A novel strategy for the identification of protein–DNA contacts by photocrosslinking and mass spectrometry

Photochemical crosslinking is a method for studying the molecular details of protein–nucleic acid interactions. In this study, we describe a novel strategy to localize crosslinked amino acid residues that combines laser-induced photocrosslinking, proteolytic digestion, Fe³⁺-IMAC (immobilized metal affinity chromatography) purification of peptide–oligodeoxynucleotide heteroconjugates and hydrolysis of oligodeoxynucleotides by hydrogen fluoride (HF), with efficient matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The new method is illustrated by the identification of the DNA-binding site of the restriction endonuclease MboI. Photoactivatable 5-iododeoxyuridine was incorporated into a single site within the DNA recognition sequence (GATC) of MboI. Ultraviolet irradiation of the protein–DNA complex with a helium/cadmium laser at 325 nm resulted in 15% crosslinking yield. Proteolytic digestion with different proteases produced various peptide–oligodeoxynucleotide adducts that were purified together with free oligodeoxynucleotide by Fe³⁺-IMAC. A combination of MS analysis of the peptide–nucleosides obtained after hydrolysis by HF and their fragmentation by MS/MS revealed that Lys²⁰⁹ of MboI was crosslinked to the MboI recognition site at the position of the adenine, demonstrating that the region around Lys²⁰⁹ is involved in specific binding of MboI to its DNA substrate. This method is suitable for the fast identification of the site of contact between proteins and nucleic acids starting from picomole quantities of crosslinked complexes.     

In vitro and in vivo irreversible plasma protein binding of beclobric acid enantiomers

Acyl glucuronides are known to be labile conjugates, which undergo hydrolysis and bind irreversibly to proteins. The lipid-regulating agent (±)-beclobrate is immediately converted to the free acid after oral administration. Further metabolism leads to formation of the corresponding diastereomeric acyl glucuronides. Beclobric acid glucuronides were quantified by indirect measurement with an HPLC method based on chiral fluorescent derivatization of the carboxylic acid and subsequent normal-phase chromatography. The renal clearance of unchanged drug is low, with almost all drug excreted into urine as glucuronic acid conjugates. Beclobric acid glucuronide is also detectable in plasma. In vitro degradation studies with beclobric acid glucuronide (at a concentration of 5 μM in 150 mM phosphate buffer pH 7.4) exhibited a minor tendency for acyl migration and hydrolysis, i.e., a higher stability than has been observed for the acyl glucuronides of most other drugs. The in vitro degradation half-lives of the two beclobric acid β-1-O-acyl glucuronides were 22.7 and 25.7 h. After incubation with pooled plasma and human serum albumin in buffer pH 7.4 irreversible binding was measured in vitro. No significant difference between the two enantiomers was detected with respect to the magnitude of in vitro irreversible binding. In 3 healthy male volunteers the extent of irreversible binding of both beclobric acid enantiomers to plasma proteins was investigated after single and multiple oral doses of racemic beclobrate (100 mg once daily). Irreversible binding of both enantiomers was observed in all volunteers. The adduct densities for (?)- and (+)-beclobric acid after single 100 mg beclobrate doses were 0.147 × 10^?4 and 0.177 × 10^?4 mol/mol protein. Multipie dosing increased irreversible binding 3- to 4-fold. © 1993 Wiley-Liss, Inc.     

Activation of cell wall-associated peroxidase isoenzymes in pea epicotyls by a xyloglucan-derived nonasaccharide

The cell wall-derived xyloglucan nonasaccharide XXFG was foundto increase the extractable activity of distinct cationic cellwall-associated peroxidase isozyme groups isolated from etiolatedpea epicotyls. Peroxidase activation occurred in the first 10h of incubation with the nonasaccharide in the pea epicotylbioassay. At the same time varying concentrations of XXFG causedgrowth inhibition up to 35%. Neither the increase of peroxidaseactivity nor the growth inhibition was restricted to a certainXXFG concentration. The increase in peroxidase activity wasnot just an oligosaccharide effect in general. The correspondingheptasaccharide XXXG neither inhibited growth nor increasedperoxidase activity. The isozymes extracted from pea epicotylswere additionally separated by cation-exchange chromatographyand submitted to isoelectric focusing. With one exception, allof the ionically-bound, cell wall-associated peroxidases presentin pea epicotyls were cationic or slightly anionic. It is proposedthat the growth inhibition caused by XXFG is at least in partthe result of peroxidasecatalysed cell wall tightening inducedby the nonasaccharide. Key words: XXFG, growth inhibition, cell wall-associated peroxidases, cell wall tightening, pea epicotyls     

Effect of chloroquine and O,O'-bis(diethylaminoethyl)hexestrol on acidic phospholipid membranes

The amphiphilic drugs chloroquine and O,O'-bis(diethylaminoethyl)hexestrol are able to form complexes with the acidic-phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol. The dissociation constants of the complexes with chloroquine are independent of pH in the range investigated here (4--7) as well as of temperature (4 degrees C--40 degrees C). The phase transition temperature of phospholipid is markedly reduced by both drugs, the effect is reversible by addition of Ca2.     

