Self-association of troponin.

Ox muscle troponin was shown by equilibrium- and velocity-sedimentation studies to undergo concentration-dependent dissociation into its constituent subunits as well as self-association in imidazole buffers, pH 6.9. The extent of troponin association was found to be strongly dependent on ionic strength and also to exhibit a dependence on pH and temperature; under conditions physiological in regard to pH, temperature and ionic strength the extent of polymerization of troponin is considerable in 2 mg/ml solutions. The ability of polymeric troponin to bind to tropomyosin has been inferred from studies of mixtures containing actin-tropomyosin and an excess of troponin over the amount required for the normal 7:1:1 actin-tropomyosin-troponin complex. These findings should be relevant to studies of reconstituted actin-tropomyosin-troponin preparations, since they signify possible chemical as well as physical differences between the gel, paracrystalline and filamentous states of the complex that result from adoption of different preparative procedures for analogues of the native thin filament.     

Self-association of the neuroregulatory peptide substance P and its C-terminal sequences

Self-association of substance P and its C-terminal partial peptide sequences was studied by CD, quasi-electric light scattering, and sedimentation experiments. CD spectra of these peptides are strongly influenced by self-association. They exhibit strong characteristic negative ellipticities, suggesting the formation of a presumably B-type ordered structure. The tendency to form multimers depends on chain length and constitution and has its maximum at the octapeptide (SP 8). The peptide multimers have a broad distribution of sizes in the range of 30- and 800-nm diameter. Subdivision of this distribution into two size classes gives mean diameters of 60–100 nm (predominating)/200–800 nm for substance P and 30–50 nm/200–800 nm for SP 8 multimers.     

Self-association of bovine immunoglobulin G1.

The self-association properties of bovine serum immunoglobulin G1 and colostral immunoglobulin G1 (IgG1) in 0.32 M-NaCl/0.01 M-Tris/HCl, pH 8.0, were investigated by analysing sedimentation data according to a monomer-dimer association model. The self-association was characterized by an equilibrium constant of 5.3 X 10(4) +/- 3.5 X 10(4) M-1 for serum IgG1 and 1.6 X 10(3) +/- 0.69 X 10(3) M-1 for colostral IgG1. The removal of the Fc portion of IgG1 by pepsin digestion abolished its property of self-aggregation. At high total protein concentrations of serum IgG1, low concentrations of the ostensible trimer species were observed. However, no self-aggregation was evident when 0.14 M-NaCl/0.01 M-sodium phosphate. pH 6.0, was used as a solvent, thus confirming results published previously [Tewari & Mukkur (1975) Immunochemistry 12, 925--930].     

Self-association of alpha-chymotrypsin at low ionic strength in the vicinity of its pH optimum.

The self-association of alpha-chymotrypsin and its di-isopropyl phosphoryl derivative in in I0.03 sodium phophate buffer, pH7,9, was investigated by velocity sedimentation, equilibrium sedimentation and difference gel chromatography. No differences between the native and chemically modified enzyme were observed in the ultracentrifuge studies, and only a marginal (0.6%) difference in weight-average elution volume was detected by difference gel chromatography of 5g/litre solutions on Sephadex G-75. From quantitative analyses of sedimentation velocity and sedimentation-equilibrium distributions obtained with iPr2P (di-isopropylphosphoryl)-chymotrypsin, the polymerizing system is postulated to involve an indefinite association of dimer (with an isodesmic association constant of 0.68 litre/g) that is formed by a discrete dimerization step with equilibrium constant 0.25 litre/g. In addition to providing the best fit of the experimental results, this model of chymotrypsin polymerization at low ionic strength is also consistent with an earlier observation that dimer formation is a symmetrical head-to-head phenomenon under conditions of higher ionic strength (I0.29, pH7.9) where association is restricted to a monomer-dimer equilibrium. It is proposed that the dimerization process is essentially unchanged by variation in ionic strength at pH7.9, and that higher polymers are formed by an entirely different mechanism involving largely electrostatic interactions between dimeric species.     

Self-association of human PCSK9 correlates with its LDLR-degrading activity

Genetic studies have demonstrated an important role for proprotein convertase subtilisin/kexin type 9 (PCSK9) as a determinant of plasma cholesterol levels. However, the underlying molecular mechanism is not completely understood. To this end, we have generated a mammalian cell expression system for human PCSK9 and its mutants and produced transgenic mice expressing human PCSK9. HEK293T cells transfected with the human PCSK9 DNA construct expressed and secreted PCSK9 and displayed decreased LDLR levels; functional PCSK9 protein was purified from the conditioned medium. In vitro studies showed that PCSK9 self-associated in a concentration-, temperature-, and pH-dependent manner. A mixture of PCSK9 monomers, dimers, and trimers displayed an enhanced LDLR degrading activity compared to monomeric PCSK9. A gain-of-function mutant, D374Y, displayed greatly increased self-association compared to wild-type PCSK9. Moreover, we demonstrated that the catalytic domain of PCSK9 is responsible for the self-association. Self-association of PCSK9 was enhanced by incubation with mouse apoE-/- VLDL and inhibited by incubation with both human and mouse HDL. When PCSK9 protein was incubated with total serum, it partially associated with LDL and HDL but not with VLDL. In transgenic mice, PCSK9 also associated with LDL and HDL but not with VLDL. We conclude that self-association is an intrinsic property of PCSK9, correlated to its LDLR-degrading activity and affected by plasma lipoproteins. These results provide a basis for developing strategies to manipulate PCSK9 activity in the circulation for the treatment of hypercholesterolemia.     

