Interaction of transmembrane-spanning segments of the α2-adrenergic receptor with model membranes

Adrenergic receptors are integral membrane proteins involved in cellular signalling that belong to the G protein-coupled receptors. Synthetic peptides resembling the putative transmembrane (TM) segments TM4, TM6 and TM7, of the human α2-adrenergic receptor subtype C10 (P08913) and defined lipid vesicles were used to assess protein-lipid interactions that might be relevant to receptor structure/function. P6 peptide contains the hydrophobic core of TM6 plus the N-terminal hydrophilic motif REKR, while peptides P4 and P7 contained just the hydrophobic stretches of TM4 and TM7, respectively. All the peptides increase their helical tendency at moderate concentrations of TFE (30–50%) and in presence of 1,2-dielaidoyl-sn-glycero-3-phosphatidylethanolamine (DEPE) lipids. However, only P6 displays up to 19% of α-helix in the presence of just the DEPE lipids, evidences a transmembrane orientation and stabilizes the Lα lipid phase. Conversely, P4 and P7 peptides form only stable β-sheet structures in DEPE and favour the non-lamellar, inverted hexagonal (H_II) phase of DEPE by lowering its phase transition temperature. This study highlights the potential of using synthetic peptides derived from the amino acid sequence in the native proteins as templates to understand the behaviour of the transmembrane segments and underline the importance of interfacial anchoring interactions to meet hydrophobic matching requirements and define membrane organization.     

Interaction of α2-macroglobulin with L-asparaginase

Summary Obvious protection of the catalytic activity of Esch. coli L-asparaginase by ₂-macroglobulin (₂M) was observed under conditions otherwise propitious to the dissociation of the tetrameric molecule into inactive subunits, i.e. very diluted enzyme solutions or the presence of either SDS or urea. The degree of protection depended on enzyme and ₂M concentrations respectively, and on the preincubation time of the ₂M-enzyme mixture prior to substrate addition. The formation of a catalytically active complex between ₂M and L-asparaginase was confirmed by gel filtration on a Sephadex-G column and by polyacrylamide gel electrophoresis. The fact that the migration distance of the active complex corresponded to the migration of ₂M and the absence in that case of a migration band corresponding to the intact molecule suggest that complexing of the enzyme with ₂M prevented its dissociation into subunits and thus its inactivation. Addition of ₂M to the already dissociated enzyme molecule did not restore its catalytic activity.Alpha₂-macroglobulin was shown to have an inhibiting effect on the proteolytic activity of almost all proteases and no effect on their esterolytic activity. Furthermore, it prevents the inhibition of esterolytic activity by some natural compounds^1–5. The effect of ₂M on other types of catalytic activity has not been investigated enough to afford a generalization of the possible role of this macroglobulin in the control of enzyme activity in the body.This paper reports the results of an in vitro study of the effect of ₂M on the catalytic activity of an important amidase, i.e. L-asparaginase (L-asparagine amidohydrolase 3.5.1.1), which in recent years has been used in the treatment of acute lymphocytic leukemia in children^6,7.Abbreviations ₂M ₂-macroglobulin - E enzyme - SDS sodium dodecylsulfate Part of the results were reported at the 10th International Congress of Biochemistry, Hamburg 1976, Abst. p. 377.     

Assay and purification of a solubilized membrane receptor that binds the lysosomal enzyme α-l-iduronidase

A receptor that binds the lysosomal enzyme α-l-iduronidase via phosphorylated mannose residues on the enzyme has been solubilized from Swarm rat chondrosarcoma membranes using a pH 9.5 buffer containing 0.1% Triton X-100. Detergent-solubilized receptor in crude and purified preparations was measured by assay of bound α-l-iduronidase after adsorbing the receptor-enzyme complex onto insoluble phospholipid vesicles (liposomes). Binding of α-l-iduronidase to the liposomes required receptor and was completely inhibited by mannose 6-phosphate but not glucose 6-phosphate, indicating that the receptor maintained specificity following solubilization. Receptors from rat chondrosarcoma and human diploid fibroblasts were purified to apparent homogeneity using a phosphomannan-Sepharose affinity column. Both had identical molecular weights in polyacrylamide gels containing sodium dodecyl sulfate (M_r = 215,000). Amino acid analysis and two-dimensional gel electrophoresis was carried out on the purified rat chondrosarcoma receptor. Two forms of the receptor with different pI's were observed (pI 5.5 and 6.2). One form (pI 5.5) was made more basic (pI 5.8) by treatment with neuraminidase.     

