Interaction of FXYD10 (PLMS) with Na,K-ATPase from shark rectal glands. Close proximity of Cys74 of FXYD10 to Cys254 in the a domain of the alpha-subunit revealed by intermolecular thiol cross-linking

FXYD domain-containing proteins are tissue-specific regulators of the Na,K-ATPase that have been shown to have significant physiological implications. Information about the sites of interaction between some FXYD proteins and subunits of the Na,K-ATPase is beginning to emerge. We previously identified an FXYD protein in plasma membranes from shark rectal gland cells and demonstrated that this protein (FXYD10) modulates shark Na,K-ATPase activity. The present study was undertaken to identify the location of the C-terminal domain of FXYD10 on the alpha-subunit of Na,K-ATPase, using covalent cross-linking combined with proteolytic cleavage. Treatment of Na,K-ATPase-enriched membranes with the homobifunctional thiol cross-linker 1,4-bismaleimidyl-2,3-dihydroxybutane resulted in cross-linking of FXYD10 to the alpha-subunit. Cross-linking was not affected by preincubation with sodium or potassium but was significantly reduced after pre-incubation with the non-hydrolyzable ATP analog beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP). A peptic assay was developed, in which pepsin treatment of Na,K-ATPase at low pH resulted in extensive cleavage of the alpha-subunit while FXYD10 was left intact. Proteolytic fragments of control and cross-linked preparations were isolated by immunoprecipitation and analyzed by gel electrophoresis. A proteolytic fragment containing FXYD10 cross-linked to a fragment from the alpha-subunit could be localized on SDS gels. Sequencing of this fragment showed the presence of FXYD10 as well as a fragment within the A domain of the alpha-subunit comprising 33 amino acids, including a single Cys residue, Cys254. Thus, regulation of Na,K-ATPase by FXYD10 occurs in part via cytoplasmic interaction of FXYD10 with the A domain of the shark alpha-subunit.     

Identification and characterization of a heterogeneous population of growth hormone receptors in mouse hepatic membranes

Covalent cross-linking of radiolabeled mouse growth hormone (125I-mGH) with the homobifunctional cross-linking agent bis(sulfosuccinimidyl)suberate (BS3) to microsomal membranes prepared from late pregnant mouse liver resulted in the labeling of three specific mGH binding proteins (receptors) with apparent Mr = 125,000, 62,000, and 56,000, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. These same three specifically labeled proteins were present, but with slightly lower apparent molecular weights, when samples were electrophoresed in the absence of reductant. Cross-linking of 125I-mGH to plasma membrane-enriched fractions of late pregnant mouse liver resulted only in the specific labeling of the two lower molecular weight receptors. Removal of all N-linked carbohydrate with peptide: N-glycosidase F resulted in decreasing the apparent molecular weights of the three receptor forms to 110,000, 50,000, and 46,000 for the 125,000, 62,000, and 56,000 molecular weight forms of the receptor, respectively. Smaller decreases in the molecular weights of all three receptor forms were also apparent after treatment with neuraminidase. However, the differences seen in the intact forms of the growth hormone receptor were also present in the deglycosylated receptors. The relationship between the three forms of the growth hormone receptor was further investigated by comparing the fragments produced by proteolytic digestion of the cross-linked receptors with Staphylococcus aureus protease and endoproteinase Lys-C. The fragments produced from all three receptor forms had very similar molecular weights, although there were slight molecular weight differences in the fragments produced by endoproteinase Lys-C digestion. The overall similarity of the fragments produced by the proteolytic digestions suggests that the three forms of the receptor are related.     

