Comparison of 252californium plasma desorption and fast atom bombardment mass spectrometry for analysis of small peptides

The data obtained with 252Cf plasma desorption (PD) and fast atom bombardment mass spectrometry of eight tri-, tetra- and pentapeptides were compared. Good spectra were obtained with 1-10 nmol of peptide. In both techniques molecular weight information was obtained. The PD mass spectra are often dominated by the cationized molecular ions in contrast to the fast atom bombardment (FAB) mass spectra, where cationization is rarely observed. Amino acid content is reflected in the immonium ions equally well in both techniques. The fragmentation patterns observed with the two techniques are almost identical. However, practical sequencing of peptides based on either FAB or PD mass spectrometry of underivatized peptides alone is difficult. This is due to the unpredictable and sometimes absent cleavage yield at certain peptide bonds. Another difficulty is the many simultaneous fragmentation pathways. However, for many peptides enough information is present to allow sequence determination for at least a major part of the molecule.     

Comparison of the NMR and X-ray structures of the HIV-1 matrix protein: evidence for conformational changes during viral assembly.

The three-dimensional solution- and solid-state structures of the human immunodeficiency virus type-1 (HIV-1) matrix protein have been determined recently in our laboratories by NMR and X-ray crystallographic methods (Massiah et al. 1994. J Mol Biol 244:198-223; Hill et al. 1996. Proc Natl Acad Sci USA 93:3099-3104). The matrix protein exists as a monomer in solution at low millimolar protein concentrations, but forms trimers in three different crystal lattices. Although the NMR and X-ray structures are similar, detailed comparisons have revealed an approximately 6 A displacement of a short 3(10) helix (Pro 66-Gly 71) located at the trimer interface. High quality electron density and nuclear Overhauser effect (NOE) data support the integrity of the X-ray and NMR models, respectively. Because matrix apparently associates with the viral membrane as a trimer, displacement of the 3(10) helix may reflect a physiologically relevant conformational change that occurs during virion assembly and disassembly. These findings further suggest that Pro 66 and Gly 71, which bracket the 3(10) helix, serve as "hinges" that allow the 3(10) helix to undergo this structural reorientation.     

Quinuclidin-2-ene-based muscarinic antagonists

A series of achiral 3-heteroaryl substituted quinuclidin-2-ene derivatives and related compounds have been synthesized by facile methods. The compounds were evaluated for muscarinic and antimuscarinic properties in receptor binding studies using (-)-[3H]-QNB as the radioligand and in a functional assay using isolated guinea pig urinary bladder. 3-(2-Benzofuranyl)-quinuclidin-2-ene (15) displayed the highest M1-receptor affinity in the present series (Ki = 9.6 nM).     

Selective 2''-benzoylation at the cis 2'',3''-diols of protected ribonucleosides. New solid phase synthesis of RNA and DNA-RNA mixtures.

5'-0-(Dimethoxytrityl)-2'-0-(benzoyl or 3,4,5-trimethoxybenzoyl)-base protected ribonucleosides have been prepared by selective benzoylation of the 2'-hydroxyl group. The isomerization of the 2'-benzoates to the 3'-benzoates was studied. The protected ribonucleosides have been converted to either methylphosphochloridites or methylphosphoamidites and used to synthesize oligoribonucleotides on silica gel solid support. The synthetic RNA were deprotected and isolated using conditions that minimize internucleotide cleavage. The use of 2'-benzoates as protecting groups for ribonucleosides has made it possible to easily prepare and isolate mixtures of DNA and RNA.     

Histone content in relation to amount of heterochromatin and developmental stage in three species of Drosophila

Relative amounts of various histone fractions in Drosophila chromatin were estimated densitometrically on electrophoretic gel separations. Several consistent and highly significant differences were obtained between larval and adult chromatin. The arginine-rich histones showed the most conspicuous changes: higher amounts of H4 in larvae, higher H3 in adults. The level of modification of these histones was clearly higher in larval than in adult chromatin. The modification of the two slower subfractions of H4 involved, in all probability, phosphorylation as well as acetylation. In all types of Drosophila chromatin studied 50% or more of the H2a molecules were phosphorylated—a remarkably high proportion. The species differences observed in relative amounts of histone were consistent in both stages of development. D. melanogaster differed from D. hydei and D. virilis in all histones except H2b, while the latter two species were generally similar. The interspecific variation in histone pattern was generally not correlated to differences in content of heterochromatin. The level of modification of H3 was, however, presumably an exception, as it was significantly lower for both larvae and adults in D. virilis than in the other two species. These differ from D. virilis in containing appreciably lower proportions of heterochromatic chromosome segments.     

