Segment Of Alpha Research Articles

Helicity of alpha(404-451) and beta(394-445) tubulin C-terminal recombinant peptides.

We have investigated the solution conformation of the functionally relevant C-terminal extremes of alpha- and beta-tubulin, employing the model recombinant peptides RL52alpha3 and RL33beta6, which correspond to the amino acid sequences 404-451(end) and 394-445(end) of the main vertebrate isotypes of alpha- and beta-tubulin, respectively, and synthetic peptides with the alpha-tubulin(430-443) and beta-tubulin(412-431) internal sequences. Alpha(404-451) and beta(394-445) are monomeric in neutral aqueous solution (as indicated by sedimentation equilibrium), and have circular dichroism (CD) spectra characteristic of nearly disordered conformation, consistent with low scores in peptide helicity prediction. Limited proteolysis of beta(394-445) with subtilisin, instead of giving extensive degradation, resulted in main cleavages at positions Thr409-Glu410 and Tyr422-Gln423-Gln424, defining the proteolysis resistant segment 410-422, which corresponds to the central part of the predicted beta-tubulin C-terminal helix. Both recombinant peptides inhibited microtubule assembly, probably due to sequestration of the microtubule stabilizing associated proteins. Trifluoroethanol (TFE)-induced markedly helical CD spectra in alpha(404-451) and beta(394-445). A substantial part of the helicity of beta(394-445) was found to be in the CD spectrum of the shorter peptide beta(412-431) with TFE. Two-dimensional 1H-NMR parameters (nonsequential nuclear Overhauser effects (NOE) and conformational C alphaH shifts) in 30% TFE permitted to conclude that about 25% of alpha(404-451) and 40% of beta(394-451) form well-defined helices encompassing residues 418-432 and 408-431, respectively, flanked by disordered N- and C-segments. The side chains of beta(394-451) residues Leu418, Val419, Ser420, Tyr422, Tyr425, and Gln426 are well defined in structure calculations from the NOE distance constraints. The apolar faces of the helix in both alpha and beta chains share a characteristic sequence of conserved residues Ala,Met(+4),Leu(+7),Tyr(+11). The helical segment of alpha(404-451) is the same as that described in the electron crystallographic model structure of alphabeta-tubulin, while in beta(394-451) it extends for nine residues more, supporting the possibility of a functional coil --> helix transition at the C-terminus of beta-tubulin. These peptides may be employed to construct model complexes with microtubule associated protein binding sites.

NMR structure and backbone dynamics of the extended second transmembrane domain of the human neuronal glycine receptor alpha1 subunit.

The structure and backbone dynamics of an extended second transmembrane segment (TM2e) of the human neuronal glycine receptor alpha(1) subunit in sodium dodecyl sulfate micelles were studied by (1)H and (15)N solution-state NMR. The 28-amino acid segment contained the consensus TM2 domain plus part of the linker between the second and third transmembrane domains. The presence of a well-structured helical region of at least 13 amino acids long and an unstructured region near the linker was evident from the proton chemical shifts and the pattern of midrange nuclear Overhauser effects (NOE). (15)N relaxation rate constants, R(1) and R(2), and (15)N-[(1)H] NOE indicated restricted internal motions in the helical region with NOE values between 0.6 and 0.8. The squared order parameter (S(2)), the effective correlation time for fast internal motions (tau(e)), and the global rotational correlation time (tau(m)) were calculated for all TM2e backbone N-H bonds using the model-free approach. The S(2) values ranged about 0.75-0.86, and the tau(e) values were below 100 ps for most of the residues in the helical region. The tau(m) value, calculated from the dynamics of the helical region, was 5.1 ns. The S(2) values decreased to 0.1, and the tau(e) values sharply increased up to 1.2 ns at the linker near the C-terminus, indicating that the motion of this region is unrestricted. The results suggest a relatively high degree of motional freedom of TM2e in micelles and different propensities of the N- and C-terminal moieties of the transmembrane domain to assume stable helical structures.

