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PubMed1991

N-ethylmaleimide and mercurials modulate inhibition of the mitochondrial inner membrane anion channel by H+, Mg2+ and cationic amphiphiles.

Previously it has been shown that the mitochondrial inner membrane anion channel is reversibly inhibited by matrix Mg2+, matrix H+ and cationic amphiphiles such as propranolol. Furthermore, the IC50 values for both Mg2+ and cationic amphiphiles are dependent on matrix pH. It is now shown that pretreatment of mitochondria with N-ethylmaleimide, mersalyl and p-chloromercuribenzenesulfonate increases the IC50 values of these inhibitors. The effect of the mercurials is most evident when cysteine or thioglycolate is added to the assay medium to reverse their previously reported inhibitory effect (Beavis, A.D. (1989) Eur. J. Biochem. 185, 511-519). Although the IC50 values for Mg2+ and propranolol are shifted they remain pH dependent. Mersalyl is shown to inhibit transport even in N-ethylmaleimide-treated mitochondria indicating that N-ethylmaleimide does not react at the inhibitory mercurial site. However, the effects of N-ethylmaleimide and mersalyl on the IC50 for H+ are not additive which suggests that mercurials and N-ethylmaleimide react at the same 'regulatory' site. It is suggested that modification of this latter site exerts an effect on the binding of Mg2+, H+ and propranolol by inducing a conformational change. It is also suggested that a physiological regulator may exist which has a similar effect in vivo.

Animals↗
PubMed1980

Subcellular distribution and some properties of N-ethylmaleimide-sensitive and-insensitive forms of glycerol phosphate acyltransferase in rat adipocytes.

1. Glycerol phosphate acyltransferase (GPAT) activities were measured in subcellular fractions obtained from rat epididymal adipocytes. These contained both N-ethylmaleimide-sensitive and N-ethylmaleimide-insensitive forms of the enzyme. 2. As shown by parallel measurements of marker enzymes, N-ethylmaleimide-insensitive GPAT is most probably a mitochondrial activity, whereas N-ethylmaleimide-sensitive GPAT is the microsomal enzyme. 3. Subcellular distributions are also reported for dihydroxyacetone phosphate acyltransferase (DHAPAT) (assayed with and without N-ethylmaleimide), monoacylglycerol phosphate acyltransferase (MGPAT) and Mg2+-dependent and Mg2+-independent forms of phosphatidate phosphohydrolase (PPH).

1-Acylglycerol-3-Phosphate O-Acyltransferase↗
PubMed1995

Structure-function analysis of human alpha 1-->3fucosyltransferases. A GDP-fucose-protected, N-ethylmaleimide-sensitive site in FucT-III and FucT-V corresponds to Ser178 in FucT-IV.

Human alpha 1-->3fucosyltransferases constitute a family of closely related membrane-bound enzymes distinguished by differences in acceptor specificities and inherent protein biochemical properties. One such biochemical property is sensitivity to enzyme inactivation by sulfhydral-group modifying reagents such as N-ethylmaleimide. The basis for this property has been studied using a fusion protein of FucT-III and FucT-V composed of Protein A coupled to the catalytic domain of the enzyme. The results indicate that modification of FucT-V by 5,5'-dithiobis(2-nitrobenzoic acid) resulted in efficient enzyme inactivation that could be reversed by excess thiol reagent suggesting that the free sulfhydral group on the enzyme was required for activity. Recombinant forms of both FucT-III and FucT-V were irreversibly inactivated by N-ethylmaleimide and could be effectively protected from inactivation by GDP-fucose and GDP but not by UDP-galactose, fucose, or N-acetyllactosamine. Analysis of the distribution of Cys residues in aligned sequences of cloned human alpha 1-->3fucosyltransferases indicated one site, Cys143 of FucT-III and Cys156 of FucT-V, corresponded to the highly conservative replacement of Ser178 in FucT-IV, an enzyme insensitive to N-ethylmaleimide. A site-directed mutagenesis experiment was performed to replace Ser178 of FucT-IV with a Cys residue. The mutant FucT-IV enzyme was active; however, the Km for GDP-fucose was increased about 3-fold compared to the native enzyme to 28 +/- 3 microM. This enzyme was N-ethylmaleimide sensitive and could be partially protected by GDP-fucose but not N-acetyllactosamine. These results support the importance of Ser178 of FucT-IV in donor substrate binding and strongly suggest analogous Cys residues are the GDP-fucose protectable, N-ethylmaleimide-sensitive sites present in FucT-III and -V.

