Engineering of functional supramacromolecular complexes of proteins (enzymes) using reversed micelles as matrix microreactors.

The size of the inner water cavity of reversed micelles formed in a triple system 'water-surfactant-organic solvent' can be widely varied by changing the degree of surfactant hydration. This gives grounds to use reversed micelles as matrix microreactors for the design of supramolecular complexes of proteins. Using ultracentrifugation analysis, it has been demonstrated that the oligomeric composition of various enzymes (ketoglutarate dehydrogenase, alkaline phosphatase, lactic dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase) solubilized in reversed micelles of Aerosol OT [sodium bis(2-ethylehexyl)sulfosuccinate] in octane changes upon variation of the degree of hydration. An oligomeric complex forms under conditions when the radius of the micelle inner cavity is big enough to incorporate this complex as a whole. At lower degrees of hydration the micelles 'uncouple' such complexes to their components. The catalytic properties of various oligomeric complexes have been studied. Possibilities of using reversed micelles for the separation of subunits of oligomeric enzymes under non-denaturating conditions have been demonstrated. In particular, the isolated subunits of alkaline phosphatase, lactic dehydrogenase and glyceraldehyde-3-phosphate have been found to be active in Aerosol OT reversed micelles. The dependences of the catalytic activity of oligomeric enzymes represent saw-like curves. The maxima of the catalytic activity observed at these curves relate to the functioning of various oligomeric forms of an enzyme. The radii of the micelle inner cavity under conditions when these maxima are observed correlate with the linear dimensions of the enzyme oligomeric forms. Correlation of the position of a maximum with the shape of an oligomeric complex is discussed.     

Activation of protein kinase B induced by H(2)O(2) and heat shock through distinct mechanisms dependent and independent of phosphatidylinositol 3-kinase

Protein kinase B (PKB) is a downstream target of phosphatidylinositol (PI) 3-kinase in the signaling pathway of growth factors, and is activated by cellular stress such as H(2)O(2) and heat shock. To study the mechanism of the stress-induced activation of PKB, PI 3-kinase products were measured in stress-stimulated cells. Both PI 3,4-bisphosphate and PI 3,4, 5-trisphosphate increased in H(2)O(2)-treated cells, and the elevation of these phospholipids and activation of PKB were concurrently blocked by wortmannin, a potent inhibitor of PI 3-kinase. In heat-shocked cells, the level of PI 3,4-bisphosphate did not change while that of PI 3,4,5-trisphosphate increased slightly, and an association between PKB molecules was observed. Two active PKB fractions, presumably monomeric and oligomeric forms, were resolved from heat-shocked cells by gel filtration column chromatography. Activation of the former was suppressed by pretreatment with wortmannin, whereas the generation and activation of the latter were not blocked by the PI 3-kinase inhibitor. Only the monomeric form, but not the oligomeric form, was recovered from H(2)O(2)-treated cells, and its activation was prevented by wortmannin. These results indicate that PKB is activated by two distinct mechanisms that are dependent and independent of PI 3-kinase in stress-stimulated cells.     

Toxicity mediated by soluble oligomers of beta-amyloid(1-42) on cholinergic SN56.B5.G4 cells

Alzheimer's disease (AD) is characterized by cholinergic dysfunction and progressive basal forebrain cell loss which has been assumed to be as a result of the extensive accumulation of beta-amyloid (Abeta). In addition to Abeta fibrillar assemblies, there are pre-fibrillar forms that have been shown to be neurotoxic, although their role in cholinergic degeneration is still not known. Using the cholinergic cell line SN56.B5.G4, we investigated the effect of different Abeta(1-42) aggregates on cell viability. In our model, only soluble oligomeric but not fibrillar Abeta(1-42) forms induced toxicity in cholinergic cells. To determine whether the neurotoxicity of oligomeric Abeta(1-42) was caused by its oxidative potential, we performed microarray analysis of SN56.B5.G4 cells treated either with oligomeric Abeta(1-42) or H(2)O(2). We showed that genes affected by Abeta(1-42) differed from those affected by non-specific oxidative stress. Many of the genes affected by Abeta(1-42) were present in the endoplasmic reticulum (ER), Golgi apparatus and/or otherwise involved in protein modification and degradation (chaperones, ATF6), indicating a possible role for ER-mediated stress in Abeta-mediated toxicity. Moreover, a number of genes, which are known to be involved in AD (clusterin, Slc18a3), were identified. This study provides important leads for the understanding of oligomeric Abeta(1-42) toxicity in cholinergic cells, which may account in part for cholinergic degeneration in AD.     