Self-association of nucleotides

The self-association of nucleosides decreases within the series adenosine>guanosine>inosine>cytidine ≈uridine. The same trend is observed for the corresponding nucleotides, though less pronounced, as the charge effect governs series like adenosine ? AMP^2?>ADP^3??ATP^4?. Protonation of adenosine considerably reduces its self-stacking tendency: this is different with ATP^4?, where a maximum is reached for H₂(ATP)^2? caused by additional ionic interactions in the [H₂(ATP)]₂ ^4? dimer. Metal ion coordination may promote self-association, e.g., of ATP^4? via (mainly) charge neutralization (Mg²⁺) and the formation of intermolecular bridges in dimeric stacks (Zn²⁺, Cd²⁺). These results allow definition of conditions with negligible self-association and thus the determination of the stability and structure of monomeric nucleotide complexes in aqueous solution, e.g., quantification of macrochelate formation in M(ATP)^2? complexes. Some biological implications of the results are indicated.     

Self-association of mucin

Aggregation phenomena in aqueous solutions of purified human tracheobronchial mucin have been studied by rheological methods, steady-state fluorescence, quasielastic light scattering, and spin probe techniques. At temperatures below 30 degrees C and concentrations above 15 mg/mL and in the absence of chaotropic agents, mucin solutions are viscoelastic gels. A gel-sol transition is observed at temperatures above 30 degrees C that is manifested by the diminishing storage modulus and a loss tangent above unity throughout the studied frequency range of the oscillatory shear. No decline in the mucin molecular weight is observed by size-exclusion chromatography above 30 degrees C in the absence of redox agents or proteolytic enzymes. Aggregation of hydrophobic protein segments of the mucin chains at 37 degrees C is indicated by QELS experiments. The decreasing polarity of the microenvironment of pyrene solubilized into mucin solutions at temperatures above 30 degrees C, concomitant with the gel-sol transition, shows the hydrophobicity of the formed aggregates. ESR spectra of the fatty acid spin probe, 16-doxylstearic acid indicate that the aggregate-aqueous interface becomes more developed at elevated temperatures.     

Self-association of lyophilized horse liver alcohol dehydrogenase.

The molecular weights of lyophilized and non-lyophilized horse liver alcohol dehydrogenase have been compared by quasi-elastic light scattering, and ultracentrifugation. Whereas the non-lyophilized enzyme has the expected molecular weight of 78 000, the lyophilized enz)me has an initial molecular weight of about 10(6) which increases with time by an endothermic process. This result shows that any physical measurement using lyophilized liver alcohol dehydrogenase to investigate the enzyme mechanism, which relies upon the molecular size, will be invalid.     

Self-association accompanies inhibition of Ca-ATPase by thapsigargin.

Recent studies have demonstrated a relationship between the activity of the Ca-ATPase of sarcoplasmic reticulum and its state of self-association. In the present study, the effects of thapsigargin (TG), a toxin that specifically inhibits the Ca-ATPase of rabbit skeletal muscle sarcoplasmic reticulum membrane, were studied by detecting the time-resolved phosphorescence anisotropy (TPA) decay of the Ca-ATPase that had been labeled with the phosphorescent probe erythrosin-isothiocyanate (ErITC). Anisotropy decays were fit to a function that consisted of three exponential decays plus a constant background, as well as to a function describing explicitly the uniaxial rotation of proteins in a membrane. In the absence of TG, the anisotropy was best-fit by a model representing the rotation of three populations, corresponding to different-sized oligomeric species in the membrane. The addition of stoichiometric amounts of TG to the Ca-ATPase promptly decreased the overall apparent rate of decay, indicating decreased rotational mobility. A detailed analysis showed that the principal change was not in the rates of rotation but rather in the population distribution of the Ca-ATPase molecules among the different-sized oligomers. TG decreased the proportion of small oligomers and increased the proportion of large ones. Preincubation of the ErITC-SR in 1 mM Ca2+, which stabilizes the E1 conformation relative to E2, was found to protect partially against the changes in the TPA associated with the presence of the inhibitor. These results are consistent with the hypothesis that TG inhibits the Ca-ATPase by stabilizing it in an E2-like conformation, which promotes the formation of larger aggregates of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)