Determination of sedimentation coefficients for small peptides.

Direct fitting of sedimentation velocity data with numerical solutions of the Lamm equations has been exploited to obtain sedimentation coefficients for single solutes under conditions where solvent and solution plateaus are either not available or are transient. The calculated evolution was initialized with the first experimental scan and nonlinear regression was employed to obtain best-fit values for the sedimentation and diffusion coefficients. General properties of the Lamm equations as data analysis tools were examined. This method was applied to study a set of small peptides containing amphipathic heptad repeats with the general structure Ac-YS-(AKEAAKE)nGAR-NH2, n = 2, 3, or 4. Sedimentation velocity analysis indicated single sedimenting species with sedimentation coefficients (s(20,w) values) of 0.37, 0.45, and 0.52 S, respectively, in good agreement with sedimentation coefficients predicted by hydrodynamic theory. The described approach can be applied to synthetic boundary and conventional loading experiments, and can be extended to analyze sedimentation data for both large and small macromolecules in order to define shape, heterogeneity, and state of association.     

The use of a transesterification technique to distinguish between certain neuraminidase resistant epithelial mucins.

The effects of potassium hydroxide and soidum methoxide treatments upon the Alcian blue staining and neuraminidase lability of certain neuraminidase resistant epithelial mucins have been studied. The results were interpreted as indicating that while the mucins of rat colon and rabbit Brunner's gland contain only 4-0-acetyl sialic acid, human colonic epithelial mucins may contain some sialic acid with esters at the C1 carboxyl group.     

Limiting sedimentation and diffusion coefficients of polyelectrolytes. The charge effect

General expressions relating the sedimentation and diffusion constants of polyelectrolytes to the friction properties of ionic components in multicomponent systems have been derived. A quantitative explanation of the charge effect with respect to the addition of a low-molecular-weight salt is given; special attention is laid on limiting laws according to the salt-to-polymer concentration ratio. The theory is applied to a simple model, the infinitely long rod-like model, to illustrate the peculiar aspect of the charge effect in presence of salt.     

The use of a transesterification technique to distinguish between certain neuraminidase resistant epithelial mucins

Summary The effects of potassium hydroxide and sodium methoxide treatments upon the Alcian blue staining and neuraminidase lability of certain neuraminidase resistant epithelial mucins have been studied. The results were interpreted as indicating that while the mucins of rat colon and rabbit Brunner's gland contain only 4-O-acetyl sialic acid, human colonic epithelial mucins may contain some sialic acid with esters at the C₁ carboxyl group. Supported by the Medical Research Council of Canada Grant MA 4376.     

The use of phosphopeptides to distinguish between protein phosphatase and acid/alkaline phosphatase activities: opposite specificity toward phosphoseryl/phosphothreonyl substrates.

The four main classes of protein phosphatases (PP-1, 2A, 2B and 2C), although differing in their ability to dephosphorylate phosphopeptide substrates, invariably display a marked preference toward phosphothreonyl peptides over their phosphoseryl counterparts. Conversely, all the acidic and alkaline phosphatases tested so far dephosphorylate phosphoseryl derivatives far more readily than phosphothreonyl ones. This opposite behaviour provides a criterion for discriminating between protein dephosphorylating activity due to authentic protein phosphatases as compared to nonspecific acid and/or alkaline phosphatases. In particular the phosphothreonyl peptides RRATPVA and RRREEETPEEEAA appear to be especially suited for detecting the activity of PP-2C and PP-2A, since they are hardly dephosphorylated by acid and alkaline phosphatases. Conversely, the phosphoseryl peptides SPEEEEE and RRASPVA can provide a sensitive evaluation of the majority of acid and alkaline phosphatases, while being refractory to protein phosphatases.     

Acetylcholine and epibatidine binding to muscle acetylcholine receptors distinguish between concerted and uncoupled models.

