Statistical properties predicted by the ball and chain model of channel inactivation.

It has been proposed that part of a voltage gated channel is a tethered ball and that inactivation occurs when this wandering ball binds to a site in the channel. In order to be able to quantitatively test this model by comparison to experiments we developed analytical solutions and numerical simulations of the distribution of times it takes the ball to reach the binding site when the motion of the ball is random and when it is also influenced by a directed force. If the motion of the ball is one-dimensional, at long times this distribution is a single exponential with a rate constant that is inversely proportional to the square of the length of the chain and does not depend on the starting position of the ball. This dependence on the chain length is not significantly altered if there are short range electrical forces between the ball and its binding site. These predictions suggest that to confirm the validity of this model additional experiments should be done to more precisely determine the form of this distribution and its dependence on the length of the chain.     

Site-specific glycation of lens crystallins by ascorbic acid.

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.     

A molecular thermodynamic approach to predict the secondary structure of homopolypeptides in aqueous systems.

Under physiological conditions, many polypeptide chains spontaneously fold into discrete and tightly packed three-dimensional structures. The folded polypeptide chain conformation is believed to represent a minimum Gibbs energy of the system, governed by the weak interactions that operate between the amino acid residues and between the residues and the solvent. A semiempirical molecular thermodynamic model is proposed to represent the Gibbs energy of folding of aqueous homopolypeptide systems. The model takes into consideration both the entropy contribution and the enthalpy contribution of folding homopolypeptide chains in aqueous solutions. The entropy contribution is derived from the Flory-Huggins expression for the entropy of mixing. It accounts for the entropy loss in folding a random-coiled polypeptide chain into a specific polypeptide conformation. The enthalpy contribution is derived from a molecular segment-based Non-Random Two Liquid (NRTL) local composition model [H. Renon and J. M. Prausnitz (1968) AIChE J., Vol. 14, pp. 135-142; C.-C. Chen and L. B. Evans (1986) AIChE J., Vol. 32, pp. 444-454], which takes into consideration of the residue-residue, residue-solvent, and solvent-solvent binary physical interactions along with the local compositions of amino acid residues in aqueous homopolypeptides. The UNIFAC group contribution method [A. Fredenslund, R. L. Jones, and J. M. Prausnitz (1975) AIChE J., 21, 1086-1099; A. Fredenslund, J. Gmehling, and P. Rasmussen (1977) Vapor-Liquid Equilibrium Using UNIFAC, Elsevier Scientific Publishing Company, Amsterdam], developed originally to estimate the excess Gibbs energy of solutions of small molecules, was used to estimate the NRTL binary interaction parameters. The model yields a hydrophobicity scale for the 20 amino acid side chains, which compares favorably with established scales [Y. Nozaki and C. Tanford (1971) Journal of Biological Chemistry, Vol. 46, pp. 2211-2217; E. B. Leodidis and T. A. Hatton (1990) Journal of Physical Chemistry, Vol. 94, pp. 6411-6420]. In addition, the model generates qualitatively correct thermodynamic constants and it accurately predicts thermodynamically favorable folding of a number of aqueous homopolypeptides from random-coiled states into alpha-helices. The model further facilitates estimation of the Zimm-Bragg helix growth parameter s and the nucleation parameter sigma for amino acid residues [B. H. Zimm and J. K. Bragg (1959) Journal of Chemical Physics, Vol. 31, pp. 526-535]. The calculated values of the two parameters fall into the ranges suggested by Zimm and Bragg.     

Unique biphasic band shape of the visible circular dichroism of bacteriorhodopsin in purple membrane: Excitons,multiple transitions or protein heterogeneity?

Over a decade and a half ago, when the first visible membrane suspension circular dichroic (CD) spectrum of the purple membrane (PM) was presented, two mechanisms were proposed to account for the observed biphasic shaped CD band: (a) excitonic interactions among the retinals of the sole protein bacteriorhodopsin (bR) in the crystalline structure of the PM, and (b) combination of CD bands with opposite rotational strengths due to a retinal-apoprotein heterogeneity of the bR molecules or due to two possible close-lying long-wavelength transitions of the retinal of the bR with opposite rotational strengths. Since that time, an impressive body of experimental and theoretical evidence has been accumulated, mostly consistent with an exciton model but many at serious odds with any heterogeneity or multiple transition model. Recently, a number of articles have appeared reporting analyses of new experimental observations which are proposed to cast serious doubts on the viability of the exciton model, and therefore, may revive the heterogeneity or multiple transition model as an explanation for the unique shape of the CD band of the PM. The intent of this article is to demonstrate that if all observations found in literature baring on this question are considered in toto and in a consistent manner, they can be interpreted without exception by excitons, and furthermore, that there is no plausible evidence available to warrant the revival of the heterogeneity or multiple transition model as an explanation for the unique shape of the biphasic CD band of the PM.     

Separation of fructose and glucose mixture by zeolite Y

Na-, K-, Ba-, and Ca-Y were employed for the separation of fructose and glucose in an adsorption column. Effects of temperature, solvent flow rate, amount of mixture injection, and exchangeable cations on the separation were investigated. Efficiency of separation was used as a criterion to characterize the effectiveness of the separation. The transport and kinetic parameters for the column separation were also presented. From simple pulse experiments and moment analysis, the obtained process information of equilibrium and dynamic parameters might be used to design, operate, and control the separation column. (c) 1992 John Wiley & Sons, Inc.     

