Rat atrial natriuretic factor: Isolation,structure and biological activities of four major peptides

Four peptides possessing both natriuretic activity and smooth muscle relaxant activity were isolated from rat atrium and their amino acid sequences determined. The four peptides designated ANF-I, ANF-II, ANF-III and ANF-IV containing 35, 31, 30 and 25 amino acid residues, respectively, were obtained in a molar ratio of 4:60:20:16. The predominant species ANF-II, which may represent the native form of ANF, has the following sequence: (H₂N)-G-P-R-S-L-R-R-S-S-C-F-G-G-R-I-D-R-I-G-A-Q-S-G-L-G-C-N-S-F-R-Y-(COOH) in which Cys-10 and Cys-26 are disulfide linked. Cleavage of the aspartyl linkage at position 16 by staphylococcal protease caused complete inactivation, indicating that the ring conformation is essential. The dose-response relationships determined for the four pepdides in relaxing norepinephrine-induced contraction of rabbit thoracic aorta showed half-maximum relaxation at concentrations ranging from 1.5 × 10^?9 to 2.5 × 10^?9 M. Comparable dose-response relationships were observed in relaxation of carbacol-induced contraction of chick rectum strips as tested with ANF-II and ANF-IV.     

Partial amino acid sequence of the preprotein form of the alpha subunit of human choriogonadotropin and identification of the site of subsequent proteolytic cleavage

Messenger RNA isolated from first trimester placentae was translated using radiolabeled amino acids in both the wheat germ and the ascites cell-free systems. The choriogonadotropin α subunit product was purified by immunoprecipitation with a subunit specific antiserum. Its amino acid sequence was partially determined by automated Edman degradation analysis. An NH₂-terminal extension of 24 amino acids was found and its partial sequence is:

The preprotein form of the subunit was cleaved by the addition of microsomal membranes resulting in a homogeneous NH₂-terminal product. Hence, it is unlikely that this processing step accounts for the heterogeneity that has been observed previously in the structure of this region of the subunit.     

Hemin as a generic and potent protein misfolding inhibitor

Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer’s disease, Parkinson’s disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.     

Carboxymethylated cyclodextrins and their complexes with Pr(III) and Yb(III) as water‐soluble chiral NMR solvating agents for cationic compounds

Cyclodextrins that are indiscriminately carboxymethylated at the 2‐, 3‐, and 6‐positions are used as chiral NMR solvating agents for cationic substrates with phenyl, naphthyl, pyridyl, indoline, and indole rings. Enantiodifferentiation with the α‐, β‐, and γ‐cyclodextrin derivatives is compared. The carboxymethylated derivatives are almost always more effective as chiral NMR solvating agents for cationic substrates than native cyclodextrins. The most effective carboxymethylated cyclodextrin varies for different substrates, and at times even different resonances of the substrate. Addition of paramagnetic praseodymium(III) or ytterbium(III) to mixtures of the carboxymethylated cyclodextrin and substrate often causes enhancements in enantiomeric discrimination and facilitates measurements of enantiomeric purity. The lanthanide ion bonds to the carboxymethyl groups and causes perturbations in the chemical shifts in the NMR spectra of substrate molecules in the cyclodextrin cavity. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Covalent linkages between cellulose and lignin in cell walls of coniferous and nonconiferous woods

Covalent linkages between wall polysaccharides and lignin, especially linkage between cellulose and lignin were discussed by carboxymethylation technique of whole cell walls of coniferous and nonconiferous woods. Hydroxyl groups of plant cell walls polysaccharides were highly substituted, but not those of lignin by carboxymethyl groups under the used conditions, and separated into water-soluble and insoluble fractions by water extraction. Carboxymethylated wall polysaccharides linked covalently with lignin were distributed into the water-insoluble fractions. Composition of carboxymethylated sugar residues in the both fractions was analyzed quantitatively by 1H NMR spectroscopy after hydrolyzation with D2SO4 in D2O. More than half of cellulose linked covalently with lignin in coniferous wood, but only one-sixth of cellulose was involved in the linkage in nonconiferous wood. The major noncellulosic wall polysaccharides of coniferous wood also linked significantly with lignin. On the other hand, noncellulosic wall polysaccharides of nonconiferous wood were involved slightly in the covalent linkage with lignin. The situation of linkage between wall polysaccharides containing cellulose and lignin was visualized by scanning electron micrographs.     

