Poly(L-lysyl-L-alanyl-α-L-glutamic acid)

Poly(Lys(Cbz)-Ala-Glu(OBzl)) was prepared by the self-condensation of Lys(Cbz)-Ala-Glu(OBzl)-ONSu in dimethylformamide. After deprotection of the side chains, the product was subjected to Sephadex G-50 chromatography. The molecular weight of unfractionated and fractionated poly(Lys-Ala-Glu) was calculated from a calibrated Sephadex G-50 column, spectrophotometrically from Dnp-(Lys-Ala-Glu), equilibrium centrifugation, and viscosity measurements. Approximately 21% of the unfractionated material was polymeric with the remaining 79% being cyclic and monomeric material. Treatment of polymer hydrolysate with L -amino acid and D -amino acid oxidase indicated poly(Lys-Ala-Glu) to be optically pure. The apparent pKa's of the two ionizable groups were 4.1 and 9.7.     

Spectroscopic and equilibrium dialysis studies of poly(α-L-glutamic acid)–Cu(II) complexes in the pH range 4–7

The formation of complex between the Cu²⁺ ion and poly(α-L -glutamic acid) [poly(Glu)] in 150 mM NaCl solutions was studied by uv–visible absorption and equilibrium dialysis methods at the mixing ratios of Glu residues to Cu²⁺, R, of 32, 16, and 8 and in the pH range 4–7. The results showed that more than 90% of Cu²⁺ ions bind to the poly(Glu) at pH > 4.9, but the bound Cu(II) begins to dissociate with a decrease in pH. The absorption spectra of bound Cu(II) varied with pH and R in a complicated manner. Three different component spectra were disclosed from the analysis of the pH dependence of the bound spectra. We concluded that poly(Glu)–Cu(II) complexes fall into three classes in the pH range 4–7, with the proportions of these complexes varying with both pH and R. The three complexes predominate either in the helix or extended-coil region, in the helix–coil transition region, or in the helix-aggregate region. The stability constant and binding mode of each Cu(II)–Glu complex were estimated from the dialysis data. With these results, the possible structure of each complex is discussed.     

Viscosity and potentiometric measurements of poly(L-histidyl-L-alanyl-α-L-glutamic acid) and poly(L-lysyl-L-alanyl-α-L-glutamic acid)

Poly(His-Ala-Glu) and poly(Lys-Ala-Glu) were examined by viscosity and potentiometric titration. These measurements were interpreted in terms of the hydrodynamic size of the above sequential polypeptides. Effects of polymer, size and concentration, and solution-salt concentration were demonstrated. Although the sequential polypeptides generally behave like polyampholytes, they do demonstrate some differences. These differences my be attributed to the ability of ionized side chains three residues apart to repel themselves, in the order His < Glu < Lys.     

Interaction of poly(α-L-glutamic acid) and sodium poly(α-L-glutamate) with solvent components in water–2-chloroethanol mixtures

The preferential interaction of sodium poly(α-L -glutamate) and poly(α-L -glutamic acid) with the solvent components in water/2-chloroethanol mixtures has been determined using density-increment measurements. The degree of preferential interaction was deduced from the density increments at constant molality of 2-chloroethanol and at constant chemical potential of 2-chloroethanol. Sodium poly(α-L -glutamate) and poly(α-L -glutamic acid) are both preferentially hydrated in the whole range of solvent composition. A dehydration process occurs during the 2-chloroethanol-induced coil-to-helix transition of sodium poly(α-L -glutamate). This dehydration process was attributed to the release of some moles of water from the neighborhood of the peptide bond during the nucleation of the helix. After the conformational transition, sodium poly(α-L -glutamate) is solvated by one 2-chloroethanol molecule. The location of water and 2-chloroethanol molecules in the different parts of the residue (more polar and less polar portions) is also discussed.     

