Interaction of alpha- and beta-subunits in native H-K-ATPase and cultured cells transfected with H-K-ATPase beta-subunit

The assembly of the -subunit of thegastric H-K-ATPase (HK) with the -subunit of the H-K-ATPase orthe Na-K-ATPase (NaK) was characterized with two anti-HKmonoclonal antibodies (MAbs). In fixed gastric oxyntic cells, inH-K-ATPase in vitro, and in Madin-Darby canine kidney (MDCK) cellstransfected with HK, MAb 2/2E6 was observed to bind to HK onlywhen interactions between - and -subunits were disrupted byvarious denaturants. The epitope for MAb 2/2E6 was mapped to thetetrapeptide S₂₂₆LHY₂₂₉ of the extracellulardomain of HK. The epitope for MAb 2G11 was mapped to the eightNH₂-terminal amino acids of the cytoplasmic domain ofHK. In transfected MDCK cells, MAb 2G11 could immunoprecipitate HK with -subunits of the endogenous cell surface NaK, as well as that from early in the biosynthetic pathway, whereas MAb 2/2E6 immunoprecipitated only a cohort of unassembled endoglycosidase H-sensitive HK. In HK-transfected LLC-PK₁ cells,significant immunofluorescent labeling of HK at the cell surfacecould be detected without postfixation denaturation or in live cells,although a fraction of transfected HK could also becoimmunoprecipitated with NaK. Thus assembly of HK with NaKdoes not appear to be a stringent requirement for cell surface deliveryof HK in LLC-PK₁ cells but may be required in MDCKcells. In addition, endogenous posttranslational regulatory mechanismsto prevent hybrid - heterodimer assembly appear to be compromisedin transfected cultured renal epithelial cells. Finally, theextracellular epitope for assembly-sensitive MAb 2/2E6 may represent aregion of HK that is associated with - interaction.

     

Glucoamylase-like domains in the alpha- and beta-subunits of phosphorylase kinase

Phosphorylase kinase is a four-subunit enzyme involved in the regulation of glycogen breakdown. The traditional textbook view is that only the gamma subunit has enzymatic activity, whereas the other three subunits have a regulatory role. Evidence from homology searches and sequence alignments, however, shows that the alpha- and beta-subunits possess amino-terminal glucoamylase-like domains and suggests that they might possess a previously overlooked amylase activity. If true, this would have important implications for the understanding, diagnosis, and management of glycogen storage diseases. There is thus a clear need to test this hypothesis through enzymatic assays and structural studies.     

Colonic H-K-ATPase beta -subunit: identification in apical membranes and regulation by dietary K depletion

P-type ATPasesrequire both - and -subunits for functionalactivity. Although an -subunit for colonic apical membraneH-K-ATPase (HKc) has been identified and studied, its -subunithas not been identified. We cloned putative -subunit rat colonicH-K-ATPase (HKc) cDNA that encodes a 279-amino-acid protein with asingle transmembrane domain and sequence homology to other rat-subunits. Northern blot analysis demonstrates that this HKc isexpressed in several rat tissues, including distal and proximal colon,and is highly expressed in testis and lung. HKc mRNA abundance is upregulated threefold compared with normal in distal colon but notproximal colon, testis, or lung of K-depleted rats. In contrast, Na-K-ATPase ₁ mRNA abundance isunaltered in distal colon of K-depleted rats. Na depletion, which alsostimulates active K absorption in distal colon, does not increaseHKc mRNA abundance. Western blot analyses using a polyclonalantibody raised to a glutathioneS-transferase-HKc fusion proteinestablished expression of a 45-kDa HKc protein in both apical andbasolateral membranes of rat distal colon, but K depletion increasedHKc protein expression only in apical membranes. Physicalassociation between HKc and HKc proteins was demonstrated byWestern blot analysis performed with HKc antibody onimmunoprecipitate of apical membranes of rat distal colon and HKcantibody. Tissue-specific upregulation of this -subunit mRNA inresponse to K depletion, localization of its protein, its upregulationby K depletion in apical membranes of distal colon, and its physicalassociation with HKc protein provide compelling evidence that HKcis the putative -subunit of colonic H-K-ATPase.     