Metrics that differentiate the origins of osmolyte effects on protein stability: a test of the surface tension proposal

Osmolytes that are naturally selected to protect organisms against environmental stresses are known to confer stability to proteins via preferential exclusion from protein surfaces. Solvophobicity, surface tension, excluded volume, water structure changes and electrostatic repulsion are all examples of forces proposed to account for preferential exclusion and the ramifications exclusion has on protein properties. What has been lacking is a systematic way of determining which force(s) is(are) responsible for osmolyte effects. Here, we propose the use of two experimental metrics for assessing the abilities of various proposed forces to account for osmolyte-mediated effects on protein properties. Metric 1 requires prediction of the experimentally determined ability of the osmolyte to bring about folding/unfolding resulting from the application of the force in question (i.e. prediction of the m-value of the protein in osmolyte). Metric 2 requires prediction of the experimentally determined ability of the osmolyte to contract or expand the Stokes radius of the denatured state resulting from the application of the force. These metrics are applied to test separate claims that solvophobicity/solvophilicity and surface tension are driving forces for osmolyte-induced effects on protein stability. The results show clearly that solvophobic/solvophilic forces readily account for protein stability and denatured state dimensional effects, while surface tension alone fails to do so. The agreement between experimental and predicted m-values involves both positive and negative m-values for three different proteins, and as many as six different osmolytes, illustrating that the tests are robust and discriminating. The ability of the two metrics to distinguish which forces account for the effects of osmolytes on protein properties and which do not, provides a powerful means of investigating the origins of osmolyte-protein effects.     

Hydrophobic interactions of the apo and holo forms of human cobalamin binding proteins

The interactions of transcobalamin II (TC II), intrinsic factor (IF) and R-type binding protein of cobalamin (Cb1, vitamin B₁₂) with the hydrophobic chromatography matrix Phenyl-Sepharose CL-4B were investigated. IF-Cb1 and R-Cb1 complexes were not adsorbed on Phenyl-Sepharose at room temperature or at 4°C with buffer containing 50 mM sodium phosphate, pH 7.4 containing 150 mM sodium chloride. The TC II-Cb1 complex adsorbed and could be eluted with buffer containing 50% $\text{v}\text{v}$ glycerol. IF without Cb1 adsorbed and was eluted with 50% glycerol at room temperature and 4°C. At room temperature, R binder without Cb1 eluted with buffer, but later than the R-Cb1 complex. At 4°C, R binder completely adsorbed to the matrix. TC II-without Cb1 bound to the matrix at 4°C and room temperature and could not be eluted with glycerol. These results suggest that Cb1 binding proteins can be separated and identified based on their hydrophobic properties. In addition, upon binding Cb1, TC II, IF and R-type binders undergo a conformational change such that the protein-Cb1 complex shows reduced hydrophobicity.     

(−)-Epigallocatechin-3-Gallate (EGCG) Maintains κ-Casein in Its Pre-Fibrillar State without Redirecting Its Aggregation Pathway

The polyphenol (−)-epigallocatechin-3-gallate (EGCG) has recently attracted much research interest in the field of protein-misfolding diseases because of its potent anti-amyloid activity against amyloid-β, α-synuclein and huntingtin, the amyloid-fibril-forming proteins involved in Alzheimer's, Parkinson's and Huntington's diseases, respectively. EGCG redirects the aggregation of these polypeptides to a disordered off-folding pathway that results in the formation of non-toxic amorphous aggregates. Whether this anti-fibril activity is specific to these disease-related target proteins or is more generic remains to be established. In addition, the mechanism by which EGCG exerts its effects, as with all anti-amyloidogenic polyphenols, remains unclear. To address these aspects, we have investigated the ability of EGCG to inhibit amyloidogenesis of the generic model fibril-forming protein RCMκ-CN (reduced and carboxymethylated κ-casein) and thereby protect pheochromocytoma-12 cells from RCMκ-CN amyloid-induced toxicity. We found that EGCG potently inhibits in vitro fibril formation by RCMκ-CN [the IC₅₀ for 50 μM RCMκ-CN is 13 ± 1 μM]. Biophysical studies reveal that EGCG prevents RCMκ-CN fibril formation by stabilising RCMκ-CN in its native-like state rather than by redirecting its aggregation to the disordered, amorphous aggregation pathway. Thus, while it appears that EGCG is a generic inhibitor of amyloid-fibril formation, the mechanism by which it achieves this inhibition is specific to the target fibril-forming polypeptide. It is proposed that EGCG is directed to the amyloidogenic sheet-turn-sheet motif of monomeric RCMκ-CN with high affinity by strong non-specific hydrophobic associations. Additional non-covalent π-π stacking interactions between the polyphenolic and aromatic residues common to the amyloidogenic sequence are also implicated.     

Competition between Folding, Native-State Dimerisation and Amyloid Aggregation in β-Lactoglobulin

We show that a series of peptides corresponding to individual β-strands in native β-lactoglobulin readily form amyloid aggregates and that such aggregates are capable of seeding fibril formation by a full-length form of β-lactoglobulin in which the disulfide bonds are reduced. By contrast, preformed fibrils corresponding to only one of the β-strands that we considered, βA, were found to promote fibril formation by a full-length form of β-lactoglobulin in which the disulfide bonds are intact. These results indicate that regions of high intrinsic aggregation propensity do not give rise to aggregation unless at least partial unfolding takes place. Furthermore, we found that the high aggregation propensity of one of the edge strands, βI, promotes dimerisation of the native structure rather than misfolding and aggregation since the structure of βI is stabilised by the presence of a disulfide bond. These findings demonstrate that the interactions that promote folding and native-state oligomerisation can also result in high intrinsic amyloidogenicity. However, we show that the presence of the remainder of the sequence dramatically reduces the net overall aggregation propensity by negative design principles that we suggest are very common in biological systems as a result of evolutionary processes.