Reaction of 1-fluoro-2,4-dinitrobenzene and 2,4,6-trinitrobenzenesulphonic acid with membranes of sarcoplasmic reticulum.

Membranes of sarcoplasmic reticulum were labelled with 1-fluoro-2,4-dinitro[3H]benzene at pH 6.5 and with 2,4,6-trinitrobenzenesulphonate at pH 9.2. Conditions were chosen to restrict reaction to amino groups, and the effect of blockings of these groups by methyl acetimidate was determined. All proteins were labelled to some extent by both reagents, but, whereas the trinitrophenylation of both lipid and protein amino groups was almost completely blocked by methyl acetimidate, the dinitrophenylation of the ATPase at pH 6.5 was much less affected. The seven amino groups on the ATPase that were labelled under these conditions did not react with methyl acetimidate. This reagent can therefore be used to enhance the specificity of fluorodinitrobenzene for amino groups in a hydrophobic environment. The amino groups on the minor proteins and on the phospholipids that reacted with fluorodinitrobenzene at pH 6.5 were probably in an aqueous environment, since the reaction was blocked by methyl acetimidate.     

Characterization of the reaction of methyl acetimidate with sperm whale myoglobin.

The effects of pH, acetimidate concentration, temperature, and reaction time of methyl acetimidate with sperm whale myoglobulin have been assessed. Reaction at pH 9.8 and 15 degrees C for 30 min with a sixfold excess of methyl acetimidate relative to each amino group yielded six acetimidomyoglobin derivatives which were separated and purified. Reaction with tetrahydrophthalic anhydride revealed the number of amino groups that remained unreacted in each separated component and made possible further subractionation. Modification at the NH2 terminus was quantitated by automated stepwise Edman degradation. The acetimidyl and tetrahydrophthalyl groups, were readily removable. The potentiometric titration of three of the completely deprotected components showed identity with the parent untreated sperm whale myoglobin. The first of two major products was acetimidated at all 19 epsilon-amino groups but not at the NH2 terminus. The second major product bore a blocked NH2 terminus but retained one unmodified epsilon-amino group, identified after modification by trinitrobenzenesulfonate as lysine residue 77. Of the minor components, one was identified as completely acetimidated at all 20 amino groups. The other three minor components appeared to contain irreversible by-products.     

Crosslinking and modification of Na,K-ATPase by ethyl acetimidate.

Monofunctional imidoesters such as ethyl acetimidate can induce crosslinking of subunits of the (Na⁺ + K⁺) ion-stimulated ATPase. The cross-linked product is shown to be composed of equal parts of two subunits: one phosphorylated by γ-[³²P]ATP, the other a glycoprotein. Because crosslinking of proteins by imidoesters normally requires reaction at both ends of a bifunctional reagent, the reaction is unexpected. A model for the reaction is proposed, in which a favorably positioned amino group on one subunit displaces the amidino group on the other, forming a covalent diamidino crosslink between the two subunits.Reaction with imidoesters also partially inhibits the Na,K-ATPase and reduces the sensitivity of the phosphorylated form of the enzyme to potassium ion. This modification resembles the effect of ouabain, a specific inhibitor of Na,K-ATPase, and is independent of crosslinking.     

Light-enhanced cross-linking of rhodopsin in rod outer segment membranes as detected by chemical probes

Bovine rod outer segment membranes were treated with cross-linking reagents before and after light exposure. Bleached membranes showed enhanced cross-linking with difluorodinitrobenzene or methyl acetimidate compared to dark-adapted membranes. The light-induced enhancement of cross-linking may be due to increased association of rhodopsin monomers in the light and/or due to increased reactivity of amino and sulfhydryl groups of bleached rhodopsin. In some instances, the band ascribed to the rhodopsin monomer in gel electrophoresis appears as a partially resolved doublet. Treatment of bleached rod outer segment membranes with methyl acetimidate improved the resolution of the doublet into two closely migrating bands.     

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.     

