Molecular forms of acetylcholinesterase from Torpedo californica: their relationship to synaptic membranes.

The 16S and 8S forms of acetylcholinesterase (AchE), which are composed of an elongated tail structure in addition to the more globular catalytic subunits, were extracted and purified from membranes from Torpedo californica electric organs. Their subunit compositions and quaternary structures were compared with 11S lytic enzyme which is derived from collagenase or trypsin treatment of the membranes and devoid of the tail unit. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of reducing agent, appreciable populations of monomeric through tetrameric species are observed for the 11S form. Under the same conditions, the 16S form yields only monomer and dimer in addition to a higher molecular weight species. If complete reduction is effected, only the 80,000 molecular weight monomer is dominant for both the 11S and 16S forms. Cross-linking of the 11S form by dimethyl suberimidate followed by reduction yields monomer through tetramer in descending frequency, while the 16S form again shows a high molecular weight species. A comparison of the composition of the 11S and 16S forms reveals that the latter has an increased glycine content, and 1.1 and 0.3 mol % hydroxyproline and hydroxylysine, respectively. Collagenases that have been purified to homogencity and are devoid of amidase and caseinolytic activity, but active against native collagen, will convert 16S acetylcholinesterase to the 11S form. Thus, composition and substrate behavior of the 16S enzyme are indicative of the tail unit containing a collagen-like sequence. A membrane fraction enriched in acetylcholinesterase and components of basement membrane can be separated from the major portion of the membrane protein. The 16S but not the 11S form reassociates selectively with this membrane fraction. These findings reveal distinct similarities between the tail unit of acetylcholinesterase and basement membrane components and suggest a primary association of AchE with the basement membrane.     

Solubilization of Collagen-Tailed Acetylcholinesterase from Chick Retina: Effect of Different Extraction Procedures

We have extracted acetylcholinesterase from young chick retinas by homogenization in different solutions combining high salt concentration, ionic and nonionic detergents, and EDTA, looking for an optimum procedure for the solubilization of collagen-tailed, asymmetric structural forms of the enzyme. High salt and EDTA seem to be the only necessary requirements for the solubilization of acetylcholinesterase as the A12 form (20S), and the presence of detergent in the homogenization medium does not significantly improve the yield of tailed enzyme. Extraction in the absence of detergent has the potential advantage of a threefold enrichment of tailed enzyme, because only about one-third of the total retinal acetylcholinesterase activity is solubilized. Divalent cations, especially Ca2+, seem to be involved in the attachment of the tailed enzyme to the retinal membranes, at the tail level. High salt-EDTA-extracted 20S acetylcholinesterase (without detergent) aggregates in the presence of exogenous Ca2+ and becomes "insoluble." However, the aggregated 20S acetylcholinesterase can be completely recovered and brought back into solution by further addition of EDTA. Besides, the aggregation can be prevented by the inclusion of Triton X-100 in the homogenization buffer or by adding the detergent concurrently with Ca2+. It is postulated that the acetylcholinesterase collagenous tail is coated by acidic lipid molecules hydrophobically bound to the tail protein so that Ca2+ ionic bridges would actually link these lipid molecules (and consequently the tail) to the membrane matrix. Removal of the lipid coat (e.g., by Triton X-100) produces tailed acetylcholinesterase molecules that no longer aggregate in the presence of Ca2+ and are fully accessible to collagenase digestion.     

Heparin and the solubilization of asymmetric acetylcholinesterase

Heparin solubilizes asymmetric acetylcholinesterase, from chick skeletal muscle and retina, as a 24 S complex which is quantitatively converted to conventional asymmetric molecular forms of the enzyme (A12 and A8, either class I or class II) upon exposure to high salt. The simultaneous presence of salt and heparin in the homogenization medium selectively prevents, however, the release of class II A-forms in both muscle and retina. Heparin may generally act by displacing native proteoglycans involved in the attachment of the enzyme tail to the extracellular matrix, or its neural equivalent, being in turn removed by salt to yield typical asymmetric enzyme forms. Heparin would also appear to displace some other molecules specifically involved in the EDTA-sensitive attachment of class II tailed forms, this effect being antagonized by salt.     

Studies on plasmid replication. V Replicative intermediates.