Factor XIIIa-mediated cross-linking of fibronectin in fibroblast cell layers. Cross-linking of cellular and plasma fibronectin and of amino-terminal fibronectin fragments

Factor XIIIa cross-links plasma fibronectin as it is being assembled into the extracellular matrix of cultured human skin fibroblasts (Barry, E. L. R., and Mosher, D. F. (1988) J. Biol. Chem. 262, 10464-10469). We have further characterized this process. Fibroblasts were metabolically labeled with proline in the presence or absence of ascorbate and Factor XIIIa. Endogenous fibronectin in the extracellular matrix was cross-linked by Factor XIIIa. There was no evidence for cross-linking of collagenous proteins. Fibro-blast cell layers were incubated with iodinated 27-kDa heparin-binding or 70-kDa collagen- and heparin-binding amino-terminal fibronectin fragments. Factor XIIa cross-linked the fragments into high molecular weight aggregates. The amounts of cross-linked fragments reaches a steady state after 1 to 2 h, whereas intact fibronectin continues to be cross-linked for 24 h. When fibroblast cell layers were pulsed with iodinated fibronectin or amino-terminal fragments and Factor XIIIa was included in the chase media, the high molecular weight aggregates were formed in a step-wise manner. The smallest cross-linking steps were to high molecular weight extracellular matrix molecules forming approximately 270-, 300-, and 440-kDa complexes for the 27-kDa fragment, 70-kDa fragment, and intact fibronectin, respectively. When iodinated fibronectin was bound to fibroblast cell layers and chased into the matrix pool in the absence of Factor XIIIa, it could also be cross-linked into high molecular weight complexes when Factor XIIIa was added to the media. These results, therefore, indicate that both cellular and plasma fibronectin and amino-terminal fragments are cross-linked specifically by Factor XIIIa, that the cross-linking is probably to other fibronectin molecules rather than to collagenous proteins, and that both assembling and assembled fibronectin are substrates for Factor XIIIa.     

Isolation and characterization of the rainbow trout erythrocyte band-3 protein

Rainbow trout (Salmo gairdneri) band-3 protein was isolated from trout erythrocyte plasma membranes by a combination of preparative SDS/PAGE and electroelution. High purity and recovery of the plasma membranes were achieved by a new method. This was demonstrated using 4,4'diiso-thiocyano[3H2]dihydro-stilbene 2,2'disulfonic acid (3H2DIDS) which specifically labels band-3 protein. On SDS/PAGE, band-3 protein yields a similarly diffuse pattern, as does mammalian band-3 protein, with an apparent Mr of 116,000. In situ chymotryptic cleavage/cross-linking experiments with 3H2DIDS reveal that the fragments cross-link as in human and mouse band-3 proteins but that there are minor differences. Treatment of trout erythrocytes with trypsin results in cleavage of the band-3 protein. Purified polyclonal antibodies raised against trout band-3 protein react with trout band-3 protein and do not crossreact with mouse or human band-3 protein. They react specifically with only one chymotryptic fragment of trout band-3 protein.     

Binding properties of the stilbene disulfonate sites on human erythrocyte AE1: kinetic, thermodynamic, and solid state deuterium NMR analyses.

A novel stilbene disulfonate, 4-trimethylammonium-4'-isothiocyanostilbene-2,2'-disulfonic acid (TIDS), has been chemically synthesized, and the interaction of this probe with human erythrocyte anion exchanger (AE1) was characterized. Covalent labeling of intact erythrocytes by [N(+)((14)CH(3))(3)]TIDS revealed that specific modification of AE1 was achieved only after removal of other ligand binding sites by external trypsinization. Following proteolysis, (1.2 +/- 0.4) x 10(6) TIDS binding sites per erythrocyte could be blocked by prior treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a highly specific inhibitor of AE1. Inhibition of sulfate equilibrium exchange by TIDS in whole cells was described by a Hill coefficient of 1.10 +/- 0.06, which reduced to 0.51 +/- 0.01 following external trypsinization. The negative cooperativity of TIDS binding following external trypsinization suggests that trypsin-sensitive proteins modulate allosteric coupling between AE1 monomers. Thermodynamic analysis revealed that TIDS binding induces smaller conformational changes in AE1 than is observed following DIDS binding. The similar inhibitory potencies of both TIDS (IC(50) = 0.71 +/- 0.48 microM) and DIDS (IC(50) = 0.2 microM) imply that there is no correlation between the ability of stilbene disulfonates to arrest anion exchange function and the magnitude of ligand-induced conformational changes in AE1. Solid state (2)H NMR analysis of a [N(+)(CD(3))(3)]TIDS-AE1 complex in both unoriented and macroscopically oriented membranes revealed that large amplitude "wobbling" motions describe ligand dynamics. The data are consistent with a model where TIDS bound to AE1 is located exofacially in contact with the bulk aqueous phase.     