Further observations on the activation of lysosomal acid α-glucosidase by cortisone derivatives

1. Cortisone acetate activates the acid alpha-glucosidase in rat liver slices and in isolated liver lysosomes. 2. The reaction is steroid specific and moreover does not occur with lysosomal acid phosphatase or beta-galactosidase. 3. After pretreatment of the lysosomes with cortisone, substrate (maltose) binding to the soluble lysosomal acid alpha-glucosidase is not affected, but the steroid does increase the V(max.) value. Membrane-bound enzyme is not activated by cortisone. 4. 4-[(14)C]Cortisone is preferentially bound to the lysosomal membrane and the possible involvement of this structure in the activation phenomenon is discussed.     

Interaction of α-tocopherol quinone, α-tocopherol and other prenyllipids with photosystem II

We have found that plastoquinone-A (PQ-A) and α-tocopherol (α-Toc) increased the reduction level of the high-potential form of cytochrome b-559 (cyt. b-559 HP) and α-tocopherol quinone (α-TQ) decreased the level of this cytochrome form in Scenedesmus obliquus wild-type, while the investigated prenyllipids were not active in the restoration of the cyt. b-559 HP form in Scenedesmus PS28 mutant and Synechococcus 6301 (Anacystis nidulans) where the cyt. b-559 HP form is naturally not present. Among the tested prenyllipids, α-TQ quenched fluorescence in thylakoids of S. obliquus wild-type, the PS28 mutant and tobacco to the highest extent, while PQ-A was less effective in this respect. α-Tocopherol showed the opposite effect to α-TQ and it was rather small. The fluorescence quenching measurements of thylakoids in the presence of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) showed that the α-Toc and FCCP (carbonylcyanide-p-trifluoromethoxy-phenyl-hydrazone) did not quench non-photochemically chlorophyll fluorescence while PQ-9 and α-TQ were effective fluorescence quenchers at higher concentrations (> 15 μM). However, at the lower α-TQ concentrations where its effective fluorescence quenching was found in DCMU-free samples, there was nearly no quenching effect by α-TQ observed in DCMU-treated thylakoids. This suggested a specific, not non-photochemical, DCMU sensitive, fluorescence quenching of photosystem II (PSII) at low α-TQ concentrations which is probably connected with the cyclic electron transport around PSII and might have a function of excess light energy dissipation. The effects of α-TQ on PSII resembled those of FCCP under many respects which might suggest similar mechanism of action of these compounds on PSII, i.e. the catalytic deprotonation and/or redox changes of some components of PSII such as the water splitting system, tyrosine D, Chl_z or cytochrome b-559.     

Regional mapping of the human gene for lysosomal α-glucosidase by in situ hybridization

Summary The current approach to the chromosomal localization of genes coding for lysosomal enzymes has been the correlation of enzymatic and karyotypic analyses of human-rodent somatic cell hybrids. The feasibility of regional mapping depends on the availability of human cells with informative chromosomal rearrangements. In this communication we report the first localization of a gene coding for a lysosomal enzyme by in situ hybridization. The application of an acid -glucosidase cDNA probe to normal human chromosomes allowed direct regional mapping of the -glucosidase locus (GAA) to the region q23q25 of chromosome 17.     

