Initial analysis of the molecular image of pamlin, a sea urchin cell adhesion protein, by transmission electron microscopy

Pamlin, an important extracellular protein required early for sea urchin embryogenesis, is readily isolated from the embryos of Hemicentrotus pulcherrimus . A molecular image analysis of pamlin was conducted using immuno-electron microscopy, rotary shadowing and negative staining technique-applied electron microscopy. The electron microscopy showed that a monoclonal antibody to the pamlin α-subunit bound to a position 13.5 nm from one end of a purified 255 kDa pamlin molecule, which is a 132 nm long and 6.8 nm wide linear structure. The pamlin structure is composed of three subunits, a 47 nm long 52 kDa α-subunit that attaches to one end of a 105 nm long 180 kDa β-subunit, and a 15.6 nm diameter globular 23 kDa γ-subunit that binds to the middle of the β-subunit. The α- and β-subunits together form a 125–140 nm linear structure. Intermolecular aggregation frequently occurred between the free end of two β-subunits of the αβγ pamlin molecule, leaving the entire α-subunit surface free. Occasionally associations between the ends of α-subunits, or between an α-subunit and the middle of a β-subunit also occurred, but no aggregations of pamlin formed through the γ-subunit. These homophilic molecular aggregations of pamlin formed a large supramolecular network. In addition, the single pamlin molecule rounded at one end under high calcium ion concentration to form a 'loop', suggesting the presence of a calcium sensitive region in the molecule.     

How to dissect the Node of Ranvier

Voltage-gated sodium channels are unique in that they combine action potential conduction with cell adhesion. Mammalian sodium channels are heterotrimers, composed of a central, pore-forming α subunit and two auxiliary β subunits. The α subunits are members of a large gene family containing the voltage-gated sodium, potassium, and calcium channels. Sodium channel α subunits form a gene subfamily with at least 11 members. Mutations in sodium channel α subunit genes have been linked to paroxysmal disorders such as epilepsy, long QT syndrome (LQT), and hyperkalemic periodic paralysis in humans, and motor endplate disease and cerebellar ataxia in mice. Three genes encode the sodium channel β subunits with at least one alternative splice product. Unlike the pore-forming α subunits, the sodium channel β subunits are not structurally related to β subunits of calcium and potassium channels. Sodium channel β subunits are multifunctional. They modulate channel gating and regulate the level of channel expression at the plasma membrane. We have shown that β subunits also function as cell adhesion molecules (CAMs) in terms of interaction with extracellular matrix molecules, regulation of cell migration, cellular aggregation, and interaction with the cytoskeleton. A mutation in SCN1B has been shown to cause GEFS + 1 epilepsy in human families. We propose that the sodium channel signalling complex at nodes of Ranvier involves β subunits as channel modulators as well as CAMs, other CAMs such as neurofascin and contactin, RPTPβ, and extracellular matrix molecules such as tenascin.     

Complete primary structure of a newly characterized galactose-specific lectin from the seeds of <Emphasis Type="Italic">Dolichos lablab</Emphasis>

A new unique lectin (galactose-specific) purified from the seeds of Dolichos lablab, designated as DLL-II is a heterodimer composed of closely related subunits α and β. These were separated by SDS-PAGE and isolated by electroelution. By ESI-MS analysis their molecular masses were found to be 30.746 kDa (α) and 28.815 kDa (β) respectively. Both subunits were glycosylated and displayed similar amino acid composition. Using advanced mass spectrometry in combination with de novo sequencing and database searches for the peptides derived by enzymatic and chemical cleavage of these subunits, the primary sequence was deduced. This revealed DLL-II to be made of two polypeptide chains of 281(α) and 263(β) amino acids respectively. The β subunit differed from the α subunit by the absence of some amino acids at the carboxy terminal end. This structural difference suggests that possibly, the β subunit is derived from the α subunit by posttranslational proteolytic modification at the COOH-terminus. Comparison of the DLL-II sequence to other leguminous seed lectins indicates a high degree of structural conservation. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dynamic inter-subunit interactions in thermophilic F<Subscript>1</Subscript>-ATPase subcomplexes studied by cross-correlated relaxation-enhanced polarization transfer NMR

