Selective alteration of erythrocyte deformabiliby by SH-reagents: evidence for an involvement of spectrin in membrane shear elasticity

In order to elucidate the molecular basis of membrane shear elasticity, the effect of membrane protein modification by SH-reaents on the deformability of human erythrocytes was studied. Deformability was quuantified by measuring the elongation of erythrocytes subjected to viscometric flow in a transparent cone plate viscometer. Impermeable SH-reagents proved to have no mechanical effect. Many, but not all, permeable SH-reagents markedly decreased the elongation. Among these, bifunctional SH-reagents (e.g. diamide, tetrathionate and N, N' -p-phenylenedimaleimide) able to cross-link membrane SH-groups were more effective than monofunctional SH-reagents (e.g. N-ethylmaleimide and ethacrynic acid). The bifunctional SH-reagents produced a 50% decrease of elongation after modification of less than 5% of the membrane SH-groups. In contrast, for a comparable effect, more than 20% of the SH-groups had to be modified by the monofunctional reagents. The effect of SH-oxidizing agents was fully reversible after treatment with disulfide-reducing agents. All bifunctional SH-reagents induced a dimerization of a small fraction of spectrin. Anaalysis of the distribution of the diamide-induced disulfide bonds among the various membrane protein fractions showed that this agent preferentially acts on the spectrin polypeptides. The results provide direct experimental evidence that the native arrangement of spectrin is essential for the shear resistance of the erythrocyte membrane and that introduction of small numbers of intermolecular cross-links as well as modification within the molecule lead to a rapid loss of this function.     

Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane

After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A₂ cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B. (1976) Biochim. Biophys. Acta 436, 353–365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys. Acta 323, 178–193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of < 10⁶ dalton.Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents.It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.     

Intra- and intermolecular cross-linking of membrane proteins in intact erythrocytes and ghosts by SH-oxidizing agents

In intact human erythrocytes, SH-oxidizing agents exclusively cross-link spectrin via disulfide bonds. In ghosts, additional dimerization of the major intrinsic protein, band 3, is observed. After blockade of intracellular GSH the agents dimerize band 3 in the intact cell too, indicating that GSH may prevent band 3 dimerization under physiological conditions. The oxidizing agents reversibly oxidize 80% of the membrane SH-groups, suggesting that these groups are arranged close enough to each other to form disulfide bonds. This arrangement may protect other cell structures against free radicals or oxidative stress.     

Erythrocyte Lii-Nao countertransport system Inhibition by N-ethylmaleimide probes for a conformational change of the transport system

Human erythrocytes were treated by a series of SH-reagents, including maleimides, iodo compounds, mercurials and oxidizing agents. Rates of Li efflux into Na-rich medium, Li leak and Li_i-Na_o countertransport were then determined. Of the 13 different reagents studied, only N-ethylmaleimide, iodoacetamide and iodoacetate inhibited selectively the countertransport activity. The effect of the various reagents indicates that the sensitive SH-groups of the countertransport system are not externally exposed. N-Ethylmaleimide was used to probe for changes elicited by substrate cations in Li_i-Na_o countertransport. In Na- and Li-free medium, inhibition of Li_i-Na_o countertransport by N-ethylmaleimide of 35% was reached within 2 s. In Na or Li medium, maximal inhibition was twice as great, but was attained much more slowly, within 10 min. Kinetic data and Hill plot analysis indicate the involvement of two classes of SH-groups: one expressed in the various media with and without substrate cations, and an additional one, which becomes specifically available to N-ethylmaleimide in the presence of external Na or Li. The affinity of Na to the site promoting inhibition by N-ethylmaleimide (apparent K_m  12 mM) is higher than the affinity of Na to its external countertransport site (apparent K_m  25 mM), as reported by Sarakadi, B., Alifimoff, J.K., Gunn, R.B. and Tosteson, D.C. (1978) J. Gen. Physiol. 72, 249–265). Reactivity of $\text{N-}\text{ethyl}\text{[}{}^{14}\text{C}\text{]}\text{maleimide}$ was not modified by the media tested. It is concluded that external Na and Li cause a conformational change in the protein(s) of the countertransport system in human erythrocytes.     

Analyses of phosphorylated tryptic peptide of spectrin from human erythrocyte membrane

Labelling of the membrane protein spectrin with ³²P in intact human erythrocytes involves a limited and specific reaction. digest of [³²P]spectrin reveals the presence of a single labelled peptide. Principal phosphate modification takes place at a serine residue (90%) but a small amount of phosphothreonine (10%) is also present.     

