PubMed Health⌕ Search

Biomedical subjects

J Eichler

Publications and source records attributed to J Eichler.

At least 19 recordsLinked to original sources
PubMed1998

Endogenous SecA catalyzes preprotein translocation at SecYEG.

SecA is found in the cytosol and bound to the plasma membrane of Escherichia coli. Binding occurs either with high affinity at SecYEG or with low affinity to lipid. Domains of 65 and 30 kDa of SecYEG-bound SecA insert into the membrane upon interaction with preprotein and ATP. Azide blocks preprotein translocation, in vivo and in vitro, through interacting with SecA and preventing SecA deinsertion. This provides a measure of the translocation relevance of each form of SecA membrane association. We now report that azide acts exclusively on SecA that is cycling at SecYEG and has no effect on SecA lipid associations. SecA molecules recovered with sucrose gradient-purified inner membrane vesicles ("endogenous" SecA) support translocation at the same rate as "added" SecA molecules bound at SecYEG. Both endogenous and added SecA yield the same proteolytic fragments, which are distinct from those obtained from SecA once it has inserted into membranes at SecYEG or from SecA at lipidic sites. Endogenous and added SecA differ, however, in their resistance to urea extraction. The translocation supported by either endogenous or added SecA is blocked by azide or by antibody to SecY. We conclude that SecA functions in preprotein translocation only through cycling at SecYEG.

Adenosine Triphosphatases↗
PubMed1998

125I-labeled fasciculin 2: a new tool for quantitation of acetylcholinesterase densities at synaptic sites by EM-autoradiography.

Radio-iodinated fasciculin 2 (Fas2), a polypeptide anticholinesterase toxin from Mamba venom, was used as a new probe for localizing and quantifying acetylcholinesterase (AChE) at mouse neuromuscular junctions (NMJs) by quantitative electron microscope autoradiography. We demonstrate that 125I-Fas2 binds very specifically to the NMJs of mouse sternomastoid muscles, with very little binding to other regions in the muscles. Junctional AChE-site densities obtained from the autoradiograms were similar to those previously obtained for the same muscles using 3H-DFP. The use of 125I-Fas2 with EM-autoradiography is simpler and provides higher resolution and sensitivity, as well as considerably lower non-specific binding than previously attainable with 3H-DFP. The advantages and limitations of this procedure are discussed.

Acetylcholinesterase↗
PubMed1998

The SecA subunit of Escherichia coli preprotein translocase is exposed to the periplasm.

SecA undergoes conformational changes during translocation, inserting domains into and across the membrane or enhancing the protease resistance of these domains. We now show that some SecA bound at SecYEG is accessible from the periplasm to a membrane-impermeant probe in cells with a permeabilized outer membrane but an intact plasma membrane.

Adenosine Triphosphatases↗
PubMed1997

Both an N-terminal 65-kDa domain and a C-terminal 30-kDa domain of SecA cycle into the membrane at SecYEG during translocation.

SecA, a 102-kDa hydrophilic protein, couples the energy of ATP binding to the translocation of preprotein across the bacterial inner membrane. SecA function and topology were studied with metabolically labeled [35S]SecA and with inner membrane vesicles from cells that overexpressed SecYEGDFyajC, the integral domain of preprotein translocase. During translocation in the presence of ATP and preprotein, a 65-kDa N-terminal domain of SecA is protected from proteolytic digestion through insertion into the membrane, as previously reported for a 30-kDa C-terminal domain [Economou, A. & Wickner, W. (1994) Cell 78, 835-843]. Insertion of both domains occurs at saturable SecYEGDFyajC sites and is rapidly followed by deinsertion. SecA also associates nonsaturably and unproductively with lipid. In the presence of ATP, yet without involvement of preprotein or SecYEG, lipid-bound SecA forms domains that are protease-resistant and that remain so even upon subsequent membrane disruption. Unlike the [35S]SecA that inserts into the membrane at SecYEGDFyajC as it promotes preprotein translocation, lipid-associated [35S]SecA does not chase from its protease-resistant state upon the addition of excess SecA. The finding that two domains of SecA (which together represent most regions of the polypeptide chain) cycle into the membrane during preprotein translocation, as well as the distinction between the membrane association of SecA at translocation sites of SecYEGDFyajC and at nonproductive lipid sites, are fundamental to the study of the role of SecA in preprotein movement.

