CcdB at pH 4 Forms a Partially Unfolded State with a Dry Core.

pH is an important factor that affects the protein structure, stability, and activity. Here, we probe the nature of the low-pH structural form of the homodimeric CcdB (controller of cell death B) protein. Characterization of CcdB protein at pH 4 and 300 K using circular dichroism spectroscopy, 8-anilino-1-naphthalene-sulphonate binding, and Trp solvation studies suggests that it forms a partially unfolded state with a dry core at equilibrium under these conditions.

Open and Closed Form of Maltose Binding Protein in Its Native and Molten Globule State As Studied by Electron Paramagnetic Resonance Spectroscopy.

An intensively investigated intermediate state of protein folding is the molten globule (MG) state, which contains secondary but hardly any tertiary structure. In previous work, we have determined the distances between interacting spins within maltose binding protein (MBP) in its native state using continuous wave and double electron-electron resonance (DEER) electron paramagnetic resonance (EPR) spectroscopy. Seven double mutants had been employed to investigate the structure within the two domains of MBP.

Methotrexate-gelonin conjugate - an inhibitor of MCF-7 cells expressing the dihydrofolate receptor.

An immunotoxin composed of gelonin, a basic ribosome-inactivating protein, type I of 30 kDa, isolated from the seeds of the Indian plant Gelonium multiflorum and methotrexate (MTX) has been studied as a potential tool of gelonin delivery into the cytoplasm of MTX-responsive cells. On the average, about five molecules of methotrexate were chemically coupled to gelonin via an N-hydroxysuccinimide ester of the drug. The MTX-gelonin conjugate was able to reduce the viability of MCF-7 cells in a dose-dependent manner with ID50 of 10 nm, whereas gelonin or MTX alone showed none or very little effects.

Chemically and biologically synthesized CPP-modified gelonin for enhanced anti-tumor activity.

The ineffectiveness of small molecule drugs against cancer has generated significant interest in more potent macromolecular agents. Gelonin, a plant-derived toxin that inhibits protein translation, has attracted much attention in this regard. Due to its inability to internalize into cells, however, gelonin exerts only limited tumoricidal effect.

Conformational changes of the chaperone SecB upon binding to a model substrate--bovine pancreatic trypsin inhibitor (BPTI).

SecB is a homotetrameric cytosolic chaperone that forms part of the protein translocation machinery in E. coli. Due to SecB, nascent polypeptides are maintained in an unfolded translocation-competent state devoid of tertiary structure and thus are guided to the translocon.

Novel immunotoxin: a fusion protein consisting of gelonin and an acetylcholine receptor fragment as a potential immunotherapeutic agent for the treatment of Myasthenia gravis.

In continuation of our attempts for antigen-specific suppression of the immune system [I.L. Urbatsch, R.

On the contentious sequence and glycosylation motif of the ribosome inactivating plant protein gelonin.

The amino acid sequence and the glycosylation motif of the ribosome inactivating protein (RIP) gelonin are identified by Fourier transform ion cyclotron resonance mass spectrometry. Intact gelonin as isolated from the seeds of Gelonium multiflorum consists of at least three different post-translational modified forms: analysis of gelonin peptides as obtained by proteolytic digestion is consistent with the amino acid sequence published by Nolan et al. High resolution mass determination established a glycosylation pattern of GlcNAc2Man(3-5)Xyl.

Soluble expression and affinity purification of functional domain of human acetylcholine receptor alpha-subunit by the modulation of maltose binding protein.

An open reading frame of the alpha-subunit 1-205 residues (alpha205) of human acetylcholine receptor (AchR) was amplified by PCR with pUC-AChR alpha205 as the template and inserted into vector pMAL-c2X. The constructed pMAR alpha205 was transferred into E. coli BL21 which were then grown in LB medium.

Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin.

An open reading frame of gelonin (Gel), one of ribosome inactivating proteins, was inserted into the vector pBSL-C which contains the coding region of chitin binding domain (CBD)-intein, resulting in the fusion expression of CBD-intein-Gel in Escherichia coli BL21 (DE3) by the induction of IPTG. The fusion product formed an aggregate of the misfolded protein, commonly referred to as inclusion bodies (IBs). The IBs were denatured and then refolded by step-wise dialysis.