Preparation and characterization of α-L-glutamic acid oligomers

The preparation and characterization of α-L -glutamic acid oligomers with degree of polymerization (DP) up to 12 are described. The preparation of polymers with low DP corresponding to various A/I ratios (where A and I are monomer and initiator concentrations, respectively) with end groups blocked is given. The conditions of the fractionation, which separates the different oligomers by ion-exchange chromatography, are discussed. Finally, the isolation from salt solutions of the pure acidic form is given. Each polymer obtained for a given A/I is characterized at the end of the polymerization by its molecular-weight distribution. The average DP values calculated are compared to the A/I values; agreement is very good. Potentiometric behaviour during neutralization is obtained as a function of the degree of polymerization and the elaboration of the polyelectrolytic phenomenon is discussed.     

Structure and conformation of peptides: N-benzyloxycarboyl-(γ-ethyl)-L-glutamyl-(γ-ethyl)-L-glutamic acid ethyl ester

C₂₄H₃₄N₂O₉, orthorhombic, P2₁2₁2₁; a = 39.432 (10), b = 14.061 (5), c = 4.850 (2) Å, M = 494 a.m.u., Z = 4, Dm = 1.22 g cm^?3, Dx = 1.22 g cm^?3, R = 0.13 for 1205 observed reflections after refinement with isotropic thermal factors. The urethane and amide bonds are in the trans configuration, as well as all the ester groups. The φ and ψ angles of the L -glutamyl residues fall in the β-structure region of the Ramachandran's plot; the molecule is rather flat with the amide plane almost parallel to the c axis along which two hydrogen bonds hold the molecules together to form long rows in a “parallel pleated-sheet” fashion.     

Studies of the collagen fold formation in aqueous solutions of α-gelatin. II

Optical rotation and density studies have been performed as a function of time on solutions of a single-strand gelatin in salt-free solutions and in solutions containing sodium bromide, tetramethyl and tetrabutylammonium bromide. The reversion to the collagen fold is shown to be first order in all cases but undergoes a change in magnitude as a function of an aggregation process which occurs at a concentration of approximately 0.1% w/v. The structuring effects of the electrolytes on the solvent are shown to relate to the extent of helix regeneration by the protein, in the presence of the electrolyte. The extent of helix regeneration is also shown to relate to the thickness of the electric double layer surrounding the protein molecule.     

Poly(L-histidyl-L-alanyl-α-L-glutamic acid). II. Catalysis of p-nitrophenyl acetate hydrolysis

The hydrolysis of p-nitrophenyl acetate is catalyzed by imidazole, free in solution or as the side chain in poly(His-Ala-Glu). This is based on the observations that the reaction is first order in ester and first order in nonprotonated imidazole. Catalysis of p-nitrophenyl acetate hydrolysis is dependent on solvent conditions. The effect of low concentrations of ethanol, dioxane, and trifluoroethanol were investigated. As the concentration of organic solvent is increased, the second-order rate constant for imidazole catalysis decreases. The decrease, however, is greater for imidazole than for poly(His-Ala-Glu). In 2% trifluoroethanol/water solution, free imidazole has twice the catalytic activity of polymeric imidazole, while in 40% trifluoroethanol/water they have equal activity. Since under the latter solvent conditions poly(His-Ala-Glu) is partially α-helical, the relative improvement in polymeric–imidazole catalysis may be attributed to imidazole hydrogen-bonded to a carboxylate ion. With this assumption the carboxylate–imidazole hydrogen-bonded system has been calculated to have three times the base catalytic activity of imidazole.     

Poly(L-histidyl-L-alanyl-α-L-glutamic acid). I. Synthesis

Poly(L -histidyl-L -alanyl-α-L -glutamic acid) has been prepared in order to test the acid–base catalytic ability of a carboxyl-imidazole hydrogen-bonded system. Two different blocked histidyl-alanyl-glutamic acid monomers were used in the polymerization step. The imidazole ring was blocked with either a dinitrophenyl or a t-butyloxycarbonyl group. The γ-carboxyl of glutamic acid was protected as the benzyl ester. Both the coupling reactions and the polymerization step were via the N-hydroxysuccinimide active ester method. Thiolysis removed the dinitrophenyl group, while hydrogen bromide removed the t-butyloxycarbonyl and the benzyl groups. The water-soluble unblocked polymers obtained were fractionated on Sephadex G-50 or Bio-Gel P30. Fractions within a range of average molecular weights of 2,000 to 25,000 were isolated. Enzymatic oxidation of the acid hydrolyzate of the polymers revealed that no detectable racemization had occurred.     