Membrane insertion of alpha- and beta-subunits of Na+,K+-ATPase

Insertion of the alpha- and beta-subunits of amphibian epithelial Na+,K+-ATPase into pancreatic microsomes in cell-free systems was shown to be the same as into membranes of intact cells. The glycoproteic beta-subunit was observed to be cotranslationally inserted into endoplasmic reticulum membranes and to adopt a different pattern of N-linked core and terminal sugars in two different amphibian species. The beta-subunit lacks a cleavable signal sequence but quantitative membrane integration required membrane addition at the start of synthesis. Proteolysis of beta-subunit assembled in vitro indicated a cleavable cytoplasmic domain of about 2000 daltons. The catalytic 98-kilodalton alpha-subunit was also membrane-associated during its synthesis in an alkali-resistant fashion and independent of newly synthesized beta-subunit. In contrast to the beta-subunit, membrane integration of the alpha-subunit was possible as late as a time point in its synthesis which corresponded to about 1/3-1/2 of completion of the nascent chain. A small 34 kDa trypsin-resistant fragment of the alpha-subunit was produced at an early stage of synthesis both in the intact cell and in the cell-free system. These results suggest that membrane insertion of both alpha- and beta-subunit occurs during their synthesis but with a different time course.     

Colonic adenoma-associated Escherichia coli express specific phenotypes

Specific Escherichia coli strains have been associated to colorectal cancer, while no data are available on genotypic and phenotypic features of E. coli colonizing premalignant adenomatous polyps and their pathogenic potential. This study was aimed at characterizing isolates collected from polyps and adjacent tissue in comparison with those from normal mucosa.From colonoscopy biopsies, 1500 E. coli isolates were retrieved and genotyped; 272 were characterized for phylogroup and major phenotypic traits (i.e., biofilm formation, motility, hemolysins, and proteases). Selected isolates were analyzed for extraintestinal pathogenic E. coli (ExPEC)-associated virulence genes and in vivo pathogenicity using Galleria mellonella.The majority of isolates collected from polyps were strong biofilm and poor protease producers, whereas those isolates from normal mucosa were highly motile, proteolytic and weak biofilm formers. Isolates from adjacent tissues shared features with those from both polyps and normal mucosa. Among selected E. coli isolates, ExPEC gene content/profile was variable and uncorrelated with the tissue of collection and larval mortality.Despite the heterogeneous virulence-gene carriage of the E. coli intestinal population, E. coli colonizing colonic adenomatous polyps express specific phenotypic traits that could represent an initial pathoadaptation to local environmental changes characterizing these lesions.     

Faulty association of alpha- and beta-subunits in some forms of beta-hexosaminidase A deficiency

We have previously described the kinetics of association of the alpha- and beta-subunits of beta-hexosaminidase A in intact cultured human fibroblasts, using biosynthetic labeling and immunoprecipitation with antisera that distinguish between monomeric and associated alpha-chains (Proia, R. L., d'Azzo, A., and Neufeld, E. F. (1984) J. Biol. Chem. 259, 3350-3354). We now show lack of alpha-beta association in fibroblasts of several individuals deficient in beta-hexosaminidase A (5 patients with nonclassic forms of Tay-Sachs disease and 2 asymptomatic siblings). Defective association was accompanied by markedly reduced (less than one-tenth of normal) conversion of the alpha-chain precursor of Mr = 67,000 to the mature lysosomal form of Mr = 54,000. Analysis by hybridization with fibroblasts lacking the alpha- or beta-chain showed that the association defect resided in the alpha-chain. Most of the cell strains studied also had decreased synthesis of the alpha-chain, suggesting compound heterozygosity with the Ashkenazi Tay-Sachs (no synthesis) allele. An unusual feature of the association defect is the variability in the resulting clinical manifestations, even within families, implying that other factors determine the adequacy of the residual associated beta-hexosaminidase A in vivo.     