Study of histamine binding to h2-receptors of gastric mucosa membranes of the frogRana ridibunda

The stability was studied of histamine H₂-receptors and of histamine-sensitive adenylyl cyclase of the crude membrane fraction of the gastric mucosa of the frogRana ridibunda to the action of exogenous hydrolases, lipase (phospholipase C), protease (papain), glycosidase (sialidase), and blockers of free SH-groups (iodoacetate and N-ethylmaleimide). The action of these agents on the free and histamine-occupied H₂ -receptors of the frog gastric mucosa was analyzed by the amount of the bound ligand. The histamine binding to receptor increased the receptor vulnerability to the effect of phospholipase C, papain, and SH-reagents. Study of the action of hydrolases on the basal and stimulated, histamine-sensitive adenylyl cyclase activity revealed that phospholipase C caused a decrease of the basal and all kinds of the stimulated activity of adenylyl cyclase, while papain and sialidase only prevented the histamine stimulation of the enzyme. The obtained data indicate changes of the surface exposure of functional groups during the specific ligand-receptor interaction.     

Study of histamine binding to h2-receptors of gastric mucosa membranes of the frogRana ridibunda

The stability was studied of histamine H₂-receptors and of histamine-sensitive adenylyl cyclase of the crude membrane fraction of the gastric mucosa of the frogRana ridibunda to the action of exogenous hydrolases, lipase (phospholipase C), protease (papain), glycosidase (sialidase), and blockers of free SH-groups (iodoacetate and N-ethylmaleimide). The action of these agents on the free and histamine-occupied H₂-receptors of the frog gastric mucosa was analyzed by the amount of the bound ligand. The histamine binding to receptor increased the receptor vulnerability to the effect of phospholipase C, papain, and SH-reagents. Study of the action of hydrolases on the basal and stimulated, histamine-sensitive adenylyl cyclase activity revealed that phospholipase C caused a decrease of the basal and all kinds of the stimulated activity of adenylyl cyclase, while papain and sialidase only prevented the histamine stimulation of the enzyme. The obtained data indicate changes of the surface exposure of functional groups during the specific ligand-receptor interaction.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.     

Evidence for the Existence of Two Essential and Proximal Cysteinyl Residues in NADP-Malic Enzyme from Maize Leaves

Incubation of maize (Zea mays) leaf NADP-malic enzyme with monofunctional and bifunctional N-substituted maleimides results in an irreversible inactivation of the enzyme. Inactivation by the monofunctional reagents, N-ethylmaleimide (NEM) and N-phenylmaleimide, followed pseudo-first-order kinetics. The maximum inactivation rate constant for phenylmaleimide was 10-fold higher than that for NEM, suggesting a possible hydrophobic microenvironment of the residue(s) involved in the modification of the enzyme. In contrast, the inactivation kinetics with the bifunctional maleimides, ortho-, meta-, and para-phenylenebismaleimide, were biphasic, probably due to different reactivities of the groups reacting with the two heads of these bifunctional reagents, with a possible cross-linking of two sulfhydryl groups. The inactivation by mono and bifunctional maleimides was partially prevented by Mg²⁺ and l-malate, and NADP prevented the inactivation almost totally. Determination of the number of reactive sulfhydryl groups of the native enzyme with [³H]NEM in the absence or presence of NADP showed that inactivation occurred concomitantly with the modification of two cysteinyl residues per enzyme monomer. The presence of these two essential residues was confirmed by titration of sulfhydryl groups with [³H]NEM in the enzyme previously modified by o-phenylenebismaleimide in the absence or presence of NADP.     

Cross bonding and stiffening of the red cell membrane

Cross bonding and stiffening of the human red cell membrane was studied using treatments with SH, amino, and carboxyl reagents, oxidizing and denaturing treatments and acidification. Membrane cross bonding was initiated when, after red cell treatment, opposite areas of the cytoplasmic face of the red cell membrane were brought into contact by cell shrinking. Membrane cross bonding was detected by light microscopy when this contact persisted upon swelling the cells in a hypotonic medium. Membrane stiffening was recorded as a decrease in elongation of red cells in the shear field of a viscous dextran solution. No correlation was found between membrane cross bonding and membrane stiffening. The results are explained by the existence of two modifications of spectrin, type I causing solely membrane stiffening, type II causing membrane cross bonding as well as membrane stiffening. The amino and carboxyl reagents caused only type I modification. The other treatments caused both types of modification although with varying proportions. The results support the previously suggested mechanism of membrane cross bonding which involves a rearrangement of spectrin similar to denaturation by heat or urea, a decrease in associations within the membrane skeletal network, and a lateral aggregation of membrane proteins. These changes are proposed to occur by the type II modification. The data further substantiate the membrane stiffening effect of inter- and intra-molecular cross linking of spectrin which is identified with the type I modification. Finally, hypotheses are presented concerning the mechanism of membrane stiffening due to type II modifications of spectrin.     