Interaction of α-N-acetyl-β-endorphin and calmodulin

Acetylation at the α-amino terminal is a common post-translational modification of many peptides and proteins. In the case of the potent opiate peptide β-endorphin, α-N-acetylation is a known physiological modification that abolishes opiate activity. Since there are no known receptors for α-N-acetyl-β-endorphin, we have studied the association of this peptide with calmodulin, a calcium-dependent protein that binds a variety of peptides, phenothiazines, and enzymes, as a model system for studying acetylated endorphin-protein interactions. Association of the acetylated peptide with calmodulin was demonstrated by cross-linking with bis(sulfosuccinimidyl)suberate; like β-endorphin, adducts containing 1 mol and 2 mol of acetylated peptide per mole calmodulin were formed. Some of the bound peptides are evidently in relatively close proximity to each other since, in the presence of amidated (i.e., lysine-blocked) calmodulin, cross-linking yielded peptide dimers. The acetylated peptide exhibited no appreciable helicity in aqueous solution, but in trifluoroethanol (TFE) considerable helicity was formed. Also, a mixture of acetylated peptide and calmodulin was characterized by a circular dichroic spectrum indicative of induced helicity. Empirical prediction rules, applied earlier to β-endorphin, suggest that residues 14–24 exhibit α-helix potential. This segment has the potential of forming an amphipathic helix; this structural unit is believed to be important in calmodulin binding. The acetylated peptide was capable of inhibiting the calmodulin-mediated stimulation of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity with an effective dose for 50% inhibition of about 3 µM; this inhibitory effect was demonstrated using both an enzyme-enriched preparation as well as highly purified enzyme. Thus, acetylation at the α-amino terminal of β-endorphin, although abolishing opiate activity, does not interfere with the binding to calmodulin. Indeed, β-endorphin and the α-N-acetylated peptide behave very similarly with respect to calmodulin association.     