Nucleotide sequence of genes for alpha- and beta-subunits of luciferase from Photobacterium leiognathi

Nucleotide sequence of the Photobacterium leiognathi DNA containing genes of alpha and beta subunits of luciferase has been determined. We also deduced amino acid sequence and molecular mass of luciferase and localized luciferase genes in the sequenced DNA fragment.     

Binding modes of L35 to alpha- and beta-semihemoglobins: structural insights into the inequivalence of alpha- and beta-subunits of hemoglobin

It has been thought for several years that the greatly lowered oxygen affinity, high cooperativity, and heterotropic modulation displayed by tetrameric human hemoglobin (Hb) was an exclusive result of the assembly of high affinity alpha(1)beta(1) dimers into alpha(2)beta(2) tetramers. However, in recent times, it has been shown that alpha- and beta-semihemoglobins, namely alpha(heme)beta(apo) and alpha(apo)beta(heme), which are dimers of Hb characterized by a high affinity for oxygen and lack of cooperativity do respond to effectors such as 2-[4-(3,5-dichlorophenylureido) phenoxy]-2-methylpropionic acid (L35), a bezafibrate (BZF) related compound, by decreasing the ligand affinity to a considerable extent (between 60- and 130-fold). In order to shed some light on the structural basis of this phenomenon, we have developed a binding mode of L35 to semihemoglobins through docking analysis using the program GRID. Molecular modelling studies did identify sites on semihemoglobins where favourable interactions with L35 can occur. We found that the effector binds differently to the two semihemoglobins exhibiting high affinity only for the alpha chain heme pocket. The proposed binding models are consistent with the experimental findings and may be rationalized in terms of different hydrophobic and hydrophilic characteristics between alpha- and beta-heme pockets of Hb.     

Association of alpha- and beta-subunits during the biosynthesis of beta-hexosaminidase in cultured human fibroblasts

Subunit association of beta-hexosaminidase was studied in intact fibroblasts using antisera that discriminate between free and associated alpha-chains. These were anti-beta-hexosaminidase A (anti-alpha beta), which precipitated all alpha-chains, free or associated; anti-beta-hexosaminidase B (anti-beta beta), which precipitated those alpha-chains that were associated with beta; and anti-alpha-chains, which recognized only monomeric alpha-chains. After biosynthetic labeling, beta-hexosaminidase or its free alpha-subunit were immuno-precipitated from extracts of cells and medium with the aid of protein A-bearing Staphylococcus aureus, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by fluorography. Pulse-chase labeling showed that the alpha-chains existed predominantly in the monomeric precursor form during the first 5 h, and then began to accumulate in the mature (lysosomal) associated alpha beta form. Precursor alpha beta complexes were secreted, along with some precursor alpha monomers; the latter were catalytically inert. Both alpha- and beta-chains were phosphorylated (a Golgi modification) prior to association. Thus alpha-beta association probably occurred in the Golgi area before transfer to lysosomes and before secretion. Cycloheximide inhibited the association and subsequent maturation of preformed alpha-chains, perhaps by causing a depletion of a pool of beta-chain precursor upstream from the site of subunit association. In fibroblasts from a patient with Sandhoff disease, that produced no beta-chains, the alpha-chains self-associated but their maturation was markedly decreased. We suggest that association with beta-chains is necessary not only for acquisition of catalytic activity but also for transport of alpha-chains to lysosomes.     

Immunological identification of G protein alpha- and beta-subunits in tail membranes of bovine spermatozoa.

Heterotrimeric G proteins are believed to play important roles as signal transducing components in various mammalian sperm functions. To assess the distribution of G proteins in bovine sperm tails, we purified membranes by hypoosmotic swelling of bovine spermatozoa followed by disruption of plasma membranes in a homogenizer and various centrifugation steps. Electron microscopy revealed highly purified membranes of bovine sperm tails. Subsequently, antisera against synthetic peptides were used to identify G proteins in immunoblots. An antiserum directed against the C-terminal decapeptide of Gi3 and detecting all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. In contrast, various other specific peptide antisera against alpha-subunits did not detect any G protein in enriched tail membranes. An antiserum recognizing the beta 2-subunit of G proteins and an antiserum reacting with both beta 1- and beta 2-subunits identified a 35-kDa protein in sperm tail membranes. In contrast, antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. Our results suggest that G proteins in membranes of tails of bovine spermatozoa most likely belong to a novel subtype of G protein alpha-subunits, whereas the putative beta-subunit could be identified as a beta 2-subunit.     