F₁-ATPase is a unique enzyme in terms of its rotational catalytic activity. The smallest unit showing this property is the α₃β₃γ complex (351 kDa). For investigation of such a huge system by means of solution NMR, we have explored a suitable NMR method using F₁-ATPase subcomplexes from a thermophilic Bacillus PS3 including an α₃β₃ hexamer (319 kDa). Pulse sequences for large molecules, effects of deuteration and simplification of the spectra were examined in this work. Since the β subunit includes the catalytic site, this was the target of the analysis in this work. The combination of [¹⁵N,¹H]-CRINEPT-HMQC-[¹H]-TROSY, deuteration of both α and β subunits, and segmental isotope-labeling was found essential to analyze such a huge and complex molecular system. Utilizing this method, subcomplexes composed of α and β subunits were investigated in terms of inter-subunit interactions. It turned out that there is equilibrium among monomers, heterodimers and the α₃β₃ hexamers in solution. The rate of exchange between the dimer and hexamer is in the slow regime on the NMR time scale. In chemical shift perturbation experiments, the N-terminal domain was found to be involved in strong inter-subunit interactions. In contrast, the C-terminal domain was found to be mobile even in the hexamer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of root-wave response by extra large and conventional G proteins in Arabidopsis thaliana

Heterotrimeric G proteins composed of α, β and γ subunits regulate a number of fundamental processes concerned with growth and development in plants. In addition to the canonical heterotrimeric G proteins, plants also contain a small family of extra large G proteins (XLGs) that show significant similarity to the G-protein α subunit in their C-terminal regions. In this paper we show that one of the three XLG genes, XLG3 , and the Gβ subunit (AGB1) of the Arabidopsis G-protein heterotrimer are specifically involved in the regulation of a subset of root morphological and growth responses. Based on analysis of T-DNA insertional mutant phenotypes, XLG3 and AGB1 each positively regulate root waving and root skewing. Since these responses are regulated by physical as well as physiological cues, we assessed the roles of AGB1 and XLG3 in gravitropism, thigmotropism and hormonal responses. Our data show that mutants lacking either XLG3 or AGB1 genes are hypersensitive to ethylene and show growth responses consistent with alterations in auxin transport, while maintaining an essentially wild-type response to the physical cues of gravity and touch. These results suggest that XLG3 and AGB1 proteins regulate the hormonal determinants of root-waving and root-skewing responses in plants and possibly interact in a tissue-specific or signal-specific manner. Because plants harboring knockout mutations in the Gα subunit gene, GPA1 , exhibit wild-type root waving and skewing, our results may indicate that the AGB1 subunit functions in these processes without formation of a classic Gαβγ heterotrimer.     

The pea chloroplast membrane-associated protein, IEP96, is a subunit of acetyl-CoA carboxylase

Two forms of acetyl-CoA carboxylase (ACCase) have been characterized in pea ( Pisum sativum L.) leaves; a heteromeric chloroplast enzyme and a homomeric, presumably cytosolic enzyme. The biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and β-carboxyltransferase (CT) subunits of the plastidial-ACCase have recently been characterized and cloned. To further characterize the carboxyl-transferase, an improved assay for CT was developed and used to follow its partial purification. CT activity co-purifies with ACCase activity during gel permeation chromatography. However, upon anion-exchange chromatography or native PAGE, CT separates from the BC and BCCP subunits of plastidiaI-ACCase and ACCase activity is lost. In addition, it is demonstrated that a previously sequenced pea chloroplast cDNA of unknown function (IEP96) with a predicted molecular weight of 91 kDa encodes the α-CT subunit of the MS-ACCase. Antibodies raised against the first 404 amino acids of IEP96 protein detected a polypeptide with molecular weight of 91 kDa that co-eluted during gel permeation chromatography with plastidial CT and ACCase activities. These antibodies also immunoprecipitated the activities of both ACCase and CT with the concomitant precipitation of the β-CT subunit. Furthermore, antibodies against β-CT immunoprecipitated the IEP96 protein. Two-dimensional PAGE and DEAE purification of ACCase protein demonstrated that the β-CT forms a tight association with the IEP96 protein. Pea leaf was fractionated into soluble and membrane fractions and the α-CT subunit was primarily associated with the membrane fraction. Together, these data demonstrate that IEP96 is the α-CT subunit of pea chloroplast ACCase.     

The α5(H105R) mutation impairs α5 selective binding properties by altered positioning of the α5 subunit in GABAA receptors containing two distinct types of α subunits

GABA_A receptors are pentameric ligand-gated ion channels that are major mediators of fast inhibitory neurotransmission. Clinically relevant GABA_A receptor subtypes are assembled from α5(1-3, 5), β1-3 and the γ2 subunit. They exhibit a stoichiometry of two α, two β and one γ subunit, with two GABA binding sites located at the α/β and one benzodiazepine binding site located at the α/γ subunit interface. Introduction of the H105R point mutation into the α5 subunit, to render α5 subunit-containing receptors insensitive to the clinically important benzodiazepine site agonist diazepam, unexpectedly resulted in a reduced level of α5 subunit protein in α5(H105R) mice. In this study, we show that the α5(H105R) mutation did not affect cell surface expression and targeting of the receptors or their assembly into macromolecular receptor complexes but resulted in a severe reduction of α5-selective ligand binding. Immunoprecipitation studies suggest that the diminished α5-selective binding is presumably due to a repositioning of the α5(H105R) subunit in GABA_A receptor complexes containing two different α subunits. These findings imply an important role of histidine 105 in determining the position of the α5 subunit within the receptor complex by determining the affinity for assembly with the γ2 subunit.     