Effect of factors of favism on the protein and lipid components of rat erythrocyte membrane

Erythrocytes prepared from riboflavin- and tocopherol-deficient (RT^?) and from control rats were used to investigate the mechanism of oxidative hemolysis by the factors of favism. RT^? erythrocytes have a defense system against the oxidative stress which is blocked either where regeneration of GSH occurs or the scavenging of the radicals from the membrane is prevented. The oxidative factors used were isouramil, divicine and diamide. When RT^? erythrocytes were treated with isouramil, GSH decreased to undetectable levels and was not regenerated. Complete hemolysis occurred, but no oxidation of SH groups of membrane proteins or formation of spectrin polymers was detected. A similar effect was observed with diamide. However, SH groups of membrane proteins were completely oxidized and spectrin polymers were formed. Extensive lipid peroxidation was also detected together with a 30% fall in the arachidonic acid level. Control erythrocytes treated with either isouramil or diamide were not hemolyzed. When treated with isouramil, after a fall in the first few minutes, the GSH level was completely regenerated after 20 min. Incubation with diamide caused extensive oxidation of SH groups of membrane proteins and formation of spectrin polymers. No lipid peroxidation was detected after treatment with isouramil, but the same decrease of arachidonic acid occurred as in RT^? erythrocytes. These results support the hypothesis that oxidative hemolysis by the factors of favism is caused by uncontrolled peroxidation of membrane lipids.     

Erythrocyte Lii-Nao countertransport system. Inhibition by N-ethylmaleimide probes for a conformational change of the transport system

Human erythrocytes were treated by a series of SH-reagents, including maleimides, iodo compounds, mercurials and oxidizing agents. Rates of Li efflux into Na-rich medium, Li leak and Lii-Nao countertransport were then determined. Of the 13 different reagents studied, only N-ethylmaleimide, iodoacetamide and iodoacetate inhibited selectively the countertransport activity. The effect of the various reagents indicates that the sensitive SH-groups of the countertransport system are not externally exposed. N-Ethylmaleimide was used to probe for changes elicited by substrate cations in Lii-Nao countertransport. In Na- and Li-free medium, inhibition of Lii-Nao countertransport by N-ethylmaleimide of 35% was reached within 2 s. In Na or Li medium, maximal inhibition was twice as great, but was attained much more slowly, within 10 min. Kinetic data and Hill plot analysis indicate the involvement of two classes of SH-groups: one expressed in the various media with and without substrate cations, and an additional one, which becomes specifically available to N-ethylmaleimide in the presence of external Na or Li. The affinity of Na to the site promoting inhibition by N-ethylmaleimide (apparent Km = 12 mM) is higher than the affinity of Na to its external countertransport site (apparent Km = 25 mM, as reported by Sarakadi, B., Alifimoff, J.K., Gunn, R.B. and Tosteson, D.C. (1978) J. Gen. Physiol. 72, 249-265). Reactivity of N-ethyl[14C]maleimide was not modified by the media tested. It is concluded that external Na and Li cause a conformational change in the protein(s) of the countertransport system in human erythrocytes.     

Role of cysteine sulfhydryl groups in activation of adenylyl cyclase signaling system by biogenic amines in molluscan and rat tissues

It has been shown that in smooth muscles of the freshwater bivalve molluscAnodonta cygnea as well as in skeletal muscles and brain striatum of rats a blocker of SH-groups,para-chlormercury benzoate (ChMB), and an alkylating agent,N-ethylmaleimide, inhibit both the basal adenylyl cyclase (AC) activity and the activity of the enzyme stimulated by non-hormonal agents (NaF, Gpp[NH]p) and by hormonal agents such as serotonin (mollusc muscles, rat brain) or isoproterenol (rat muscles and rat brain). The inhibitory effects of ChMB andN-ethylmaleimide on AC are partly eliminated by an SH-group containing reagent, β-mercaptoethanol (ME, 5 mM). Restoration of the basal and of the stimulated enzyme activity inhibited by ME is better in the case of the ChMB than of theN-ethylmaleimide action. It has also been found that ME stimulates both the basal and the stimulated by non-hormonal agents AC activity. In the presence of ME the hormonal stimulating effects on the enzyme are also preserved, except for the effect of isoproterenol on AC in rat skeletal muscles; this effect is inhibited by ME. Potentiation of the stimulating effect of the hormones on AC by Gpp[NH]p is only preserved in the molluscan smooth muscles (the effect of serotonin—90%). The data obtained indicate that cysteine sulfhydryl groups play a key role in hormonal regulation of the functional activity of the components of the hormone-sensitive adenylyl cyclase signaling system.     