Application of denaturing gradient gel electrophoresis (DGGE) to screen for mutations of the human glucocorticoid receptor α gene (hGRα)

Objectives In a previous publication, we had presented a sensitive method to detect mutations of the segment of the human glucocorticoid receptor alpha (hGRα) gene encoding the ligand binding domain (LBD) and part of the DNA binding domain (DBD) of hGRα, as several types of glucocorticoid resistance syndromes have been correlated with mutations in the respective nucleotide sequences. However, mutations affecting various regions covering the whole length of hGRα are increasingly reported in a variety of disease states. We now present an expanded screening methodology to detect mutations covering the whole length of hGRα. Design and methods We developed a sensitive, simple screening PCR-DGGE method to detect mutations in the aminoterminal domain and DNA-binding domain of the hGRα. Wild type hGRα cDNA and mutant samples were included in the analysis to ensure the accuracy and sensitivity of the method. Results The PCR-DGGE method identified the mutant samples and discriminated them from wild type hGRα. Conclusions The method described is accurate, sensitive, simple, cheap and fulfills the critera for a screening method which will be useful in delineating possible involvement of hGRα mutations in the aetiopathology of diseases correlated to derangements of glucocorticoid action.

Adsorption column for myasthenia gravis treatment: Medisorba MG-50.

Adsorption column Medisorba MG-50 (Kuraray Medical Inc.) for the treatment of myasthenia gravis (MG) is introduced. The adsorbent in this column is composed of cellulose beads as carrier material and covalent-bound synthetic peptide as a ligand that has a specific affinity to the pathogenic anti-acetylcholine receptor antibody of MG. The amino acid sequence of the peptide is modified from the segment of alpha 183-200 of the torpedo acetylcholine receptor (AChR) protein, and the segment is the acetylcholine binding site on AChR and the target site of anti-AChR antibody. The adsorbent showed specific adsorption characteristics to the anti-ACHR antibody (blocking antibody) in vitro. Clinically, MG-50 is used in plasma-perfusion therapy, and it is recognized that MG-50 specifically reduces blocking antibody titer and improves MG symptoms. MG-50 is approved in Japan.

Fast Inactivation of Voltage-dependent Calcium Channels: A HINGED-LID MECHANISM?

We recently described domains II and III as important determinants of fast, voltage-dependent inactivation of R-type calcium channels (Spaetgens, R. L., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 22428-22438). Here we examine in greater detail the structural determinants of inactivation using a series of chimeras comprising various regions of wild type alpha(1C) and alpha(1E) calcium channels. Substitution of the II S6 and/or III S6 segments of alpha(1E) into the alpha(1C) backbone resulted in rapid inactivation rates that closely approximated those of wild type alpha(1E) channels. However, neither individual or combined substitution of the II S6 and III S6 segments could account for the 60 mV more negative half-inactivation potential seen with wild type alpha(1E) channels, indicating that the S6 regions contribute only partially to the voltage dependence of inactivation. Interestingly, the converse replacement of alpha(1E) S6 segments of domains II, III, or II+III with those of alpha(1C) was insufficient to significantly slow inactivation rates. Only when the I-II linker region and the domain II and III S6 regions of alpha(1E) were concomitantly replaced with alpha(1C) sequence could inactivation be abolished. Conversely, introduction of the alpha(1E) domain I-II linker sequence into alpha(1C) conferred alpha(1E)-like inactivation rates, indicating that the domain I-II linker is a key contributor to calcium channel inactivation. Overall, our data are consistent with a mechanism in which inactivation of voltage-dependent calcium channels may occur via docking of the I-II linker region to a site comprising, at least in part, the domain II and III S6 segments.

Packing of the transmembrane helices of Na,K-ATPase: direct contact between beta-subunit and H8 segment of alpha-subunit revealed by oxidative cross-linking.

