Synthesis and structural characterization of bioactive peptide conjugates using thioether linkage approaches.

Applications of cysteine-insertion and thioether linkage approaches to the preparation of a number of bioactive peptide conjugates are reported. Peptides containing epitopes from (i) herpes simplex virus type 1 glycoprotein D, (ii) a specific N-terminal beta-amyloid epitope recognized by therapeutically active antibodies, and (iii) a GnRH-III peptide from sea lamprey with antitumour activity, were elongated with Cys residues and attached to a chloroacetylated tetratuftsin derivative carrier via a thioether linkage either directly, or by insertion of a spacer. The structures and molecular homogeneity of all the peptide conjugates were ascertained by HPLC, MALDI and electrospray mass spectrometry. The use of a spacer such as an oligoglycine or GFLG-tetrapeptide gave an increased yield in the conjugation reaction and enhanced reaction rates. In the formation of cysteinyl-thioether linkages, it was found that the position of flanking Cys residues markedly influenced the conjugation reaction and the formation of intermolecular epitope disulfide-dimers. C-terminal Cys residues gave thioether conjugates with significantly diminished epitope-dimerization, while Cys at the N-terminal caused rapid disulfide-dimerization, thereby preventing efficient conjugation.     

Chromogranin A from bovine adrenal medulla: molecular characterization of glycosylations, phosphorylations, and sequence heterogeneities by mass spectrometry.

Chromogranin A (CGA) is a member of a family of acidic glycoproteins present in endocrine and neuroendocrine tissues. One of its suggested physiological roles is being a precursor molecule for several peptide hormons. Further interest in this protein has recently originated from its potential role in pathophysiological processes of Alzheimer's disease. The concentration of CGA in the brain has been used for diagnosis of this disease, and CGA as an insoluble deposit has been found in the extracellular beta-amyloid plaques. By developing a new purification procedure we were able to isolate abundant CGA in high purity from bovine chromaffin cells. A MALDI-MS analysis of the intact protein revealed a heterogeneous molecular mass of ca. 50 kDa, indicating several structure modifications. By use of several subsequent proteolytic/chemical cleavage steps, HPLC isolation, a newly developed deglycosylation procedure, and several MS and MS-MS fragmentation approaches, the complete primary structure of CGA including four sequence heterogeneities, two O-glycosylations, five phosphorylations, and one disulfide bridge could be characterized. For both glycans six different forms could be identified. Ser167 was found to be mainly glycosylated by a trisaccharide, and Thr231 was found to be mainly glycosylated by a tetrasaccharide. Ser81, Ser124, and Ser297 residues were partially phosphorylated, whereas Ser372 and Ser377 were found completely phosphorylated. Sequence heterogeneities were identified in positions 293 (H/R), 301 (K/E), and 373 (Q/R) and at the partly missing C-terminal residue. Furthermore, a disulfide bridge between Cys17 and Cys38 was ascertained.     

Multinuclear magnetic resonance, electrospray ionization-mass spectroscopy, and parametric method 5 studies of a new derivative of gossypol with 2-thiophenecarbohydrazide as well as its complexes with LI+, Na+, K+, RB+, and Cs+ cations

A new derivative of racemic gossypol with 2-thiophenecarbohydrazide (GHHT) and its complexes with monovalent cations have been synthesized and studied by electrospray ionization-mass spectroscopy (ESI-MS), multinuclear nuclear magnetic resonance (NMR), as well as by the Parametric Method 5 (PM5) methods. It is demonstrated that GHHT forms stable complexes of 1:1 stoichiometry with monovalent metal cations. The structures of the complexes are stabilized by three types of intramolecular hydrogen bonds. The spectroscopic methods have provided clear evidence that GHHT and its complexes exist in the DMSO-d6 solution in the N-imine-N-imine tautomeric forms. The structures of the GHHT and its complexes with Li+, Na+, K+, Rb+, and Cs+ cations are visualized and discussed in detail.     

The role of intraspecific competition in the dispersal of an invasive fish

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Specific nitration at tyrosine 430 revealed by high resolution mass spectrometry as basis for redox regulation of bovine prostacyclin synthase

Treatment of bovine aortic microsomes containing active prostacyclin synthase (PGI(2) synthase) with increasing concentrations of peroxynitrite (PN) up to 250 microm of PN yielded specific staining of this enzyme on Western blots with antibodies against 3-nitrotyrosine (3-NT), whereas above 500 microm PN staining of additional proteins was also observed. Following treatment of aortic microsomes with 25 microm PN, PGI(2) synthase was about half-maximally nitrated and about half-inhibited. It was then isolated by gel electrophoresis and subjected to proteolytic digestion with several proteases. Digestion with thermolysin for 24 h provided a single specific peptide that was isolated by high performance liquid chromatography and identified as a tetrapeptide Leu-Lys-Asn-Tyr(3-nitro)-COOH corresponding to positions 427-430 of PGI(2) synthase. Its structure was established by precise mass determination using Fourier transform-ion cyclotron resonance-nanoelectrospray mass spectrometry and Edman microsequencing and ascertained by synthesis and mass spectrometric characterization of the authentic Tyr-nitrated peptide. Complete digestion by Pronase to 3-nitrotyrosine was obtained only after 72 h, suggesting that the nitrated Tyr-430 residue may be embedded in a tight fold around the heme binding site. These results provide evidence for the specific inhibition of PGI(2) synthase by nitration at Tyr-430 that may occur already at low levels of PN as a consequence of endothelial co-generation of nitric oxide and superoxide.     

Variation in fish growth characteristics along a river course

Variation in the growth patterns of roach, Rutilus rutilus (L.), pike, Esox lucius L., and chub, Leuciscus cephalus (L.) was examined along the upper Warta River, where human impact (mostly pollution) has influenced the longitudinal zonation on the fish assemblage. Significant differences were found in the exponent of weight-length relationships for roach and chub populations occupying different zones of the river, but no such variation was observed in pike. Moreover, pike growth was isometric, whereas roach and chub grew allometrically, with regression coefficients (slope) above 3. Although the length-at-age data were similar for each zone, the von Bertalanffy parameters (L _inf, K and t ₀) suggest that there may exist some inter-zone variation in the overall growth patterns of these species. All the species grew better in the zone where the index of relative abundance (relating dominance of a particular species to its maximum abundance in river system) achieved its highest value. The results suggest that a relative abundance index expressed in this way can be a good index of habitat quality.     

Calcium regulation of skeletal myogenesis: IV A defined culture medium permissive for myotube formation and the use of the calcium antagonist lanthanum

Summary Lanthanum has been used effectively in studies of calcium physiology in experiments of short duration. In experiments of longer duration, we report that solutions, such as cell culture medium, containing lanthanum (La⁺⁺) undergo a decrease in pH on the time scale of hours. Presumaly, the decrease in pH is a consequece of the hydrolysis of water by the solution-active La⁺⁺⁺ ions. We have devised a defined culture medium without serum and chick embryo extract which is permissive for myotube formation. This defined medium is also useful for studies of La⁺⁺⁺ as a calcium antagonist. with Ca⁺⁺ to low-Ca⁺⁺ fusion-blocked cultures. This study was supported in part by NIH grants NS 10196 and AM 25202 and The Muscular Dystrophy Association.