Adenosine Triphosphatases↗
PubMed1997

The protease-protected 30 kDa domain of SecA is largely inaccessible to the membrane lipid phase.

SecA binds to the inner membrane of Escherichia coli through low affinity lipid interactions or with high affinity at SecYEG, the integral domain of preprotein translocase. Upon addition of preprotein and nucleotide, a 30 kDa domain of SecYEG-bound SecA is protected from proteolysis via membrane insertion. Such protection could result from some combination of insertion into the lipid phase, into a proteinaceous environment or across the membrane. To assess the exposure of SecYEG-bound SecA to membrane lipids, a radiolabeled, photoactivatable and lipid-partitioning crosslinker, 3-trifluoromethyl-3-(m[125I]iodophenyl) diazirine benzoic acid ester, was incorporated into inner membrane vesicles. The 30 kDa domain of SecYEG-bound SecA, inserted into the membrane in response to translocation ligands, is 18-fold less labeled than SecY, which is labeled effectively. In contrast, incorporation of the purified 30 kDa SecA fragment into crosslinker-containing detergent micelles or addition of detergent to crosslinker-containing membranes bearing the protease-protected SecA domain readily allows for labeling of this domain. We propose that the protease-inaccessible 30 kDa SecA domain is shielded from the fatty acyl membrane phase by membrane-spanning SecYEG helices and/or is largely exposed to the periplasm.

Adenosine Triphosphatases↗
PubMed1996

Novel alpha-glucosidase inhibitors identified using multiple cyclic peptide combinatorial libraries.

Twenty-six cyclic synthetic peptide combinatorial libraries (disulfides and lactams) of varying size and composition, representing 6.8 x 10(3) to 4.7 x 10(7) individual peptides, were synthesized along with their respective linear analogs. One of the hexapeptide lactam libraries (cyclo[xXxXxN]) was found to have significant alpha-glucosidase inhibitory activity. This library was carried through an iterative process of synthesis and screening, during which all of the five mixture positions (x and X) were successively defined. As the result of this process, potent and selective alpha-glucosidase inhibitors were identified.

Amino Acid Sequence↗
PubMed1996

Libraries from libraries: generation and comparison of screening profiles.

A positional scanning tetrapeptide library was chemically modified through alkylation and/or reduction of the amide bonds, thus generating three new combinatorial libraries with physico-chemical properties very different from the parent peptide library ('libraries from libraries'). Specific results were obtained with each of these libraries upon screening in kappa-opioid receptor binding and microdilution antimicrobial assays, illustrating the potential of the 'libraries from libraries' concept for the efficient generation of a variety of chemically diverse combinatorial libraries.

Animals↗
PubMed1995

SecYEG and SecA are the stoichiometric components of preprotein translocase.

The transport of large preproteins across the Escherichia coli plasma membrane is catalyzed by preprotein translocase, comprised of the peripherally bound SecA subunit and an integrally bound heterotrimeric domain consisting of the SecY, SecE, and SecG subunits. We have now placed the secY, secE, and secG genes under the control of an arabinose-inducible promoter on a multicopy plasmid. Upon induction, all three of the proteins are strongly overexpressed and recovered in the plasma membrane fraction. These membranes show a strong enhancement of 1) translocation ATPase activity, 2) preprotein translocation, 3) capacity for SecA binding, and 4) formation of the membrane-inserted form of SecA. These data establish that SecY, SecE, and SecG constitute the integral membrane domain of preprotein translocase.