Spatial relationships among the transmembrane (TM) segments of alpha- and beta-subunits of the Na,K-ATPase molecule have been investigated using oxidative induction of disulfide bonds. The catalytic alpha-subunit contains 10 TM alpha-helices (H1-H10) with 9 Cys residues located within or close to the membrane moiety. There is one Cys residue in the single TM segment of beta-subunit (Hbeta). Previously, the cross-linking products containing the beta-subunit and two fragments of alpha-subunit (the N-terminal containing H1-H2 helices and the C-terminal containing H7-H10 helices) have been identified in experiments with membrane-bound or detergent-solubilized preparations of the membrane moiety of trypsin-digested Na,K-ATPase [Sarvazyan, N. A., Modyanov, N. N., and Askari, A. (1995) J. Biol. Chem. 270, 26528-26532 and Sarvazyan, N. A., Ivanov, A., Modyanov, N. N., and Askari, A. (1997) J. Biol. Chem. 272, 7855-7858]. Here, we have shown that Cu(2+)-phenanthroline treatment of digitonin-solubilized preparation provides the most efficient formation of intersubunit cross-linked product that is predominantly a dimer of beta-subunit and a 22-kDa C-terminal alpha-fragment containing H7-H10 helices. This cross-linked product was isolated and subjected to CNBr cleavage. The resulting fragments were electrophoretically separated and sequenced. A 17-kDa peptide composed of Ile853-Met942 alpha-fragment and Ala5-Met56 beta-fragment was identified as a product of intersubunit disulfide cross-link between Cys44 of Hbeta and either Cys911 or Cys930, located in H8. This provides the first direct experimental evidence of the juxtaposition of Hbeta and H8 within the Na,K-ATPase molecule. The second detected cross-linked product was composed of alpha-fragments Lys947-Met963 and Tyr974-Tyr1016 linked by induced disulfide bridge between Cys964 (H9) and Cys983 (H10). The spatial proximity of these Cys residues defines the mutual orientation of H9 and H10 helices of alpha-subunit.

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.

Amino acid sequences within the alpha subunit of integrin alpha M beta 2 (Mac-1) critical for specific recognition of C3bi.

Phagocytosis of opsonized particles by neutrophils and monocytes plays a central role in host defense mechanisms against foreign pathogens. This process depends on the interaction between C3bi, a degradation product derived from activation of the complement system, and the alpha M beta 2 (CD11b/CD18, Mac-1) receptor, the major integrin on neutrophils. Previous studies had established a central role for the I domain, a stretch of approximately 200 amino acids within the alpha M subunit in the binding of C3bi, as well as many other alpha M beta 2 ligands. The present study was undertaken to establish the molecular basis of C3bi recognition by alpha M beta 2. The strategy employed the use of a series of mutant receptors in which short segments of the I domain of alpha M were switched to the corresponding segments of alpha L, which is structurally very similar but does not bind C3bi. We report three major findings: (1) The C3bi binding pocket is composed of three regions, P147-R152, P201-K217, and K245-R261 of alpha M, which surround the cation binding site within the MIDAS motif of the I domain. (2) Within the latter segment, K245 plays a critical role in mediating C3bi binding to alpha M beta 2. Mutation of K245 to Ala significantly reduced C3bi binding but had no effect on binding of another alpha M beta 2 I domain ligand, NIF. (3) Blocking of C3bi binding to alpha M beta 2 by monoclonal antibodies is achieved through two different mechanisms: direct competition for the ligand binding site or induction of conformational changes. Overall, these studies support the hypothesis that many of the ligands of alpha M beta 2 bind to overlapping but not identical sites within the I domain. Although the same short structural segments within the I domain may be involved in binding, different amino acids within these segments may contact different ligands.

Chemistry of the "molecular trap" of protease-catalyzed splicing reaction of complementary segments of alpha-subunit of hemoglobin A.

The complementary fragments of human Hb alpha, alpha1-30, and alpha31-141 are spliced together by V8 protease in the presence of 30% n-propanol to generate the full-length molecule (Hb alpha-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb alpha is facilitated by the organic cosolvent induced alpha-helical conformation of product acting as the "molecular trap" of the splicing reaction. The segments alpha24-30 and alpha31-40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) alpha24-40 has been manipulated by engineering the amino acid replacements to the positions alpha27 and alpha31 to delineate the structural basis of the molecular trap. The location of Glu27 and Arg31 residues in the contiguous segment alpha24-40 (as well as in other larger segments) is ideal to generate (i, i + 4) side-chain carboxylate-guanidino interaction in its alpha-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at alpha27 and alpha31 facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The gamma-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an alpha-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu30-Arg31 peptide bond. The second structural aspect is a site-specific event, an i, i + 4 side-chain interaction in the alpha-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven "molecular traps" of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.

Contribution of the beta subunit M2 segment to the ion-conducting pathway of the acetylcholine receptor.

