Competitive interactions of serum protein fractions during complex formation with polycations

Interaction of gamma-globulin with quaternized poly-4-vinylpyridine in water solutions at pH 7 has been studied. Formation of soluble stable cooperative complexes has been observed in a wide range of component ratios. Protein globules are distributed unevenly between adsorbing polycations. Soluble complexes are rod-like particles assembled from the globules which are stabilized by polycation chains. Complex formation in the system gamma-G + PE is similar to that in the system BSA + PE. Competitive interaction of serum protein fractions was studied at the interacting with polycation. It has been shown that selectivity at binding protein fractions is observed in both artificially prepared systems (BSA + gamma-G, beta1-G + gamma-G, BSA + gamma-G + beta1-G), and in serum and whole blood. In those ratios where uneven distribution of protein molecules is observed the soluble complexes protein-PE are formed by separate distribution of individual proteins at the matrix. Decrease of PE concentration in the systems results in the formation of a soluble complex of mixed composition. When an insoluble complex is formed in the system serum-PE selective sorbtion of beta 2-globulin fractions is observed. The reasons for the selective sorbtion of various protein fractions are described, structural models of the soluble complexes protein-PE are suggested.     

Cellular mechanism of fibril formation from serum amyloid A1 protein

Serum amyloid A1 (SAA1) is an apolipoprotein that binds to the high‐density lipoprotein (HDL) fraction of the serum and constitutes the fibril precursor protein in systemic AA amyloidosis. We here show that HDL binding blocks fibril formation from soluble SAA1 protein, whereas internalization into mononuclear phagocytes leads to the formation of amyloid. SAA1 aggregation in the cell model disturbs the integrity of vesicular membranes and leads to lysosomal leakage and apoptotic death. The formed amyloid becomes deposited outside the cell where it can seed the fibrillation of extracellular SAA1. Our data imply that cells are transiently required in the amyloidogenic cascade and promote the initial nucleation of the deposits. This mechanism reconciles previous evidence for the extracellular location of deposits and amyloid precursor protein with observations the cells are crucial for the formation of amyloid.     

Effect of rhythmic alternation of protein depletion and repletion on serum protein synthesis.

The authors attempted to determine whether repeated protein depletion would produce changes in the organism's response to this unfavourable situation indicative of the preservation of information on past depletion and of its inclusion in the formation of the organism's defences against repetition of this unphysiological state. It can be concluded from the results that the anticipated trend was demonstrated. The question of the site of origin, storage and action of this information was not resolved.     

Effect of electroconvulsive therapy on serum myelin basic protein immunoreactivity.

A sensitive radioimmunoassay that can detect brain damage in cases of head injury and stroke was applied to blood samples from 13 patients before and after they received multiple treatments with electroconvulsive therapy for psychiatric disorder. None of the patients showed a significant increase in serum myelin basic protein immunoreactivity. As increased serum myelin basic protein immunoreactivity may reflect myelin damage it is apparent that in these patients electroconvulsive therapy did not cause measureable breakdown of myelin.     

Cartilage proteoglycan aggregate formation. Role of link protein.

Cartilage proteoglycan aggregate formation was studied by zonal rate centrifugation in sucrose gradients. Proteoglycan aggregates, monomers and proteins could be resolved. It was shown that the optimal proportion of hyaluronic acid for proteoglycan aggregate formation was about 1% of proteoglycan dry weight. The reaggregation of dissociated proteoglycan aggregate A1 fraction was markedly concentration-dependent and even at 9 mg/ml only about 90% of the aggregates were reformed. The lowest proportion of link protein required for maximal formation of link-stabilized proteoglycan aggregates was 1.5% of proteoglycan dry weight. It was separately shown that link protein co-sedimented with the proteoglycan monomer. By competition with isolated hyaluronic acid-binding-region fragments, a proportion of the link proteins was removed from the proteoglycan monomers, indicating that the link protein binds to the hyaluronic acid-binding region of the proteoglycan monomer.     

Multivalent ligand binding by serum mannose-binding protein.

The serum-type mannose-binding protein (MBP) is a defense molecule that has carbohydrate-dependent bactericidal effects. It shares with mammalian and chicken hepatic lectins similarity in the primary structure of the carbohydrate-recognition domain, as well as the ligand-binding mode: a high affinity (KD approximately nM) is generated by clustering of approximately 30 terminal target sugar residues on a macromolecule, such as bovine serum albumin, although the individual monosaccharides have low affinity (KD 0.1-1 mM). On the other hand, MBP does not manifest any significant affinity enhancement toward small, di- and trivalent ligands, in contrast to the hepatic lectins whose affinity toward divalent ligands of comparable structures increased from 100- to 1000-fold. Such differences may be explained on the basis of different subunit organization between the hepatic lectins and MBP.     

alpha-helix formation: discontinuous molecular dynamics on an intermediate-resolution protein model.

An intermediate-resolution model of small, homogeneous peptides is introduced, and discontinuous molecular dynamics simulation is applied to study secondary structure formation. Physically, each model residue consists of a detailed three-bead backbone and a simplified single-bead side-chain. Excluded volume and hydrogen bond interactions are constructed with discontinuous (i.e., hard-sphere and square-well) potentials. Simulation results show that the backbone motion of the model is limited to realistic regions of Phi-Psi conformational space. Model polyalanine chains undergo a locally cooperative transition to form alpha-helices that are stabilized by backbone hydrogen bonding, while model polyglycine chains tend to adopt nonhelical structures. When side-chain size is increased beyond a critical diameter, steric interactions prevent formation of long alpha-helices. These trends in helicity as a function of residue type have been well documented by experimental, theoretical, and simulation studies and demonstrate the ability of the intermediate-resolution model developed in this work to accurately mimic realistic peptide behavior. The efficient algorithm used permits observation of the complete helix-coil transition within 15 min on a single-processor workstation, suggesting that simulations of very long times are possible with this model.     