A procedure has been developed for the study of rapidly labeled intermediates in plasmid replication in normally growing bacteria. Pulse-labeled cells are enzymatically lysed on top of a neutral sucrose gradient and centrifuged so that the chromosomal DNA forms a pellet and the plasmids (and other smaller DNA molecules) form bands in the gradient. Analysis of penicillinase plasmid replication in Staphylococcus aureus has revealed that although pulse-labeled intermediates sediment faster than the 60 S circular duplex monomeric plasmid molecules, they do not have stable superhelical structure. The conversion of partially polymerized molecules, having a sedimentation coefficient of about 58 S, to fully polymerized terminal forms appears to involve a progressive change in sedimentation rate from 58 S to 67 S. Conversion of the presumably dimeric terminal forms to mature closed circular monomers is a slow and rate-limiting multi-step process (taking some 3 to 4 min at 37 °C).     

Three-dimensional structure of recombinant human muscle fatty acid-binding protein.

The three-dimensional structure of recombinant human muscle fatty acid-binding protein with a bound fatty acid has been solved and refined with x-ray diffraction data to 2.1 A resolution. The refined model has a crystallographic R factor of 19.5% for data between 9.0 and 2.1 A (7243 unique reflections) and root-mean-square deviations in bond length and bond angle of 0.013 A and 2.7 degrees. The protein contains 10 antiparallel beta-strands and two short alpha-helices which are arranged into two approximately orthogonal beta-sheets. Difference electron density maps and a multiple isomorphous derivative electron density map showed the presence of a single bound molecule of a long chain fatty acid within the interior core of the protein. The hydrocarbon tail of the fatty acid was found to be in a "U-shaped" conformation. Seven ordered water molecules were also identified within the interior of the protein in a pocket on the pseudo-si face of the fatty acid's bent hydrocarbon tail. The methylene tail of the fatty acid forms van der Waals interactions with atoms from 13 residues and three ordered waters. The carboxylate of the fatty acid is located in the interior of the protein where it forms hydrogen bonds with the side chains of Tyr128 and Arg126 and two ordered water molecules. A comparison of the three-dimensional structure of human muscle fatty acid-binding protein and rat intestinal fatty acid-binding protein shows strong similarity. Both proteins bind a single fatty acid within their interior cores, but the bound fatty acids are very different in their conformations and interactions. These findings suggest that the intestinal and muscle fatty acid-binding proteins have evolved distinct binding sites in order to satisfy different requirements within the tissues where they are expressed.     

Negative staining of myosin molecules

A reproducible method has been developed for the negative staining of myosin molecules. The dimensions of stained molecules are in close agreement with those obtained by metal shadowing. Sharp bends in the tail, indicative of hinge regions, were observed at two positions 44 nm and 76 nm from the head-tail junction. The tail was often ill-defined at the position of the first (44 nm) bend. The bend positions may be sites of proteolytic cleavage that result in the production of long and short myosin subfragment S2. About half the molecules exhibited bending to various degrees at one or both of these positions, but cases where the tail folded back on itself in a 180 degrees bend were comparatively rare (approximately equal to 10%). However, in the absence of EGTA, a large fraction of the molecules (approximately equal to 80%) exhibited 180 degrees bends. A small region, approximately 20 nm long, at the tip of the tail often appears to be significantly different from the rest. The heads are about 19 nm long and roughly pear-shaped. Although sometimes straight, more often they show a pronounced curvature. Both senses of curvature were observed, but those curved in a clockwise manner were the most common, indicating preferential binding of one side of the head to the carbon substrate. An analysis of the different combinations of head shapes in individual molecules indicates that each head can rotate independently around its long axis. No preferred angle of orientation between the two heads in a molecule, or between either head and the tail could be found. Substructure has been observed within the heads.     

