Electrophoretic variants of alpha 2u-globulin in the livers of adult male rats: a possible polymorphism.

Two-dimensional polyacrylamide gel electrophoresis has been used to examine the microsomal fractions from the livers of 32 adult male Alpk/AP (Wistar-derived) rats for the presence of alpha 2u-globulin variants of differing isoelectric point. Three major such isoelectric variants are described. Different combinations of these three forms were found in the population examined, with one-half of the animals expressing all three variants in approximately equal proportions and with one variant being present in all the animals examined. An understanding of the relevance of such different alpha 2u-globulin profiles to the individual animal must now await the assignment of a biological role for alpha 2u-globulin.

Double-beam flying spot scanner for two-dimensional polyacrylamide gel electrophoresis.

The construction of a double-beam photometer in which the light source is a cathode ray oscilloscope is described. The light spot from the oscilloscope was focused and reduced in size at the gel plane to give a diameter of less than 0.15 mm and make it possible to scan over a 50 X 59-mm rectangle; using reduced spatial resolution (spot less than 0.2 mm) the area scanned becomes 70 X 90 mm. The light from the CRT was divided into two beams; one was directed through the transparent object to a photomultiplier and the other to a reference photomultiplier. The signals from these two detectors were converted to the logarithm of the ratio by a logging amplifier to give a direct measure of absorbance. Positioning of the spot, control of light intensity, and measurement of absorbance were carried out through an interface to a 16-bit computer. The relationship between measured and actual absorbance was linear over the range of absorbance 0 to 2, which could be raised to 1 to 3 by placing a neutral filter in the reference beam. The system generated an image containing 256 X 256 pixels in about 5 min, the scanning speed was determined by the persistence time of the P4 phosphor on the cathode ray tube, and faster scans can be made using A6 phosphor.

The temperature dependence of the fluorescence decay of low-density lipoproteins.

The decay of the intrinsic tryptophan fluorescence of low-density lipoproteins (LDL) is measured using a picosecond laser system with an excitation wavelength of 295 nm. The emission wavelengths were observed at 320, 340 and 360 nm. The measurements were performed in the temperature range 10-45 degrees C. The fluorescence decay data are analysed in terms of: (i) three exponentials; (ii) a continuous distribution of exponentials; and (iii) the average decay time. The results indicate that the tryptophans are in a non-polar environment and that the fluorescence is not affected by phase transitions found to occur in the lipid core of the LDL molecule.

An optical comparator for measuring two-dimensional polyacrylamide gel electrophoresis records using an on-line microcomputer.

A comparator which makes it possible to compare two wet gels or photographic negatives or autoradiograms through a flickering light system has been built. The system consists of two special-purpose projectors which combine the images on a digitizing platform. When the lights are switched On and off out of phase, the positions of the common components remain unchanged, whereas those that are spatially displaced appear to jump from side to side and those present in one image but not the other switch on and off. This produces a flickering image in which differences are readily seen. Commercial camera lenses were used to construct the projectors and the overall specifications for the system are given. The coordinates of both the displaced components, as well as the selected standards from the two images, are digitized and entered automatically into an on-line microcomputer. By using an iterative procedure for collecting records from several superimposable records of the gel, it is possible to compensate for the lack of total reproducibility over the whole gels. These coordinates are then normalized and superimposed on a master map through a television display using a curser to adjust the coordinates. The whole procedure can be repeated for many gels using a common reference gel in the comparator, and the result is a set of normalized coordinates which can be plotted on a single map to provide a final record of the experiments.

Phospholipid-nucleoside conjugates. The aggregational characteristics and morphological aspects of selected 1-beta-D-arabinofuranosylcytosine 5'-diphosphate-L-1,2-diacylglycerols.

1-beta-D-Arabinofuranosylcytosine 5'-diphosphate-1,2-diacylglycerols have previously been shown to be promising candidates as prodrugs of the clinically useful antileukemic agent 1-beta-D-arabinofuranosylcytosine. Because of the amphipathic nature of these liponucleotides and the potential that their morphological state may mediate their biological activity, it was necessary to undertake detailed studies of their aggregational and morphological characteristics. When samples of 1-beta-D-arabinofuranosylcytosine 5'-diphosphate-L-1,2-diacylglycerols (containing either dimyristoyl, dipalmitoyl or distearoyl fatty acid side chains) were prepared in buffered saline solutions using sonication methods, the morphological nature of the resulting aggregate was shown to be related to temperature and the length of the side chain. When sonicated at low temperatures all the above-mentioned derivatives gave turbid solutions containing large bilayer sheets. As the temperature was raised, a transition temperature was reached at which a stable three-dimensional cross-linked network of small interlocking bilayer stacks was formed. This turbidity transition temperature was directly related to the chain length of the fatty acid side chain. Sonication at temperatures close to this turbidity transition temperature produced small disc-shaped micellar structures. These micelles were shown to exist in another aggregational equilibrium consisting of a stacking-destacking process, the position within this equilibrium being dependent upon the concentration. In contrast, a sample of 1-beta-D-arabinofuranosylcytosine 5'-diphosphate-L-1,2-dioleoylglycerol (which contains an unsaturated carbon-carbon bond in each of the fatty acid side chains) was shown to give a multilamellar liposome structure when sonicated in buffered saline at temperatures above its turbidity transition temperature.