Adenosine Triphosphatases↗
PubMed1995

The activity of an endoplasmic reticulum-localized pool of acetylcholinesterase is modulated by heat shock.

Primary cultures prepared from embryonic chick pectoral muscle were subjected to heat shock, and the effect on acetylcholinesterase activity in the cultures was examined. A rapid recovery in enzyme activity was observed soon after an initial heat shock-induced drop and was shown to be independent of de novo synthesis of protein, since it could occur in the presence of an inhibitor of protein synthesis. Lectin binding and sucrose gradient centrifugation studies suggested that molecular monomers and dimers found in the endoplasmic reticulum are involved in the observed recovery of acetylcholinesterase activity. Enhanced activation of a pre-existing pool of inactive enzyme was clearly not the main agent of the recovery in enzymic activity. Recovery relied principally on restoration of the activity of previously active, heat-denatured acetylcholinesterase molecules found in the endoplasmic reticulum. Possible agents involved in the recovery of enzymatic activity might be heat shock proteins acting as molecular chaperones.

Acetylcholinesterase↗
PubMed1995

A review of the utility of soluble peptide combinatorial libraries.

This paper reviews the preparation and use of soluble synthetic combinatorial libraries (SCLs) made up of millions of peptide and nonpeptide sequences for the identification of highly active individual compounds. First presented in 1991, SCLs have been prepared in a number of different lengths and formats, and are composed entirely of L-, D-, and unnatural amino acids. Also, existing peptide libraries have been chemically transformed to yield large diversities of nonpeptidic compounds. This review encompasses the published work from this laboratory using SCLs for the identification of antigenic sequences recognized by monoclonal antibodies, novel peptide agonists and antagonists to opioid receptors, new trypsin inhibitors, novel antibacterials, and compounds that inhibit melittin's hemolytic activity. SCLs offer a fundamental, practical advance in the study of interactions between peptide and nonpeptide sequences and their biochemical or pharmacological targets.

Amino Acid Sequence↗
PubMed1995

Generation and utilization of synthetic combinatorial libraries.

The use of combinatorial chemistry is fundamentally changing the pace and scope of basic research and drug discovery. Since the introduction of synthetic peptide libraries several years ago, combinatorial chemistry has proven to be a powerful tool for the generation of immense molecular diversities of peptides, peptidomimetics and new organic compounds. This article briefly reviews methods for the generation and application of combinatorial libraries, with particular emphasis on soluble synthetic combinatorial libraries. The utility of these molecular diversities for basic research and drug discovery has been demonstrated through the identification of numerous highly active compounds such as antigenic peptides, receptor ligands, antimicrobial compounds and enzyme inhibitors.

Amino Acid Sequence↗
PubMed1994

A "molten globule" of Torpedo acetylcholinesterase undergoes thiol-disulfide exchange.

Torpedo acetylcholinesterase is a disulfide-linked homodimer containing three intramolecular disulfide bonds, as well as a single free thiol on Cys-231. We report that in a "molten globule" state, produced by 1.5 M guanidine hydrochloride, this enzyme undergoes rapid intramolecular thiol-disulfide exchange, in the absence of reducing agents, resulting in the production of novel species. Most strikingly, this results in appearance of enzyme monomers. Chemical modification of the free thiol group prevents these changes. Unfolded acetylcholinesterase, namely in 5 M guanidine hydrochloride, also undergoes intramolecular thiol-disulfide exchange, including production of enzyme monomers, but at a much lower rate. Our data show that the molten globule state, in contrast to the native and unfolded states, is both compact and flexible, thus being especially amenable to thiol-disulfide exchange.

Acetylcholinesterase↗
PubMed1994

Two-site immunoradiometric assay of chicken acetylcholinesterase: active and inactive molecular forms in brain and muscle.