IgG1 cytoplasmic tail is essential for cell surface expression in Igβ down-regulated cells

It has been shown that cytoplasmic tail of the IgG1 B cell receptors (BCRs) are essential for the induction of T-dependent immune responses. Also it has been revealed that unique tyrosine residue in the cytoplasmic tail of IgG2a has the potential of being phosphorylated at tyrosine and that this phosphorylation modulates BCR signaling. However, it still remains unclear whether such phosphorylation of IgG cytoplasmic tail is involved in the regulation of BCR surface expression. In order to approach the issue, we established and analyzed the cell lines which express wild-type or mutated forms of IgG1 BCR. As the result, we found that IgG1 BCR expressed normally on the surface of A20 B cell line independent of the cytoplasmic tail. In contrast, IgG1 BCR whose cytoplasmic tyrosine was replaced with glutamic acid which mimics phosphorylated tyrosine, was expressed most efficiently on the surface of non-B lineage cells and Igβ-down-regulated B cell lines. These results suggest that tyrosine residue in IgG cytoplasmic tail is playing a essential role for the efficient expression of IgG BCR on the cell surface when BCR associated signaling molecules, including Igβ, are down-regulated.     

Solubilization of the membrane proteins from Semliki Forest virus with Triton X100

Increasing concentrations of Triton X100 have been found to cause stepwise dissociation of the membrane of Semliki Forest virus. The final stage of the breakdown process leads to solubilization of the membrane proteins which can be separated from the membrane lipids and the viral nucleocapsid by density gradient centrifugation in the presence of 0.05% Triton X100. Two different forms of Semliki Forest virus protein have been observed with sedimentation coefficients of approximately 4 S and 23 S. The 4 S aggregate appears to consist of two polypeptide chains complexed with about 75 molecules of Triton X100. The 23 S form is a rosette-like aggregate containing about 16 polypeptide chains and about 260 molecules of Triton X100. Sucrose alters the equilibrium between the 4 S and 23 S forms: removal of sucrose leads to association of the 4 S form to the 23 S form and addition of sucrose to dissociation.A scheme for the dissociation of the Semliki Forest virus membrane is presented which is discussed with reference to other biological membranes. It is suggested that Triton X100 and deoxycholate solubilize amphipathic membrane proteins by binding to the hydrophobic segments of these proteins.     

Identification of discrete disulfide-linked oligomers which distinguish 18 S from 14 S acetylcholinesterase

Acetylcholinesterase (EC 3.1.1.7) purified by affinity chromatography from 1.0 m ionic strength extracts of electric organ from the eel Electrophorus electricus consists of a mixture of 18 and 14 S enzyme forms. When examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate without exposure to disulfide reducing agents, these purified preparations show two major high molecular weight bands (>300,000), labeled oligomers A and B, in addition to a major band corresponding to catalytic subunit dimers (150,000 M_r). All these major bands reflect intersubunit disulfide bonding. The 18 and 14 S forms in purified preparations were separated by extensive sucrose gradient centrifugation. Gel analyses of the isolated 18 and 14 S pools indicated that the larger oligomer A derives from the 18 S pool, while oligomer B is found primarily in the 14 S pool. These observations support a previous model for 18 S acetylcholinesterase (T. L. Rosenberry and J. M. Richardson (1977) Biochemistry, in press) which considers this molecule to consist of one oligomer A unit, composed of three pairs of catalytic subunits disulfide-bonded to a collagen-like tail structure, and three catalytic subunit dimers. Proteolytic cleavage of the tail structure in the 18 S form can occur to release an 11 S enzyme tetramer containing a residual tail fragment and to leave a 14 S form. We propose this 14 S form to consist of one oligomer B unit, composed of two pairs of catalytic subunits disulfide-bonded to the remaining tail structure, and two catalytic subunit dimers.     

The binding of 18S acetylcholinesterase to sphingomyelin and the role of the collagen-like tail

The “native” forms of acetylcholinesterase (EC 3.1.1.7) from $\text{Electrophorus electricus}$ have sedimentation coefficients of 18S, 14S, and 8.5S (1) and have been shown to possess a collagen-like tail structure thought to function in the immobilization of the enzyme on a membrane matrix. We report that collagenase treatment of the enzyme purified by affinity chromatography yields three products with sedimentation coefficients of 21.4S, 17.1S, and 11.8S. It is suggested that these species are tailless analogs of the 18S 14S, and 8.5S species, respectively.The 18S acetylcholinesterase species is shown to bind to sphingomyelin at high (μ=1.0) or low (μ=0.1) ionic strength, but not to phosphatidylcholine. The failure of the corresponding tailless analog, the 21.4S species, to bind to sphingomyelin suggests that the sphingomyelin binding site or sites reside on the tail structure.     