Parallel evolution in the major haemoglobin genes of eight species of Andean waterfowl

Theory predicts that parallel evolution should be common when the number of beneficial mutations is limited by selective constraints on protein structure. However, confirmation is scarce in natural populations. Here we studied the major haemoglobin genes of eight Andean duck lineages and compared them to 115 other waterfowl species, including the bar-headed goose ( Anser indicus ) and Abyssinian blue-winged goose ( Cyanochen cyanopterus ), two additional species living at high altitude. One to five amino acid replacements were significantly overrepresented or derived in each highland population, and parallel substitutions were more common than in simulated sequences evolved under a neutral model. Two substitutions evolved in parallel in the αA subunit of two (Ala-α8) and five (Thr-α77) taxa, and five identical βA subunit substitutions were observed in two (Ser-β4, Glu-β94, Met-β133) or three (Ser-β13, Ser-β116) taxa. Substitutions at adjacent sites within the same functional protein region were also observed. Five such replacements were in exterior, solvent-accessible positions on the A helix and AB corner of the αA subunit. Five others were in close proximity to inositolpentaphosphate binding sites, and two pairs of independent replacements occurred at two different α¹β¹ intersubunit contacts. More than half of the substitutions in highland lineages resulted in the acquisition of serine or threonine (18 gains vs. 2 losses), both of which possess a hydroxyl group that can hydrogen bond to a variety of polar substrates. The patterns of parallel evolution observed in these waterfowl suggest that adaptation to high-altitude hypoxia has resulted from selection on unique but overlapping sets of one to five amino acid substitutions in each lineage.     

Evidence that GABAA Receptor Subunit mRNA Expression During Development Is Regulated by GABAA Receptor Stimulation

Abstract: The expression of six mRNA species (α2, α3, α5, β2, β3, and γ2) encoding for GABA_A receptor subunits was followed in cultured early postnatal cortical neurons by in situ hybridization histochemistry. In untreated control cultures it was found that these subunit mRNA expression profiles closely follow those seen during development in vivo. α3, α5, and β3 subunit expression declined, α2 expression increased, whereas β2 and γ2 subunit mRNA expression remained relatively constant. To test the hypothesis that GABA_A receptor stimulation regulates these expression profiles, we tested the effect of a GABA_A receptor positive modulator, allopregnanolone, and a GABA_A receptor noncompetitive antagonist, tert -butylbicyclophosphorothionate (TBPS). It was found that allopregnanolone augmented the rate at which the α3, α5, or β3 subunit mRNA expression declined and prevented the increase in α2 subunit mRNA expression. As well, allopregnanolone down-regulated β2 subunit mRNA expression. TBPS, on the other hand, up-regulated α3, α5, β2, and β3 subunit mRNA expression. It also down-regulated the expression of α2 subunit mRNA. Both allopregnanolone and TBPS had no effect on γ2 subunit mRNA expression. These results imply that the developmental switchover of GABA receptor subunit mRNA expression is regulated by GABA_A receptor activity.     

Construction of a cosmid library of Spirulina platensis as an approach to DNA physical mapping

Abstract A Spirulina platensis gene library has been constructed using cosmid vector pMMB34. The cosmid bank was controlled for its random gene distribution by colony hybridization. Genes were identified using either homologous or heterologous probes of genes involved in photosynthesis (large and small subunit of d -ribulose 1,5-bisphosphate carboxylase, 32 kDa thylakoid protein, α, β subunits of C-phycocyanin) and protein synthesis (elongation factors EF-Tu, EF-G).     