Interaction of αVβ3 and αVβ6 Integrins with Human Parechovirus 1

Human parechovirus (HPEV) infections are very common in early childhood and can be severe in neonates. It has been shown that integrins are important for cellular infectivity of HPEV1 through experiments using peptide blocking assays and function-blocking antibodies to α_V integrins. The interaction of HPEV1 with α_V integrins is presumably mediated by a C-terminal RGD motif in the capsid protein VP1. We characterized the binding of integrins α_Vβ₃ and α_Vβ₆ to HPEV1 by biochemical and structural studies. We showed that although HPEV1 bound efficiently to immobilized integrins, α_Vβ₆ bound more efficiently than α_Vβ₃ to immobilized HPEV1. Moreover, soluble α_Vβ₆, but not α_Vβ₃, blocked HPEV1 cellular infectivity, indicating that it is a high-affinity receptor for HPEV1. We also showed that HPEV1 binding to integrins in vitro could be partially blocked by RGD peptides. Using electron cryo-microscopy and image reconstruction, we showed that HPEV1 has the typical T=1 (pseudo T=3) organization of a picornavirus. Complexes of HPEV1 and integrins indicated that both integrin footprints reside between the 5-fold and 3-fold symmetry axes. This result does not match the RGD position predicted from the coxsackievirus A9 X-ray structure but is consistent with the predicted location of this motif in the shorter C terminus found in HPEV1. This first structural characterization of a parechovirus indicates that the differences in receptor binding are due to the amino acid differences in the integrins rather than to significantly different viral footprints.Picornaviruses consist of a positive-sense, single-stranded infectious RNA genome of approximately 7.3 kb enclosed in a capsid composed of 60 copies of each of the three or four capsid proteins (VP1 to VP4). Human parechovirus 1 (HPEV1) is a member of the Parechovirus genus of the Picornaviridae family (38, 70). There are currently eight completely sequenced human parechovirus types and 14 described types (4, 19, 24, 30, 38, 39, 51, 58, 78). In addition, the Parechovirus genus currently has four Ljungan virus members that infect rodents. HPEV1 exhibits several distinct molecular characteristics compared to other picornaviruses (38, 71). These include the lack of the maturation cleavage of the capsid proteins VP0 to VP4 (N-terminal) and VP2 (C-terminal), existence of an approximately 30-amino-acid-long extension to the N terminus of VP3, a unique nonstructural protein 2A, and a 5′ untranslated region that is more closely related to picornaviruses infecting animals than those infecting humans.HPEV infections are common during the first years of life and are often mild or asymptomatic (20, 28, 42, 73, 80). Recently, a number of new types have been identified, and their prevalence in stool samples, for example, highlights their clinical importance. Normally, they cause gastroenteritis and respiratory infections, but severe illnesses, such as infections of the central nervous system, generalized infections of neonates, and myocarditis, have also been associated with HPEV infections (1, 8, 10, 28, 80). Currently, the role of the unique molecular, structural, and antigenic characteristics of HPEVs in the pathogenesis of infection is unknown.HPEV types 1, 2, 4, 5, and 6 are known to possess an RGD motif near the C terminus of VP1 that is known to facilitate binding of cellular ligands (e.g., fibronectin) to α_v integrins. The motif is in an analogous position to motifs in coxsackievirus A9 (CAV9) and echovirus 9 (EV9; Barty strain) (Fig. ​(Fig.1).1). The role of the RGD sequence in cellular entry and subsequent replication of HPEV1 has been shown through blocking assays with RGD-containing peptides, mutation of the sequence, and function-blocking antibodies to α_v integrins (11, 43, 62, 71). These results strongly suggested that α_v integrins play a central role in the initiation of HPEV1 infection. Direct involvement of α_v integrins in the infectious entry of HPEV1 was further confirmed by overexpression of human α_vβ₁ and α_vβ₃ integrins in Chinese hamster ovary (CHO) cells, allowing successful virus infection (74). There are no reports yet on the identification of receptors for the HPEV types lacking the RGD motif (HPEV3, HPEV7, and HPEV8) (19, 39, 51).Open in a separate windowFIG. 1.Sequence alignments. Amino acid sequence alignment of the viral coat protein VP1 from different picornaviruses with the CAV9 secondary structure derived from the atomic model displayed above the alignment (34). The columns boxed in blue with red letters signify similarity, and the red column signifies identity. There is limited similarity between HPEV and other picornaviruses. C-terminal RGD motifs are boxed in red.Although the crystal structures of several picornaviruses have been determined (3, 26, 34, 35, 44, 57, 59, 65, 68, 72) and the receptor interactions have been studied in detail by X-ray crystallography, electron cryo-microscopy (cryo-EM), and three-dimensional (3D) image reconstruction (6, 9, 23, 31, 32, 47, 83), there is no structural information available for the parechoviruses or parechovirus-receptor complexes. Here, we compare the binding of α_Vβ₃ and α_Vβ₆ to HPEV1 in vitro by biochemical assays and determine the structures of HPEV1 and the corresponding HPEV1-integrin complexes.     

Synthesis and activity of α-glucosidase produced byBifidobacterium pseudolongum

Bifidobacterium pseudolongum NCFB 2244 grew on starch as sole source of carbon and energy, but cell yields and specific growth rates were considerably lower than on glucose (=0.19±0.04 and 0.38±0.09 respectively). Amylase activity was not detected in cultures of this bacterium, but cell-associated -glucosidase was constitutively produced. Analysis of -glucosidase activity from cell extracts by preparative isoelectric focusing gave two peaks of activity with apparent isoelectric points of 3.9 (Enzyme I) and 4.2 (Enzyme II), corresponding to threefold and fourfold purification factors respectively. No -glucosidase activity was detected with Enzyme I after gel-filtration chromatography on Sephadex G150. However, activity was recovered in samples containing Enzyme II, indicating the protein had a molecular mass of approximately 126 kDa. This was subsequently confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). These results show that the restricted ability ofB. pseudolongum to utilize starch as a carbohydrate source is owing to synthesis of at least one, and possibly two, -glucosidase(s).     

