Biophysical characterization of a soluble CD40 ligand (CD154) coiled-coil trimer: evidence of a reversible acid-denatured molten globule.

The CD40 ligand molecule is unique, consisting of a receptor-binding domain anchored by an isoleucine zipper moiety. Exact determination of the multimeric state and its tendency to form molten globules has not been elucidated. Corroborating evidence of a trimerized molecule in aqueous solution was obtained from size-exclusion chromatography, laser light scattering, and analytical ultracentrifugation. A reversible acid-denatured molten globule state was observed from circular dichroism and fluorescence spectroscopy data. The molten globule state was characterized by a loss of tertiary structure with associated retention of secondary structure near pH 3. Once returned to pH 7, the acid-denatured state refolded over the course of 7 days resulting in approximately 90% recovery of the native structure. The molten globule state was characterized by a broadening of structural features in the second-derivative spectra of Fourier transform infrared spectroscopy. A component band at 1650 cm(-1) was shown to be alpha-helix and originate from amide carbonyl vibrations of the isoleucine zipper. Differential scanning calorimetry measurements characterized the pH-sensitive molten globule state at pH 3.3 as one lacking a well-defined unfolding transition with an accompanying baseline shift at 58 degrees C (a consequence of increased heat capacity). The tendency to form molten globules during acid denaturation stress permits an opportunity to study the process of partial protein unfolding with implications concerning stability. Although reversible molten globules can be formed, it is important to recognize the unusual nature since the molten globule state is formed exclusively within the beta-sheet receptor-binding region.

In vitro release kinetics of gentamycin from a sodium hyaluronate gel delivery system suitable for the treatment of peripheral vestibular disease.

For certain patients who experience intense vertigo arising from unilateral vestibular lesions, the primary therapy is a vestibular nerve section, an intracranial surgical procedure. One alternative to this treatment is therapeutic ablation of vestibular function on the unaffected side using an ototoxic agent. We prepared a biodegradable sustained-release gel delivery system using sodium hyaluronate that can be administered into the middle ear using only a local anesthetic. The gel contains gentamycin sulfate, the ototoxic agent of choice for treatment of unilateral vestibulopathy, and it exhibits diffusion-controlled release of the drug over a period of hours. The released gentamycin could then diffuse into the inner ear through the round membrane. This represents an important advance over previous formulations, which used only gentamycin sulfate solutions, in that it should allow more careful control of the dose, it should reduce loss of the drug from the middle ear site, and it should maintain intimate contact with the round membrane. By carefully controlling the dose, it should be possible to inhibit vestibular function while minimizing hearing loss. Herein we describe the in vitro release kinetics of gentamycin sulfate from sodium hyaluronate gels and find that the system obeys Fickian behavior.

Hydrophobic ion pairing as a method for enhancing structure and activity of lyophilized subtilisin BPN' suspended in isooctane.

The use of enzymes in low water environments permits reactions to occur that are difficult or impossible in aqueous solution. In this manner, proteases can be used to form, rather than hydrolyze, ester and amide linkages. Presumably, the native-like structure of the enzyme must remain intact for catalysis to transpire. However, little is known regarding the integrity of the overall structure of lyophilized proteins suspended in organic media. In this study, the structural changes that occur during the freeze-drying process and those effected by suspension in the organic solvent were examined. Using Fourier-transform infrared spectroscopy, the secondary structure of lyophilized subtilisin BPN' was monitored and correlated to the level of enzymatic activity when suspended in isooctane. In addition, the ability of ionic detergents to stabilize subtilisin BPN' via ion pairing was evaluated. It was found that subtilisin unfolds to some degree during lyophilization, whether it is ion paired or not. Furthermore, there are structural changes observed when the enzyme is placed in isooctane, although the effects are less with ion-paired subtilisin. This higher level of retention of secondary structure results in increased enzymatic activity.

Structure-activity studies on position 14 of human alpha-calcitonin gene-related peptide.

A structure-activity study was performed to examine the role of position 14 of human alpha-calcitonin gene-related peptide (h-alpha-CGRP) in activating the CGRP receptor. Interestingly, position 14 of h-alpha-CGRP contains a glycyl residue and is part of an alpha-helix spanning residues 8-18. Analogues [Ala14]-h-alpha-CGRP, [Aib14]-h-alpha-CGRP, [Asp14]-h-alpha-CGRP, [Asn14]-h-alpha-CGRP, and [Pro14]-h-alpha-CGRP were synthesized by solid phase peptide methodology and purified by RP-HPLC. Secondary structure was measured by circular dichroism spectroscopy. Agonist activities were determined as the analogues' ability to stimulate amylase secretion from guinea pig pancreatic acini and to relax precontracted porcine coronary arteries. Analogues [Ala14]-h-alpha-CGRP, [Aib14]-h-alpha-CGRP, [Asp14]-h-alpha-CGRP, and [Asn14]-h-alpha-CGRP, all containing residues with a high helical propensity in position 14, were potent full agonists compared to h-alpha-CGRP in both tissues. Interestingly, replacement of Gly14 of h-alpha-CGRP with these residues did not substantially increase the helical content of these analogues. [Pro14]-h-alpha-CGRP, predictably, has significantly lower helical content and is a 20-fold less potent agonist on coronary artery, known to contain CGRP-1 receptor subtypes, and an antagonist on pancreatic acini, known to contain CGRP-2 receptor subtypes. In conclusion, the residue in position 14 plays a structural role in stabilizing the alpha-helix spanning residues 8-18. The alpha-helix is crucial for maintaining highly potent agonist effects of h-alpha-CGRP at CGRP receptors. The wide variety of functional groups that can be tolerated in position 14 with no substantial modification of agonist effects suggests the residue in this position is not in contact with the CGRP receptor. [Pro14]-h-alpha-CGRP may be a useful pharmacological tool to distinguish between CGRP-1 and CGRP-2 receptor subtypes.

