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.     

The organisation of methanol dehydrogenase and c-type cytochromes on the respiratory membrane of Methylophilus methylotrophus

The membrane-impermeant, protein-labelling reagent [¹⁴C]isethionyl acetimidate has been used to investigate the detailed topography of the membrane-associated methanol oxidase system in the methylotrophic bacterium Methylophilus methylotrophus. The results show that methanol dehydrogenase and cytochrome c₁ are significantly less exposed on the periplasmic surface of the membrane than either cytochrome C_H or an as yet unidentified 12-kDa protein. The partial crypticity of the methanol dehydrogenase has been confirmed by fingerprinting with V8 protease. The results are discussed in terms of the electron transfer functions of the various redox proteins.     

The asymmetric orientation of cytochrome b561 in bovine chromaffin granule membranes

The topological arrangement of cytochrome b561 in the bovine adrenal medullary chromaffin granule membrane was investigated by radiolabeling and immunoprecipitation techniques using antibody raised against the purified cytochrome. The first labeling procedure involved a membrane-permeable amino group labeling reagent, ethyl acetimidate, and two membrane-nonpermeable amino group labeling reagents, isethionyl acetimidate and trinitrobenzenesulfonic acid. The second radiolabeling procedure involved lactoperoxidase-catalyzed iodination of the exposed tyrosines on the membrane-bound proteins. The labeled cytochrome b561 was isolated by immunoprecipitating detergent extracts of treated membranes, followed by electrophoresis of the precipitated cytochrome in polyacrylamide-dodecyl sulfate. From the analysis of both labeling techniques, cytochrome b561 appeared to be a transmembrane protein and a major portion of this protein was cytoplasmically exposed.     

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.     

Identification of the lysine residue modified during the activation of acetimidylation of horse liver alcohol dehydrogenase.

A single amino group in horse liver alcohol dehydrogenase was modified with methyl(14C)acetimidate by a differential labeling procedure. Lysine residues outside the active site were modified with ethyl acetimidate while a lysine residue in the active site was protected by the formation of an enzyme-NAD+-pyrazole complex. After the protecting reagents were removed, the enzyme was treated with methyl(14C)acetimidate. Enzyme activity was enhanced 13-fold as 1.1 (14C)acetimidyl group was incorporated per active site. A labeled peptide was isolated from a tryptic-chymotryptic digest of the modified enzyme in 35% overall yield. Amino acid composition and sequential Edman degradations identified the peptide as residues 219-229; lysine residue 228 was modified with the radioactive acetimidyl group.     

Asymmetry in the renewal of molecular classes of phosphatidylcholine in the rat-erythrocyte membrane

1. 1. Rat-blood phospholipids were labeled in vivo with [³²P]phosphate. The erythrocytes were treated with phospholipase A₂ plus sphingomyelinase to discriminate between the labeling patterns of the phospholipids from the inner and outer layer of the membrane.

2. 2. The specific activities of the more unsaturated classes of phosphatidylcholine were higher in the outer layer of the erythrocyte membrane than in the inner layer. The disaturated class, however, had the highest specific activity in the inner layer.

3. 3. After incubating ³²P-labeled erythrocytes in unlabeled plasma, the labeling pattern recovered in the molecular classes of plasma phosphatidylcholine was very similar to that of the phosphatidylcholines in the outer layer of the erythrocyte membrane.

4. 4. It is proposed that the exchange of phosphatidylcholines between plasma and the outer layer of the erythrocyte is mainly responsible for the renewal of the unsaturated phosphatidylcholines of the erythrocyte, and that the acylation activity of the erythrocyte is directed towards the formation of disaturated phosphatidylcholines at the inside of the membrane.

Asymmetric distribution of the phosphatidylinositol-linked phospho-oligosaccharide that mimics insulin action in the plasma membrane

We have investigated the topography of a glycosyl-phosphatidylinositol implicated in insulin action by a combination of two complementary methods: (a) chemical labelling with a non-permeable (isethionyl acetimidate) and a permeable (ethyl acetimidate) probe; and (b) enzymatic modifications with beta-galactosidase (EC 3.2.1.23) or phosphatidylinositol-specific phospholipase C (EC 3.1.4.3). Using the first approach the majority of the glycosyl-phosphatidylinositol is found in the outer surface of intact hepatocytes, adipocytes, fibroblasts and lymphocytes, but not in erythrocytes which presented only a 20% of the total labelled glycosyl-phosphatidylinositol to the exterior. Upon insulin addition (10 nM), about 60% of the total glycosyl-phosphatidylinositol was hydrolysed in both hepatocytes and adipocytes but not in erythrocytes. In agreement with the extracellular localization in hepatocytes and with the proposed role of this glycolipid in insulin action, treatment of rat hepatocytes with beta-galactosidase from Escherichia coli, an enzyme that hydrolyses the oligosaccharide moiety of the glycosyl-phosphatidylinositol, cleaved 65% of the total glycophospholipid and blocked the effect of insulin (but not of glucagon) on pyruvate kinase (EC 2.7.1.40). Similar treatment with phosphatidylinositol-specific phospholipase C from Bacillus cereus hydrolysed 62% of the total glycosyl-phosphatidylinositol. From the various approaches used it is concluded that the majority of this glycophospholipid is at the outer surface in a variety of insulin-sensitive cells.     