Base Sequence↗
PubMed2006

Single and repeated morphine administrations differently regulate expression of N-ethylmaleimide-sensitive factor gene in the rat brain.

The present study investigated the potential effects of single and repeated morphine treatments on gene expression of N-ethylmaleimide-sensitive protein, a key regulatory protein of neurotransmitter release and synaptic plasticity. After a single morphine injection (10 mg/kg), N-ethylmaleimide-sensitive factor gene expression increased significantly in the cortex, hippocampus and periaqueductal gray. In contrast, N-ethylmaleimide-sensitive factor gene expression was not changed after repeated morphine exposures in these brain regions. Sixteen hours after cessation of repeated morphine administration, N-ethylmaleimide-sensitive factor mRNA level increased again to a level approximate to that of the single morphine injection group in the hippocampus, periaqueductal gray and cerebral cortex. These findings suggest that the N-ethylmaleimide-sensitive factor may be involved in the maladaptive changes in the nervous system after opiate treatment.

Analysis of Variance↗
PubMed1991

The effect of N-ethylmaleimide on K+ and Cl- transport pathways in the lamprey erythrocyte membrane: activation of K+/Cl- cotransport.

The effect of the sulphydryl reagent N-ethylmaleimide on the K+ and Cl- transport pathways of the lamprey erythrocyte membrane was found to be quite complex. N-Ethylmaleimide inhibited the Ba(2+)-sensitive pathway that mediates most of the ouabain-resistant influx of K+ into the cell under physiological conditions but stimulated a Cl(-)-dependent, B(2+)-resistant K+ transport pathway that was inhibited by compounds that inhibit Cl(-)-dependent K+ transport in the human erythrocyte. N-Ethylmaleimide (in most cases) reduced the total influx of Cl- into the lamprey erythrocyte but (in all cases) introduced a K(+)-dependent component into the measured Cl- uptake; this was explained in terms of N-ethylmaleimide having inhibited the pathway primarily responsible for Cl- influx under physiological conditions but having stimulated a second, K(+)-dependent Cl- transport pathway. Although the magnitude of the K+ and Cl- fluxes stimulated by N-ethylmaleimide varied widely between cells from different lampreys, there was, in each individual case, a close similarity between the magnitude of the Cl(-)-dependent K+ influx (calculated from the 86Rb+ uptake) and the K(+)-dependent Cl- influx; the mean value for the ratio of the former to the latter was 1.01 +/- 0.03 (N = 5). The results are therefore consistent with the sulphydryl reagent having activated a K+/Cl- cotransport system similar to that present in erythrocytes from many mammalian species. This raises the possibility that the lamprey red cell may be a uniquely suitable system in which to study the characteristics of Cl- transport by this pathway.

Animals↗
PubMed1990

An investigation of placental transferrin processing: influence of N-ethylmaleimide.

The uptake and processing of 125I-59Fe labelled diferric transferrin was studied with the single bolus technique using the isolated guinea-pig placenta. Following preperfusion of the placenta with the SH-alkylating agent N-ethylmaleimide preferential iron uptake was inhibited and a total blockade of transplacental iron transfer was obtained. It can be shown by means of subcellular fractionation studies that neither transferrin binding nor endocytosis of the transferrin receptor complex were affected by N-ethylmaleimide. Additional experiments performed with microvillous membrane vesicles isolated from control placentas or from placentas preperfused with N-ethylmaleimide demonstrated that N-ethylmaleimide does not affect the affinity of the transferrin receptor for its ligand. The number of receptors per mg membrane protein remains unchanged as well. Ka = 1.4 x 10(8) M-1, n = 3.6 x 10(12). The results show that the blockade is located at the level of endosomal iron release. Since it is known that N-ethylmaleimide inhibits the endosomal proton pump, our results strongly suggest that the endocytotic pathway is a necessary route in transferrin mediated transplacental iron transfer.

Animals↗
PubMed1975

Influence of adenine nucleotides on the inhibition of photophosphorylation in spinach chloroplasts by N-ethylmaleimide.