Flexibility of the molecular forms of acetylcholinesterase measured with steady-state and time-correlated fluorescence polarization spectroscopy

Steady-state and time-correlated fluorescence polarizations have been examined for selected complexes and covalent conjugates of the 11S and (17 + 13)S forms of Torpedo acetylcholinesterase. The 11S form exists as a tetramer of apparently identical subunits, whereas the (17 + 13)S forms contain two or three sets of tetramers disulfide-linked to an elongated collagen-like tail unit. Pyrenebutyl methylphosphonofluoridate and (dansylsulfonamido)pentyl methylphosphonofluoridate were conjugated at the active center serine whereas propidium was employed as a fluorescent ligand for the spatially removed peripheral anionic site. Steady-state polarization of the pyrenebutyl conjugates indicates rotational correlation times of approximately 400 ns for the 11S species and greater than 1100 ns for the (17 + 13)S species. Hence, the tail unit severely restricts rotational motion of the catalytic subunits. Time-correlated fluorescence polarization analysis of the 11S species indicates multiple rotational correlation times. Anisotropy decay of the propidium complex (tau = 6 ns) occurs in exponential manner with a rotational correlation time of approximately 150 ns, while covalent adducts at the active center exhibit rotational correlation times greater than or equal to 300 ns. Anisotropy decay of the (dansylsulfonamido)pentyl conjugate (tau = 16 ns) appears exponential with a correlation time of approximately 320 ns, whereas decay of the pyrenebutyl conjugate (tau = 100 ns) is described by two correlation times, phi S = 18 ns and phi L = 320 ns, of small (15%) and large (85%) amplitudes, respectively. Two limiting models have been considered to explain the results.(ABSTRACT TRUNCATED AT 250 WORDS)     

A conformational transition in gizzard heavy meromyosin involving the head-tail junction, resulting in changes in sedimentation coefficient, ATPase activity, and orientation of heads

Gizzard heavy meromyosin (HMM) sediments in the ultracentrifuge as a single peak, whose sedimentation coefficient (S20,w) decreases from 9 to 7.5 S upon increasing the NaCl concentration from 0.02 to 0.3 M. This decrease is accompanied by a parallel increase in Mg2+-ATPase activity, suggesting that both changes have a common molecular basis. Phosphorylation decreases S20,w and increases ATPase activity, while ATP increases S20,w. Sedimentation equilibrium studies indicate that HMM undergoes no detectable aggregation at 0.02 or 0.4 M NaCl, remaining monomeric with a molecular weight of 3.4 X 10(5). In contrast, S20,w of subfragment 1 does not change with changes in ionic strength, and its ATPase activity does not decrease at low ionic strengths. Electron micrographs of samples of HMM prepared at low ionic strength show that up to half of the molecules are flexed, i.e. the heads are bent at the neck and project back toward the tail, while the remaining molecules have either one or both of the heads pointing away from the tail. In samples prepared at high ionic strength only about 10% of the molecules are flexed. There is a linear relationship between the fraction of flexed molecules and S20,w, with no significant bending or folding of the tail and no detectable change in the shape of the heads. This correlation suggests that the changes in ATPase activity and S20,w may be a result of the reorientation of the heads.     

S1核酸酶作用与微环DNA分子的克隆策略

米曲霉来源的S1 核酸酶具有降解单链DNA或RNA的作用。在适当的条件下 ,该酶能将不同的环形DNA分子从超螺旋转变成开环和线形结构 ,对质粒pUC19的实验证明 ,S1 核酸酶的这种转变作用与加入的酶量呈正相关。在 2 5 μL总反应体积中 ,按 10 0ngDNA加入 5u至 17u的S1 核酸酶 ,能获得较高比例的线形DNA。由于微环DNA分子太小 ,单酶切位点的出现率较低 ,很难用常规方式进行克隆 ,以S1 核酸酶进行线形化是微环DNA克隆的途径。pC3是已知最小的真核生物线粒体DNA类质粒 (5 37bp) ,经S1 核酸酶线形化后 ,成功地克隆到pMD18 T载体上。     