Localization of the major dehydrogenases in two methylotrophs by radiochemical labeling.

The localization of prominent proteins in intact cells of two methylotrophic bacteria, Hyphomicrobium sp. strain X and bacterium W3A1, was investigated by radiochemical labeling with [14C]isethionyl acetimidate. In bacterium W3A1, trimethylamine dehydrogenase was not labeled by the reagent and is, therefore, an intracellular protein, whereas the periplasmic location of the methylamine and methanol dehydrogenases was evidenced by being readily labeled in intact cells. Similarly, an intracellular location of the trimethylamine and dimethylamine dehydrogenases in Hyphomicrobium sp. strain X was indicated, whereas methanol dehydrogenase was periplasmic.     

In situ incorporation of Fatty acids into lipids of the outer and inner envelope membranes of pea chloroplasts

When incubated with [1-¹⁴C]acetate and cofactors (ATP, Coenzyme A, sn-glycerol-3-phosphate, UDPgalactose, and NADH), intact chloroplasts synthesized fatty acids that were subsequently incorporated into most of the lipid classes. To study lipid synthesis at the chloroplast envelope membrane level, ¹⁴C-labeled pea (Pisum sativum) chloroplasts were subfractionated using a single flotation gradient. The different envelope membrane fractions were characterized by their density, lipid and polypeptide composition, and the localization of enzymic activities (UDPgalactose-1,2 diacylglycerol galactosyltransferase, Mg²⁺-dependent ATPase). They were identified as very pure outer membranes (light fraction) and strongly enriched inner membranes (heavy fraction). A fraction of intermediate density, which probably contained double membranes, was also isolated. Labeled glycerolipids recovered in the inner envelope membrane were phosphatidic acid, phosphatidyl-glycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol. Their ¹⁴C-fatty acid composition indicated that a biosynthetic pathway similar to the prokaryotic pathway present in cyanobacteria occurred in the inner membrane. In the outer membrane, phosphatidylcholine was the most labeled glycerolipid. Phosphatidic acid, phosphatidylglycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol were also labeled. The ¹⁴C-fatty acid composition of these lipids showed a higher proportion of oleate than palmitate. This labeling, different from that of the inner membrane, could result either from transacylation activities or from a biosynthetic pathway not yet described in pea and occurring partly in the outer chloroplast envelope membrane. This metabolism would work on an oleate-rich pool of fatty acids, possibly due to the export of oleate from chloroplast toward the extrachloroplastic medium. The respective roles of each membrane for chloroplast lipid synthesis are emphasized.     

Light-dependent chemical modification of thylakoid membrane proteins with carboxyl-directed reagents

Spinach chloroplast thylakoid membranes were chemically modified with membrane penetrating reagents reactive toward protein carboxyl groups, a carbodiimide and the nucleophiles [¹⁴C]glycine ethyl ester or [³H]serotonin. The reagents, being weak bases, were accumulated within the inner aqueous space in the light, due to the low pH inside. Both the accumulation and the low pH stimulating effect on the carbodiimide activation step contributed to a greater labeling in the light compared to dark, and uncouplers inhibited most of the light-dependent increase. Hence, it is likely that the proteins showing the light-dependent, uncoupler-sensitive labeling have those parts located within the inner aqueous space or within the membrane itself. While many membrane proteins which separated on sodium dodecyl sulfate-polyacrylamide gels (12.5–25% gradient) showed some increased labeling in the light, the most conspicuous were the four polypeptides of the chlorophyll $\text{a}\text{b}$ light-harvesting complex. The light-harvesting complex was purified from dark- and light-treated, labeled membranes. The resultant preparation showed about a sixfold, light-dependent, uncoupler-sensitive labeling increase compared to dark conditions. Polypeptides near 6 and 8 kdalton showed light-dependent, uncoupler-resistent increases in carboxyl group modification, which could be due to localized acidic conditions near sites of proton release.     

Cytochrome b5 induced flip-flop of phospholipids in sonicated vesicles

Cytochrome b5 induced flip-flop of phosphatidylethanolamine (PE) in sonicated vesicles prepared from a 9:1 mixture of phosphatidylcholine (PC) to phosphatidylethanolamine was determined as follows. First, vesicles having a nonequilibrium distribution of PE across the bilayer were prepared by amidinating the external amino groups with isethionyl acetimidate. Amidinated cytochrome b5 was then added, and after the protein was completely bound, the rate of appearance of fresh PE on the outer surface was determined by removing aliquots at timed intervals and titrating the external amino groups with trinitrobenzenesulfonic acid. The results show an initial rapid phase of flip-flop (especially in the presence of salt) followed by a very slow phase, at 25 degrees C. Similar results were obtained when cytochrome b5 was introduced into the amidinated vesicles by spontaneous transfer from PC donor vesicles. These results indicate that the accumulation of the transferable ("loose") form of cytochrome b5 on the outer surface of a vesicle causes a transient, global destabilization of the bilayer that is relieved by lipid flip-flop. We speculate that this mechanism may be a significant driving force for the transfer of amphipathic molecules across membranes.     