The incubation of spinach chloroplasts with 1 mM N-ethylmaleimide in light for 60 to 90 s results in a partial, irreversible inhibition of photophosphorylation. The inhibition was not overcome at infinite light intensity or at infinite concentrations of the phosphorylation substrates. Although the inhibition diminished with decreasing concentrations of adenosine diphosphate in the assay of phosphorylation, the inhibition of guanosine diphosphate phosphorylation was independent of the concentration of this nucleotide. Although adenosine di- or triphosphate (10 to 30 muM) alone partially prevented the development of the N-ethylmaleimide inhibition of phosphorylation, these nucleotides were more effective when either 1 mM inorganic phosphate or arsenate was also present. The light-dependent incorporation of N-ethylmaleimide into chloroplast-bound coupling factor 1 was affected by adenosine triphosphate and inorganic phosphate in a manner similar to the onset of N-ethylmaleimide inhibition. Since guanosine diphosphate did not protect phosphorylation from N-ethylmaleimide inhibition but is phosphorylated at rapid rates, it is apparent that coupling factor 1 in chloroplasts has multiple nucleotide recognition sites.

Adenine Nucleotides↗
PubMed1981

Localization and characterization of N-ethylmaleimide sensitive inhibitor(s) of thiol cathepsin activity from cultured nil and polyoma virus-transformed nil hamster cells.

Exposure of cultured Nil (a stable line of fibroblast cells from Syrian hamsters) or polyoma virus-transformed (PyNil) hamster fibroblasts to 0.5 mM N-ethylmaleimide for 5 minutes resulted in striking increases in thiol cathepsin activity in unfractionated cell-free lysates. The paradoxical increase in activity of the normally N-ethylmaleimide-sensitive cathepsins apparently occurred as the result of the protective compartmentalization of the cathepsins in the lysosomes (20,000 X g sedimented fraction) and the unprotected localization of an inhibitor(s) in the soluble cytoplasm (175,000 X g supernatant fraction). Under continuous exposure of the cells to N-ethylmaleimide, a rapid increase in cathepsin activity (seen in the first 5 minutes) was followed by a steady decrease in activity (half inactivation time, 90 minutes). The relative difference in rates of N-ethylmaleimide inactivation of thiol cathepsins and thiol cathepsin inhibitors provides a means for estimating lysosomal cathepsin activity in whole cell extracts without the need for more time-consuming fractionation procedure. In reciprocal inhibition tests, it was found that, regardless of the source of cathepsins, the Nil and PyNil cathepsin inhibitor(s) inactivated the cathepsins to approximately the same extent. The inhibitors were heat stable (90-100 degrees C for 15 minutes) at pH 4, but were totally inactivated when boiled at pH 8.5. On a calibrated Sephadex G-100 column, the relative molecular weight (Mr) of the inhibitor(s) was 13,000 daltons. On the same column, the Mr of the cathepsins was 24,000 daltons. Compared with the cathepsin activity from Nil cells, there was about five times less cathepsin activity recoverable from the PyNil cells.

Aminopeptidases↗
PubMed1992

Intragastric nicotine protects against 40% ethanol-induced gastric mucosal injury despite pretreatment with propranolol or N-ethylmaleimide in rats.

We tested the hypotheses that the protective effect of intragastric nicotine against ethanol-induced gastric mucosal injury is dependent on propranolol- or N-ethylmaleimide-sensitive mechanisms. Propranolol was administered in doses (2 and 20 mg/kg) that provided dose-related blockade of beta-adrenoceptors (significant decreases in heart rate). N-Ethylmaleimide was administered in doses that previously had been shown to increase gastric vascular permeability (10 mg/kg) or inhibit gastric mucosal sulfhydryl compounds (50 mg/kg). At 0.5 hr after these or control subcutaneous pretreatments, the rats received intragastric nicotine (4 mg/kg) or vehicle. One hour later 40% ethanol was given intragastrically. The gastric corpus mucosal lesions were recorded by polaroid photographs after another hour, and their areas measured unbiasedly by computerized image analysis. The results showed that N-ethylmaleimide, but not propranolol, aggravated ethanol-induced gastric mucosal injury. The protective effect of intragastric nicotine was not modified by either pretreatment. We conclude that the mechanism mediating intragastric nicotine protection against 40% ethanol-induced gastric mucosal injury is independent of propranolol- or N-ethylmaleimide-sensitive mechanisms.