Detection of proteins in human amniotic fluid using two-dimensional gel electrophoresis.

Samples of human amniotic fluid from 48 pregnancies were examined by high resolution two-dimensional gel electrophoresis and the positions of the major peptides were mapped. Many of the proteins in amniotic fluid also occur in adult and fetal serum. Three regions in the amniotic fluid maps could be defined containing peptides which were not found in adult or fetal serum. The concentration of these peptides is variable and their origin is as yet unknown. Many differences were seen between adult and fetal serum proteins.

Some measurements of the shape and hydrodynamic properties of yeast phosphoglycerate kinase (E.C.2.7.2.3).

Using values obtained for sedimentation and diffusion constants the relative mass of phosphoglycerate kinase was calculated to be 45 800 +/- 1700. This value is higher than was previously estimated and the difference is thought to be caused by contamination of earlier crystalline preparations. Using the coordinates from X-ray crystallography it was found possible to calculate a frictional ratio for a linear dumb-bell (1.115) which compared well with the ratio calculated from diffusion (1.114 +/- 0.033). Since the calculated ratio for a bent molecule was 1.020 the natural state of the molecule in solution is essentially linear. From the concentration dependence of sedimentation and diffusion was calculated the effective interactive radius which resembles haemoglobin in its relationship to the molecular radius.

Malate dehydrogenase of the cytosol. Preparation and reduced nicotinamide-adenine dinucleotide-binding studies.

1. Two methods of preparing pig heart soluble malate dehydrogenase are described. A slow method yields an enzyme composed of three electrophoretically separable subforms. The more rapid method reproducibly gives a high yield of an enzyme that consists predominantly of the least acid subform. 2. The A(1%) (1cm) of the protein was redetermined as 15 at 280nm. By using this value the enzyme molecule was found to contain two independent and indistinguishable NADH-binding sites in titrations with NADH. 3. No evidence was found for the dissociation of the enzyme in the concentration range 0.02-7.2mum. 4. l-Malate (0.1m) tightened the binding of NADH to both pig and ox heart enzyme (2-fold), but, in contrast with the report by Mueggler, Dahlquist & Wolfe [(1975) Biochemistry14, 3490-3497], did not cause co-operative interactions between the binding sites. 5. Fructose 1,6-bisphosphate had no effect on the binding of NADH to the pig heart enzyme, but with the ox heart enzyme the NADH is slowly oxidized. This slow oxidation explains the ;sigmoidal' binding curves obtained when NADH was added to ox heart soluble malate dehydrogenase in the presence of fructose 1,6-bisphosphate [Cassman (1973) Biochem. Biophys. Res. Commun.53, 666-672] without the postulate of site-site interactions. 6. It is concluded that neither l-malate nor fructose 1,6-bisphosphate could in vivo modulate the activity of soluble malate dehydrogenase and alter the rates of transport of NADH between the cytosol and the mitochondrion. 7. Details of the preparation of soluble malate dehydrogenase have been deposited as Supplementary Publication SUP 50080 (8 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1978) 169, 5.

A comparison of the association of yeast phosphoglycerate mutase (EC2.7.5.3) with that of haemoglobin. An ultracentrifuge study.

1. Previous work showed that yeast phosphoglycerate mutase (EC 2.7.5.3) has a mol.wt. of between 107000 and 110000. Preliminary examination showed that at dilutions less than 0.1 g/1 the enzyme dissociated into its subunits. 2. This dissociation was quantitatively examined by both equilibrium and velocity centrifugation. 3. The mathematical analysis of the equilibrium records was tested against oxyhaemoglobin in a variety of ionic strengths and at two temperatures. 4. The estimated L2,4 (interaction coefficient) for oxyhaemoglobin generally agreed with published values except at 6 degrees C in 0.9 M-NaCl, when it was 2.5 times larger than the published value. 5. Statistical analysis of ultracentrifugal-equilibrium experiments showed that the predominant reaction for phosphoglycerate mutase was monomer in equilibrium tetramer, to give an L1,4 of 40.3+/-23.4 (S.D.)1(3)-g(-3) at 20 degrees C. Decreasing the temperature decreased the association to given an enthalpy of between 40 and 60kJ/mol. 6. Analysis of velocity experiments carried out with concentrations varying from 0.3 to 17 g/1 gave an L1,4 of 3111(3)-g(-3). Incorporating errors from estimating S20,w into the analysis showed that this estimate could range from 893 to 1421(3)-g(-3). 7. The concentration-dependence of S20,w was 0.95 litre-g-1 and s020,w for the tetramer was 66.9ps. 8. These results are discussed in relation to the activity of the enzyme.