Protoporphyrin-induced photodynamic effects on band 3 protein of human erythrocyte membranes

In previous studies it has been shown that protoporphyrin-induced photodynamic effects on red blood cells are caused by photooxidation of amino acid residues in membrane proteins and by the subsequent covalent cross-linking of these proteins. Band 3, the anion transport protein of the red blood cell membrane, has a relatively low sensitivity to photodynamic cross-linking. This cannot be attributed to sterical factors inherent in the specific localization of band 3 in the membrane structure. Solubilized band 3, for instance, showed a similar low sensitivity to cross-linking. By extracellular chymotrypsin cleavage of band 3 into fragments of 60 000 and 35 000 daltons it could be shown that both fragments were about equally sensitive to photodynamic cross-linking. The 17 000 dalton transmembrane segment, on the other hand, was completely insensitive. Inhibition of band 3-mediated sulfate transport proceeded much faster than band 3 interpeptide cross-linking, presumably indicating that the inhibition of transport is caused by photooxidation of essential amino acid residues or intrapeptide cross-linking. A close parallel was observed between photodynamic inhibition of anion transport and decreased binding of 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonate (H₂DIDS), suggesting that a photooxidation in the immediate vicinity of the H₂DIDS binding site may be responsible for transport inhibition.     

Identification of the V1 vasopressin receptor by chemical cross-linking and ligand affinity blotting

Chemical and photoaffinity cross-linking experiments as well as ligand affinity blotting techniques were used to label the V1 vasopressin receptor. In order to determine the optimal reaction conditions, pig liver membranes were incubated with 5 nM [8-lysine]vasopressin (LVP) labeled with 125I and then cross-linked with the use of DMS (dimethyl suberimidate), EGS [ethylene glycol bis(succinimidyl succinate)] or HSAB (hydroxysuccinimidyl p-azidobenzoate) at different final concentrations. Consistently, EGS was found to label with high yield one band of Mr 60,000 in rat and pig liver membranes when used at a final concentration between 0.05 and 0.25 mM. The protein of Mr 60,000 is labeled in a concentration-dependent manner when pig liver membranes are incubated with increasing concentrations of 125I-LVP and then cross-linked with EGS. The label was displaced by increasing concentrations of unlabeled LVP or d(CH2)5 [Tyr2(Me),-Tyr9(NH2)]AVP (V1/V2 antagonist). A protein band of similar molecular mass was cross-linked with 125I-LVP in rat liver membranes. The reaction was specific since the incorporation of label into the protein of Mr 60,000 was inhibited by LVP, [8-arginine]vasopressin (AVP), the V1/V2-antagonist, and the specific V1-antagonist d(CH2)5 [Tyr2(Me)]AVP, only partially by [des-Gly9]AVP (V2-agonist) and by oxytocin, and not at all by angiotensin II. Incubation of nitrocellulose containing membrane proteins from pig liver with 125I-LVP showed the labeling of a band of Mr 58,000 that is inhibited by an excess of unlabeled LVP. This band of Mr 58,000 seems to correspond with the protein of Mr 60,000 revealed by the cross-linking experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of the permeation properties of glomerular basement membrane: cross-linking renders glomerular basement membrane permeable to protein.

Cross-linking glomerular basement membrane (GBM) has been shown to render it more permeable to protein. Isolated pig GBM was cross-linked with dimethylmalonimidate which reacts selectively with lysine epsilon-NH2 groups or with glutaraldehyde, a less selective cross-linking agent. Studies of the ultrafiltration properties of these materials in vitro using cytochrome c, myoglobin, bovine serum albumin and immunoglobulin showed that cross-linking had markedly increased solvent and protein fluxes as compared with native membranes particularly at higher pressures. Filtration studies with serum demonstrated that the cross-linked membranes were more permeable to serum proteins. Thickness measurements under pressure indicated that cross-linked membrane was less compressed than native membrane as pressure was increased. Pore theory did not provide a suitable model for analysis of the results, but analysis of the results using the fibre-matrix hypothesis indicated that cross-linking had the effect of bundling together the fibres (type IV collagen) in the GBM matrix. The effect of cross-linking on filtration could be explained by a combination of contraction of the membrane, fibre bundling and increased rigidity compared with native membrane. Cross-linking of GBM might lead to long-term damage of the glomerular capillary wall in nephritis, so promoting proteinuria.     

