Gas phase studies of N-nitrosamines by ion cyclotron resonance spectroscopy

The gas phase basicities of a series of cyclic and acyclic N-nitrosamines were determined by ion cyclotron resonance techniques. The fragmentation pattern and ion-molecule reactions of these compounds are discussed in terms of high resolution mass spectrometry and deuterium labeling. The presence of a unique rearrangement of the molecular ion is discussed in terms of specific ion-molecule proton transfer reactions and fragment ion products.     

LIGHT-INDUCIBLE ABSORBANCE CHANGES AND VANADATE-SENSITIVE ATPase ACTIVITY ASSOCIATED WITH THE PRESUMPTIVE PLASMA MEMBRANE FRACTION FROM CAULIFLOWER INFLORESCENCES

Sucrose density gradient centrifugation of a microsomal membrane fraction of cauliflower inflorescences showed a strong correlation between a blue light mediated cytochrome b reduction (LIAC) and an ion stimulated nitrate-insensitive but a vanadate-sensitive ATPase activity at 38-40% sucrose. LIAC activity and vanadate-sensitive ATPase might be assigned to the same type of membrane different from ER, Golgi, tonoplast and mitochondria. The Mg²⁺-dependent ATP-hydrolytic activity obtained after purification of the microsomal fraction on an aqueous polymer two phase system was partially characterized. Temperature optimum (40°C), pH optimum (pH 7.0), vanadate inhibition (I₅₀ at 20 μ M ), substrate kinetics ( K _m= 1.37 m M Mg.ATP) and inhibitor studies all point to the presence of the frequently described plasma membrane ATPase. Potassium and Na⁺ stimulated the enzyme activity (20-40%). In general our data arc strongly in favour of the hypothesis that LIAC activity is localized on the plant plasma membrane. The cytochrome b involved in the light reaction has a midpoint potential near +150 mV. This cytochrome which has been previously shown in a cauliflower microsomal fraction is a constituent of the plasma membrane.     

Reactant adsorption in differential pulse polarography: Effects of adsorptive depletion of reactant,non-linear adsorption isotherms and uncompensated resistance

Digital simulation techniques are applied to the diagnosis of the ways in which the peak currents and wave shapes of differential pulse polarograms are affected by the adsorption of reactants or products. The changes in the polarograms introduced by the items mentioned in the title are examined and their magnitudes calculated and compared with experimental polarograms. Some implications of the results in analytical applications of differential pulse polarography are described.     

Die Analyse aller bei der Stahlbereitung in Anwendung kommenden Materialien

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Cis→Trans Isomerization of Pro<Superscript>7</Superscript> in Oxytocin Regulates Zn<Superscript>2+</Superscript> Binding

Ion mobility/mass spectrometry techniques are employed to investigate the binding of Zn²⁺ to the nine-residue peptide hormone oxytocin (OT, Cys¹-Tyr²-Ile³-Gln⁴-Asn⁵-Cys⁶-Pro⁷-Leu⁸-Gly⁹-NH₂, having a disulfide bond between Cys¹ and Cys⁶ residues). Zn²⁺ binding to OT is known to increase the affinity of OT for its receptor [Pearlmutter, A. F., Soloff, M. S.: Characterization of the metal ion requirement for oxytocin-receptor interaction in rat mammary gland membranes. J. Biol. Chem. 254, 3899–3906 (1979)]. In the absence of Zn²⁺, we find evidence for two primary OT conformations, which arise because the Cys⁶–Pro⁷ peptide bond exists in both the trans- and cis-configurations. Upon addition of Zn²⁺, we determine binding constants in water of K_A = 1.43 ± 0.24 and 0.42 ± 0.12 μM^?1, for the trans- and cis-configured populations, respectively. The Zn²⁺ bound form of OT, having a cross section of Ω = 235 Ų, has Pro⁷ in the trans-configuration, which agrees with a prior report [Wyttenbach, T., Liu, D., Bowers, M. T.: Interactions of the hormone oxytocin with divalent metal ions. J. Am. Chem. Soc. 130, 5993–6000 (2008)], in which it was proposed that Zn²⁺ binds to the peptide ring and is further coordinated by interaction of the C-terminal, Pro⁷-Leu⁸-Gly⁹-NH₂, tail. The present work shows that the cis-configuration of OT isomerizes to the trans-configuration upon binding Zn²⁺. In this way, the proline residue regulates Zn²⁺ binding to OT and, hence, is important in receptor binding.

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Coupling protein complex analysis to peptide based proteomics

Proteolysis is a central component of most proteomics methods. Unfortunately much of the information relating to the structural diversity of proteins is lost during digestion. This paper describes a method in which the native proteome of yeast was subjected to preliminary fractionation by size exclusion chromatography (SEC) prior to trypsin digestion of SEC fractions and reversed phase chromatography-mass spectral analysis to identify tryptic peptides thus generated. Through this approach proteins associated with other proteins in high molecular mass complexes were recognized and identified. A focus of this work was on the identification of Hub proteins that associate with multiple interaction partners. A critical component of this strategy is to choose methods and conditions that maximize retention of native structure during the various stages of analysis prior to proteolysis, especially during cell lysis. Maximum survival of protein complexes during lysis was obtained with the French press and bead-beater methods of cell disruption at approximately pH 8 with 200 mM NaCl in the lysis buffer. Structure retention was favored by higher ionic strength, suggesting that hydrophobic effects are important in maintaining the structure of protein complexes. Recovery of protein complexes declined substantially with storage at any temperature, but storage at -20°C was best when low temperature storage was necessary. Slightly lower recovery was obtained with storage at -80°C while lowest recovery was achieved at 4°C. It was concluded that initial fractionation of native proteins in cell lysates by SEC prior to RPC-MS/MS of tryptic digests can be used to recognize and identify proteins in complexes along with their interaction partners in known protein complexes.     

Thiaminase I (42 kDa) heterogeneity,sequence refinement,and active site location from high-resolution tandem mass spectrometry

Thiaminase I (E.C. 2.5. 1.2) from Bacillus thiaminolyticus catalyzes the degradation of thiamin (vitamin B₁). Unexpected mass heterogeneity (MW 42,127, 42,197, and 42,254; 1:2:1) in recombinant thiaminase I from Escherichia coli was detected by electrospray ionization Fourier-transform mass spectrometry, resolving power 7×10⁴. Nozzle-skimmer fragmentation data reveal an extra Ala (+71.02; 71.04=theory) and GlyAla (+128.04; 128.06=theory) on the N-terminus, in addition to the fully processed enzyme. However, the fragment ion masses were consistent only with this sequence through 330 N-terminal residues; resequencing of the last 150 bps of the thiaminase I gene yields a sequence consistent with the molecular weight values and all 61 fragment ion masses. Covalently labeling the active site with a 108-Da pyrimidine moiety via mechanism-based inhibition produces a corresponding molecular weight increase in all three thiaminase I components, which indicates that they are all enzymatically active. Inspection of the fragment ions that do and do not increase by 108 Da indicates that the active site nucleophile is located between Pro⁷⁹ and Thr¹⁷⁷ in the 379 amino acid enzyme.