The muscle acetylcholine receptor (AChR) has served as a prototype for understanding allosteric mechanisms of neurotransmitter-gated ion channels. The phenomenon of cooperative agonist binding is described by the model of Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118; MWC model), which requires concerted switching of the two binding sites between low and high affinity states. The present study examines binding of acetylcholine (ACh) and epibatidine, agonists with opposite selectivity for the two binding sites of mouse muscle AChRs. We expressed either fetal or adult AChRs in 293 HEK cells and measured agonist binding by competition against the initial rate of 125I-alpha-bungarotoxin binding. We fit predictions of the MWC model to epibatidine and ACh binding data simultaneously, taking as constants previously determined parameters for agonist binding and channel gating steps, and varying the agonist-independent parameters. We find that the MWC model describes the apparent dissociation constants for both agonists but predicts Hill coefficients that are far too steep. An Uncoupled model, which relaxes the requirement of concerted state transitions, accurately describes binding of both ACh and epibatidine and provides parameters for agonist-independent steps consistent with known aspects of AChR function.     

The calculation of sedimentation coefficients in vertical rotors

A program for the calculation of sedimentation coefficients of molecules centrifuged in sucrose in vertical rotors has been developed. The program has been tested with both protein and RNA of known sedimentation coefficients. The preparation can accept any shape of gradient in the 0–70% sucrose and any temperature in the range of 0–60°C. The program can be used with any vertical rotor for which the dimensions are known.     

The effect of rotor velocity on sedimentation coefficients

Sedimentation-coefficient measurements on human IgG, fibrinogen and alpha(2)-macroglobulin and pig thyroglobulin were made at rotor velocities of 15220-59780 rev./min and 25.0 degrees C in capillary-type synthetic-boundary cells or ordinary cells. At the lowest velocity, IgG and fibrinogen gave results several per cent higher than at other velocities, whereas alpha(2)-macroglobulin and thyroglobulin gave values only about 1.5% higher. This behaviour of IgG and fibrinogen is attributed primarily to imperfect initial boundaries.     

Anomalies in sedimentation. IV. Decrease in sedimentation coefficients of chains at high fields

A theory is presented for the decrease of sedimentation coefficient at high centrifugal fields recently reported for samples of DNA by Rubenstein and Leighton and others. The theory uses the model of a chain of beads and springs to represent the molecule. Kirkwood's approximation is used for the sedimentation coefficient. The decrease in sedimentation coefficient with field comes about as a result of the uneven frictional forces in the chain, which on the average are less on segments near the center of the chain than on those near the ends. As a result the ends of the chain tend to drag behind the center, and the average intersegment distances are increased; consequently the hydrodynamic shielding of one segment by another is reduced, and the average friction is increased. The effect is thus characteristic of single molecules; intermolecular interaction is not involved. The sedimentation coefficient, S, varies as a power series in a parameter y that measures the distortion produced by the uneven friction: S = S⁰(1?D₂y² + D₄y⁴ ? ·). where S⁰ is the limiting value of S at zero centrifugal field and D₂ and D₄ are constants; y is proportional to the cen speed squared tunes the molecular weight squared divided by S⁰. It has been observed that the effects of centrifuge speed on S are negligible below certain critical values of the speed and molecular weight, but increase dramatically immediately above these values; this follows naturally from the high powers of the speed and molecular weight that appear in the above equation.     

Monoclonal antibodies that distinguish between subspecies of human interferon-alpha and that detect interferon oligomers

Monoclonal antibodies to human interferons (HuIFN) of the alpha-class have been prepared by screening against 125I-labeled IFN in a rapid liquid-phase radioimmunoassay. All of the six antibodies produced react with HuIFN-alpha 2 and with some components of HuIFN-alpha N (Namalwa); three of the antibodies also bind HuIFN-alpha 1, and these either do not bind or bind very weakly the 25K component of Namalwa. Reaction of the antibodies with IFN components blotted onto nitrocellulose after separation on reducing gels suggests that two of the antibodies are against conformational determinants, whereas the epitopes recognized by the other antibodies are not destroyed by reduction or SDS treatment; these antibodies can be used to detect the presence of oligomers in IFN preparations. From the reaction of the antibodies with different alpha-IFN in immunoblots, in an antiviral assay, and in an ELISA, it was concluded that at least five different epitopes are recognized by the six antibodies, only one of which is non-neutralizing.     