The comparative effects of a mono- and a bi-functional alkylating agent on recombination inChlamydomonas reinhardi

Summary Synchronous populations of diploid cells ofChlamydomonas reinhardi were exposed to a bifunctional (Nitrogen Mustard) and a monofunctional (Diethyl Sulphate) alkylating agent at various stages of the pre-meiotic interphase and meiosis, and the effect on recombination in the arg 1/pab 2 region of linkage group I assessed. Both compounds affected recombination—the monofunctional one depressing it during two short periods, one just pre-meiosis when the main DNA duplication for meiosis is thought to occur, and the other in early prophase. The bifunctional compound on the other hand depressed recombination over an extended period. These effects have been related to the ability of the agents used to affect DNA synthesis and the different temporal patterns of response interpreted in terms of differential repair activities. The results provide additional evidence of the existence of two periods during which recombination can be affected.     

Hemolysis of human erythrocytes under hydrostatic pressure is suppressed by cross-linking of membrane proteins

The effects of cross-linking of membrane proteins on hemolysis of human erythrocytes under high pressure (2.0 kbar) were examined. The membrane proteins were cross-linked by oxidation of their SH-groups with diamide (0.05-0.5 mM) under different pressures (1-1,000 bar) at which no hemolysis occurs. As the pressure during diamide treatment was raised, the degree of hemolysis under 2.0 kbar and the quantity of cytoskeletal proteins extracted in a low ionic strength medium were gradually decreased. However, both values were increased by reduction with dithiothreitol. From the determination of membrane SH-groups, it was found that cross-linking of membrane proteins by diamide was accelerated under pressure. Only in erythrocytes treated with diamide under pressure were parts of spectrin and ankyrin, in addition to band 3 and band 4.2 proteins, extracted by using Triton X-100. One- and two-dimensional SDS-PAGE of membrane proteins showed that cross-linking of the membrane with cytoskeletal meshwork through linking proteins, in addition to that of membrane proteins themselves, was formed only in the diamide treatment under pressure. These results indicate that pressure-induced hemolysis is greatly suppressed by the supramolecular-weight polymers formed among membrane proteins, and that the high pressure technique is useful for cross-linking membrane proteins with diamide.     

Spectrin, red cell shape and deformability. II. The antagonistic action of spectrin and sialic acid residues in determining membrane curvature in genetic spectrin deficiency in mice

In a companion paper, the shapes of spectrin deficient mouse erythrocytes were described; in contrast to previous assumptions, spherules with tethered microvesicles rather than true "spherocytes" were found. Thence, spectrin deficient mouse erythrocytes are endowed with an excess of surface area for the given volume but the membrane is assuming a highly positive curvature. Observations during and after the action of enzymes cleaving the red cell surface charge (Neuraminidase, Trypsin, Chymotrypsin) showed that the previously positive membrane curvature, as well as the tendency of the membrane to flow into fingerlike protrusions was completely abolished. The erythrocytes of the spectrin deficient, desialylated mouse erythrocytes assumed a variety of shapes, often discocytic or even stomatocytic, i.e. their membrane presented with negative curvature. However, while these desialylated membranes could be easily deformed (elongated) by shear flow they did not recoil elastically into any definitive configuration after removal of the deforming forces. It is concluded from these observations that spectrin (acting on the inner interface between membrane and cytoplasm) and sialic acid residues (acting on the outer interface between membrane and plasma) exert antagonizing effects on membrane curvature and membrane bending elasticity. Sialic acid residues, strongly charged and situated on the outer side of the cell, produce positive membrane curvature; this observation can most readily be explained by assuming that this mechanical effect is caused by repulsive coulombic forces expanding the outer half of the bilayer. To explain the effect of the spectrin-complex in counteracting positive or in producing negative membrane curvature, a similar expansive coulombic force acting between the highly charged residues has been postulated. Thence, a model for explaining the overall elastic behaviour of the normal mammalian red cell is developed which is based on the assumption of elastic interactions of proteinacous membrane components coupled to the lipid bilayer of the membrane.     

What we can learn from the effects of thiol reagents on transport proteins.