Formation of optically active ether lipids from race-mic-1-O-tetradecylglycerol in plant cell cultures

Photomixotrophic rape cells in culture specifically incorporate 1-O-tetradecyl-sn-glycerol from a racemic mixture into complex alkyl glycerolipids. Thus, both neutral and ionic 1-O- alkyl-2-O-acyl-sn-glycerolipids with defined alkyl moieties can be prepared from racemic mixtures of alkylglycerols.     

Inducing activity of subcellular fractions from amphibian embryos

Summary The homogenate from unfertilized eggs, gastrulae, neurulae and hatched embryos ofXenopus laevis was fractionated by differential centrifugation and subsequent repeated centrifugation on discontinuous sucrose gradients. A high archencephalic-neural inducing activity was found in RNP particles, which were released from the high-speed (microsomal) sediment by treatment with EDTA, and in a fraction of heterogeneous small vesicles. The highest archencephalic inducing activity was observed in RNP particles from unfertilized eggs and from gastrulae. RNP particles isolated from hatched embryos had a lower inducing activity. The neuralizing factor can be extracted from the small vesicles with pyrophosphate buffer at pH 8.6, but it is not solubilized with a non-ionic detergent (Triton X 100). The high-speed supernatant from the gastrula homogenate contains soluble neuralizing factor, whereas the supernatant from egg homogenate has a low inducing activity. The plasma membrane fraction (isolated from gastrulae) also has only a low inducing activity. The possible significance of the subcellular distribution of neuralizing factors for the transmission of neuralizing inducer from the mesoderm to competent gastrula ectoderm and the processing of signals which are generated on the plasma membrane of induced cells is discussed.     

Lysoplasmalogenase–A Microsomal Enzyme from Rat Brain

Abstract: An enzymic activity of rat brain that liberates radioactive free aldehydes from 1-[1-¹⁴C]alk-1'-enyl- sn -glycero-3-phosphoethanolamine (lysoplasmalogen) is described. It was present mainly in microsomal fractions (crude) of brains of rats of different ages. The highest specific enzyme activity was found in 21-day-old animals. The formation of free aldehyde was dependent on the amount of enzyme protein as well as the amount of substrate added, and was linear to the incubation time up to 60 min. The pH optimum was between 7.1 and 7.3. Bivalent cations (Mg²⁺, Ca²⁺) and detergents inhibited the reaction. However, the same cell fractions as well as extracts of acetone-dried powder of brain from young or old rats possessed no enzyme activity for liberating the aldehyde from the acylated substrates: 1-[1-¹⁴C]alk-1'-enyl-2-acyl- sn -glycero-3-phosphoethanolamine (plasmalogen) or plasmalogen of ox corpus callosum.     

Capillary gas chromatography of methylhexitol acetates obtalned upon methylation of N-glycosidically linked glycoprotein oligosaccharides

The 30 O-methylated hexitol and 2-deoxy-2-(N-methyl)acetamidohexitol acetates which commonly may be obtained during methylation analysis of N-glycosidically linked glycoprotein oligosaccharides or their biosynthetic precursors, were subjected to chromatography through glass capillary columns, wall-coated with either Silar 9CP, Dexsil 410, SE-30, or OV 101. All 22 methylhexitol acetates (e.g., 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol and -mannitol) were optimally resolved on the (most polar) Silar column, whereas the 8 aminohexitol derivatives (as well as the corresponding unmethylated hexitol and aminohexitol acetates) were best separated on either Dexsil 410 or OV 101.     

Influence of chloroquine treatment on enzymes and phospholipids from rat liver cell fractions

Rats, treated for 12 days with chloroquine show a threefold increase of arylsulfatase activity in the mitochondrial-lysosomal mixed fraction, whereas the succinate: cytochrome c reductase activity is decreased to about 50% in this fraction. Purified lysosomes possess a 35 fold higher arylsulfatase activity, compared with homogenate, whereas neither NADPH: - nor succinate: cytochrome c reductase activity can be detected. In these lysosomes, one third of the phospholipids consists of bis (monoacylglycero) phosphate. The neutral phospholipids — mainly phosphatidylethanolamine — are drastically reduced in these cell organelles during the treatment. Our results indicate that chloroquine is nearly exclusively present in the lysosomal fraction. Furthermore we conclude from our data that bis (monoacylglycero) phosphate — isolated from lysosomal phospholipids — forms complexes with chloroquine.