Assembly of Adenoviruses

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified incomplete particles of adenoviruses type 2 and 3 revealed that core proteins V and VII and capsid proteins VI, VIII, and X were absent in these particles, but they contained polypeptides not present in complete particles. Two types of incomplete particles were observed in the electron microscope, appearing as deoxyribonucleic acid-less particles with single discontinuities in the capsid structure (about 80%), or more amorphous particles resembling hexon aggregates (about 20%). The amount of incomplete and complete particles increased in parallel during the infectious cycle. Detectable amounts were found at 13 h with a maximum rate of synthesis for both particles at 24 h after infection. (3)H-labeled amino acids were incorporated into incomplete particles without a detectable lag period, but the label appeared in complete particles with a 60- to 80-min lag. Early after the pulse in pulse-chase experiments, the radioactivity was higher for incomplete particles than for complete particles and leveled off before the activity of complete particles reached a maximum. In the adenovirus type 2 system, pulse-chase experiments suggested a precursor-product relationship between incomplete and complete particles. After a short pulse, 19 h postinfection, entrance of (3)H-labeled amino acids into the hexon polypeptide of complete particles was delayed for 80 min, but no delay was observed for the labeling of the hexon polypeptide of incomplete particles. The core polypeptides appear in complete particles without a delay, also suggesting that incomplete particles were precursors to complete particles. Incorporation of (3)H-labeled amino acids into the hexon polypeptide of complete and incomplete particles was drastically decreased by inhibition of protein synthesis with emetine. However, the uptake of label into core proteins of complete particles was only decreased to 50% on inhibition of protein synthesis. The results suggest that incomplete particles are intermediates in virus assembly in vivo and that the assembly of capsid polypeptides into incomplete and complete particles is dependent on continuing protein synthesis.     

Plasma-desorption mass spectrometry. Study of the desorption mechanism and prospects for use]

Particle impact mass spectrometry and in particular the use of MeV particles as in plasma desorption mass spectrometry (PDMS) applied to biomolecules is described. Experimental and theoretical studies of the mechanisms involved for large molecular ion ejection are treated in some detail. Applications of PDMS mass spectrometry to proteins are discussed.     

The α1-adrenergic receptor in human erythrocyte membranes mediates interaction in vitro of epinephrine and thyroid hormone at the membrane Ca-ATPase

Membrane Ca²⁺-ATPase activity was stimulated in vitro separately by T₄ (10⁻¹⁰ M) and by epinephrine (10⁻⁶ M). In the presence of a fixed concentration of T₄, additions of 10⁻⁸ and 10⁻⁶ M epinephrine reduced the T₄ effect on the enzyme. β-Adrenergic blockade with propranolol (10⁻⁶ M) prevented stimulation by epinephrine of Ca²⁺-ATPase activity, but did not prevent the suppressive action of epinephrine on T₄-stimulable Ca²⁺-ATPase. In contrast α₁-adrenergic blockade with unlabelled prazosin restored the effect of T₄ on Ca²⁺-ATPase activity in the presence of epinephrine. Like propranolol, prazosin prevented enhancement of enzyme activity by epinephrine in the absence of thyroid hormone. Neither prazosin nor propranolol had any effect on the stimulations by T₄ of red cell Ca²⁺-ATPase in the absence of epinephrine. Analysis of radiolabelled prazosin binding to human red cell membranes revealed the presence of a single class of high-affinity binding sites (K_d, 1.2 × 10⁻⁸ M; B_max, 847 fmol/mg membrane protein). Thus, the human erythrocyte membrane contains α₁-radrenergic receptor sites that are capable of regulating Ca²⁺-ATPase activity.     

Optimization of assay conditions for, and the selected tissue distribution of, alanine aminotransferase and aspartate aminotransferase of English sole, Parophrys vetulus Girard

Optimization of assay conditions for serum alanine aminotransferase (ALAT, GPT) and aspartate aminotransferase (ASAT, GOT) are described for English sole, Parophrys vetulus . Although most conditions for measurement of these fish enzymes parallel conditions in mammals, the optimal pH is lower for both these enzymes. Liver represents a potentially large source of ASAT activity, potential sources for ALAT activity are liver and kidney. As in mammals, the use of these enzymes as indicators of fish liver and kidney dysfunction may be warranted.     

Xylanases in bleaching: From an idea to the industry

Abstract: The utilization of hemicellulases in bleaching of kraft (sulphate) pulp is considered as one of the most important new large-scale industrial applications of enzymes. This is partly due to the great potential of an environmentally safe method. This method has in a short period also proven to be economically realistic. The main enzymes needed in the enzyme-aided bleaching have been shown to belong to the group of endo-/gb-xylanases. Xylanases act mainly on the relocated, reprecipitated xylan on the surface of the pulp fibres. Enzymatic hydrolysis of this specific type of xylan renders the structure of the fibres more permeable. The hydrolysis of xylan or mannan in the inner fibre layers may also enhance the bleachability. In practical process conditions, properties of the enzymes such as substrate specificity and the pH and temperature optima are of utmost importance. The benefits obtained by enzymes are dependent on the chemical bleaching sequence used as well as on the residual lignin content of pulp. The main goals in the enzyme-aided bleaching of kraft pulps have been the reduction of consumption of chlorine chemicals in the bleaching process and consequently lowering the AOX of the effluents. Enzymes have been applied as a pretreatment both in conventional (C/D)EDED and in ECF (elementary chlorine-free) bleaching sequences. In the production of TCF (totally chlorine-free) pulps, enzymes have also been successfully used for increasing the brightness of pulp.