High-dose intravenous vitamin C is not associated with an increase of pro-oxidative biomarkers.

Objective: High-dose vitamin C therapy might mediate beneficial clinical effects by counteracting reactive oxygen species. However, concerns are raised whether this approach might provoke diametrical (ie pro-oxidative) effects. The objective was to determine ascorbyl free radical (AFR) concentrations and potential variables of pro-oxidative damage.

Association of alpha-subunits with nucleotide-modified beta-subunits induces asymmetry in the catalytic sites of the F1-ATPase alpha3beta3-hexamer.

The photoaffinity spin-labeled ATP analog, 2-N3-SL-adenosine triphosphate (ATP), was used to covalently modify isolated beta-subunits from F1-ATPase of the thermophilic bacterium PS3. Approximately 1.2 mol of the nucleotide analog bound to the isolated subunit in the dark.

Vitamin E supplementation does not increase the vitamin C radical concentration at rest and after exhaustive exercise in healthy male subjects.

Background: Extensive exercise may promote the formation of reactive oxygen species and subsequently contribute to tissue damage. A compound which can protect cells and tissues is vitamin E. The vitamin E radical, formed during the radical scavenging process, can be reduced by vitamin C resulting in a higher level of the vitamin C radical (semidehydroascorbate free radical).

Lipid peroxidation-independent mechanisms of vitamin e-mediated protection against cyclosporine a-induced hepatocellular toxicity and apoptosis.

Cyclosporine A (CsA) produces oxidative stress and apoptosis in rat hepatocytes, but it is not known whether membrane lipid peroxidation plays a role in CsA toxicity. The objective of the present study was to determine whether iron-catalyzed hydroxyl or membrane alkoxyl radical formation is causally involved in the prooxidative cell injury and apoptosis. As previously reported, cultured rat hepatocytes exposed to CsA exhibited concentration-dependent signs of apoptotic cell injury, including chromatin condensation and fragmentation, increased caspase-3 activity, and release of cytosolic lactate dehydrogenase.

Role of antioxidants in the O-hydroxyethyl-D-(Ser)8-cyclosporine A (SDZ IMM125)-induced apoptosis in rat hepatocytes.

The mechanisms underlying the apoptotic activity of the immunosuppressive drug cyclosporine A and its O-hydroxyethyl-D-(Ser)(8)-derivative SDZ IMM125 in rat hepatocytes are not yet fully understood. It was the purpose of the present study to investigate the role of anti- and pro-oxidants and of caspase-3 and intracellular Ca(2+) in SDZ IMM125-induced apoptosis in rat hepatocytes. SDZ IMM125 induced an increase in chromatin condensation and fragmentation, and the activation of caspase-3.

Electron spin resonance and fluorescence studies of the bound-state conformation of a model protein substrate to the chaperone SecB.

SecB is a homotetrameric, cytosolic chaperone that forms part of the protein translocation machinery in Escherichia coli. We have investigated the bound-state conformation of a model protein substrate of SecB, bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin-labeling and pyrene fluorescence methods. BPTI is a 58-residue protein and contains three disulfide groups between residues 5 and 55, 14 and 38, as well as 30 and 51.

Nucleotide-binding sites in the functional unit of sarcoplasmic reticulum Ca2+-ATPase as studied by photoaffinity spin-labeled 2-N3-SL-ATP.

2-N3-SL-ATP [2-azido-2',3'-O-(1-oxyl-2,2,5,5-tetramethyl-3-carbonyl-pyrroline) adenosine triphosphate], a photoaffinity spin-labeled derivative of ATP with a nitroxide moiety attached to the ribose ring and an azido group attached to C2 of the adenine ring, was used to study the nucleotide-binding site stoichiometry of sarcoplasmic reticulum (SR) Ca2+-ATPase. The label was shown to bind at the catalytic site of the enzyme, even though the rate of hydrolysis was poor. A maximal binding ratio of 1 mol/mol of ATPase was found.

Protective effects of vitamins C and E on the number of micronuclei in lymphocytes in smokers and their role in ascorbate free radical formation in plasma.