Anion exchanger is present in both luminal and basolateral renal membranes

Binding of the anion-exchange inhibitor 3H2-labeled 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) to highly purified luminal and basolateral beef kidney tubular membranes was characterized. Specific binding of [3H2]DIDS is present in both luminal and basolateral membranes. Scatchard analysis revealed a Kd for [3H2]DIDS of 5.5 microM and 19.3 microM and a maximal number of binding sites of 10.9 nmol and 31.7 nmol DIDS/mg protein in basolateral and luminal membranes, respectively. To assess the role of this putative anion exchanger on transport we measured 35SO4 uptake by luminal and basolateral membranes. In both luminal and basolateral membranes sulfate uptake was significantly greater in the presence of an outward-directed Cl gradient, OH gradient or HCO3 gradient than in the absence of these gradients. There was an early anion-dependent sulfate uptake of five to ten times the equilibrium uptake at 60 min. The sulfate taken in could be released by lysis of the vesicles indicating true uptake and not binding of sulfate. No significant difference in SO4 uptake was found in the presence and in the absence of valinomycin, indicating that the anion exchanger is electroneutral. The anion-dependent sulfate uptake was completely inhibited by either DIDS or furosemide in both luminal and basolateral membranes. Dixon analysis of HCO3-dependent SO4 uptake by luminal membranes in the presence of different concentrations of DIDS revealed a Ki for DIDS of 20 microM. The similar values of the Kd for [3H2]DIDS binding and the Ki for DIDS inhibition of SO4 uptake might suggest an association between DIDS binding and the inhibition of SO4 transport. In addition, an inward-directed Na gradient stimulated sulfate uptake in luminal but not in basolateral membranes. The Na-dependent sulfate uptake in luminal membranes was also inhibited by DIDS. We conclude that, in addition to the well-known Na-dependent sulfate uptake in luminal membranes, there exists an anion exchanger in both basolateral and luminal membranes capable of sulfate transport.     

A new variant of the anion transport protein in human erythrocytes

The major plasma membrane protein of human erythrocytes is the anion transport protein, termed protein 3. We previously reported a variant form of protein 3 that is elongated on the amino-terminal end of the molecule, which is exposed on the cytoplasmic side of the membrane, but otherwise its features are identical with those of the normal molecule. We have termed this molecule protein 3 variant 1. We now report a new variant form, protein 3 variant 2. The erythrocyte donor was a double heterozygote whose red cells possess a normal protein 3 and a protein 3 variant which is elongated and possesses a second variation at the 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) reactive site. Variant 2 reacts with 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid (H2DIDS) more readily than does the normal molecule. At high pH values, H2DIDS acts as a bifunctional cross-linking agent; it cross-links the proteolytic products generated by Pronase (or chymotrypsin) treatment of variant 2 less efficiently than noted for normal protein 3 or the first variant. Thus, the newly identified molecule has an alteration at the DIDS reactive site, which is near the outer surface of the membrane. The results can be interpreted as indicating that the DIDS binding site of variant 2 is more exposed than the normal molecule, but further removed from the site on the carboxyl-terminal fragment involved in cross-linking. Although there is a difference in the reactivity of the two protein 3 chains in variant 2, the reaction of variants 1 and 2 and normal cells with varying concentrations of [3H]H2DIDS results in the same amount of incorporation in all cells. Since protein 3 exists as a dimer or higher aggregate in the membrane, these results may indicate an interaction between monomers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of the permeation properties of glomerular basement membrane: cross-linking renders glomerular basement membrane permeable to protein