Influence of the carboxy terminus of serum amyloid A on protein oligomerization, misfolding, and fibril formation

The fibrillar deposition of serum amyloid A (SAA) has been linked to the disease amyloid A (AA) amyloidosis. We have used the SAA isoform, SAA2.2, from the CE/J mouse strain, as a model system to explore the inherent structural and biophysical properties of SAA. Despite its nonpathogenic nature in vivo, SAA2.2 spontaneously forms fibrils in vitro, suggesting that SAA proteins are inherently amyloidogenic. However, whereas the importance of the amino terminus of SAA for fibril formation has been well documented, the influence of the proline-rich and presumably disordered carboxy terminus remains poorly understood. To clarify the inherent role of the carboxy terminus in the oligomerization and fibrillation of SAA, we truncated the proline-rich final 13 residues of SAA2.2. We found that unlike full-length SAA2.2, the carboxy-terminal truncated SAA2.2 (SAA2.2ΔC) did not oligomerize to a hexamer or octamer, but formed a high molecular weight soluble aggregate. Moreover, SAA2.2ΔC also exhibited a pronounced decrease in the rate of fibril formation. Intriguingly, when equimolar amounts of denatured SAA2.2 and SAA2.2ΔC were mixed and allowed to refold together, the mixture formed an octamer and exhibited rapid fibrillation kinetics, similar to those for full-length SAA2.2. These results suggest that the carboxy terminus of SAA, which is highly conserved among SAA sequences in all vertebrates, might play important structural roles, including modulating the folding, oligomerization, misfolding, and fibrillation of SAA.     

Effects of inert volume-excluding macromolecules on protein fiber formation. I. Equilibrium models

The equilibrium Oosawa-Asakura model for nucleated assembly of rod-like protein fibers is recast in terms of dimensionless (scaled) quantities. The model is then generalized to treat arbitrarily large deviations from thermodynamic ideality arising from high fractional volume occupancy by an inert protein or polymer. Each state of association of the self-associating protein is modeled as an equivalent rigid convex particle (sphere or spherocylinder) and the crowding species is modeled either as an equivalent sphere or cylindrical rod. The resulting conservation of mass relation is readily solved to yield the fractional abundance of monomer, from which the entire equilibrium distribution of oligomeric species can be calculated, either directly or through the use of an additional scaling relationship. Results indicating the potential effect of volume occupancy on the equilibrium solubility of the self-associating protein and upon the equilibrium distribution of polymer size are presented. It is found that the fractional (logarithmic) change in both solubility and in the breadth of the polymer size distribution scale almost linearly with the fractional (logarithmic) change in the thermodynamic activity of monomer.     

Structural characteristics of green tea catechins for formation of protein carbonyl in human serum albumin

Catechins are polyphenolic antioxidants found in green tea leaves. Recent studies have reported that various polyphenolic compounds, including catechins, cause protein carbonyl formation in proteins via their pro-oxidant actions. In this study, we evaluate the formation of protein carbonyl in human serum albumin (HSA) by tea catechins and investigate the relationship between catechin chemical structure and its pro-oxidant property. To assess the formation of protein carbonyl in HSA, HSA was incubated with four individual catechins under physiological conditions to generate biotin-LC-hydrazide labeled protein carbonyls. Comparison of catechins using Western blotting revealed that the formation of protein carbonyl in HSA was higher for pyrogallol-type catechins than the corresponding catechol-type catechins. In addition, the formation of protein carbonyl was also found to be higher for the catechins having a galloyl group than the corresponding catechins lacking a galloyl group. The importance of the pyrogallol structural motif in the B-ring and the galloyl group was confirmed using methylated catechins and phenolic acids. These results indicate that the most important structural element contributing to the formation of protein carbonyl in HSA by tea catechins is the pyrogallol structural motif in the B-ring, followed by the galloyl group. The oxidation stability and binding affinity of tea catechins with proteins are responsible for the formation of protein carbonyl, and consequently the difference in these properties of each catechin may contribute to the magnitude of their biological activities.     

Glycosylation influences cross-species formation of protease-resistant prion protein.

A key event in the transmissible spongiform encephalopathies (TSEs) is the formation of aggregated and protease-resistant prion protein, PrP-res, from a normally soluble, protease-sensitive and glycosylated precursor, PrP-sen. While amino acid sequence similarity between PrP-sen and PrP-res influences both PrP-res formation and cross-species transmission of infectivity, the influence of co- or post-translational modifications to PrP-sen is unknown. Here we report that, if PrP-sen and PrP-res are derived from different species, PrP-sen glycosylation can significantly affect PrP-res formation. Glycosylation affected PrP-res formation by influencing the amount of PrP-sen bound to PrP-res, while the amino acid sequence of PrP-sen influenced the amount of PrP-res generated in the post-binding conversion step. Our results show that in addition to amino acid sequence, co- or post-translational modifications to PrP-sen influence PrP-res formation in vitro. In vivo, these modifications might contribute to the resistance to infection associated with transmission of TSE infectivity across species barriers.     

The action of ionizing radiation on protein. III. Radical formation in L-phenylalanine.HCl.

The radicals produced by X-irradiation in L-phenylalanine.HCl crystals have been analysed by electron-spin-resonance (e.s.r.) spectroscopy. Four radicals have been identified: the radical resulting from electron capture by the carboxylic group, the radical resulting from deamination, an hydrogen addition radical to the benzene ring, and the radical resulting from hydrogen abstraction from the methylene group bonded to Cbeta. These identifications are supported by INDO calculations.