Acridine orange–poly (α-L-glutamic acid) complexes. I. Stoichiometry and stacking coefficients

By means of fluorescence, absorption, and acid-base titrations, it has been shows that there is a one-to-one correspondence between free carboxylates and bound acridine orange in the dye-polyacid complex. Contrary to expections, stacking coefficients for the dye were found to be virtually the same on binding to the helical or coiled polyacid, indicating a strong similarity in the binding sites for both forms of the polyelectrolyte.     

Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Two γ-aminobutyric acid_A (GABA_A) receptor chimeras were designed in order to elucidate the structural requirements for GABA_A receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [³H]muscimol and [³H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

Conformation of poly(γ‐glutamic acid) in aqueous solution

Local conformation and overall conformation of poly(γ‐DL‐glutamic acid) (PγDLGA) and poly(γ‐L‐glutamic acid) (PγLGA) in aqueous solution was studied as a function of degree of ionization ε by ¹H‐NMR, circular dichroism, and potentiometric titration. It was clarified that their local conformation is represented by random coil over an entire ε range and their overall conformation is represented by expanded random‐coil in a range of ε > ε^*, where ε^* is about 0.3, 0.35, 0.45, and 0.5 for added‐salt concentration of 0.02M, 0.05M, 0.1M, and 0.2M, respectively. In a range of ε < ε^*, however, ε dependence of their overall conformation is significantly differentiated from each other. PγDLGA tends to aggregate intramolecularly and/or intermolecularly with decreasing ε, but PγLGA still behaves as expanded random‐coil. It is speculated that spatial arrangement of adjacent carboxyl groups along the backbone chain essentially affects the overall conformation of PγGA in acidic media. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 191–198, 2016.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

Specific aggregation of poly(α-L-glutamic acid) and hysteresis effects in aqueous solutions. I. Influence of temperature-dependent aggregation on the optical rotation of PGA

The instability of aqueous solutions of poly(α-L -glutamic acid) (PGA) at low pH is due to two distinguishable phenomena: precipitation, favored above 40°C., and aggregation, favored below 20°C. The aggregated form of PGA can be isolated by gel permeation chromatography. Both aggregation and precipitation increase with decreasing pH, i.e., with decreasing ionization of the side chain carboxyl groups. Temperature-induced aggregation and disaggregation give rise to a reproducible hysteresis loop which can be followed by optical rotation, light scattering, sedimentation, viscosity, and chromatography. Hysteresis has been observed with different PGA samples, and in several aqueous buffered or unbuffered solvents and organic-aqueous solvent mixtures and in the pH range 4.1–4.5. Aggregation manifests itself as an increase in negative optical rotation in the visible and ultraviolet spectral range. The specific relation at 233 mμ is sensitive to aggregation and also reflects the hysteresis. Measurements of optical rotatory dispersion indicate that a₀ reflects the hysteresis but b₀ does not, the latter revealing only reversible changes with aggregation and disaggregation. The helix-coil equilibrium is apparently unperturbed by aggregation, as is the thermal stability of the helix structure. For fully aggregated PGA it is estimated that a₀ increases by about 300 degrees, which suggests that a₀ may be a sensitive parameter to measure aggregation in other systems. The rate of aggregation increases with decreasing temperature. The disaggregation, upon heating, is more rapid. However, kinetics measurements have not yet been done. The temperature M at which all aggregates are disrupted increases with decreasing pH, but is independent of total PGA concentration, at constant pH. No molecular weight dependence of M was detected in the range 20–100 × 10³. The shape and size of the hysteresis loop depends upon pH and molecular weight, which is interpreted as a dependence on the extent of aggregation. One branch of the loop, representing the helix–coil transition of isolated molecules, is reversible, while the others, representing the formation and disruption of the aggregates, are not. The system exhibits both ascending and descending scanning curves, which are typical of a true hysteresis.     

Blood Platelets Contain and Secrete Laminin-8 (α4β1γ1) and Adhere to Laminin-8 via α6β1 Integrin

Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin γ1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to γ1 and β1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin β1 antibody–Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to β1 and γ1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin α4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20–35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to β1 and α6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (α4β1γ1) and that the cells adhere to the protein by using α6β1 integrin.