Cross-linking glomerular basement membrane (GBM) has been shown to render it more permeable to protein. Isolated pig GBM was cross-linked with dimethylmalonimidate which reacts selectively with lysine ?-NH₂ groups or with glutaraldehyde, a less selective cross-linking agent. Studies of the ultrafiltration properties of these materials in vitro using cytochrome c, myoglobin, bovine serum albumin and immunoglobulin showed that cross-linking had markedly increased solvent and protein fluxes as compared with native membranes particularly at higher pressures. Filtration studies with serum demonstrated that the cross-linked membranes were more permeable to serum proteins. Thickness measurements under pressure indicated that cross-linked membrane was less compressed than native membrane as pressure was increased. Pore theory did not provide a suitable model for analysis of the results, but analysis of the results using the fibre-matrix hypothesis indicated that cross-linking had the effect of bundling together the fibres (type IV collagen) in the GBM matrix. The effect of cross-linking on filtration could be explained by a combination of contraction of the membrane, fibre bundling and increased rigidity compared with native membrane. Cross-linking of GBM might lead to long-term damage of the glomerular capillary wall in nephritis, so promoting proteinuria.     

Cross-linking of the skeletal myosin subfragment 1 heavy chain to the N-terminal actin segment of residues 40-113

Glutaraldehyde (GA) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), a hydrophobic, carboxyl group directed, zero-length protein cross-linker, were employed for the chemical cross-linking of the rigor complex between F-actin and the skeletal myosin S-1. The enzymatic properties and structure of the new covalent complexes obtained with both reagents were determined and compared to those known for the EDC-acto-S-1 complex. The GA- or EEDQ-catalyzed covalent attachment of F-actin to the S-1 heavy chain induced an elevated Mg2+-ATPase activity. The turnover rates of the isolated cross-linked complexes were similar to those for EDC-acto-S-1 (30 s-1). The solution stability of the new complexes is also comparable to that exhibited by EDC-acto-S-1. The proteolytic digestion of the isolated AEDANS-labeled covalent complexes and direct cross-linking experiments between actin and various preformed proteolytic S-1 derivatives indicated that, as observed with EDC, the COOH-terminal 20K and the central 50K heavy chain fragments are involved in the cross-linking reactions of GA and EEDQ. KI-depolymerized acto-S-1 complexes cross-linked by EDC, GA, or EEDQ were digested by thrombin which cuts only actin, releasing S-1 heavy chain-actin peptide cross-linked complexes migrating on acrylamide gels with Mr 100K (EDC), 110K and 105K (GA), and 102K (EEDQ); these were fluorescent only when fluorescent S-1 was used. They were identified by immunostaining with specific antibodies directed against selected parts of he NH2-terminal actin segment of residues 1-113.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains cross-linked by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide.

The structural organization of photosystem II proteins has been investigated by use of the zero-length protein cross-linking reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and monoclonal and polyclonal antibody reagents. Photosystem II membranes were treated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide which cross-links amino groups to carboxyl groups which are in van der Waals contact. This treatment did not affect the oxygen evolution rates of these membranes and increased the retention of oxygen evolution after CaCl2 washing. Analysis of the proteins cross-linked by this treatment indicated that two cross-linked species with apparent molecular masses of 95 and 110 kDa were formed which cross-reacted with antibodies against both the 33-kDa manganese-stabilizing protein and the chlorophyll protein CPa-1. Cleavage of the 110-kDa cross-linked species with cyanogen bromide followed by N-terminal sequence analysis was used to identify the peptide fragments of CPa-1 and the manganese-stabilizing protein which were cross-linked. Two cyanogen bromide fragments were identified with apparent molecular masses of 50 and 25 kDa. N-Terminal sequence analysis of the 50-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa fragment of CPa-1 and the intact manganese-stabilizing protein. This strongly suggests that the manganese-stabilizing protein is cross-linked to the large extrinsic loop domain of CPa-1. N-Terminal analysis of the 25-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa peptide of CPa-1 and the N-terminal 8-kDa peptide of the manganese-stabilizing protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-linking of actin to myosin subfragment 1: course of reaction and stoichiometry of products