Sequences in the Amino Termini of GABA ρ and GABAA Subunits Specify Their Selective Interaction In Vitro

Abstract: Molecular cloning has revealed that there are six classes of subunits capable of forming GABA-gated chloride channel receptors. GABA_A receptors are composed of α, β, γ, δ, and ε/χ subunits, whereas GABA_C receptors appear to contain ρ subunits. However, retinal cells exhibiting GABA_C responses express α, β, and ρ subunits, raising the possibility that GABA_C receptors may be a mixture of subunit classes. Using in vitro translated protein, we determined that human GABA_A receptor subunits α1, α5, and β1 did not coimmunoprecipitate with full-length ρ1, ρ2, or the N-terminal domain of ρ1 that contains signals for ρ-subunit interaction. To explore the molecular mechanism underlying these apparently exclusive combinations, chimeric subunits were created and tested for interaction with the wild-type subunits. Transfer of the N terminus of β1 to ρ1 created a β1ρ1 chimera that coimmunoprecipitated with the α1 subunit but not with the ρ2 subunit. Furthermore, exchanging the N terminus of the ρ1 subunit with the corresponding region of β1 produced a ρ1β1 chimera that interfered with ρ1 receptor expression in Xenopus oocytes, whereas the full-length β1 subunit had no effect. Together, these results indicate that sequences in the N termini direct assembly of ρ subunits and GABA_A subunits into GABA_C and GABA_A receptors, respectively.     

Tubulin: An Integral Protein of Mammalian Synaptic Vesicle Membranes

Abstract: The major protein in isolated synaptic vesicles from bovine cerebral cortex has been compared to tubulin by sodium dodecyl sulphate-urea polyacrylamide gel electrophoresis, by two-dimensional gel electrophoresis and by peptide mapping following limited proteolysis of the protein by Staphylococcus aureus protease. The results establish in purified synaptic vesicles the presence of tubulin, which is composed of the α and β subunits. In the presence of ethyleneglycol bis (aminoethyl ether)- N, N' -tetraacetic acid (EGTA) or magnesium in the isolation buffers, the synaptic vesicles contained mainly the α-tubulin whereas the β subunit was less abundant. Similarly, synaptosomal plasma membranes that were prepared in the presence of EGTA also contained more of α-tubulin than of the β subunit. Non-ionic detergents such as Triton X-100 or Nonidet P-40 failed to solubilize the tubulin from the synaptic vesicles. Ionic detergents such as deoxycholate and sodium dodecyl sulphate solubilized all the vesicle proteins, including tubulin. The results indicate that α-tubulin is an integral vesicle membrane protein, whereas most of the β sub-unit is peripherally attached and can be easily dissociated from the vesicle membrane with EGTA.     

Microbial nitrilases: versatile, spiral forming, industrial enzymes

The nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The superfamily is characterized by having a homodimeric building block with a αββα–αββα sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications.     

Purification and Characterization of Two Casein Kinase Type II Isozymes from Bovine Brain Gray Matter

Abstract: Highly purified casein kinase II (CK II) isozymes from bovine brain gray matter (BBGM) were obtained by means of a new purification procedure consisting of one phosphocellulose and three Mono-Q steps. The phosphocellulose eluate showed two BBGM-CK II activities. The first minor component (BBGM-CK IIa) was eluted with 0.9 M NaCl and the major component was eluted at 1.1 M NaCl (BBGM-CK IIb). The protein complexes responsible for these two activities were comprised of three subunits, i.e., α (40 kDa), α' (38 kDa), and β (28 kDa), with various subunit ratios. The two isozymes displayed the same behavior on Superose 12 fast protein liquid chromatographic gel filtration and sucrose density centrifugation. BBGM-CK IIa and b showed chromatographic and biochemical differences including differing K _m for ATP and GTP and K _i for heparin and 2,3-bisphosphoglycerate. The properties of the main peak (BBGM-CK IIb) were studied in detail. The stimulatory effect of Mg²⁺, Mn²⁺, and Co²⁺ was highly dependent both on the nature of the substrate and on ionic type and concentration. It is surprising that with phosvitin as substrate, BBGM-CK IIb was fully active even in the absence of Mg²⁺ and NaCl. The inhibitory effect of heparin and the stimulatory effects of NaCl, KCl, spermine, and polylysine were highly dependent on the ionic strength, buffer type, and substrate. BBGM-CK II isozymes phosphorylated stathmine in the presence of polylysine, but the requirement for polybasic compounds was not absolute, as is the case with calmodulin and clathrin β-light chain. The unusual chromatographic behavior and biochemical properties of these BBGM-CK II isozymes, compared with the classical CK II, could be explained at least in part by their subunit ratios.     