The use of thioglycolate to distinguish between 3'' AP (apurinic/apyrimidinic) endonucleases and AP lyases.

Addition of thioglycolate and DEAE-Sephadex chromatography were used to analyze the cleavage of the C(3')-O-P bond 3' to AP (apurinic/apyrimidinic) sites in DNA and to distinguish between a mechanism of hydrolysis (which would allow the nicking enzyme to be called 3' AP endonuclease) or beta-elimination (so that the nicking enzyme should be called AP lyase). For this purpose, DNA labelled in the AP sites was first cleaved by rat-liver AP endonuclease, then with the 3' nicking catalyst in the presence of thioglycolate and the reaction products were analyzed on DEAE-Sephadex: deoxyribose-5-phosphate (indicating a 3' cleavage by hydrolysis) and the thioglycolate:unsaturated sugar-5-phosphate adduct (indicating a cleavage by beta-elimination) are well separated allowing to eventually easily discard the hypothesis of a hydrolytic process and the appellation of 3' AP endonuclease. We have shown that addition of thioglycolate to the unsaturated sugar resulting from nicking the C(3')-O-P bond 3' to AP sites by beta-elimination is an irreversible reaction. We have also shown that the thioglycolate must be present from the beginning of the reaction with the nicking catalyst to prevent the primary 5' product of the beta-elimination reaction from undergoing other modifications that complicate the interpretation of the results.     

The use of analytical sedimentation velocity to extract thermodynamic linkage

For 25 years, the Gibbs Conference on Biothermodynamics has focused on the use of thermodynamics to extract information about the mechanism and regulation of biological processes. This includes the determination of equilibrium constants for macromolecular interactions by high precision physical measurements. These approaches further reveal thermodynamic linkages to ligand binding events. Analytical ultracentrifugation has been a fundamental technique in the determination of macromolecular reaction stoichiometry and energetics for 85 years. This approach is highly amenable to the extraction of thermodynamic couplings to small molecule binding in the overall reaction pathway. In the 1980s this approach was extended to the use of sedimentation velocity techniques, primarily by the analysis of tubulin-drug interactions by Na and Timasheff. This transport method necessarily incorporates the complexity of both hydrodynamic and thermodynamic nonideality. The advent of modern computational methods in the last 20 years has subsequently made the analysis of sedimentation velocity data for interacting systems more robust and rigorous. Here we review three examples where sedimentation velocity has been useful at extracting thermodynamic information about reaction stoichiometry and energetics. Approaches to extract linkage to small molecule binding and the influence of hydrodynamic nonideality are emphasized. These methods are shown to also apply to the collection of fluorescence data with the new Aviv FDS.     

Ultracentrifugal studies in capillary cells. I. Determination of sedimentation coefficients

A technique has been developed which is based on transport method equations and allows determination of sedimentation coefficients using less than 5 ng of a biological active principle. Glass capillaries of 0.30 mm inside diameter and 3.0 mm length are used as sedimentation cells. About 200 nl of pure or impure solutions with concentration as low as 0.05 mg/ml are ultracentrifuged in a swinging-bucket rotor with a conventional preparative ultracentrifuge. The capillary microcells are easily sectioned after centrifugation through a plane previously marked on the glass surface. The technique was successfully extended to the use of larger glass capillaries, up to 1.25 mm inside diameter, and plastic centrifuge tubes of 5.0 mm diameter and 0.40 ml capacity. The method has been experimentally verified with proteins and protein-polysaccharides of known sedimentation constants.