The cross-linking of actin to myosin subfragment 1 (S-1) with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide was reexamined by using two cross-linking procedures [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306; Sutoh, K. (1983) Biochemistry 22, 1579-1585] and two independent methods for quantitating the reaction products. In the first approach, the cross-linked acto-S-1 complexes were cleaved with elastase at the 25K/50K and 50K/22K junctions in S-1. This enabled direct measurements of the cross-linked and un-cross-linked fractions of the 50K and 22K fragments of S-1. We found that in all cases actin was preferentially cross-linked to the 22K fragment and that the overall stoichiometry of the main cross-linked products was that of a 1:1 complex of actin and S-1. In the second approach, actin was cross-linked to tryptically cleaved S-1, and the course of these reactions was monitored by measuring the decay of the free 50K and 20K fragments and the formation of cross-linked products. After selecting the optimal cross-linking procedure and conditions, we determined that the rate of actin cross-linking to the 20K fragment of S-1 was 3-fold faster than the reaction with the 50K peptide. The overall rate of cross-linking actin to S-1 corresponded to the sum of the individual reactions of the 50K and 20K fragments, indicating their mutually exclusive cross-linking to actin. Thus, the reactions with tryptically cleaved S-1 were consistent with the 1:1 stoichiometry of actin and S-1 in the main cross-linked products and verified the preferential cross-linking of actin to the 20K fragment of S-1. These results are discussed in the context of the binding of actin to S-1.     

Cl(-)/HCO(3)(-) exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca(2+)](i) increase in salivary acinar cells

Large volumes of saliva are generated by transepithelial Cl(-) movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl(-) uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na(+) independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an approximately 170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive Cl(-)/HCO(3)(-) exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the Cl(-)/HCO(3)(-) exchanger activity in acinar cells from both glands. Intracellular Ca(2+) chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca(2+) concentration with the Ca(2+)-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating Cl(-)/HCO(3)(-) exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca(2+)-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.     

The chicken receptor for endocytosis of glycoproteins contains a cluster of N-acetylglucosamine-binding sites

The oligomeric state of the chicken hepatic receptor for N-acetylglucosamine-terminated glycoproteins (the chicken hepatic lectin) has been examined in detergent solution, in various membrane preparations, and in hepatocytes. In detergent solution, the cross-linking reagent, 1,5-difluoro-2,4-dinitrobenzene produces covalent complexes containing up to six receptor polypeptides. This result, along with hydrodynamic studies of the receptor-detergent complex, indicates that the purified receptor is a hexamer. Analysis of large proteolytic fragments of the receptor reveals that portions of the receptor polypeptide near the membrane anchor are essential for hexamer stability. This analysis also demonstrates that each receptor polypeptide has an N-acetylglucosamine-binding site, indicating that the native hexameric receptor contains a cluster of six such sites. Immunoblot analysis of membrane fractions and cells cross-linked with 1,5-difluoro-2,4-dinitrobenzene or dimethyl adipimidate reveals that the receptor is also oligomeric in intact cells and in subcellular fractions representing cell surface and internalized receptor. Although the pattern of cross-linking observed in membranes differs from that observed with purified receptor, experiments indicate that the differences may be explained by the presence of membrane components which compete with receptor for reaction with cross-linking reagent. The presence of a cluster of carbohydrate-binding sites in the hepatocyte membrane can account for the preferential endocytosis of multivalent glycoprotein ligands by hepatocytes.     