Inhibition of Na+-Ca2+ exchange in heart sarcolemmal vesicles by phosphatidylethanolamine N-methylation

The effect of phosphatidylethanolamine N-methylation on Na+-Ca2+ exchange was studied in sarcolemmal vesicles isolated from rat heart. Phosphatidylethanolamine N-methylation following incubation of membranes with S-adenosyl-L-methionine, a methyl donor for the enzymatic N-methylation, inhibited Nai+-dependent Ca2+ uptake by about 50%. The N-methylation reaction did not alter the passive permeability of the sarcolemmal vesicles to Na+ and Ca2+ and did not modify the electrogenic characteristics of the exchanger. The depressant effect of phosphatidylethanolamine N-methylation on Nai+-dependent Ca2+ uptake was prevented by S-adenosyl-L-homocysteine, an inhibitor of the N-methylation. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino-group-blocking agent, also prevented methylation-induced inhibition of Ca2+ uptake. In the presence of exogenous phospholipid substrate, the phospholipid N-methylation process in methyl-acetimidate-treated sarcolemmal vesicles was restored and the inhibitory effect on Ca2+ uptake was evident. These results suggest that phosphatidylethanolamine N-methylation influences the heart sarcolemmal Na+-Ca2+ exchange system.     

Transbilayer exchange of phosphatidylethanolamine for phosphatidylcholine and N-acetimidoylphosphatidylethanolamine in single-walled bilayer vesicles.

A preparation of small single-walled liposome vesicles containing a 9:1 mole ratio of phosphatidylcholine to phosphatidylethanolamine was subjected to reaction with the membrane-impermeable reagent, isethionyl acetimidate hydrochloride. This reagent converted 90% of the external phosphatidylethanolamine groups to the amidine derivative, leaving the mole ratio of unreacted phosphatidylethanolamine to phosphatidylcholine on the outside surface of the vesicle much lower than that on the inside surface. Equilibration of phosphatidylethanolamine across the bilayer was then measured as a function of time by monitoring the appearance of phosphatidylethanolamine on the outside surface utilizing the reaction of the amino groups with 2, 4, 6-trinitrobenzenesulfonic acid. The results show that no new phosphatidylethanolamine appeared on the external surface of the vesicles over a period of 12 days at 22 degrees. A conservative estimate of the precision of the measurements is +/- 10%. On this basis, the estimated half-time for the equilibration of phosphatidylethanolamine across the bilayer of these vesicles must be at least 80 days at 22 degrees.     

Elution of interferon beta from blue sepharose by poly(vinylpyrrolidone)

Pre-equilibration of erythrocytes with the membrane-impermeable aldehyde, pyridoxal 5'-phosphate, for 30 min at 22 degrees C, prior to the addition of methyl acetimidate to the incubation mixture has been shown to prevent agglutination of acetamidinated cells which were resuspended in immune serum (Chao, T.L. and Berenfeld, M.R. (1981) J. Biol. Chem. 256, 5324-5326). This observation led to the possibility that the immune reaction, observed in some sickle cell anemia patients to reinfused cells which had been reacted with methyl acetimidate, could be prevented. The present communication further evaluates that reaction sequence and shows that while the pre-equilibration of cells with pyridoxal 5'-phosphate does protect membrane amines from reaction with methyl acetimidate, the protection is not extensive enough to prevent an immune response in a sickle cell anemia patient who had already been sensitized against acetamidinated cells. It is apparent that the design of antisickling agents which covalently modify hemoglobin must take into account protection of functional groups in the erythrocyte membrane, modification of which could produce an immunogenic response.     

Properties of methyl acetimidate and its use as a protein-modifying reagent.

The rate of hydrolysis of the imido ester methyl acetimidate and its rate of amidination of denatured aldolase were investigated under different conditions of temperature, pH and ionic strength. Both rate constants increase greatly with temperature, whereas ionic strength has no effect on either. The effect of pH is more complex. Between pH 6.8 and 8.8 the rate of hydrolysis decreases and the rate of amidination increases. These results are discussed in terms of the reaction mechanisms involved.     