Collecting and processing records from the ultracentrifuge in "real-time" using an on-line computer.

An analytical system consisting of an analytical cantrifuge coupled 'on-line' to a computer was assembled and tested. Collection of records from up to 9 solutions was achieved through programmes which sum readings to reduce noise as well as controlling the positioning of the scanner. With this system it was found that the limit on accuracy for molecular weights at concentrations less than 0.01 g cm-3 was +/- 3% estimated from sedimentation equilibrium experiments. The same system was used to collect records for similar concentrations from velocity experiments by employing a scanning schlieren. In this case the accuracy in estimating sedimentation coefficients was similar to those found when measuring photographs. Since the collection yields detailed information about the shape of the sedimenting boundary, the centroids of the boundary were routinely computed by second moment analysis rather than relying on the position of the maximum of the schlieren peak. In the same analysis estimates of diffusion coefficients were made routinely by calculating corrected height/area ratios for each scan. These calculations were made during the real-time of the experiment, so making available molecular parameters rather than records which must be evaluated some time after stopping the experiment.

The association of yeast phosphoglycerate kinase (EC 2.7.2.3).

1. A mol.wt. of 40030 +/- 830 has been estimated for phosphoglycerate kinase in concentrations less than 0.1 g/100 cm3 comparing favourably with expected values from X-ray diffraction measurements by 10% lower than the previously reported molecular weights made at higher concentrations. 2. The so20w, was estimated to be 3.12(+/-0.02)x10(-13)s and the coefficient had a low concentration dependency giving a g value (concentration-dependency) of 2.3 +/- 1.6cm3 .g-1. This agrees with previous qualitative observations. 3. By using fluctuation-intensity spectroscopy, the D20,w was estimated to be 7.4(+/-0.2)x10(-11)m2.s-1, and this was indistinguishable from the D20,w calculated from ultracentrifuge results. The water of hydration was estimated to be 0.46 g/g of protein. 4. It is inferred from the estimates that phosphoglycerate kinase associates with an interaction coefficient at 20 degrees C for monomer/dimer of between 10 and 12 cm3.g-1. 5. The ratio of molecular asymmetry (a/b) was estimated to be 2.5+/-0.2 from the values of D20,w and water of hydration. This compares favourably with the ratio from the overall dimensions estimated from X-ray diffraction measurements.

The statistical estimation of molecular weights from normal and difference ultracentrifuge boundaries.

1. To determine molecular weights from boundary data taken from a sedimentation velocity experiment in an ultracentrifuge, the parameter s/D must be estimated. This can be obtained by using non-linear statistical methods to fit a mathematical model [the Fujita & MacCosham (1959) equation] to the results. 2. The statistical method chosen was the simplex method of Nelder & Mead (1965), which was found to be ideal for this problem. Internal errors were calculated at the end of the search for the minimum in the residuals, but in general these errors were found to not represent the overall true error of the experiment. 3. Calculations of molecular weights of myoglobin showed that instabilities at low concentrations of protein (less than 0.8mg/ml) disturbed the calculation of s/D. If 1% (w/v) sucrose was included in the solvent, these instabilities were decreased, and extrapolating to infinite time the linear function of s versus 1/(time) gave an acceptable value for s with an error of +/-4.8%. The estimates of the molecular weights were less well-defined and the mean value was low by 8%, with an estimated error of the mean of +/-3%. The conclusion was that vibration was responsible for the instabilities without sucrose. 4. The Fujita-MacCosham equation can be extended to make it possible to estimate ratios of sedimentation and molecular weights for difference boundaries. Tests using two solutions of orosomucoid in which a 2% decrease in velocity of one boundary was achieved by adding a calculated quantity of sucrose showed that the analysis gave realistic values for the two ratios, and the error for the ratio of sedimentation coefficients was +/-10%. The error was larger for the estimated ratio of the molecular weights, but the analysis gave the expected value for the ratio.

The thermal polymerization of orosomucoid.

1. Orosomucoid was prepared from the urine of a nephrotic patient and polymerized by heating it in a range of salt concentrations at pH4.1. 2. Heating at low ionic strengths produced a ;chain' polymer of indefinite length but having the same width as the diameter of the monomer (5.0nm.). Similar treatment in high ionic strengths also produced a spherical (;ball') polymer of limited diameter (14.8nm.). 3. The size and shape of both polymers were determined from ultra-centrifuge, gel-filtration and electron-microscope results. The results suggest that eight monomer units condense to form the ball polymer. 4. Heating orosomucoid at pH1.8 hydrolysed the N-acetylneuraminic acid off the molecule; only chains could then be formed, even in high ionic strengths. 5. Both polymers were stable under normal conditions but could be depolymerized in 3m-guanidine hydrochloride. The monomer could be repolymerized on heating: the ;chain monomer' only formed chains at all ionic strengths, but the ;ball monomer' was indistinguishable from the original monomer in its immunological properties and polymerization reaction.