Interdependent domains controlling the enzymatic activity of mitogen-activated protein kinase kinase 1.

The activation of human mitogen-activated protein kinase kinase 1 (MKK1) is achieved by phosphorylation at Ser218 and Ser222 within a regulatory loop. Partial activation was achieved by replacing these residues with aspartic/glutamic acid. Higher activity was obtained by introducing four acidic residue substitutions in the regulatory loop, indicating that acidic residues in the loop stabilize an active configuration by the introduction of negative charge. Activation of MKK1 is also achieved by deleting residues 44-51, N-terminal to the consensus catalytic core. Although substitution of residues within this segment by alanine does not affect activity, introduction of proline residues elevates kinase activity, indicating that activation results from perturbation of secondary structure within residues 44-51. Pseudosubstrate inhibition, a commonly observed mechanism of kinase regulation, is not operative in this process. Both the acidic substitutions and the N-terminal deletion increase Vmax, V/K(m),ERK2, and V/K(m),ATP, as is also observed following phosphorylation of wild-type MKK1. A synergistic enhancement of these steady-state rate parameters occurs upon combining the mutations, suggesting that conformational changes induced by mutagenesis together mimic those seen upon phosphorylation.

Generation of soluble and active subtilisin and alpha-chymotrypsin in organic solvents via hydrophobic ion pairing.

With very low concentrations of anionic detergents, such as sodium dodecyl sulfate (SDS) and Aerosol OT (AOT), it is possible to solubilize proteases in organic solvents, while retaining enzymatic activity. For example, the SDS-subtilisin BPN' complex catalyzes transesterification of Ac-Phe-OMe in ethanol with a kcat/Km of 36 M-1 s-1 for mutant M1 and 39 M-1 s-1 for the wild type. By comparison, M1 suspended in ethanol is approximately 1000-fold less active, with a kcat/Km of 0.03 M-1 s-1. Similarly, AOT complexes of alpha-chymotrypsin were found to be approximately 1000 times more active (kcat/Km = 100-350 M-1 s-1) than the suspended enzyme.

Approaches for increasing the solution stability of proteins.

Stabilization of proteins through proper formulation is an important challenge for the pharmaceutical industry. Two approaches for stabilization of proteins in solution are discussed. First, work describing the effect of additives on the thermally induced denaturation and aggregation of low molecular weight urokinase is presented. The effects of these additives can be explained by preferential exclusion of the solute from the protein, leading to increased thermal stability with respect to denaturation. Diminished denaturation leads to reduced levels of aggregation. The second approach involves stoichiometric replacement of polar counter ions (e.g., chloride, acetate, etc.) with anionic detergents, in a process termed hydrophobic ion pairing (HIP). The HIP complexes of proteins have increased solubility in organic solvents. In these organic solvents, where the water content is limited, the thermal denautration temperatures greatly exceed those observed in aqueous solution. In addition, it is possible to use HIP to selectively precipitate basic proteins from formulations that contain large amounts of stabilizers, such as human serum albumin (HSA), with a selectivity greater than 2000-fold. This has been demonstrated for various mixtures of HSA and interleukin-4. (c) 1995 John Wiley & Sons, Inc.

Selective precipitation of interleukin-4 using hydrophobic ion pairing: a method for improved analysis of proteins formulated with large excesses of human serum albumin.

In order to ensure the stability of protein pharmaceuticals, human serum albumin (HSA) is often added as an excipient, frequently in large excess. This makes chromatographic analysis of the stability of the active protein difficult. In the case of interleukin-4 (IL-4), separation from HSA can be achieved to some degree by size exclusion chromatography, but some HSA co-elutes with the IL-4. Hydrophobic ion pairing provides a method for selective precipitation of IL-4 from HSA. Hydrophobic ion pairing involves the electrostatic interaction of ionic detergents with oppositely charged polypeptides. Even when HSA is present in fifty-fold excess (w/w), the resulting precipitate contains greater than 70% of the IL-4. Selective precipitation with SDS produces enhancements in IL-4 over HSA of more than 2000-fold. This approach permits subsequent facile analysis of IL-4 by conventional reverse phase HPLC.

Expression of the mature and the pro-form of human sterol carrier protein 2 in Escherichia coli alters bacterial lipids.