Direct binding of human immunodeficiency virus type 1 Nef to the major histocompatibility complex class I (MHC-I) cytoplasmic tail disrupts MHC-I trafficking

Nef, an essential pathogenic determinant for human immunodeficiency virus type 1, has multiple functions that include disruption of major histocompatibility complex class I molecules (MHC-I) and CD4 and CD28 cell surface expression. The effects of Nef on MHC-I have been shown to protect infected cells from cytotoxic T-lymphocyte recognition by downmodulation of a subset of MHC-I (HLA-A and -B). The remaining HLA-C and -E molecules prevent recognition by natural killer (NK) cells, which would otherwise lyse cells expressing small amounts of MHC-I. Specific amino acid residues in the MHC-I cytoplasmic tail confer sensitivity to Nef, but their function is unknown. Here we show that purified Nef binds directly to the HLA-A2 cytoplasmic tail in vitro and that Nef forms complexes with MHC-I that can be isolated from human cells. The interaction between Nef and MHC-I appears to be weak, indicating that it may be transient or stabilized by other factors. Supporting the fact that these molecules interact in vivo, we found that Nef colocalizes with HLA-A2 molecules in a perinuclear distribution inside cells. In addition, we demonstrated that Nef fails to bind the HLA-E tail and also fails to bind HLA-A2 tails with deletions of amino acids necessary for MHC-I downmodulation. These data provide an explanation for differential downmodulation of MHC-I allotypes by Nef. In addition, they provide the first direct evidence indicating that Nef functions as an adaptor molecule able to link MHC-I to cellular trafficking proteins.     

Steered molecular dynamics simulations on the "tail helix latch" hypothesis in the gelsolin activation process

The molecular basis of the "tail helix latch" hypothesis in the gelsolin activation process has been studied by using the steered molecular dynamics simulations. In the present nanosecond scale simulations, the tail helix of gelsolin was pulled away from the S2 binding surface, and the required forces were calculated, from which the properties of binding between the tail helix and S2 domain and their dynamic unbinding processes were obtained. The force profile provides a detailed rupture mechanism that includes six major unbinding steps. In particular, the hydrogen bonds formed between Arg-207 and Asp-744 and between Arg-221 and Leu-753 are of the most important interaction pairs. The two hydrogen bond "clamps" stabilize the complex. The subsequent simulation on Arg-207-Ala (R207A) mutation of gelsolin indicated that this mutation facilitates the unbinding of the tail helix and that the contribution of the hydrogen bond between Arg-207 and Asp-744 to the binding is more than 50%, which offers a new clue for further mutagenesis study on the activation mechanism of gelsolin. Surrounding water molecules enhance the stability of the tail helix and facilitate the rupture process. Additionally, temperature also has a significant effect on the conformation of the arginine and arginine-related interactions, which revealed the molecular basis of the temperature dependence in gelsolin activation.     

Interaction of hirudin with the dysthrombins Quick I and II

The interaction of hirudin with the dysfunctional enzymes thrombin Quick I and II has been investigated. Natural and recombinant hirudin caused nonlinear competitive inhibition of thrombin Quick I. The results were consistent with thrombin Quick I existing in two forms that have different affinities for hirudin. The affinities of these forms for natural hirudin were respectively 10(4)- and 10(6)-fold lower than that of alpha-thrombin. In contrast, truncated hirudin molecules lacking the C-terminal tail of the molecule caused linear inhibition of thrombin Quick I. These results indicate that different modes of interaction of the two forms of thrombin Quick I with the C-terminal tail of hirudin were the cause of the nonlinear inhibition. Comparison of the dissociation constants of thrombin Quick I with the truncated and full-length forms of hirudin suggested that the interactions that normally occur between the C-terminal tail of hirudin and thrombin were completely disrupted with the low-affinity form of thrombin Quick I. Thrombin Quick II displayed an affinity for natural hirudin that was 10(3)-fold lower than that observed with alpha-thrombin. In contrast, it bound a mutant hirudin with altered N-terminal amino acids only 16-fold less tightly. These results are discussed in terms of structural alterations in the active-site cleft in thrombin Quick II.     