Activation of Ca2+-stimulated ATPase by phospholipid N-methylation in cardiac sarcoplasmic reticulum

Incubation of cardiac sarcoplasmic reticulum (SR) in the presence of S-adenosyl-L-methionine, a methyl donor for the enzymatic N-methylation of phosphatidylethanolamine, increased Ca2+-stimulated ATPase activity. The increase in Ca2+-ATPase activity was not due to changes in the affinity for Ca2+ and was prevented by methyl acetimidate, an inhibitor of phospholipid N-methylation. The results suggest a possible regulatory role of phospholipid N-methylation in SR Ca2+-pump mechanism.     

Modification of sodium and gating currents by amino group specific cross-linking and monofunctional reagents.

To test the possible role of lysine residues in Na channel function the effects of several imidoesters on Na and gating currents were studied in voltage-clamped single frog nerve fibers. Mono- and bisimidoesters were used. These reagents modify amino groups exclusively and do not change the net charge. The three bisimidoesters used easily introduce cross-links between neighboring amino groups. Their structure is almost identical; only the length of the spacers between the two amino-reactive groups is different. An irreversible reduction of Na currents and gating currents was observed with the longest (dimethyl suberimidate [DMS]) and the shortest (dimethyl adipimidate [DMA]) of the cross-linkers used. Of the three cross-linking reagents only the shortest made Na current inactivation slow and incomplete. The steady-state inactivation curve, h infinity (E), was shifted by greater than 25 mV in the hyperpolarizing direction by each of the reagents. The voltage dependence of activation, however, remained unchanged. Furthermore, the effects of two different monoimidoesters (ethyl acetimidate [EAI] and isethionyl acetimidate [IAI]) on gating currents were tested. EAI can penetrate a membrane, whereas IAI is membrane impermeant. IAI was almost without effect, whereas EAI caused a considerable reduction of the gating currents. EAI and DMS reduced the Qoff/Qon ratio without affecting the decay of the Na currents. The results show that lysine residues are critically involved in Na channel gating.     

Activation/inactivation of human angiotensin I converting enzyme following chemical modifications of amino groups near the active site

We have studied the effects of amidination of lysyl residues on the activity of angiotensin I converting enzyme isolated from human kidney. Anion concentration was an important reaction variable. In 4 M chloride or acetate, amidination with methyl acetimidate produced derivatives with up to a 4-fold increase in activity with hippuryl-glycyl-glycine as substrate. Modification with methyl p-hydroxybenzimidate also increased activity while treatment with methyl 4-mercaptobutyrimidate resulted in a 90% loss of activity. The effects of amidination were partially prevented when the reactions were carried out in the presence of the inhibitors, captopril or 5S-benzamido-4-oxo-6-phenyl-hexanoyl-L-proline. These results suggest that lysyl residues are present near the active site while different amino groups have a role in anion activation.     

Preparation and properties of a holo-lactate dehydrogenase enzyme reactor

NAD+ was the base material for syntheses of coenzyme analogs with reactive groups bound to N6 of the adenine moiety via spacers that are 3-17 A long. These analogs were used for the modification of dehydrogenases. Aromatic imidoesters and acyl azides are suitable reactive groups, which form covalent amidinium or amide bonds with amino acid residues such as the epsilon-amino groups of lysines. The catalytic function of the modified protein decreased only slightly. Coenzymes that are linked via a spacer to carboxyl and amino groups are fixed to the protein by means of carbodiimides and hydroxysuccinimide. Coenzyme-bound aromatic imidoesters with spacer lengths of more than 12 A were incorporated to the extent of 60% at the active site. Aliphatic imidoesters proved to be inefficient for protein modification because of fast hydrolysis. Fixing of coenzyme analogs containing appended carboxyl or amino groups to enzyme in the presence of carbodiimides resulted in a decrease of enzyme activity. Modified lactate dehydrogenase and L-alanine dehydrogenase formed an enzyme reactor for the production of L-alanine in the absence of free NAD+. Both enzymes were cross-linked by dimethyl suberimidate in the presence or absence of NAD+, bis-NAD+, pyruvate, and oxamate. Site-to-site directed cross-linking yielded a reaction mixture from which four protein fractions were obtained by isoelectric focusing; one of these showed a cycling rate of 600 h-1.     