Spectroscopic studies of aqueous gallium(III) and aluminum(III) citrate complexes

Aqueous gallium(III) citrate complexes have been studied in the 10(-2) M concentration range with extended X-ray absorption fine structure (EXAFS) and FTIR techniques. From EXAFS data, one mononuclear and one oligomeric species were identified at different Ga(III) to citrate ratios. The first shell of the mononuclear complex was found to be distorted, with average Ga-O bond lengths of 1.95 and 2.06 A, in agreement with the solid-state structure of Ga(Cit)2(3-) (Cit=citrate). Also the oligomeric species was found to have a distorted first shell, with average Ga-O bond lengths of 1.95 and 2.04 A. This complex was found to contain two Ga-Ga distances at 3.03 and 3.56 A, typical for edge and corner sharing GaO6 octahedra, respectively. The gallium(III) and aluminum(III) citrate systems were compared by means of FTIR, and were found to be analogous. The IR results suggest that the bond lengths derived from EXAFS for the 1:2 gallium(III) citrate complex also provide a good estimate of the corresponding distances in the mononuclear 1:1 complex. Direct coordination of citrate to the metal ions in the oligomeric gallium(III) citrate complex was indicated from both EXAFS and IR results, and this complex is stoichiometrically analogous to the Al3(H-1Cit)3(OH)(H2O)4- complex, which has been structurally determined. However, while the formation of the aluminum trimer has been shown to be slow, the gallium trimer was significantly more labile with a rate of formation indicated to be in the order of seconds or faster.     

A Monomeric Variant of Insulin Degrading Enzyme (IDE) Loses Its Regulatory Properties

Background

Insulin degrading enzyme (IDE) is a key enzyme in the metabolism of both insulin and amyloid beta peptides. IDE is unique in that it is subject to allosteric activation which is hypothesized to occur through an oligomeric structuture.

Methodology/Principal Findings

IDE is known to exist as an equilibrium mixture of monomers, dimers, and higher oligomers, with the dimer being the predominant form. Based on the crystal structure of IDE we deleted the putative dimer interface in the C-terminal region, which resulted in a monomeric variant. Monomeric IDE retained enzymatic activity, however instead of the allosteric behavior seen with wild type enzyme it displayed Michaelis-Menten kinetic behavior. With the substrate Abz-GGFLRKHGQ-EDDnp, monomeric IDE retained ∼25% of the wild type activity. In contrast with the larger peptide substrates β-endorphin and amyloid β peptide 1–40, monomeric IDE retained only 1 to 0.25% of wild type activity. Unlike wild type IDE neither bradykinin nor dynorphin B-9 activated the monomeric variant of the enzyme. Similarly, monomeric IDE was not activated by polyphosphates under conditions in which the activity of wild type enzyme was increased more than 50 fold.

Conclusions/Significance

These findings serve to establish the dimer interface in IDE and demonstrate the requirement for an oligomeric form of the enzyme for its regulatory properties. The data support a mechanism where the binding of activators to oligomeric IDE induces a conformational change that cannot occur in the monomeric variant. Since a conformational change from a closed to a more open structure is likely the rate-determining step in the IDE reaction, the subunit induced conformational change likely shifts the structure of the oligomeric enzyme to a more open conformation.     