Synthesis of lysine-containing fragments of the Proteus mirabilis O27 O-specific polysaccharide and neoglycoconjugates therefrom.

Amide-linked lysine mono- and di-uronic acid fragments of the O-specific polysaccharide from P. mirabilis O27 have been synthesised. N epsilon-Boc-L-lysine tert-butyl ester was condensed with 2-azidoethyl glycosides of glucuronic acid and beta-D-GlcpNAc-(1----3)-beta-D-GlcpA. Transformation of the products into 2-acrylamidoethyl glycosides, followed by deprotection using trifluoroacetic acid, gave the target monomers that were converted into high-molecular-weight copolymer-type neoglycoconjugates.     

Heterogeneity of natural killer cell subsets in NK-1.1+ and NK-1.1- inbred mouse strains and their progeny.

The 4LO3311 monoclonal antibody, a new NK-specific reagent recently produced in our laboratory, reacts with spleen cells of 11 mouse strains, most of which do not express the NK-1.1 alloantigen recognized by the PK136 mAb. Among positive strains, the susceptibility of spleen cells to the complement-dependent NK-inhibiting activity of the 4LO3311 mAb was variable but independent of the initial NK cell activity level of cells tested. This property was furthermore not modified after poly(I:C) stimulation. The susceptibility of spleen cells to the in vitro 4LO3311 mAb plus complement treatment is however influenced by the absolute number of 4LO3311+ cells as well as by the density of the corresponding alloantigen at the cell surface. Moreover, it was established that the strain-related variations observed also depended upon the relative size of the 4LO3311 cell subset within the lytic NK cell population. Indeed, when C3H (NK-1.1-4LO3311+) mice were inoculated with the 4LO3311 mAb, the lytic activity of their spleen cells was almost unaltered but 4LO3311-reactive cells were no longer detected in the spleen of treated animals and remaining NK cells were totally resistant to the in vitro 4LO3311 mAb plus complement treatment. These findings indicate that the 4LO3311 mAb identifies a subset rather than all NK cells, even in a NK-1.1- strain. Since a NK-1.1-unreactive cell subset was identified in NZB (NK-1.1+4LO3311-) mice inoculated with the PK136 mAb, the NK-1.1+ cell population is not necessarily responsible for all the splenic NK cell activity in all NK-1.1+ strains. In B6C3F1 hybrid mice, a relatively large subset of NK-1.1-4LO3311- cells was found in addition to those expressing the NK-1.1, the 4LO3311 alloantigen, or both. According to these results, NK cell heterogeneity should thus be taken as an evolving concept whose resolution appears more and more complex with the identification of new NK-specific reagents.     

Changes in Electrophoretic Patterns of Oocyte Proteins during Oocyte Maturation in Oryzias latipes

Changes in the two-dimensional SDS-electrophoretic patterns of extracts of maturing denuded oocytes of the medaka ( Oryzias latipes ) were surveyed. In oocytes without follicular constituents several proteins became detectable in the area between the acidic and slightly basic proteins on the two-dimensional electrophoretograms, while a few of the protein spots disappeared during the process of oocyte maturation. The former proteins were detected also in oocytes that were induced to mature in vivo without breakdown of the germinal vesicle. Several proteins newly observed in extracts of post-vitellogenic oocytes during maturation after breakdown of the germinal vesicle were also identified by two-dimensional electrophoresis. Of several proteins that exhibited noticeable changes in maturing oocytes, only one spot incorporated ¹⁴C-labeled amino acid during maturation, suggesting that post-translational modification of many proteins occurred during oocyte maturation.     

Catalysis by alpha-chymotrypsin entrapped into surface-modified polymeric nanogranules in organic solvent.

Physicochemical characteristics of previously suggested surface-modified polymeric nanogranules (SMPN) and catalytic and stability properties of alpha-chymotrypsin entrapped into such nanogranules in a nonpolar solvent were investigated in more details. SMPN were obtained by polymerization of an acrylamide/N,N'-methylene-bisacrylamide mixture in a mixed reversed micellar system composed of Aerosol OT [sodium di(2-ethylhexyl)sulfosuccinate] and the polymeric surfactant Pluronic F-108 modified with polymerizable groups, followed by the chromatographic removal of the auxiliary surfactant, Aerosol OT. An optimal solvent system was found providing the required orientation of the polymeric surfactant in starting mixed micelles, i.e. with polar fragments immersed into the micellar interior and apolar fragments protruding into organic solvent. The hydrodynamic diameter of SMPN in benzene solution was estimated by means of quasi-elastic light scattering to be 84 +/- 1 nm. Catalytic and stability properties of alpha-chymotrypsin entrapped into SMPN strongly depended on conditions of preparation of SMPN. The optimal concentration of acrylamide monomers in the micellar interior and hydration degree of starting reversed micelles were found to be 20% by mass and wo = 15, respectively. alpha-Chymotrypsin-containing SMPN were used as a catalyst in the synthesis of N-acetyl-L-tyrosine ethyl ester from N-acetyl-L-tyrosine and ethanol, performed in a membrane reactor.