Several monoclonal antibodies were raised against chicken acetylcholinesterase (AChE; EC 3.1.1.7). Some of these antibodies react with quail AChE but not with AChEs from nonavian vertebrates or invertebrates and not with butyrylcholinesterase. They may be classified in several mutually compatible groups, i.e., that can bind simultaneously to the monomeric form of AChE. Most antibodies recognize a peptidic domain that does not exist in mammalian AChE and that may be digested by trypsin without loss of activity or dissociation of quaternary structure. The only exception is the antibody C-131, which is conformation dependent and preferentially recognizes active AChE. We have set up two-site immunoradiometric assays, using an immobilized capture antibody, C-6 or C-131, and a radiolabeled antibody, 125I-C-54. The C-6/C-54 assay quantifies the totality of inactive and active AChE subunits: It detects 10(-3) Ellman unit (approximately 40 pg of protein) and yields a linear response up to at least 25 10(-3) Ellman units. An analysis of gradient fractions, using C-6/C-54 and C-131/C-54 assays as well as activity determination, shows that the A12 and G4 forms are exclusively composed of active subunits, whereas inactive molecules cosediment with the active G2 and G1 forms. Both active and inactive G2 and G1 forms are amphiphilic, as indicated by the influence of detergents on their sedimentation coefficients and Stokes radii. In brain, the proportion of inactive forms decreases from 40% at embryonic day 11 (E11) to 20% at birth [day 1 (D1)]. In muscle, we observed no inactive AChE at E11 and a small proportion of inactive G1 at D1.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1994

Differential effects of "peripheral" site ligands on Torpedo and chicken acetylcholinesterase.

Comparison of the effect of three 'peripheral' site ligands, propidium, d-tubocurarine, and gallamine, on acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) of Torpedo and chicken shows that all three are substantially more effective inhibitors of the Torpedo enzyme than of the chicken enzyme. In contrast, edrophonium, which is directed to the "anionic" subsite of the active site, inhibits the chicken and Torpedo enzymes equally effectively. Two bisquaternary ligands, decamethonium and 1,5-bis(4-allydimethylammoniumphenyl)pentan-3-one dibromide, which are believed to bridge the anionic subsite of the active site and the "peripheral" anionic site, are much weaker inhibitors of the chicken enzyme than of Torpedo acetylcholinesterase, whereas the shorter bisquaternary ligand hexamethonium inhibits the two enzymes similarly. The concentration dependence of activity towards the natural substrate acetylcholine is almost identical for the two enzymes, whereas substrate inhibition of chicken acetylcholinesterase is somewhat weaker than that of the Torpedo enzyme. The experimental data can be rationalized on the basis of the three-dimensional structure of the Torpedo enzyme and alignment of the chicken and Torpedo sequences; it is suggested that the absence, in the chicken enzyme, of two aromatic residues, Tyr-70 and Trp-279, that contribute to the peripheral site of Torpedo acetylcholinesterase is responsible for the differential effects of peripheral site ligands on the two enzymes.

Acetylcholinesterase↗
PubMed1993

Identification of substrate-analog trypsin inhibitors through the screening of synthetic peptide combinatorial libraries.

Synthetic peptide combinatorial libraries (SPCLs), which are made up in total of tens to hundreds of millions of peptides, enable the systematic screening for biologically active peptides in virtually all in vitro and even in vivo assay systems. In the current study, the applicability of this method to the identification of peptide enzyme inhibitors was investigated using trypsin as the model enzyme. A specifically designed library of hexapeptide mixtures was synthesized on cotton carriers and screened. The synthetic approach, using cotton as a solid support, was modified so that the deprotected peptides remained attached to the cotton carrier until they were released into solution directly prior to being assayed. Following an iterative process of synthesis and screening, in which all of the positions of the sequence were successively defined, a number of individual hexapeptides with trypsin inhibitory activity were identified. The most active, defined individual peptide sequence was then reincorporated into a new library, now made up of dodecapeptide mixtures. The iterative screening and synthesis of this library led to a dodecapeptide with improved inhibitory activity when compared to the hexapeptide from which it was derived.

Amino Acid Sequence↗