Template-directed synthesis on the oligonucleotide d(C7-G-C7)

When the deoxynucleotide template d(C7-G-C7) is incubated with the activated nucleotides 2-MeImpG and 2-MeImpC, a series of oligomers of G up to the sevenmer and a series of copolymers of composition GnC with n = 3 to 13 are formed. Oligomers GnC with n greater than 7 are completely degraded by pancreatic ribonuclease, establishing that they contain a 3' to 5' internucleotide bond between 5'-C and 3'-G within a sequence of the form (pG)ipC(pG)j. As expected, (pG)7-Cp and (pG)6-Cp are major hydrolysis products. Detailed analysis of the product distribution shows that a substantial fraction of the oligomeric products are of the type (pG)ipC(pG)j with i less than 7. This shows that product synthesis does not necessarily begin at the 3' terminus of the template. The significance of this finding in terms of the origin of molecular replication is discussed.     

Catalysis of accurate poly(C)-directed synthesis of 3'-5'-linked oligoguanylates by Zn2+

Zn²⁺ is an efficient catalyst for the oligomerization of guanosine 5′-phosphorimidazolide on a polycytidylic acid template. Up to 75% of the input ImpG² is converted to oligomers with a mean chain length up to 10. Material longer than (pG)₃₀ can be detected. The oligomeric products are predominantly 3′-5′-linked.If poly(C) is incubated with ImpG and an equimolar quantity of the 5′-phosphorimidazolides of adenosine, uridine or cytidine, in the presence of Zn²⁺, ImpG is incorporated at least 200 times more efficiently than “incorrect” nucleotides.     

The oligomeric Rep protein of Mungbean yellow mosaic India virus (MYMIV) is a likely replicative helicase

Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT downward arrow AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring > or =6 nt space for its efficient activity, translocates in the 3'-->5' direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation ( approximately 24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV).     

Oligomeric forms of peptide fragment PrP(214-226) in solution are preferentially recognized by PrP(Sc)-specific antibody

A specific monoclonal antibody (mAb) V5B2 that discriminates between brain tissue of Creutzfeldt-Jakob disease patients and that from normal controls without proteinase K digestion has been prepared using a 13-residue synthetic peptide P1 from the primary structure of human PrP. In the light of the specific interaction between mAb V5B2 and the pathological isoform of PrP (PrP(Sc)), we investigated the solution behavior of antigen P1 and its interactions with mAb V5B2. Our results show that V5B2 recognizes epitope P1 in dimeric/oligomeric forms in solution and in the fibril-like aggregates, as well as in PrP(Sc) aggregates, and demonstrate that the specific epitope is present in all of these forms, but not in PrP(C).     

Silicone fouling-release coatings: effects of the molecular weight of poly(dimethylsiloxane) and tetraethyl orthosilicate on the magnitude of pseudobarnacle adhesion strength

A series of poly(dimethyl siloxane) (PDMS)/silica nanocomposites were synthesized utilizing a sol gel method. The samples were evaluated using pseudobarnacle adhesion and tensile strength tests. The effects of the molecular weight of the PDMS and the size and structure of the silica domains on biofouling release and the mechanical behavior of the PDMS/silica materials were investigated. Three different molecular weights (18,000, 49,000 and 79,000 g mol(-1)) of hydroxyl-terminated PDMS (HT-PDMS) were used to prepare the nanocomposites with three different weight ratios (1:1, 3:1 and 5:1) of HT-PDMS to tetraethyl orthosilicate (TEOS). TEOS served as a crosslinker to form PDMS networks and as a precursor to form silica domains. Two different variants of TEOS with regard to its degree of polymerization (n) (monomeric type: n ≈= 1 and oligomeric type: n ≈= 5) were used for in situ formation of silica particles via the sol-gel process. The mechanical properties of the composites were characterized using stress-strain isotherms. All the mechanical properties evaluated (Young's modulus, tensile strength, energy required for rupture, elongation at break) improved with increases in the molecular weight of the HT-PDMS and the silica content. The pseudobarnacle adhesion test was used to examine the fouling- release (FR) properties of coatings applied on aluminum plates. The rupture energy and tensile strength increased substantially when oligomeric TEOS was employed in the PDMS/silica composites. Scanning electron microscopy (SEM) was used to investigate the structure of the silica domains. It was found that the use of oligomeric TEOS in higher molecular weight PDMS samples with higher PDMS/TEOS weight ratios led to low pseudobarnacle adhesion strengths of ≈ 0.3 MPa, which is in the range of commercial FR coatings.     