Conformational changes in the alpha- and beta-subunits of the insulin receptor identified by anti-peptide antibodies

The structure of the insulin receptor was studied with polyclonal antibodies obtained from rabbits which were immunized with synthetic peptides having a sequence identity to three regions of the alpha-subunit and five regions of the beta-subunit. None of the alpha-subunit antibodies including alpha-Pep8 (residues 40-49 (Ullrich, A., Bell, J.R., Chen, E.Y., Herrera, R., Petruzzelli, L.M., Dull, T.J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P.H., Grunfeld, C., Rosen, O.M., and Ramachandran, J. (1985) Nature 313, 756-761), alpha-Pep7 (12 amino acid C-terminal extension (Ebina, Y., Ellis, L., Jarnagin, K., Ederly, M., Graf, L., Clauser, E., Ou, J.-H., Masiar, F., Kan, Y.W., Goldfine, I.D., Roth, R.A., and Rutter, W.J. (1985) Cell 313, 747-758], or alpha-Pep6 (residues 1-7, 9) immunoprecipitated the human insulin receptor solubilized from IM-9 lymphocytes; however, alpha-Pep8 immunoprecipitated the dithiothreitol-reduced receptor. Antibodies prepared against the N terminus of the beta-subunit (alpha-Pep5, residues 780-790) and the ATP binding site (alpha-Pep3, residues 1013-1022) did not react with the intact receptor under any conditions; however, antibodies to the C terminus of the beta-subunit (alpha-Pep1, residues 1314-1324) and to the juxta-membrane region (alpha-Pep3, residues 952-962) immunoprecipitated the solubilized receptor in both its phosphorylated and nonphosphorylated forms. In contrast, the antibody reactive with the regulatory region of the beta-subunit which contains the major autophosphorylation sites (alpha-Pep2, residues 1143-1154) only precipitated the phosphorylated form. Thus the conformation of the extracellular domain of the receptor is rigid and stabilized by disulfide bonds, whereas several regions of the intracellular domain are accessible to antibodies and undergo conformational changes during autophosphorylation.     

Down-regulation of the alpha- and beta-subunits of the calcium-activated potassium channel in human myometrium with parturition

Large-conductance, calcium-dependent potassium (BKCa) channels are implicated in maintaining uterine quiescence during pregnancy. The mechanisms whereby calcium sensitivity of the BKCa channel is dramatically removed at parturition remain unknown. The aim of the present study was to investigate whether this loss of calcium sensitivity of the BKCa channel with the onset of labor is associated with changes in the protein expression of the alpha- and/or beta-subunit or arises from a physical dissociation of the alpha-subunit from the beta-subunit. The beta-subunit is a key determinant of BKCa-channel Ca2+ sensitivity. Western blot analysis, using alpha- and beta-subunit-specific antibodies, detected bands of 110-125 and 36 kDa, respectively. Protein expression levels of the alpha-subunit in term labor myometrium were significantly reduced compared with term pregnancy without labor. Furthermore, alpha-subunit levels at term pregnancy were significantly increased relative to the nonpregnant state, whereas levels at preterm gestations were unchanged. Densitometric analysis demonstrated significantly decreased beta-subunit levels in term and preterm labor samples compared with term nonlabor samples. Immunoprecipitation studies revealed the presence of both the alpha- and beta-subunits in samples taken before or after the onset of labor. We conclude that during labor, the alpha-subunit is not physically uncoupled from the beta-subunit, but a decline occurs in the level of beta-subunit protein, which may underlie the loss of calcium and voltage sensitivity of the BKCa channel with labor. Furthermore, reduced beta-subunit protein in preterm labor myometrium implies that ion channels may also contribute to pathophysiological labor.     