Dα3, a New Functional α Subunit of Nicotinic Acetylcholine Receptors from Drosophila

Abstract: Nicotinic acetylcholine (ACh) receptors (nAChRs) are important excitatory neurotransmitter receptors in the insect CNS. We have isolated and characterized the gene and the cDNA of a new nAChR subunit from Drosophila . The predicted mature nAChR protein consists of 773 amino acid residues and has the structural features of an ACh-binding α subunit. It was therefore named Dα3, for D rosophila α -subunit 3 . The dα3 gene maps to the X chromosome at position 7E. The properties of the Dα3 protein were assessed by expression in Xenopus oocytes. Dα3 did not form functional receptors on its own or in combination with any Drosophila β-type nAChR subunit. Nondesensitizing ACh-evoked inward currents were observed when Dα3 was coexpressed with the chick β2 subunit. Half-maximal responses were at ∼0.15 µ M ACh with a Hill coefficient of ∼1.5. The snake venom component α-bungarotoxin (100 n M ) efficiently but reversibly blocked Dα3/β2 receptors, suggesting that Dα3 may be a component of one of the previously described two classes of toxin binding sites in the Drosophila CNS.     

The alpha4beta2alpha5 nicotinic cholinergic receptor in rat brain is resistant to up-regulation by nicotine in vivo

We used immunoprecipitation with subunit-specific antibodies to examine the distribution of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) that contain the α5 subunit in the adult rat brain. Among the regions of brain we surveyed, the α5 subunit is associated in ∼37% of the nAChRs in the hippocampus, ∼24% of the nAChRs in striatum, and 11–16% of the receptors in the cerebral cortex, thalamus, and superior colliculus. Sequential immunoprecipitation assays demonstrate that the α5 subunit is associated with α4β2* nAChRs exclusively. Importantly, in contrast to α4β2 nAChRs, which are increased by 37–85% after chronic administration of nicotine, the α4β2α5 receptors are not increased by nicotine treatment. These data thus indicate that the α4β2α5 nAChRs in rat brain are resistant to up-regulation by nicotine in vivo , which suggests an important regulatory role for the α5 subunit. To the extent that nicotine-induced up-regulation of α4β2 nAChRs is involved in nicotine addiction, the resistance of the α4β2α5 subtype to up-regulation may have important implications for nicotine addiction.     

Subunits of Xanthine Dehydrogenase and Their Differential Appearance in Chick Tissues During Development

Xanthine dehydrogenase (XDH) from adult chick liver comprises two polypeptide chains of different size in a molar ratio of 1: 1. The molecular weights of these subunits were estimated to be 155K (α) and 135K (β) daltons (1). However, XDH isolated from the liver of newly hatched chick was not found to represent the equimolar ratio of these two subunits; that is, the amount of subunit β was lower than that of subunit α. While examamining electrophoretically the change in the amounts of these subunits in the liver, the subunit α was found to appear earlier in the embryonic stage, but β only after hatching. In the kidney, however, both subunits were detected before hatching, being consistent with the fact that XDH exists before hatching in the kidney. The two subunits also appeared differentially in the kidney; i.e., subunit α appeared earlier than subunit β. In either tissue, the rate of increase in XDH activity corresponded to that of subunit β. Thus, the synthesis of two subunits of XDH are separately regulated at least until just after hatching.     

Nicotine Enhances the Cyclic AMP-Dependent Protein Kinase-Mediated Phosphorylation of α4 Subunits of Neuronal Nicotinic Receptors

Abstract: Studies determined whether α4β2 or α3β2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP-dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 n M for α4β2 and 500 n M for α3β2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing α4β2 receptors were incubated with [γ-³²P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the α4 subunit was present. Phosphorylation of α4 subunits of α4β2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing α3β2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the α3 subunit. Results suggest that the PKA-mediated phosphorylation of α4 and not α3 subunits may explain the differential inactivation by nicotine of these receptors subtypes expressed in oocytes.     

On the Identification of α- and β-Tubulin Subunits

Abstract: Confusion appears to have arisen in the literature regarding the designation of α-and β-tubulin in polyacrylamide gels. The presence or absence of 8 M-urea in sodium dodecyl sulfate (SDS) polyacrylamide gels leads to different patterns for unalkylated tubulin subunits (and other proteins), making difficult the designation of the α and β subunits by original definition using electrophoretic mobility in the molecular weight dimension. The specific biochemical property of posttranslational tyrosylation of the α subunit has been used to identify further this subunit. Under all conditions tested, the β subunit has been found to be more acidic than the α subunit, with isoelectric point differences that agree with theoretical and published values. If the tubulin subunits are reduced and alkylated, the β subunit migrates more rapidly in SDS polyacrylamide gels, with or without urea present. However, unalkylated tubulin subunits can comigrate or even reverse their relative mobility if 8 M-urea-SDS polyacrylamide gels are used for subunit separation. The results also confirm the earlier reports that the post-translational tyrosylation of protein appears exclusively restricted to α-tubulin and can be demonstrated in an in vivo situation. In addition, the results suggest that only the α₂ subunit of tubulin is tyrosylated.