A 54-kDa fragment of the Poly(A)-specific ribonuclease is an oligomeric, processive, and cap-interacting Poly(A)-specific 3' exonuclease

We have previously identified a HeLa cell 3' exonuclease specific for degrading poly(A) tails of mRNAs. Here we report on the purification and identification of a calf thymus 54-kDa polypeptide associated with a similar 3' exonuclease activity. The 54-kDa polypeptide was shown to be a fragment of the poly(A)-specific ribonuclease 74-kDa polypeptide. The native molecular mass of the nuclease activity was estimated to be 180-220 kDa. Protein/protein cross-linking revealed an oligomeric structure, most likely consisting of three subunits. The purified nuclease activity released 5'-AMP as the reaction product and degraded poly(A) in a highly processive fashion. The activity required monovalent cations and was dependent on divalent metal ions. The RNA substrate requirement was investigated, and it was found that the nuclease was highly poly(A)-specific and that only 3' end-located poly(A) was degraded by the activity. RNA substrates capped with m(7)G(5')ppp(5')G were more efficiently degraded than noncapped RNA substrates. Addition of free m(7)G(5')ppp(5')G cap analogue inhibited poly(A) degradation in vitro, suggesting a functional link between the RNA 5' end cap structure and poly(A) degradation at the 3' end of the RNA.     

Stable octameric structure of recombinant hemoglobin alpha(2)beta(2)83 Gly-->Cys

We have engineered a recombinant hemoglobin (rHb betaG83C) based on the variant Hb Ta-Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb betaG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide-binding properties similar to those of natural human hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto-oxidation rate, and gel filtration experiments on the oligomeric betaG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric betaG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer betaG83C was not modified after incubation with fresh plasma.     

Versatility of the small heat shock protein HSPB6 (Hsp20)

The recently published review by Dreiza et al. (Cell Stress and Chaperones DOI ) dealing with the functional role of HSPB6 in muscle regulation is critically analyzed. Published data indicate that the chaperone-like activity of HSPB6 is comparable with that of HSPB5 and that phosphorylation of HSPB6 does not affect its oligomeric structure. Different hypotheses concerning the molecular mechanisms of HSPB6 action on smooth muscle contraction and on the reorganization of the cytoskeleton are compared, and it is concluded that although HSPB6 is not a genuine actin-binding protein, it can affect the actin cytoskeleton indirectly. Phosphorylated HSPB6 interacts with 14-3-3 and thereby displaces other binding partners of 14-3-3; among them, certain phosphatases, protein kinases, and various actin-binding proteins, which can participate in the reorganization of the actin cytoskeleton. In addition, HSPB6 seems to regulate the activity of certain protein kinases. All of these processes are dependent on HSPB6 phosphorylation which in turn might be regulated by the formation of heterooligomeric complexes of HSPB6 with other small heat shock proteins.     

Studies of copper(II) and nickel(II) complexation by bis(1,4,7-triazacyclononane) ligands containing alkene and alkyne bridging groups

The copper(II) and nickel(II) complexes of three new 1,2-bis(1,4,7-triazacyclononane) ligands containing unsaturated four carbon bridging groups is studied by continuous variation UV-Vis spectroscopic and pH potentiometric equilibrium experiments. The cis-butene-2 (L_C) linked ligand may form monomeric MN₆-type complexes while the trans-butene-2 (L_T) and butyne-2 (L_Y) ligands are prevented by their stereochemistry from forming monomeric complexes and form oligomeric complexes. It is determined that the stability of the CuL_C²⁺ complex is not appreciably different from the oligomeric complexes of L_T and L_Y. Single-crystal X-ray structure determinations are made on three square pyramidal Cu₂L⁴⁺ complexes: [Cu₂L_CCl₄] (1), [Cu₂L_YCl₄] (2), and [Cu₂L_T(NO₃)₂(H₂O)₂](NO₃)₂ (3). The structure of [Ni₂(L_C)₂](ClO₄)₄ · 2H₂O (4) is a binuclear dimer that contains two nickel(II) ions sandwiched between two ligands, indicating that bis([9]aneN₃) ligands with four linker atom chains may form either monomeric or oligomeric structures.     