We have applied the substituted-cysteine-accessibility method (SCAM) to the M2 segment and the M1-M2 loop of the acetylcholine (ACh) receptor beta subunit. Each residue from beta P248 to beta D273 was mutated one at a time to Cys, and the mutant beta subunits were expressed together with wild-type alpha, beta, and delta subunits in Xenopus oocytes. For each of the mutants, the ACh-induced current was near wild-type. The accessibility of the substituted Cys was inferred from the irreversible inhibition or potentiation of ACh-induced current by methanethiosulfonate (MTS) derivatives added extracellularly. Inhibition by MTSethylammonium of beta G255C, in the narrow part of the channel, was mainly due to a reduction in the single-channel conductance. Conversely, potentiation by MTSethylammonium of beta V266C, in a wider part of the channel, was mainly due to an increase in channel open-time. Two substituted Cys at the intracellular end of M2 and three at the extracellular end were accessible to MTSethylammonium in the absence of ACh. Three additional Cys in the middle of M2 and three in the M1-M2 loop were accessible in the presence of ACh. In the presence of ACh, the secondary structure of beta M2 is alpha-helical from beta G255 to beta V266 and extended from beta L268 to beta D273. The accessible residues in beta M2 are remarkably hydrophobic, while the accessible residues in the M1-M2 loop are charged. beta M2, like alpha M2, alpha M1, and beta M1, undergoes widespread structural changes concomitant with gating, but the gate itself is close to the intracellular end of the channel. Many aligned residues in the M2 segments of alpha and beta are not identically accessible, indicating that the two subunits contribute differently to the channel lining.

Diversity and prevalence of somatic mutations in the thyrotropin receptor and Gs alpha genes as a cause of toxic thyroid adenomas.

A total of 33 different autonomous hot nodules from 31 patients, originating mainly from Belgium, were investigated for the presence of somatic mutations in the TSH receptor and Gs alpha genes. This constitutes an extension of our previous study, including the first 11 nodules of the series. The complete coding sequence of the TSH receptor gene and the segments of Gs alpha known to harbor mutations impairing guanosinetriphosphotase activity were studied by direct sequencing of genomic DNA extracted from the nodules. DNA from the juxtanodular tissue or peripheral white blood cells was analyzed in all patients to confirm that the mutations identified were somatic. Twenty-seven mutations (82%) were found in the TSH receptor gene, affecting a total of 12 different residues or locations. All these mutations but 2 (see below) have been identified previously as activating mutations. Only 2 mutations were found in Gs alpha (6%). In 4 nodules, no mutation was detected. Five residues (Ser281, Ile486, Ile568, Phe631, and Asp633) were found mutated in 3 or 4 different nodules, making them hot spots for activating mutations. Phe631 and Asp633 belong to a cluster of 5 consecutive residues (629-633) in the N-terminal half of transmembrane segment VI; which harbor together 44% of the mutations identified in this cohort. Two novel mutations were identified: a point mutation causing substitution of Phe for Leu at position 629 (L629F); and a deletion of 12 bases removing residues 658-661 at the C-terminal portion of exoloop 3 (del658-661). When tested by transfection in COS-7 cells, both mutant receptors display increase in constitutive stimulation of basal cAMP accumulation. Although it is still capable of binding TSH, the del658-661 mutant has completely lost the ability to respond to the stimulation by the hormone. Our results demonstrate that, in a cohort of patients from a moderately iodine deficient area, somatic mutations increasing the constitutive activity of the TSH receptor are the major cause of autonomous hot nodules.

Polymorphism in the alpha 1 helix of the HLA-B heavy chain can have an overriding influence on peptide-binding specificity.

Previously, we reported overlap in the repertoires of peptides endogenously bound by a group of HLA-B allotypes related to HLA-B7. Extending such analysis to four members of the B17 family and seven members of the B15 family shows that allotypes that share sequence identity in the alpha 1 helix of the class I heavy chain possess markedly similar peptide-binding specificities. Members of the B17 family share a preference for peptides with serine, threonine, or alanine at position 2 and aromatic residues at the carboxyl terminus. Strikingly, the presence of a segment of the B17 alpha 1 helix in B*1516 and B*1517 confers the B17-like peptide-binding motif. The strong influence of natural variation in the alpha 1 helix is exemplified by the differences in peptide-binding specificity of B15 allotypes related by conversion events that replaced segments of the alpha 1 helix. In contrast, evolutionary changes that are confined to the alpha 2 domain confer less dramatic change. They do not perturb the primary anchors of the peptide-binding motif but can modulate the specificity through development and diversification of secondary anchors. Our results, in combination with those obtained previously for other HLA-B allotypes, suggest a general trend whereby polymorphism in the alpha 1 helix is the overriding influence on peptide-binding specificity of HLA-B allotypes, while amino acid substitutions in the alpha 2 domain play a more modulatory role.