Sterol carrier protein 2 (SCP2) is a protein that is believed to be involved in the intracellular transport of cholesterol and phospholipids. Expression in mammalian COS cells of a cDNA encoding SCP2 revealed that the mature protein is synthesized as a pro-form containing a 20-residue amino-terminal leader sequence. The function of this presequence is currently not known, and pro-SCP2 is generally not detected in tissues. In order to obtain large quantities of pro-SCP2 as well as the mature form of human SCP2, Escherichia coli expression plasmids were constructed. Both proteins were produced in high yield (10-30% of the total cell protein) and were found in the supernatant fraction after cell lysis. Recombinant human SCP2 and pro-SCP2 were purified to homogeneity by acid precipitation followed by ion-exchange chromatography. Both recombinant human SCP2 and pro-SCP2 had sterol exchange activity similar to that seen with SCP2 purified from rat liver. In addition, the lipid content of SCP2- and pro-SCP2-producing E. coli was analyzed. Acidic lipids were significantly increased in the transfected cells. Specifically, fatty acids were increased 2-3-fold, phosphatidylglycerol was increased 2-fold, and lipid A was increased 3-4-fold, while neutral lipids were decreased 2-3-fold as compared to control cells. This alteration of the lipid composition of E. coli expressing SCP2 or pro-SCP2 is consistent with the proposed role for SCP2 in intracellular lipid movement.

Properties of phosphatidylethanolamine-containing phospholipid-apolipoprotein complexes modified by lecithin-cholesterol acyltransferase.

The effect of the inclusion of phosphatidylethanolamine (PE), a phospholipid with unusual packing properties, on the substrate properties of protein-lipid complexes toward lecithin-cholesterol acyltransferase (LCAT) has been studied. Recombinant particles of apolipoprotein A-I with dimyristoylphosphatidylcholine (DMPC), dilauroylphosphatidylethanolamine (DLPE) and cholesterol were prepared at a molar ratio of 1:140:14 (A-I/DMPC/cholesterol) or 1:70:70:14 (A-I/DMPC/DLPE/cholesterol); the efficiency of cholesterol incorporation into complexes containing phosphatidylethanolamine was found to be very pH-dependent, with enhanced cholesterol incorporation at elevated pH values. By incubating the complexes with either purified human LCAT or the d greater than 1.21 g/ml fraction of rat serum as a source of LCAT activity, it was found that a high degree of cholesterol esterification could be achieved with either complex; however, the DLPE-containing complex possessed a much smaller Stokes' diameter than the DMPC-only particle despite compositional similarities between these complexes. With respect to particle diameter the DLPE-containing particles behaved more like complexes prepared with egg yolk lecithin than did complexes prepared with DMPC alone. When human LDL was added to the incubations to provide a source of additional cholesterol, the products were markedly different. Concomitant with an increased cholesteryl ester core was an increase in the protein stoichiometry in both types of particles, from 2 to 3 or 4 apo A-I per particle. The proportion of DLPE to DMPC in the products was reduced from 1:1 to 0.3:1, reflecting a preferential hydrolysis of PE by LCAT, and the Stokes' diameters of the DMPC-only and the DLPE-containing complexes were closely similar. We conclude that the presence of elevated proportions of certain phospholipid species may significantly alter both the physical properties of the particles and their substrate properties with regard to reactions with enzymes of lipid metabolism.

High density lipoprotein subpopulations from galactosamine-treated rats and their transformation by lecithin:cholesterol acyltransferase.

It is known that an acute hepatotoxicity is produced in rats by intraperitoneal administration of galactosamine; a consequence of this treatment is a marked deficiency of lecithin:cholesterol acyltransferase (LCAT) activity in the plasma compartment. In this study high density lipoprotein (HDL) from galactosamine-treated rats was isolated, resolved into subpopulations, and characterized. In contrast to HDL from control rats, which elutes from gel filtration columns as a single peak and has a diameter of 13.1 nm, HDL from the galactosamine-treated animals was found to elute in five major zones with diameters of 7.8-35 nm. Characterization of these subpopulations has revealed that the larger fractions are enriched in apolipoprotein E, phospholipid, and cholesterol, but contain little cholesteryl ester, while the smallest two fractions contain mainly apolipoprotein A-I, are enriched in phospholipid, and have 50-60% of their cholesterol in the ester form. Incubation of HDL from treated rats with a source of LCAT activity plus low and very low density lipoproteins caused transformation of these subpopulations into a species which, by size and composition, was essentially identical to control rat HDL. In addition, when the subpopulations were individually incubated with purified human lecithin:cholesterol acyltransferase and bovine serum albumin, there was a similar convergence toward a moderate particle size approximating control rat HDL. Cross-linking studies showed that incubation with LCAT activity reduced the heterogeneity of the treated rat HDL. We conclude that the galactosamine treatment induces a complex mixture of HDL that bears strong similarities to the small, apoA-I rich and large, apoE-rich particles seen in LCAT deficiency or secreted by hepatic cells in culture. Furthermore, these species appear to coalesce in the presence of the d greater than 1.21 g/ml fraction of control serum to yield a fairly homogeneous population that resembles control rat HDL in size, composition, and apoprotein content.