Cigarette smoke is widely believed to increase free radical concentrations causing subsequent oxidative processes that lead to DNA damage and hence, to several diseases including lung cancer and atherosclerosis. Vitamin C is a reducing agent that can terminate free-radical-driven oxidation by being converted to a resonance-stabilized free radical. To investigate whether short-term supplementation with the antioxidants vitamin C and E decreases free-radical-driven oxidation and thus decreases DNA damage in smokers, we determined the frequency of micronuclei in lymphocytes in 24 subjects and monitored the electron paramagnetic resonance signal of ascorbate free radical formation in plasma.

Phosphatidylcholine activation of human heart (R)-3-hydroxybutyrate dehydrogenase mutants lacking active center sulfhydryls: site-directed mutagenesis of a new recombinant fusion protein.

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. A plasmid has been constructed to express human heart (HH) BDH in Escherichia coli as a hexahistidine-tagged fusion protein (HH-Histag-BDH). A rapid two-step affinity purification yields active HH-Histag-BDH (and six mutants) with high specific activity ( approximately 130 micromol of NAD(+) reduced.

A thermodynamic coupling mechanism for the disaggregation of a model peptide substrate by chaperone secB.

Molecular chaperones prevent protein aggregation in vivo and in vitro. In a few cases, multichaperone systems are capable of dissociating aggregated state(s) of substrate proteins, although little is known of the mechanism of the process. SecB is a cytosolic chaperone, which forms part of the precursor protein translocation machinery in Escherichia coli.

Induction of apoptosis by the O-hydroxyethyl-D(Ser)(8)-cyclosporine A derivative SDZ IMM 125 in rat hepatocytes.

The immunosuppressive cyclosporine A derivative, O-hydroxyethyl-D(Ser)(8)-cyclosporine (SDZ IMM 125), was examined for its ability to induce apoptosis in rat hepatocytes cultured for 4 or 20 h. Four hours after SDZ IMM 125 treatment, chromatin condensation and fragmentation, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeled and Annexin V-positive cells increased dose dependently without any observable lactate dehydrogenase leakage. The activity of the cysteine protease, caspase-3, was increased, but not that of caspase-1 and -6.

Mechanisms of cyclosporine A-induced apoptosis in rat hepatocyte primary cultures.

In rat hepatocytes and isolated liver mitochondrial fractions, Cyclosporine A (CsA) is often used as a specific inhibitor of mitochondrial Ca(2+) release and as a specific blocker of mitochondrial membrane potential and permeability transition (MPT), which are all processes involved in the inhibition of apoptosis. However, neither inhibition nor induction of apoptosis by CsA has yet been described in the rat hepatocyte primary culture during incubation for 4 and 20 h. It was the purpose of the present study to examine by means of morphological and biochemical criteria the effects of CsA on apoptosis and to characterize the underlying mechanisms.

Synthesis and application of novel bifunctional spin labels.

The synthesis of new bifunctional spin-labeled cross-linking reagents is described. Covalent attachment to papain was achieved via a thiol-specific thiosulfonate residue and, for the second anchor point, via a nonspecific photoreactive azido function. The thiosulfonate formed a reversible disulfide linkage, which could be cleaved again reductively by dithiothreitol.

EPR kinetic studies of the LDL oxidation process driven by free radicals.

Oxidation is widely assumed to play a causal role in the pathogenesis of atherosclerosis. Oxidative modification of low density lipoprotein (LDL) can be followed by analyzing the lag phase of the conjugated diene formation at 234 nm in LDL exposed to Cu2+. This procedure is restricted to isolated LDL fractions.

Hepatocellular effects of cyclosporine A and its derivative SDZ IMM 125 in vitro.

The novel immunosuppressive drug O-hydroxyethyl-D(Ser)8-cyclosporine (SDZ IMM 125) and cyclosporine A (CyA) were compared in different in vitro models with respect to hepatocellular side effects. SDZ IMM 125 was less lipophilic than CyA and also decreased liposomal membrane anisotropy less. Furthermore, SDZ IMM 125 increased Na+ and Ca++ permeability across the liposomal membranes significantly more than CyA.