Gastric H-K-ATPase and acid-resistant surface proteins

Despite the fact that mucus and bicarbonate are important macroscopic components of the gastric mucosal barrier, severe acidic and peptic conditions surely exist at the apical membrane of gastric glandular cells, and these membranes must have highly specialized adaptations to oppose external insults. Parietal cells abundantly express the heterodimeric, acid-pumping H-K-ATPase in their apical membranes. Its beta-subunit (HKbeta), a glycoprotein with >70% of its mass and all its oligosaccharides on the extracellular side, may play a protective role. Here, we show that the extracellular domain of HKbeta is highly resistant to trypsin in the native state (much more than that of the structurally related Na-K-ATPase beta-subunit) and requires denaturation to expose tryptic sites. Native HKbeta also resists other proteases, such as chymotrypsin and V8 protease, which hydrolyze at hydrophobic and anionic amino acids, respectively. Removal of terminal alpha-anomeric-linked galactose does not appreciably alter tryptic sensitivity of HKbeta. However, full deglycosylation makes HKbeta much more susceptible to all proteases tested, including pepsin at pH <2.0. We propose that 1) intrinsic folding of HKbeta, 2) bonding forces between subunits, and 3) oligosaccharides on HKbeta provide a luminal protein domain that resists gastric lytic conditions. Protein folding that protects susceptible charged amino acids and is maintained by disulfide bonding and hydrophilic oligosaccharides would provide a stable structure in the face of large pH changes. The H-K-ATPase is an obvious model, but other gastric luminally exposed proteins are likely to possess analogous protective specializations.     

Altered glycosylation is induced in both alpha- and beta-subunits of human chorionic gonadotropin produced by choriocarcinoma

The two subunits of human chorionic gonadotropin (hCG) purified from the urine of a patient with choriocarcinoma were successfully separated by SDS-polyacrylamide gel electrophoresis. A comparative study of the oligosaccharides released from the two subunits by hydrazinolysis revealed that altered glycosylation occurs in both subunits and possibly at all four asparagine sites of the choriocarcinoma hCG molecule.     

Identification of the geometric requirements for allosteric communication between the alpha- and beta-subunits of tryptophan synthase

The pyridoxal 5'-phosphate-dependent tryptophan synthase alpha2beta2 complex is a paradigmatic protein for substrate channeling and allosteric regulation. The enzymatic activity is modulated by a ligand-mediated equilibrium between open (inactive) and closed (active) conformations of the alpha- and beta-subunit, predominantly involving the mobile alpha loop 6 and the beta-COMM domain that contains beta helix 6. The alpha ligand-triggered intersubunit communication seems to rely on a single hydrogen bond formed between the carbonyl oxygen of betaSer-178 of beta helix 6 and the NH group of alphaGly-181 of alpha loop 6. We investigated whether and to what extent mutations of alphaGly-181 and betaSer-178 affect allosteric regulation by the replacement of betaSer-178 with Pro or Ala and of alphaGly-181 with either Pro to remove the amidic proton that forms the hydrogen bond or Ala, Val, and Phe to analyze the dependence on steric hindrance of the open-closed conformational transition. The alpha and beta activity assays and the equilibrium distribution of beta-subunit catalytic intermediates indicate that mutations do not significantly influence the intersubunit catalytic activation but completely abolish ligand-induced alpha-to beta-subunit signaling, demonstrating distinct pathways for alpha-beta-site communication. Limited proteolysis experiments indicate that the removal of the interaction between betaSer-178 and alphaGly-181 strongly favors the more trypsin-accessible open conformation of the alpha-active site. When the hydrogen bond cannot be formed, the alpha-subunit is unable to attain the closed conformation, and consequently, the allosteric signal is aborted at the subunit interface.     