Stimulation of Ca2+-pump in rat heart sarcolemma by phosphatidylethanolamine N-methylation

Incubation of purified cardiac sarcolemmal vesicles (SL) in the presence of S-adenosyl-L-methionine, a methyl donor for the enzymatic N-methylation of phosphatidylethanolamine (PE), increased the Ca2+-stimulated ATPase and ATP-dependent Ca2+ accumulation activities. Quantitative analysis of the methylated phospholipids revealed that maximal increase of Ca2+-pump activities was associated with predominant synthesis and intramembranal accumulation of phosphatidyl-N,N-dimethylethanolamine. The stimulation of SL Ca2+-pump activities was prevented by inhibitors of PE N-methylation such as S-adenosyl-L-homocysteine and methyl acetimidate hydrochloride. The results suggest a possible role of PE N-methylation in the regulation of Ca2+-transport across the heart SL membrane.     

Enhanced Cofactor and Allosteric Effector Activity of 3′-Pyrophospho Coenzyme A and Its Acetate

To prepare a chemically modified urokinase that does not dissociate into two peptide fragments upon reduction of its disulfide bridge, we cross-linked the enzyme intramolecularly with various bifunctional imidoesters. The enzyme underwent the intramolecular cross-linking most moderately by the reaction at 4^QC for 5 hr with 3mm dimethyl suberimidate in 0.1 M potassium phosphate buffer (pH 9.0). The cross-linked urokinase isolated by gel filtration with a yield of 25 % showed a specific activity of 76,000 International Units/mg protein, which corresponds to 53% of that of the native enzyme. Although the modified enzyme was similar to the native urokinase in some properties such as the autocatalytic self-digestion and the low affinity to fibrin, it showed higher in vivo and in vitro stabilities than the native one.     

Site-directed chemical modification and cross-linking of a monoclonal antibody using equilibrium transfer alkylating cross-link reagents

A new, more reactive group of protein cross-linkers in the class of equilibrium transfer alkylating cross-link (ETAC) reagents has been synthesized. These compounds include alpha,alpha-bis[(p-chlorophenyl)methyl]- and alpha,alpha-bis[(p-tolylsulfonyl)methyl]acetophenones substituted in the acetophenone ring with chloro, nitro, amino, and carboxyl groups and derivatives. Included are an 125I-labeled ETAC reagent and a 111In-labeled DTPA (diethylenetriaminepentaacetic acid) ETAC for site direction and biodistribution studies. These ETAC compounds were reacted with unreduced and partially reduced antibody under mild pH (pH 4-8) and room temperature conditions to give cross-linked structures. Examination of resultant cross-linked antibody via size-exclusion HPLC, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and an enzyme linked immunosorbent assay revealed that (1) both interantibody as well as intraantibody cross-linking had occurred; (2) the level of inter- and intraantibody cross-linking varied with the substituent on the ETAC; (3) the stability of the cross-links on the reducing SDS gels varied with substituents on the ETAC; (4) little if any immunoreactivity was lost after reaction with one of the more effective ETAC cross-linking compounds; (5) the 125I-labeled ETAC sulfhydryl cross-linking in partially reduced antibody increased with pH whereas amine cross-linking with the unreduced antibody decreased with pH; (6) the optimum pH for sulfhydryl site direction was pH 5.0; (7) the 111In DTPA ETAC labeled antibody had a biodistribution in CD1 mice similar to that of the 111In bis cyclic anhydride DTPA labeled antibody.     

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 hydrophobic 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⁺ permeabilty. The data also suggest that these amino groups are in a hydrophobic domain.     

Folding domains and intramolecular ionic interactions of lysine residues in glyceraldehyde 3-phosphate dehydrogenase.