Giant extracellular Glossoscolex paulistus Hemoglobin (HbGp) upon interaction with cethyltrimethylammonium chloride (CTAC) and sodium dodecyl sulphate (SDS) surfactants: Dissociation of oligomeric structure and autoxidation

The effects of two ionic surfactants on the oligomeric structure of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy - form have been studied through the use of several spectroscopic techniques such as electronic optical absorption, fluorescence emission, light scattering, and circular dichroism. The use of anionic sodium dodecyl sulphate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) has allowed to differentiate the effects of opposite headgroup charges on the oligomeric structure dissociation and hemoglobin autoxidation. At pH 7.0, both surfactants induce the protein dissociation and a significant oxidation. Spectral changes occur at very low CTAC concentrations suggesting a significant electrostatic contribution to the protein–surfactant interaction. At low protein concentration, 0.08 mg/ml, some light scattering within a narrow CTAC concentration range occurs due to protein–surfactant precipitation. Light scattering experiments showed the dissociation of the oligomeric structure by SDS and CTAC, and the effect of precipitation induced by CTAC. At higher protein concentrations, 3.0 mg/ml, a precipitation was observed due to the intense charge neutralization upon formation of ion pair in the protein–surfactant precipitate. The spectral changes are spread over a much wider SDS concentration range, implying a smaller electrostatic contribution to the protein–surfactant interactions. The observed effects are consistent with the acid isoelectric point (pI) of this class of hemoglobins, which favors the intense interaction of HbGp with the cationic surfactant due to the existence of excess acid anionic residues at the protein surface. Protein secondary structure changes are significant for CTAC at low concentrations while they occur at significantly higher concentrations for SDS. In summary, the cationic surfactant seems to interact more strongly with the protein producing more dramatic spectral changes as compared to the anionic one. This is opposite as observed for several other hemoproteins. The surfactants at low concentrations produce the oligomeric dissociation, which facilitates the iron oxidation, an important factor modulating further oligomeric protein dissociation.     

Identification of the ouabain-binding peptide of (nak)-atpase).

The [3H]-ouabain-(NaK)-ATPase complex when treated in the cold with sodium dodecyl sulfate (SDS) dissociates into a larger and smaller peptide called alpha and beta, resp.. Analysis of the released peptides by SDS-polyacrylamide gel electrophoresis reveals that [3H]-ouabain co-migrates with the alpha-peptide only, being apparently identic with the ouabain receptor molecule. The percentage occupancy of the receptor peptide with [3H]-ouabain can be increased up to 90% evidencing the stabilization of the [3H]-ouabain-alpha-peptide complex by SDS-exposure and release from the oligomeric enzyme. A hypothetic explanation for the seemingly paradoxical stabilising effect of SDS on the complex is offered.     

Purification and properties of extracellular poly(3-hydroxybutyrate) depolymerases from Pseudomonas lemoignei

Extracellular poly(3-hydroxybutyrate) depolymerase was purified from the culture medium of Peudomonas lemoignei and separated into four isozymes (A1, A2, B1 and B2) by CM-Sepharose CL-6B chromatography. The molecular weights of A1 and A2 and those of B1 and B2 were estimated to be 54 000 and 58 000, respectively, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric points of A1, A2, B1 and B2 were found to be approximately pH 9.7, 10.0, 10.0 and 10.6, respectively, by isoelectric focusing. All four enzymes hydrolyzed poly(3-hydroxybutyrate) and oligomeric esters of D-(-)-3-hydroxybutyrate, but showed no activity toward the dimeric ester. Analysis of hydrolytic products of the oligomeric esters with A1 and B2 suggested that the enzymes hydrolyzed mainly the second and third ester bonds from the free hydroxy terminus at different frequencies, depending upon the chain length of the substrates.     

