3'-dithiobispropionimidate (DTBP). These modified hemoglobins were separated into monomer, dimer and polymer fractions by gel filtration. All of these modified hemoglobins showed extremely left-shifted oxygen equilibrium curves with no cooperativity. The stabilities of these hemoglobins were also decreased. The solubilities of these modified hemoglobins in high-phosphate buffers were lower than those of native Hb S. Studies on the kinetics of the aggregation of these modified hemoglobins showed that intracross-linked Hb S with DMA and DTBP (DMA- and DTBP-modified monomeric Hb S) still retained the capability of aggregation with a delay time, while intercross-linked Hb S with DMA and DTBP (DMA- and DTBP-modified oligomeric Hb S) aggregated without a delay time. When the kinetics of aggregation was measured for mixtures of modified and native deoxy-Hb S, DMA-modified monomeric deoxy-Hb S shortened the delay time prior to aggregation of native deoxy-Hb S. The other modified deoxy-Hb S did not affect the delay time, suggesting that these modified oligomeric hemoglobins neither participate in the formation of nuclei nor copolymerize with native deoxy-Hb S.
Cross-Linking Reagents↗
Cross-linking of nucleosomal histones with monofunctional imidoesters.
Cross-linking experiments with the MONOfunctional imidoester methyl-acetimidate, in the pH range 7.0 - 8.0, on rat liver nucleosomes generate a cross-linking pattern almost identical with the one observed for much longer BIfunctional reagents (e.g. dimethylsuberimidate). Combined cross-linking and trypsin digestion experiments suggest that all or at least the great majority of this cross-linking occurs on trypsin digestible segments (or "tails") of the histones. The formation of oligomers over such extremely short distances and especially the observation of an H3 homodimer suggests a very close proximity of half-nucleosomes.
Animals↗
The synthesis of mono- and bifunctional nitriles and imidoesters carrying a fluorescent group.
The synthesis of nitriles and dinitriles carrying fluorescent dansyl groups is described. From these compounds the corresponding mono- and bifunctional imidoesters were prepared. They are suitable for labelling or crosslinking, respectively, of proteins under mild conditions.
Binding Sites↗
Imidoester iodination of immunoglobulins to high specific activity.
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Animals↗