Intersubunit and Intrasubunit Contact Regions of Na+/K+-ATPase Revealed by Controlled Proteolysis and Chemical Cross-linking

To identify interfaces of alpha- and beta-subunits of Na+/K(+)-ATPase, and contact points between different regions of the same alpha-subunit, purified kidney enzyme preparations whose alpha-subunits were subjected to controlled proteolysis in different ways were solubilized with digitonin to disrupt intersubunit alpha,alpha-interactions, and oxidatively cross-linked. The following disulfide cross-linked products were identified by gel electrophoresis, staining with specific antibodies, and N-terminal analysis. 1) In the enzyme that was partially cleaved at Arg438-Ala439, the cross-linked products were an alpha,beta-dimer, a dimer of N-terminal and C-terminal alpha fragments, and a trimer of beta and the two alpha fragments. 2) From an extensively digested enzyme that contained the 22-kDa C-terminal and several smaller fragments of alpha, two cross-linked products were obtained. One was a dimer of the 22-kDa C-terminal peptide and an 11-kDa N-terminal peptide containing the first two intramembrane helices of alpha (H1-H2). The other was a trimer of beta, the 11-kDa, and the 22-kDa peptides. 3) The cross-linked products of a preparation partially cleaved at Leu266-Ala267 were an alpha,beta-dimer and a dimer of beta and the 83-kDa C-terminal fragment. Assuming the most likely 10-span model of alpha, these findings indicate that (a) the single intramembrane helix of beta is in contact with portions of H8-H10 intramembrane helices of alpha; and (b) there is close contact between N-terminal H1-H2 and C-terminal H8-H10 segments of alpha; with the most probable interacting helices being the H1,H10-pair and the H2,H8-pair.

Identity of the Segment of Human Complement C8 Recognized by Complement Regulatory Protein CD59

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The inhibitory function of CD59 derives from its capacity to interact with both the C8 and C9 components of MAC, preventing assembly of membrane-inserted C9 polymer. MAC-inhibitory activity of CD59 is species-selective and is most effective when both C8 and C9 derive from human or other primate plasma. Rabbit C8 and C9, which can substitute for human C8 and C9 in MAC, mediate virtually unrestricted lysis of human cells expressing CD59. In order to identify the segment of human C8 that is recognized by CD59, recombinant peptides containing human or rabbit C8 sequence were expressed in Escherichia coli and purified. CD59 was found to specifically bind to a peptide corresponding to residues 334-385 of the human C8 alpha-subunit, and to require a disulfide bond between Cys345 and Cys369. No specific binding was observed to the corresponding sequence from rabbit C8 alpha (residues 334-386). To obtain functional evidence that this segment of human C8 alpha is selectively recognized by CD59, recombinant C8 proteins were prepared by co-transfecting COS-7 cells with human/rabbit chimeras of the C8 alpha cDNA, and cDNAs encoding the C8 beta and C8 gamma chains. Hemolytic activity of MAC formed with chimeric C8 was analyzed using target cells reconstituted with CD59. These experiments confirmed that CD59 recognizes a conformationally sensitive epitope that is within a segment of human C8 alpha internal to residues 320-415. Our data also suggest that optimal interaction of CD59 with this segment of human C8 alpha is influenced by N-terminal flanking sequence in C8 alpha and by human C8 beta, but is unaffected by C8 gamma.