Inversion and circularization of the varicella-zoster virus genome.

The genome of varicella-zoster virus (VZV) is a linear, double-stranded molecule of DNA composed of a long (L) region covalently linked to a short (S) region. The S region is capable of inverting relative to a fixed orientation of the L region, giving rise to two equimolar populations. We have investigated other forms of the VZV genome which are present in infected cells and packaged into nucleocapsids. That a small proportion of nucleocapsid DNA molecules also possess inverted L regions has been verified by the identification of submolar restriction fragments corresponding to novel joints and novel ends generated by such an inversion. The presence of circular molecules has been investigated by agarose gel electrophoresis. Bands corresponding to circular forms were present in small amounts in both VZV-infected cell DNA and nucleocapsid DNA. Southern blot analysis verified that these bands contained VZV sequences. We therefore conclude that the VZV genome may occasionally contain an inverted L region or exist in a circular configuration.     

Morphogenesis of the tail of bacteriophage lambda. III. Morphogenetic pathway.

Precursors of the tail of bacteriophage λ have been detected by measurements of in vitro complementation activities and serum blocking activity in sucrose gradients of lysates defective in tail genes.On the basis of these measurements, a pathway for the assembly of the λ tail is proposed:The morphogenesis of the λ tail starts from the tail fiber (product of gene J) located at the distal end of the tail, and proceeds to the proximal end. Gene J by itself produces a 15 S structure with serum blocking activity but without any detectable in vitro complementation activity, which may be the least advanced precursor of the λ tail or an abortive product. Functions of genes J, I, K, L are required for the formation of a 15 S precursor that has in vitro complementation activities with J⁻, I⁻, K⁻ and L⁻ lysates and serum blocking activity. If the products of genes G and H act on the latter 15 S precursor, a 25 S precursor is made, but this precursor seems either to be in equilibrium with the 15 S precursor or to degrade easily into the 15 S precursor. Gene M has a function of stabilizing the 25 S precursor. After the action of gene M product, the 25 S precursor is ready to serve as a nucleus on which the product of gene V (the major tail protein) assembles. However, gene U product is also necessary at this step for the correct assembly of the major tail protein on the 25 S precursor. Without gene U product the assembly of the major tail protein does not stop at the correct length and a polytail is formed instead of a morphologically normal tail. Finally, gene Z product acts on the morphologically normal tail and makes it a biologically active tail. Without the action of gene Z product, the defective tail binds to a head and forms a phage-like particle which is only very weakly infectious. (The position of gene T in the pathway is not determined, because no sus mutant is available in gene T.)Two abnormal, less efficient pathways are also present in vitro. (1) If gene U product acts on a polytail in an U⁻ lysate, the polytail finally binds to a head and forms a phage particle with an extra long tail which is infectious to a small extent. (2) The function of gene K seems to be bypassed to some extent: K⁻ lysates accumulate particles which sediment as fast as normal phage and which are complemented by other tail⁻ lysates.     

Long‐range molecular dynamics show that inactive forms of Protein Kinase A are more dynamic than active forms

Many protein kinases are characterized by at least two structural forms corresponding to the highest level of activity (active) and low or no activity, (inactive). Further, protein dynamics is an important consideration in understanding the molecular and mechanistic basis of enzyme function. In this work, we use protein kinase A (PKA) as the model system and perform microsecond range molecular dynamics (MD) simulations on six variants which differ from one another in terms of active and inactive form, with or without bound ligands, C‐terminal tail and phosphorylation at the activation loop. We find that the root mean square fluctuations in the MD simulations are generally higher for the inactive forms than the active forms. This difference is statistically significant. The higher dynamics of inactive states has significant contributions from ATP binding loop, catalytic loop, and αG helix. Simulations with and without C‐terminal tail show this differential dynamics as well, with lower dynamics both in the active and inactive forms if C‐terminal tail is present. Similarly, the dynamics associated with the inactive form is higher irrespective of the phosphorylation status of Thr 197. A relatively stable stature of active kinases may be better suited for binding of substrates and detachment of the product. Also, phosphoryl group transfer from ATP to the phosphosite on the substrate requires precise transient coordination of chemical entities from three different molecules, which may be facilitated by the higher stability of the active state.