Animals↗
PubMed1983

Isolation and reconstitution of an N-ethylmaleimide-sensitive phosphate transport protein from rat liver mitochondria.

An N-ethylmaleimide-sensitive phosphate transport protein has been isolated from rat liver mitochondria, substantially purified, and reconstituted into phospholipid vesicles. Purified inner mitochondrial membrane vesicles depleted of F1-ATPase by urea treatment proved to be the most satisfactory starting material. Treatment of these membrane vesicles with Triton X-100 resulted in solubilization of the phosphate transport protein. Further purification was achieved using hydroxylapatite powder. Polyacrylamide gel electrophoresis of the purified fraction in sodium dodecyl sulfate indicated the presence of two Coomassie blue-staining bands with apparent Mr's of 30,000 and 35,000. Labeling of the 35,000 Mr band by the Pi transport inhibitor diazobenzene sulfonate was reduced markedly by prior treatment of the mitochondria with the inhibitor N-ethylmaleimide. The purified fraction containing both proteins could be reconstituted into liposomes prepared from purified asolectin. Phosphate efflux from these vesicles was inhibited by N-ethylmaleimide, by the impermeant mercurial agent, p-chloromercuribenzoate, and by diazobenzene sulfonate. Treatment of the purified fraction with N-ethylmaleimide prior to incorporation into liposomes resulted in a reconstituted system incapable of catalyzing Pi efflux. These studies summarize the first detailed attempt to purify the Pi/H+ transport system from rat liver mitochondria and emphasize the need to commence the purification with purified inner membrane vesicles depleted of F1-ATPase. In addition, these studies show that the final fraction contains a reconstitutively active transport system which when incorporated into phospholipid vesicles has its essential sulfhydryl groups oriented outward. Finally, it is shown that the purified fraction also contains a 30,000 Mr component.

Animals↗
PubMed1983

Effect of N-ethylmaleimide on leucine transport in the Chang liver cell. II. Effect on the kinetics of Na+-independent transport.

The Na+-independent leucine transport system is resolved into two components by their different affinity (Km about 44 microM and 8.0 mM) for leucine in the Chang liver cell. Treatment of the cells with N-ethylmaleimide (1 mM) specifically stimulates the high-affinity component of the Na+-independent system by greatly increasing its Vmax value, whereas the Vmax value of the low-affinity component is markedly lowered. The stimulatory effect of N-ethylmaleimide on leucine transport is reduced by prior treatment of the cells with 2,4-dinitrophenol, but this phenomenon seems to be irrelevant to the ATP-depleting action of the uncoupler. The treatment with 2,4-dinitrophenol has been found not to be inhibitory on the subsequent Na+-independent leucine uptake itself. Treatment with dibucaine, a phospholipid-interacting drug, also reduces to varying degrees (depending on its concentration) the stimulatory effect of N-ethylmaleimide on the subsequent leucine uptake, although pretreatment with dibucaine can stimulate the Na+-independent leucine uptake itself. We conclude that the stimulatory effect of N-ethylmaleimide on leucine transport is not correlated with the energy level of cell, but involves the perturbation of the membrane bilayer structures.

2,4-Dinitrophenol↗
PubMed2000

Chromate-induced human erythrocytes haemoglobin oxidation and peroxidation: influence of vitamin E, vitamin C, salicylate, deferoxamine, and N-ethylmaleimide.

In order to attenuate or to prevent chromate-induced human erythrocytes injury, the influence of vitamin E, vitamin C, salicylate, deferoxamine, and N-ethylmaleimide on chromate-induced human erythrocytes haemoglobin oxidation and peroxidation were investigated. It was observed that pretreatment of human erythrocytes with vitamin E (20 microM), vitamin C (1 mM), salicylate (3 mM), and deferoxamine (4 mM) significantly increased (P=0.0001) chromate-induced human erythrocytes haemoglobin oxidation in a time dependent manner, while it was significantly decreased (P=0.0001) by pretreatment with N-ethylmaleimide (1 mM). In contrast, pretreatment of human erythrocytes with deferoxamine (4 mM) immediately inhibited (P=0.0001) chromate-induced human erythrocytes peroxidation, while it was significantly increased (P=0.0001) by pretreatment with N-ethylmaleimide (1 mM) during the first 4 h of cells exposition to chromate. For time periods superior to 6 h pretreatment with N-ethylmaleimide (1 mM) significantly decreased (P=0.0001) chromate-induced human erythrocytes peroxidation. It was concluded that care must be taken as these drugs are used to prevent against toxicity induced by chromium(VI) compounds.