Further purification and characterization of the acid α-glucosidase

1. Centrifugation of rat liver acid glucosidase, which had been purified by adsorption on dextran gel, on a density gradient of sucrose showed the enzyme to be impure. 2. Preliminary purification of the enzyme before the gel filtration improved the final degree of purity of this preparation. Disc gel electrophoresis of this preparation showed a single band of protein. 3. The sedimentation co-efficient and the molecular weight determined on a sucrose gradient were 4.9-5.1s and 76000-83000 respectively for the rat liver enzyme, and 5.6s and 97000 for the acid alpha-glucosidase purified by means of the same procedure from the human kidney. 4. The Michaelis constants of rat liver and human kidney enzyme were 4.7x10(-3)m and 13.6x10(-3)m respectively with maltose as substrate. 5. The enzyme from both tissues was inhibited by tris and by erythritol. The inhibition of the rat liver acid glucosidase by erythritol was competitive.     

Purification and characterization of α-glucosidase from Aspergillus carbonarious

Summary An -glucosidase was purified from Aspergillus carbonarious CCRC 30414 over 20 fold with 37 % recovery. Its molecular mass was estimated to be 328 kDa by gel filtration with an optimum pH from 4.2 to 5.0, and pI=5.0. The optimum temperature is at 60°C over 40 min. The enzyme was partially inhibited by 5 mM Ag⁺, Hg²⁺, Ba²⁺, Pb²⁺, and Aso₄ ⁺.     

Synthesis and α-glucosidase inhibition activity of dihydroxy pyrrolidines

A new series of Deacetylsarmentamide A and B derivatives, amides and sulfonamides of 3,4-dihydroxypyrrolidines as α-glucosidase inhibitors were designed and synthesized. The biological screening test against α-glucosidase showed that some of these compounds have the positive inhibitory activity against α-glucosidase. Saturated aliphatic amides were more potent than the olefinic amides. Among all the compounds, 5o/6o having polar –NH₂ group, 10f/11f having polar –OH group on phenyl ring displayed 3–4-fold more potent than the standard drugs. Acarbose, Voglibose and Miglitol were used as standard references. The promising compounds 6i, 5o, 6o, 10a, 11a, 10f and 11f have been identified. Molecular docking simulations were done for compounds to identify important binding modes responsible for inhibition activity of α-glucosidase.     

Photoactivatable Analogues of α-Conotoxins GI and MI and Their Interaction with Nicotinic Acetylcholine Receptor

Two photoactivatable analogues of -conotoxin GI with the benzoylphenylalanine residue (Bpa) substituted for His10 or Tyr11 were synthesized using the method of solid-phase peptide synthesis. In addition, -conotoxin MI was chemically modified by placing an azidobenzoyl or a benzoylbenzoyl photo label at N of Gly1 or N of Lys10. All the photoactivatable analogues were purified by HPLC, their structures were confirmed by MALDI MS, and the label positions in their molecules were localized by MS of their trypsinolysis fragments. All the analogues interacted with the nicotinic acetylcholine receptor (AChR) from Torpedo californica as efficiently as the native -conotoxins, with the differences in the inhibition constants being within one order of magnitude under the same conditions. [¹²⁵I] Derivatives prepared from all the analogues retained the ability to be bound by AChR and were used in the photoinduced AChR crosslinking. All the AChR subunits were found to be crosslinked to the photoactivatable analogues, with the linking depending on both the chemical nature of label and its position in the -conotoxin molecule.     

Inhibitors of α-glucosidase, α-amylase and lipase from Chrysanthemum morifolium

A new endoperoxysesquiterpene lactone, 10α-hydroxy-1α,4α-endoperoxy-guaia-2-en-12,6α-olide (1), together with a flavanone, eriodictyol (2), and two flavone glycosides, acacetin-7-O-β-d-glucopyranoside (3) and acacetin-7-O-α-l-rhamopyranoside (4), were isolated from the methanol extract of Chrysanthemum morifolium flowers by a bioassay-guided fractionation. Compound 1 showed strong inhibitory effects against α-glucosidase and lipase activities, with IC₅₀ values of 229.3 and 161.0 μM, respectively. The flavone glycosides 3 and 4 inhibited both α-glucosidase and α-amylase, while flavanone 2 was only effective against α-amylase.     