Mice with a targeted disruption of the AE2 Cl-/HCO3- exchanger are achlorhydric

The AE2 Cl-/HCO3- exchanger is expressed in numerous cell types, including epithelial cells of the kidney, respiratory tract, and alimentary tract. In gastric epithelia, AE2 is particularly abundant in parietal cells, where it may be the predominant mechanism for HCO3- efflux and Cl- influx across the basolateral membrane that is needed for acid secretion. To investigate the hypothesis that AE2 is critical for parietal cell function and to assess its importance in other tissues, homozygous null mutant (AE2(-/-)) mice were prepared by targeted disruption of the AE2 (Slc4a2) gene. AE2(-/-) mice were emaciated, edentulous (toothless), and exhibited severe growth retardation, and most of them died around the time of weaning. AE2(-/-) mice exhibited achlorhydria, and histological studies revealed abnormalities of the gastric epithelium, including moderate dilation of the gastric gland lumens and a reduction in the number of parietal cells. There was little evidence, however, that parietal cell viability was impaired. Ultrastructural analysis of AE2(-/-) gastric mucosa revealed abnormal parietal cell structure, with severely impaired development of secretory canaliculi and few tubulovesicles but normal apical microvilli. These results demonstrate that AE2 is essential for gastric acid secretion and for normal development of secretory canalicular and tubulovesicular membranes in mouse parietal cells.     

Evidence for the development of an intermonomeric asymmetry in the covalent binding of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate to human erythrocyte band 3

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies have identified two oligomeric forms of band 3 whose proportions on gel profiles were modulated by the particular ligand occupying the intramonomeric stilbenedisulfonate site during intermonomeric cross-linking by BS3 [bis-(sulfosuccinimidyl) suberate] [Salhany et al. (1990) J. Biol. Chem. 265, 17688-17693]. When DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) was irreversibly attached to all monomers, BS3 covalent dimers predominated, while with DNDS (4,4'-dinitrostilbene-2,2'-disulfonate) present to protect the intramonomeric stilbenedisulfonate site from attack by BS3, a partially cross-linked band 3 tetramer was observed. In the present study, we investigate the structure of the protected stilbenedisulfonate site within the tetrameric complex by measuring the ability of patent monomers to react irreversibly with DIDS. Our results show two main populations of band 3 monomers present after reaction with DNDS/BS3: (a) inactive monomers resulting from the displacement of reversibly bound DNDS molecules and subsequent irreversible attachment of BS3 to the intramonomeric stilbenedisulfonate site and (b) residual, active monomers. All of the residual activity was fully inhibitable by DIDS under conditions of reversible binding, confirming expectations that all of the monomers responsible for the residual activity have patent stilbenedisulfonate sites. However, within this active population, two subpopulations could be identified: (1) monomers which were irreversibly reactive toward DIDS and (2) monomers which were refractory toward irreversible binding of DIDS at pH 6.9, despite being capable of binding DIDS reversibly. Increasing the pH to 9.5 during treatment of DNDS/BS3-modified cells with 300 microM DIDS did not cause increased irreversible transport inhibition relative to that seen for cells treated at pH 6.9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization, size estimation and solubilization of alpha-macroglobulin complex receptors in liver membranes

Receptors for alpha 2-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of 125I-labelled alpha 2-macroglobulin.trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8-9.0. The half-time for association was about 5 min at 37 degrees C in contrast to about 5 h at 4 degrees C. The half-saturation constant was about 100 pM at 4 degrees C and 1 nM at 37 degrees C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 +/- 71 kDa (S.D., n = 7) for alpha 2-macroglobulin.trypsin binding activity. Affinity cross-linking to rat and human membranes of 125I-labelled rat alpha 1-inhibitor-3.chymotrypsin, a 210 kDa analogue which binds to the alpha 2-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55-60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked alpha 2-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound 125I-alpha 1-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400-500 kDa alpha 2-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Depth-dependent analysis of membranes using benzophenone-based phospholipids

Any attempt to probe the membrane hydrophobic core with chemical reagents necessitates the use of reactive intermediates like carbenes and nitrenes, which can insert into C-H bonds. Several photoactivable reagents based on carbenes and nitrenes have been reported. However, the high reactivity of these reagents, often leads to very low insertion yields. We report here a high degree of cross-linking (35-40%) achieved with three benzophenone-based phospholipids and analyze the carbon functionalization data using a multiple Gaussian function. These phospholipids are so designed so as to permit depth-dependent labeling in membranes. Single bilayer vesicles were prepared from these phospholipids and dimyristoylphosphatidylcholine. The cross-linked product was isolated and characterized by mass spectroscopy. The results obtained indicated that the cross-linked product was dominated by dimeric product formed by intermolecular cross-linking. The Gaussian analysis used here provides insight into the relative depths of the probes inside the membrane.