Pyrophosphate-dependent phosphofructokinase. Conservation of protein sequence between the alpha- and beta-subunits and with the ATP-dependent phosphofructokinase

Full-length cDNA clones for the alpha- and beta-subunits of pyrophosphate-fructose 6-phosphate 1-phosphotransferase have been isolated from a cDNA expression library derived from potato tuber poly(A)+ RNA. The nucleotide sequences indicate that the alpha- and beta-subunits are related with about 40% of amino acid residues being identical. A comparison of the deduced amino acid sequences of both subunits of this enzyme with that of the major ATP-dependent fructose 6-phosphate 1-phosphotransferase from Escherichia coli (Shirakihara, Y., and Evans, P. R. (1988) J. Mol. Biol. 204, 973-994) showed little homology between the proteins except for regions involved in the binding of fructose 6-phosphate/fructose, 1,6-bisphosphate and possibly between regions binding pyrophosphate and the beta- and gamma-phosphates of ADP/ATP. A comparison of the derived secondary structures of the two subunits of the PPi-dependent enzyme with the known secondary structure of the E. coli ATP-dependent enzyme indicated that the overall structure of these enzymes is similar. These data suggest that catalytic activity resides on the beta-subunit of the pyrophosphate-dependent enzyme.     

Primary structures of alpha- and beta-subunits of alpha-amylase inhibitors from seeds of three cultivars of Phaseolus beans

The primary structures of three -amylase inhibitors (TAI, DAI, and MAI-2) consisting of glycoprotein subunits and from the respective seeds of three cultivars of Phaseolus beans, Toramame (Phaseolus vulgaris L.), Daifukumame (Phaseolus vulgaris L.), and Murasakihanamame (Phaseolus coccineus L.) were determined by sequencing the peptide fragments derived from their enzymatic digestions. Major sugar chains of the inhibitors were also assessed by analyzing glycopeptides in the enzymatic digests. The subunits, and , were shown to be composed of 76 and 139 amino acid residues, respectively, in each inhibitor. The overall amino acid sequences of the inhibitors were slightly different from one another. Furthermore, the sequence of TAI was the same as that deduced from a cDNA clone encording -amylase inhibitor-1 from the common bean (Phaseolus vulgaris L.). It was also revealed that there were two N-glycosylation sites in each -subunit: PA-derivatives of the major N-glycans were estimated to be M6B at Asn(12) and M9A at Asn(65). Each -subunit of TAI and MAI-2 had two N-glycosylation sites, while the -subunit of DAI had only one site. The major N-glycans pyridylaminated were estimated to be M3X at Asn(63) in each -subunit and M3FX at Asn(83) in -subunits of TAI and MAI-2.     

Fluorometric measurements of intermolecular distances between the alpha- and beta-subunits of the Na+/K+-ATPase

The Na+/K+-ATPase maintains the physiological Na+ and K+ gradients across the plasma membrane in most animal cells. The functional unit of the ion pump is comprised of two mandatory subunits including the alpha-subunit, which mediates ATP hydrolysis and ion translocation, as well as the beta-subunit, which acts as a chaperone to promote proper membrane insertion and trafficking in the plasma membrane. To examine the conformational dynamics between the alpha- and beta-subunits of the Na+/K+-ATPase during ion transport, we have used fluorescence resonance energy transfer, under voltage clamp conditions on Xenopus laevis oocytes, to differentiate between two models that have been proposed for the relative orientation of the alpha- and beta-subunits. These experiments were performed by measuring the time constant of irreversible donor fluorophore destruction with fluorescein-5-maleimide as the donor fluorophore and in the presence or absence of tetramethylrhodamine-6-maleimide as the acceptor fluorophore following labeling on the M3-M4 or M5-M6 loop of the alpha-subunit and the beta-subunit. We have also used fluorescence resonance energy transfer to investigate the relative movement between the two subunits as the ion pump shuttles between the two main conformational states (E1 and E2) as described by the Albers-Post scheme. The results from this study have identified a model for the orientation of the beta-subunit in relation to the alpha-subunit and suggest that the alpha- and beta-subunits move toward each other during the E2 to E1 conformational transition.