1. Treatment with methyl acetimidate was used to probe the topography of several tetrameric glyceraldehyde 3-phosphate dehydrogenases, in particular the holoenzymes from rabbit muscle and Bacillus stearothermophilus. During the course of the reaction with the rabbit muscle enzyme, the number of amino groups fell rapidly from the starting value of 27 per subunit to a value of approx. five per subunit. This number could be lowered further to values between one and two per subunit by a second treatment with methyl acetimidate. The enzyme remained tetrameric throughout and retained 50% of its initial catalytic activity at the end of the experiment. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that only one amino group per subunit, that of lysine-306, was completely unavailable for reaction with imido ester in the native enzyme. This results is consistent with the structure of the highly homologous glyceraldehyde 3-phosphate dehydrogenase of lobster muscle deduced from X-ray-crystallographic analysis, since lysine-306 can be seen to form an intrachain ion-pair with aspartic acid-241 in the hydrophobic environment of a subunit-subunit interface. 3. Several other amino groups in the rabbit muscle enzyme that reacted only slowly with the reagent were also identified chemically. These were found to be located entirely in the C-terminal half of the polypeptides chain, which comprises a folding domain associated with catalytic activity and subunit contact in the three-dimensional structure. Slow reaction of these 'surface' amino groups with methyl acetimidate is attributed to intramolecular ionic interactions of the amino groups with neighbouring side-chain carboxyl groups, a conclusion that is compatible with the reported three-dimensional structure and with the dependence of the reaction of ionic stength. 4. Very similar results were obtained with the enzymes from B. stearothermophilus and from ox muscle and ox liver, supporting the view that the ion-pair involving lysine-306 and aspartic acid-241 will be a common structural feature in glyceraldehyde-3-phosphate dehydrogenases. The B. stearothermophilus enzyme was fully active after modification. 5. No differences could be detected between the enzymes from ox muscle and ox liver, in accord with other evidence that points to the identify of these enzymes. 6. The pattern of slowly reacting amino groups in the enzyme from B. stearothermophilus, although similar to that of the mammalian enzymes, indicated one or two additional intramolecular ionic interactions of lysine residues that might contribute to the thermal stability of this enzyme.     

Synthesis and characterization of novel thiol-reactive poly(ethylene glycol) cross-linkers for extracellular-matrix-mimetic biomaterials

Synthetic extracellular matrix hydrogels can be used for three-dimensional cell culture, wound repair, and tissue engineering. Using the bifunctional electrophile poly(ethylene glycol) diacrylate (PEGDA), thiol-modified glycosaminoglycans and polypeptides can be cross-linked into biocompatible materials in the presence of cells or tissues. However, the rate of in situ cross-linking with PEGDA under physiological conditions may occur too slowly for clinical applications requiring a fast-curing preparation. To explore a wider range of cross-linking time courses, five homo-bifunctional PEG derivatives were synthesized and examined as cross-linking agents for thiol-modified derivatives of hyaluronan (HA). Thiol reaction rate constants were measured over a pH range of 7.4 to 8.6. The order of reactivity for the functional groups used was determined to be maleimide > iodoacetate > bromoacetate > iodoacetamide > acrylate > bromoacetamide, with rates increasing exponentially with increasing pH. The range of gelation times at physiological pH varied from less than 1 min to over 2 h. Addition of the cross-linkers to cell culture medium showed minimal cytotoxicity toward primary human dermal fibroblasts at concentrations anticipated during in situ cross-linking. Moreover, hydrogels prepared from thiol-modified gelatin and thiol-modified HA were biocompatible and supported attachment and proliferation of fibroblasts and hepatocytes.     

Orientation of ferrochelatase in bovine liver mitochondria

The orientation of ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, was examined in bovine liver mitochondria. The ability of a membrane-impermeable sulfhydryl reagent, 4,4'-dimaleimidylstilbene-2,2'-disulfonic acid, to inactivate ferrochelatase in intact or disrupted mitochondria and mitoplasts was examined. Using succinate dehydrogenase as an internal marker, it was found that ferrochelatase was inactivated only in disrupted mitochondria and mitoplasts, suggesting an internal location for the active site of the enzyme. In addition, antibodies raised against purified ferrochelatase were found to inhibit activity only in disrupted but not in intact mitoplasts. These data demonstrate that in bovine liver mitochondria ferrochelatase is located on the matrix side of the inner mitochondrial membrane. Data obtained with the membrane-impermeable amino reagent isethionyl acetimidate indicate that ferrochelatase physically spans the inner mitochondrial membrane with portions of the protein exposed on both sides of the membrane.