Processing of ADP-ribosylated Integrin α7 in Skeletal Muscle Myotubes

Integrin alpha 7 is a major substrate in skeletal muscle cells for the cell surface, glycosylphosphatidylinositol-anchored, arginine-specific ADP-ribosyltransferase. Since ADP-ribosylarginine hydrolase, the enzyme responsible for cleavage of the ADP-ribosylarginine bond and a component with the transferase of a putative ADP-ribosylation cycle, is cytosolic, the processing of ADP-ribosylated integrin alpha 7 was investigated. Following incubation of differentiated mouse C2C12 myoblasts with [adenylate-32P]NAD and analysis by SDS-polyacrylamide gel electrophoresis under reducing conditions, two [32P]ADP-ribosylated forms of integrin alpha 7 were resolved. By pulse-chase and purification of the radiolabeled proteins on a laminin affinity column, it was demonstrated that a 105-kDa ADP-ribosylated form originated from a mono-ADP-ribosylated 102-kDa form and represented integrin alpha 7 modified at more than one site. The additional site(s) of modification, utilized at higher NAD concentrations, were located in the 63-kDa N-terminal segment of integrin alpha 7. Both [32P]ADP-ribosylated integrins were loosely associated with the cytoskeleton, bound to laminin affinity columns, and immunoprecipitated with antibodies to integrin beta 1. 32P label was rapidly removed from [32P]ADP-ribosylated integrin alpha 7 at either site of modification, a process inhibited by free ADP-ribose or p-nitrophenylthymidine-5'-monophosphate, an alternative substrate of 5'-nucleotide phosphodiesterase. The processed integrin alpha 7 was unavailable for subsequent ADP-ribosylation, although the amount of surface integrin alpha 7 remained constant. During the processing, no loss of label was observed from integrin alpha 7 radiolabeled with [14C]NAD, containing 14C in the nicotinamide proximal ribose, consistent with degradation of the ADP-ribose moiety by a cell surface 5'-nucleotide phosphodiesterase. Thus, cell surface ADP-ribosylation, in contrast to intracellular ADP-ribosylation, is not readily reversed by ADP-ribosylarginine hydrolase and seems to operate outside the postulated ADP-ribosylation cycle.

Topology of the Na,K-ATPase: EVIDENCE FOR EXTERNALIZATION OF A LABILE TRANSMEMBRANE STRUCTURE DURING HEATING

The topological organization of the Na,K-ATPase alpha subunit is controversial. Detection of extracellular proteolytic cleavage sites would help define the topology, and so attempts were made to find conditions and proteases that would permit digestion of Na,K-ATPase in sealed right-side-out vesicles from renal medulla. The beta subunit is predominantly extracellular and could mask the surface of the alpha subunit. Most of the tested proteases cleaved beta, and some digested it extensively. However, without further disruption of structure, there was still no digestion of the alpha subunit. Reduction (at 50 degrees C) of disulfide bonds that might stabilize the beta subunit fragments, or heating alone at 55 degrees C, permitted tryptic digestion of alpha at a site close to the C terminus, while simultaneously increasing digestion of beta. A 90-kDa N-terminal fragment of alpha was recovered, but the C-terminal fragment was further digested. Heating and reduction resulted in the extracellular exposure of a protein kinase A phosphorylation site, Ser-938, and the C terminus, both of which have been proposed to be located on the intracellular surface. At the same time, access to a distant protein kinase C phosphorylation site was not increased. The data suggest that the harsh treatment simultaneously resulted in alteration of the beta subunit and the extrusion of a segment of alpha that normally spans the membrane, without causing complete denaturation or opening the sealed vesicles. Preincubation with Rb+ was protective, consistent with prior evidence that it stabilizes the protein segments in the C-terminal third of alpha. We conclude that this portion of the alpha subunit contains a transmembrane structure with unique lability to heating.

Formation, analysis, structure and properties of type III enzyme resistant starch

Abstract During the retrogradation of starch a fraction becomes resistant to amylolytic enzymes. This undigestible fraction is described as enzyme resistant starch (RS) type III. RS type III is composed of short linear segments of alpha-(1–4)-glucans arranged in an A or B-type crystalline structure and is thermally very stable. Different factors have an impact on the formation (crystallisation) of these structures. Apart from the starch type, which defines the amylose/amylopectin ratio, polymer chain length and lipid content, process conditions following starch gelatinisation and the presence of other components, have an influence on the amount and on the quality of RS formed. Several in vitro and in vivo procedures to quantify RS have been used. However, RS levels may be affected by the analytical procedure.

Aspartate-bond isomerization affects the major conformations of synthetic peptides.