Ascorbic Acid↗
PubMed1982

Influence of N-ethylmaleimide on cholinoceptors and responses in longitudinal muscles from guinea-pig ileum.

1 The binding of carbamylcholine to membranes prepared from the longitudinal muscle of guinea-pig ileum was determined from its inhibition of the binding of [3H]-3-quinuclidinyl benzilate. Carbamylcholine binding was resolved into high and low affinity components with apparent dissociation constants of 0.11 +/- 0.02 and 11 +/- 1 microM; 42% of the receptors displayed high affinity carbamylcholine binding. 2 Alkylation of longitudinal muscle membranes with N-ethylmaleimide increased muscarinic receptor affinity for carbamylcholine in a manner consistent with a conversion of low affinity to high affinity receptors. After exposure the muscle membrane fragments to 1 mM N-ethylmaleimide for 20 min at 35 degrees C, carbamylcholine binding was resolved into two components with apparent dissociation constants of 0.11 +/- 0.01 and 9 +/- 2 microM, with 74% of the receptors displaying the higher affinity. 3 Exposure of longitudinal membranes mounted in an organ chamber to 1 mM N-ethylmaleimide for 30s depressed isometric contractions in response to acetylcholine by 80%, while contractions induced by K+ and Ba2+ were reduced by less than 20% and 10%, respectively. Acetylcholine dose-response curves were shifted to the right while Ba2+ curves were unaffected. 4 It is suggested that N-ethylmaleimide has a selective effect on muscarinic responses in the longitudinal muscle by disrupting processes occurring after receptor occupancy but before the induction of phospholipid turnover or calcium influx in the postsynaptic membrane.

Acetylcholine↗
PubMed1984

Activated complex of L-cells and Rickettsia prowazekii with N-ethylmaleimide-insensitive phospholipase A.

The interaction of large numbers of viable Rickettsia prowazekii cells with L-cells results in the expression of a phospholipase A activity with the concomitant release of free fatty acids and lysophosphatides from the phospholipids of the L-cell. About 50% of rickettsiae present in the suspension that was centrifuged onto an L-cell monolayer at 0 degree C to effect this interaction formed a tight L-cell-rickettsiae association from which the rickettsiae could not be removed by simple washing. Both the L-cell-rickettsiae association and the rickettsiae before association with L-cells interact with N-ethylmaleimide, so that the subsequent expression of the phospholipase A activity was inhibited (treatment of the L-cells with N-ethylmaleimide before centrifugation does not inhibit phospholipase activity). However, treatment of this association with 2,4-dinitrophenol and KCN caused much less inhibition of this phospholipase A activity than did treatment of the rickettsiae with these agents before centrifugation onto the L-cells. Incubation of the L-cell-rickettsiae association for a short time at 35 degrees C resulted in a very low level of free fatty acid formation and changed this association to an activated complex in which the phospholipase A activity was no longer sensitive to the inhibitory effects of N-ethylmaleimide. The characteristics of the association and activated complex were stable: after a 2-h incubation at 0 degrees C, the association and the activated complex retained both their basal phospholipase A activities and their characteristic responses to N-ethylmaleimide treatment. In scanning electron micrographs of the activated complexes, the rickettsiae that were initially attached were no longer visible after 45 min at 35 degrees C, and the surface of the L-cell appeared to have been etched away. These activated complexes provide a system in which modulators of the phospholipase A can be investigated without the confusion caused by the first-step receptor interaction between rickettsiae and their host cells.

Animals↗
PubMed1988

N-ethylmaleimide inhibition of the DNA-binding activity of the herpes simplex virus type 1 major DNA-binding protein.

The major herpes simplex virus DNA-binding protein, designated ICP8, binds tightly to single-stranded DNA and is required for replication of viral DNA. The sensitivity of the DNA-binding activity of ICP8 to the action of the sulfhydryl reagent N-ethylmaleimide has been examined by using nitrocellulose filter-binding and agarose gel electrophoresis assays. Incubation of ICP8 with N-ethylmaleimide results in a rapid loss of DNA-binding activity. Preincubation of ICP8 with single-stranded DNA markedly inhibits this loss of binding activity. These results imply that a free sulfhydryl group is involved in the interaction of ICP8 with single-stranded DNA and that this sulfhydryl group becomes less accessible to the environment upon binding. Agarose gel electrophoretic analysis of the binding interaction in the presence and absence of N-ethylmaleimide indicates that the cooperative binding exhibited by ICP8 is lost upon treatment with this reagent but that some residual noncooperative binding may remain. This last result was confirmed by equilibrium dialysis experiments with the 32P-labeled oligonucleotide dT10 and native and N-ethylmaleimide-treated ICP8.