Non-enzymatic template-directed synthesis on RNA random copolymers. Poly(C, U) templates

Poly(C, U) random copolymer templates direct the oligomerization of 2-MeImpG and 2-MeImpA, resulting in the production of a variety of oligo/(G,A)s. The efficiency of monomer incorporation into newly synthesized oligomers is greater for 2-MeImpG than for 2-MeImpA, and decreases for both monomers as the uracil content of the template increases. The relatively poor incorporation of adenine is partly due to an intrinsically less efficient incorporation reaction, and partly due to the masking of uracil sites by G X U non-complementary pairing. The efficiency of adenine incorporation can be improved by decreasing the concentration of 2-MeImpG and increasing the concentration of 2-MeImpA in the reaction mixture. The oligomeric product distribution can be characterized in detail using high-pressure liquid chromatography on an RPC-5 column. Oligomers are separated on the basis of chain length, base composition, and phospho-diester-linkage isomerism. The 3'----5' regiospecificity of monomer addition to template-bound oligomers is lower for 2-MeImpA than for 2-MeImpG. The presence of an adenine residue at the 2'(3') terminus of the acceptor strand lowers the regiospecificity of 2-MeImpA addition even further.     

Non-enzymic template-directed synthesis on RNA random copolymers. Poly(C, G) templates

Poly(C, G) random copolymer templates direct the oligomerization of 2-Me-ImpG and 2-MeImpC, resulting in the production of a variety of oligo(G, C)s. The efficiency of monomer incorporation into newly synthesized oligomers is greater for 2-MeImpG than for 2-MeImpC, and decreases for both monomers as the guanine content of the template increases. The relatively low efficiency of oligomerization on guanine-rich templates is largely a consequence of intra- and intermolecular template self-structure. The problem of template self-structure is clearly a major obstacle to the development of a system of self-replicating polynucleotides. The distribution of oligomeric products can be characterized in detail using high-pressure liquid chromatography on an RPC-5 column. Oligomers are separated on the basis of chain length, base composition and phosphodiester-linkage isomerism. Oligomers up to about the 12-mer, with base composition Gn, Gn-1C and Gn-2C2, have been identified. The 3' to 5' regiospecificity of the products is high, particularly for oligomers with base composition Gn.     

Active (9.6 s) and inactive (21 s) oligomers of NHE3 in microdomains of the renal brush border

We have previously shown that Na(+)-H(+) exchanger isoform NHE3 exists as both 9.6 and 21 S (megalin-associated) oligomers in the renal brush border. To characterize the oligomeric forms of the renal brush border Na(+)-H(+) exchanger in more detail, we performed membrane fractionation studies. We found that similar amounts of NHE3 were present in microvilli and a nonmicrovillar membrane domain of high density (dense vesicles). Horseradish peroxidase-labeled endosomes were not prevalent in the dense membrane fraction. However, megalin, which localizes primarily to the intermicrovillar microdomain of the brush border, was enriched in the dense vesicles, implicating this microdomain as the likely source of these membranes. Immunolocalization of NHE3 confirmed that a major fraction of the transporter colocalized with megalin in the intermicrovillar region of the brush border. Immunoprecipitation studies demonstrated that in microvilli the majority of NHE3 was not bound to megalin, while in the dense vesicles most of the NHE3 coprecipitated with megalin. Moreover, sucrose velocity gradient centrifugation experiments revealed that most NHE3 in microvilli sedimented with an S value of 9.6, while the S value of NHE3 in dense vesicles was 21. Finally, we examined the functional state of NHE3 in both membrane fractions. As assayed by changes in acridine orange fluorescence, imposing an outwardly directed Na(+) gradient caused generation of an inside acid pH gradient in the microvilli, indicating Na(+)-H(+) exchange activity, but not in the dense vesicles. Taken together, these data demonstrate that renal brush border NHE3 exists in two oligomeric states: a 9.6 S active form present in microvilli and a 21 S, megalin-associated, inactive form in the intermicrovillar microdomain of the apical plasma membrane. Thus, regulation of renal brush border Na(+)-H(+) exchange activity may be mediated by shifting the distribution between these forms of NHE3.