The aspartic acid bond changes to an beta-aspartate bond frequently as a side-reaction during peptide synthesis and often as a post-translational modification of proteins. The formation of beta-asparate bonds is reported to play a major role not only in protein metabolism, activation and deactivation, but also in pathological processes such as deposition of the neuritic plaques of Alzheimer's disease. Recently, we reported how conformational changes following the aspartic-acid-bond isomerization may help the selective aggregation and retention of the amyloid beta peptide in affected brains (Fabian et al., 1994). In the current study we used circular dichroism, Fourier-transform infrared spectroscopy, and molecular modeling to characterize the general effect of the beta-aspartate-bond formation on the conformation of five sets of synthetic model peptides. Each of the non-modified, parent peptides has one of the major secondary structures as the dominant spectroscopically determined conformation: a type I beta turn, a type II beta turn, short segments of alpha or 3(10) helices, or extended beta strands. We found that both types of turn structures are stabilized by the aspartic acid-bond isomerization. The isomerization at a terminal position did not affect the helix propensity, but placing it in mid-chain broke both the helix and the beta-pleated sheet with the formation of reverse turns. The alteration of the geometry of the lowest energy reverse turn was also supported by molecular dynamics calculations. The tendency of the aspartic acid-bond isomerization to stabilize turns is very similar to the effect of incorporating sugars into synthetic peptides and suggests a common feature of these post-translational modifications in defining the secondary structure of protein fragments.

A partial structural repeat forms the heterodimer self-association site of all beta-spectrins

The self-polymerization of alpha beta-spectrin heterodimers to form tetramers and higher oligomers is central to its role as a membrane stabilizer and organizer. Mutations near the amino terminus of alpha I sigma 1-spectrin or the COOH terminus of beta I sigma 1-spectrin often lead to profound impairment heterodimer polymerization and to hemolytic disease of varying severity. Previous studies using an 80-kDa univalent fragment of alpha I sigma 1-spectrin have established that the amino-terminal segment of alpha I sigma 1-spectrin mediates the association of the alpha subunit with either intact heterodimers or with isolated beta-spectrin (beta I sigma 1). However, the nature of the self-association site in beta-spectrin has remained unclear. In the present study, native beta-spectrin and recombinant beta-spectrin peptides representing COOH-terminal portions of two alternative transcripts of the gene on chromosome 2 (beta I sigma 1 or "erythrocyte" spectrin and beta I sigma 2 or "muscle" spectrin), and one transcript of the gene on chromosome 14 (beta II sigma 1 or "beta G-fodrin") have been examined for their ability to bind either intact alpha beta-spectrin or the alpha I-spectrin 80-kDa univalent fragment. Deletion of the nonhomologous beta-spectrin sequence downstream of repeat 17 (spectrin domain III) had no discernible effect on binding. Truncations proximal to codon 2085 of beta I sigma 1-spectrin demonstrated a precipitous loss of activity, accounted for by a loss of both binding affinity and capacity. Further truncations to repeat 16 (codon 1979) restored binding activity to levels approximating that of the intact molecule. Repeat 16/17 and 17/16 chimeras displayed reduced binding activity. Collectively, these data indicate that the beta-subunit self-association site is highly sensitive to conformation, involves widespread interactions within the 17th repeat unit, is largely independent of sequences in domain III, and can be recreated by the deletion of all residues distal to the COOH end (codon 1979) of the 16th and presumably other spectrin sequence repeat units. All beta-spectrins appear to use this binding motif, regardless of the nature of the nonhomologous sequence in domain III.

Amino acids lining the channel of the gamma-aminobutyric acid type A receptor identified by cysteine substitution.

The binding of gamma-aminobutyric acid (GABA) to gamma-aminobutyric acid type A (GABAA) receptors triggers the opening of an anion-selective channel. To identify amino acid residues that line the channel, we combined cysteine mutagenesis and covalent chemical modification. We mutated, one at a time, four consecutive residues (268-271) in the M2 membrane-spanning segment of the rat GABAA receptor alpha 1 subunit to cysteine and expressed the mutant alpha 1 subunits, together with either the beta 1 subunit or the beta 1 and gamma 2 subunits, in Xenopus oocytes. We probed the susceptibility of the cysteine substitution mutants to covalent modification by charged, sulfhydryl reagents added extracellularly. We assumed that among the residues in membrane-spanning segments, only those lining the channel would be susceptible to modification by polar reagents and that such modification would irreversibly alter conduction. We infer that the residues Thr-268 and Ile-271 are exposed in the channel in both the open and closed states but that Leu-269 and Ser-270 are not exposed. The susceptibility of Thr-268 and Ile-271 in the closed state implies that the gate must be closer to the cytoplasmic end of the channel than Thr-268.