Comparison of α-glucosidase and α-mannosidase in bloodstream forms of Trypanosoma brucei brucei

• 1.1. The physicochemical and kinetic properties of the two major trypanosomal glycosidases, α-glucosidase (EC 3.2.1.20) and α-mannosidase (EC 3.2.1.24), were compared in bloodstream forms of Trypanosoma brucei brucei S42.
• 2.2. Both enzymes are membrane-bound and located intracellularly.
• 3.3. The results are discussed in relation to the possible role of α-glucosidase and α-mannosidase in the processing or catabolism of trypanosomal glycoproteins.

     

Interaction of α-thalassemia genes with each other and with HbC in an American black family

The relative rates of in vitro synthesis of hemoglobin chains have been studied in an American black family in which the mother is doubly heterozygous for alpha-thalassemia and HbC and the father is heterozygous for alpha-thalassemia. The alpha/non-alpha synthetic ratio was equally unbalanced in both the bone marrow and the peripheral blood of the mother. Although HbC comprised 35% of her hemoglobin (compared to 42.2 +/- 2.2 in individuals with HbC trait and balanced globin synthesis), synthetic data showed that the newly synthesized beta C chain was 44% of the total newly synthesized beta chains. Isolated membranes contained more newly synthesized beta C than beta A chains. Three of the offspring were within the normal range, and the remaining three had alpha-thalassemia. There were two spontaneous abortions during the second trimester of pregnancy. Hydrops fetalis did not occur, and none of the children had HbH disease or HbC trait.     

Expression of enzymatically active,recombinant barley α-glucosidase in yeast and immunological detection of α-glucosidase from seed tissue

An -glucosidase cDNA clone derived from barley aleurone tissue was expressed in Pichia pastoris and Escherichia coli. The gene was fused with the N-terminal region of the Saccharomyces cerevisiae -factor secretory peptide and placed under control of the Pichia AOX1 promoter in the vector pPIC9. Enzymatically active, recombinant -glucosidase was synthesized and secreted from the yeast upon induction with methanol. The enzyme hydrolyzed maltose > trehalose > nigerose > isomaltose. Maltase activity occurred over the pH range 3.5–6.3 with an optimum at pH 4.3, classifying the enzyme as an acid -glucosidase. The enzyme had a K_m of 1.88 mM and V_max of 0.054 µmol/min on maltose. The recombinant -glucosidase expressed in E. coli was used to generate polyclonal antibodies. The antibodies detected 101 and 95 kDa forms of barley -glucosidase early in seed germination. Their levels declined sharply later in germination, as an 81 kDa -glucosidase became prominent. Synthesis of these proteins also occurred in isolated aleurones after treatment with gibberellin, and this was accompanied by a 14-fold increase in -glucosidase enzyme activity.Abbreviations: AGL, barley seed -glucosidase; rAGL, recombinant barley seed -glucosidase; BMGY, buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; GA, gibberellic acid; UTR, untranslated region.     

Interaction of α-synuclein with vesicles that mimic mitochondrial membranes

α-Synuclein, an intrinsically-disordered protein associated with Parkinson's disease, interacts with mitochondria, but the details of this interaction are unknown. We probed the interaction of α-synuclein and its A30P variant with lipid vesicles by using fluorescence anisotropy and (19)F nuclear magnetic resonance. Both proteins interact strongly with large unilamellar vesicles whose composition is similar to that of the inner mitochondrial membrane, which contains cardiolipin. However, the proteins have no affinity for vesicles mimicking the outer mitochondrial membrane, which lacks cardiolipin. The (19)F data show that the interaction involves α-synuclein's N-terminal region. These data indicate that the middle of the N-terminal region, which contains the KAKEGVVAAAE repeats, is involved in binding, probably via electrostatic interactions between the lysines and cardiolipin. We also found that the strength of α-synuclein binding depends on the nature of the cardiolipin acyl side chains. Eliminating one double bond increases affinity, while complete saturation dramatically decreases affinity. Increasing the temperature increases the binding of wild-type, but not the A30P variant. The data are interpreted in terms of the properties of the protein, cardiolipin demixing within the vesicles upon binding of α-synuclein, and packing density. The results advance our understanding of α-synuclein's interaction with mitochondrial membranes.