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.     

Backbone resonance assignments for the periplasmic chaperone LolA from Escherichia coli

LolA is an essential periplasmic protein in Gram-negative bacteria and plays a role in transporting lipoproteins through periplasmic space from the inner to the outer membrane. We established backbone resonance assignments of ²H/¹³C/¹⁵N labeled LolA from Escherichia coli.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

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.     

Stimulation of the biosynthesis of membrane glycoproteins from zajdela ascites hepatoma cells by Robinia lectin

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [¹⁴C]glucosamine and [³H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated ceils by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [¹⁴C]glucosamine and [³H]leucine and having different molecular weights, one over 200 000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [³H]glucosamine and from lectin stimulated cells labeled with [¹⁴C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

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.     

Trans-bilayer movement of added phosphatidylcholine and lysophosphatidylcholine species with various acyl chain lengths in plasma membrane of intact human erythrocytes

14C-Labeled phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) species with two homologous saturated acyl chains and of a saturated acyl chain of various lengths, respectively, were each incorporated into the outer leaflet of the membrane lipid bilayer of intact human erythrocytes, and the transbilayer movement into the inner leaflet during incubation at 37 degrees C of the lipid-loaded erythrocytes was followed. The labeled PC and lysoPC molecules present in the outer leaflet were extracted with egg-yolk PC liposome suspension and BSA solution, respectively, and the amount which moved into the inner leaflet during the incubation was measured by determining the residual amount of the labeled lipid in the membrane. Translocation of lysoPC molecules was also measured by assaying the decrease in the amount of the added labeled lysoPC in the membrane during the incubation on the basis of the previously reported fact that lysoPC molecules are all converted metabolically to PC or glycerylphosphorylcholine plus fatty acid as soon as they are translocated from the outer to the inner leaflet. Every lipid tested showed significant transbilayer movement during the course of the incubation for up to 10 h. With the C8, C10, and C12 species of PC the rate of the transbilayer movement increases with decreasing acyl chain length. The same is true with the C14, C16, and C18-lysoPC species.     

Specific protein synthesis in cellular differentiation: III. The eggshell proteins of Drosophila melanogaster and their program of synthesis

The eggshell of Drosophila melanogaster is composed of a set of proteins synthesized by the follicular epithelium during the last third of oogenesis and organized into an inner zone (vitelline membrane) and an outer zone (chorion). To study these proteins, the authors developed techniques for mass-isolating follicles of mixed stages, mature (stage 14) follicles, chorion from stage 14 follicles, and chorion and vitelline membrane from laid eggs. The eggshell is composed mainly of protein and is unusually rich in proline and alanine. Six proteins of the chorion have been identified on polyacrylamide gels. The program of synthesis of these proteins was studied by incubating follicles of different developmental stages in culture with ³H-labeled amino acids and displaying the labeled proteins on gels with the aid of autofluorography. The proteins are synthesized in a specific overlapping sequence during stages 10–14, a period when chorion deposition is known to occur. In addition, putative vitelline membrane proteins have been identified by their preferential incorporation of [³H]proline and [³H]alanine during stages of active vitelline membrane synthesis.     

Import of phosphatidylethanolamine for the assembly of rat brain mitochondrial membranes

Mitochondria can synthesize phosphatidyl-ethanolamine (PE) through phosphatidylserine decarboxylase (PS decarboxylase) activity or can import this lipid from the endoplasmic reticulum. In this work, we studied the factors influencing the import of PE in brain mitochondria and its utilization for the assembly of mitochondrial membranes. Incubation of rat brain homogenate with [1-³H]ethanolamine resulted in the synthesis and distribution of ³H-PE to subcellular fractions. T-wenty-one percent of labeled PE was recovered in purified mitochondria. The import of PE in mitochondria was studied in a reconstituted system made of microsomes (donor particles) and purified mitochondria (acceptor particles). Ca⁺² and nonspecific lipid transfer protein purified from liver tissue (nsL-TP) enhanced the translocation process. ³H-PE synthesized in membrane associated to mitochondria (MAM) could also translocate to mitochondria in the reconstituted system. Exposure of mitochondria to trinitrobenzensulfonic acid (TNBS) resulted in the reaction of more than 60% of ³H-PE imported from endoplasmic reticulum and of about 25% of ¹⁴C-PE produced in mitochondria by decarboxylation of ¹⁴C-PS. Moreover, the removal of the outer mitochondrial membrane by digitonin treatment, resulted in the loss of ³H-PE, but not ¹⁴C-PE. These results indicate that labeled PE imported in mitochondria is mainly localized in the outer mitochondrial membrane, whereas PE produced by PS decarboxylase activity is confined to the inner mitochondrial membrane. Phospholipase C hydrolyzed 25–30% of both PE radioactivity and mass of the outer mitochondrial membrane indicating an asymmetrical distribution of this lipid across the membrane.Mr. Carlo Ricci is thanked for his skillful technical assistance. This work has been supported by a grant from the Ministry of Education, Rome, Italy.