Many secondary membrane transport systems contain reactive sulfhydryl groups. In this review the applications of SH reagents for analyzing the role of sulfhydryl groups in membrane transport systems will be discussed. First an overview will be given of the more important reagents, that have been used to study SH-groups in membrane transport systems, and examples will be given of transport proteins in which the role of cysteines have been analyzed. An important application of SH-reagents to label transport proteins using various SH-reagents modified with fluorescent- or spin-label moieties will be discussed. Two general models are shown which have been proposed to explain the role of sulfhydryl groups in some membrane transport systems.     

Phosphorylation and dephosphorylation of spectrin

The phosphorylation of spectrin polypeptide 2 is thought to be involved in the metabolically dependent regulation of red cell shape and deformability. Spectrin phosphorylation is not affected by cAMP. The reaction in isolated membranes resembles the cAMP-independent, salt-stimulated phosphorylation of an exogenous substrate, casein, by enzyme(s) present both in isolated membranes and cytoplasmic extracts. Spectrin kinase is selectively eluted from membranes by 0.5 M NaCl and co-fractionates with eluted casein kinase. Phosphorylation of band 3 in the membrane is inhibited by salt, but the band 3 kinase is otherwise indistinguishable operationally from spectrin kinase. The membrane-bound casein (spectrin) kinase is not eluted efficiently with spectrin at low ionic strength; about 80% of the activity is apparently bound at sites (perhaps on or near band 3) other than spectrin. Partitioning of casein kinase between cytoplasm and membrane is metabolically dependent; the proportion of casein kinase on the membrane can range from 25% to 75%, but for fresh cells is normally about 40%. Dephosphorylation of phosphorylated spectrin has not been studied intensively. Slow release of ³²Pi from [³²P] spectrin on the membrane can be demonstrated, but phosphatase activity measured against solubilized [³²P] spectrin is concentrated in the cytoplasm. The crude cytoplasmic phosphospectrin phosphatase is inhibited by various anions – notably, ATP and 2,3-DPG at physiological concentrations. Regulation of spectrin phosphorylation in intact cells has not been studied. We speculate that spectrin phosphorylation state may be regulated (1) by metabolic intermediates and other internal chemical signals that modulate kinase and phosphatase activities per se or determine their intracellular localization and (2) by membrane deformation that alters enzyme–spectrin interaction locally. Progress in the isolation and characterization of spectrin kinase and phosphospectrin phosphatase should lead to the resolution of major questions raised by previous work: the relationships between membrane-bound and cytoplasmic forms of the enzymes, the nature of their physical interactions with the membrane, and the regulation of their activities in defined cell-free systems.     

Mechanisms of catalase activity of heme peroxidases

In the absence of exogenous electron donors monofunctional heme peroxidases can slowly degrade hydrogen peroxide following a mechanism different from monofunctional catalases. This pseudo-catalase cycle involves several redox intermediates including Compounds I, II and III, hydrogen peroxide reduction and oxidation reactions as well as release of both dioxygen and superoxide. The rate of decay of oxyferrous complex determines the rate-limiting step and the enzymes’ resistance to inactivation. Homologous bifunctional catalase-peroxidases (KatGs) are unique in having both a peroxidase and high hydrogen dismutation activity without inhibition reactions. It is demonstrated that KatGs follow a similar reaction pathway as monofunctional peroxidases, but use a unique post-translational distal modification (Met⁺-Tyr-Trp adduct) in close vicinity to the heme as radical site that enhances turnover of oxyferrous heme and avoids release of superoxide. Similarities and differences between monofunctional peroxidases and bifunctional KatGs are discussed and mechanisms of pseudo-catalase activity are proposed.     

Crosslinking of membranes: the effect of dimethylsuberimidate, a bifunctional alkylating agent, on mitochondrial electron transport and ATPase.

Isolated mitochondrial inner membranes were treated with dimethylsuberimidate, a bifunctional alkylating agent, and the effect on electron transport and ATPase activity was determined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that treatment resulted in the polymerization of membrane polypeptides. Electron transport and ATPase activity was strongly inhibited by this reagent. However, at comparable levels of amidination, a monofunctional imidate, ethylacetimidate, was less inhibitory than the bifunctional reagent. Furthermore, dimethylsuberate, a hydrolysis product of dimethylsuberimidate, caused minimal inhibition of activity. Dimethylsuberimidate treatment resulted in a decrease in the apparent K_m of the ATPase, while the monoimidate did not alter the K_m. The results obtained suggest that inhibition by dimethylsuberimidate may be due, in part, to the molecular crosslinking of inner membrane components.