DNA, Viral↗
PubMedDate not supplied

[N-ethylmaleimide inhibits polymerization of myorod, a contractile protein of thick filaments of molluscan smooth muscles].

The effect of N-ethylmaleimide on the polymerization of myorod, a protein of molluscan smooth muscles, which is colocalized with myosin on the surface of paramyosin core of thick filaments and is a product of the alternative splicing of the gene of heavy myosin chains, was studied. It was shown that myorod modified by N-ethylmaleimide completely loses the polymerization ability but acquires the ability to aggregate in the presence of Mg2+. At the same time, treatment of molluscan myosin with N-ethylmaleimide did not affect its polymerization. It was supposed that the effect of N-ethylmaleimide on myorod polymerization is related to the modification of the myorod SH domain containing Cys722.

Actin Cytoskeleton↗
PubMed1977

Investigation of the relation of the pH-dependent dissociation of malate dehydrogenase to modification of the enzyme by N-ethylmaleimide.

The pH-dependent dissociation of porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been further characterized using the technique of sedimentation velocity ultracentrifugation. The increased rate and specificity of the inactivation of mitochondrial malate dehydrogenase by the sulfhydryl reagent N-ethylmaleimide has been correlated with the pH-dependent dissociation of the enzyme. Data obtained using NAD+ and its component parts to reassociate the enzyme and also to protect the enzyme from inactivation by N-ethylmaleimide suggest that the sulfhydryl residues being modified by N-ethylmaleimide are inaccessible when the enzyme is in its dimeric form. A dissociation curve for the pH-dependent dissociation suggests that a limited number of residues are being protonated concomitant with dissociation of the enzyme. An apparent pKa of 5.3 has been determined for this phenomenon. Studies using enzyme modified by the sulfhydryl reagent N-ethylmaleimide indicate that selective modification of essential sulfhydryl residues alters the proper binding of NADH.

Animals↗
PubMed1990

Mitochondrial phosphate transport. N-ethylmaleimide insensitivity correlates with absence of beef heart-like Cys42 from the Saccharomyces cerevisiae phosphate transport protein.

The mitochondrial phosphate transport protein (PTP) has been purified in a reconstitutively active form from Saccharomyces cerevisiae and Candida parapsilosis. ADP/ATP carriers that copurify have been identified. The PTP from S. cerevisiae migrates as a single band (35 kDa) in sodium dodecyl sulfate gels with the same mobility as the N-ethylmaleimide-alkylated beef heart PTP. It does not cross-react with anti-sera against beef heart PTP. The CNBr peptide maps of the yeast and beef proteins are very different. The rate of unidirectional phosphate uptake into reconstituted proteoliposomes is stimulated about 2.5-fold to a Vmax of 170 mumol of phosphate min-1 (mg PTP)-1 (22 degrees C) by increasing the pHi of the proteoliposomes from 6.8 (same as pHe) to 8.0. The Km for Pi of this reconstituted activity is 2.2 mM. The transport is sensitive to mersalyl (50% inhibition at 60 microM) and insensitive to N-ethylmaleimide. We have purified peptides matching the highly conserved motif Pro-X-(Asp/glu)-X-X-(Lys/Arg)-X-(Arg/lys) (X is an unspecified amino acid) of the triplicate gene structure sequence of the beef heart PTP. The N-ethylmaleimide-reactive Cys42 of the beef heart protein, located between the two basic amino acids of this motif (Lys41-Cys42-Arg43), is replaced with a Thr in the yeast protein. This substitution most likely is responsible for the lack of N-ethylmaleimide sensitivity of the yeast protein and mersalyl thus reacts with another cysteine to inhibit the transport. Finally it is concluded that Cys42 has no essential role in the catalysis of inorganic phosphate transport by the mitochondrial phosphate transport protein.

Amino Acid Sequence↗