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PubMed1976

A television scanner for the ultracentrifuge. II. Multiple cell operation.

The "Optical Multichannel Analyzer" (OMA) is a commercially available instrument that with the absorption optical system of the ultracentrifuge, provides an entire 500 channel intensity profile of a cell in real time. With its own analog-todigital converter, the OMA integrates a selectable number of 32.8 msec scans to provide a time-averaged image in digital form. This paper describes an interface-controller for operation of the OMA with single- and double-sector cells in multi-cell rotors, simulating double-beam measurement required for absorbance determinations. The desired sector is selected by "gating" the intensifier stage of a "Silicon Intensified Target" vidicon (SIT) used as the light detector. The cell location in the rotor and the position of the gate relative to the cell centerline is obtained from a phase-locked loop circuit which divides each rotation of the rotor into 3600 parts independent of rotor speed. (This circuit employed with photo-multiplier scanners would select the gate position for integration of photomultiplier pulses.) From examination of appropriate signals with an oscilloscope, it was verified that gate positions and widths are located with an accuracy of 0.1degree or better and with a precision of +/- 0.1 mus. The light intensity profile for any desired cell can be examined in "real time", even during acceleration of the rotor. Additional circuits employing a 10 MHz crystal clock 1) control the automatic collection of data for all sectors in multicell rotors at digitally selected time intervals, 2) display the rotor speed, and 3) indicate the elapsed time of the experiment. Constructed but not tested are additional circuits for pulsing a laser into the absorption or Rayleigh optical system. The accuracy of the pulsed SIT has been demonstrated by measurement of absorbances of solutions and also by sedimentation equilibrium experiments with myoglobin. The estimated error is 0.003 for absorbances ranging from 0 to 1. The interface-controller operates extremely well, but problems related to the pulsed SIT (optimum gate position relative to the sector opening shape of high-voltage pulse, slight pincushion distortion) require more work.

Autoanalysis↗

PubMed1976

MaD, an automated precise analytical ultracentrifuge scanner system.

The various hardware components of this scanning system, a mechanical split-beam scanner with its photomultiplier measuring slit, minimum analog electronics and an important digital part (computer, interface), are intimately interwoven by an elaborate software. A high degree of automation and sophistication is thus reached. On the one hand, all the photoelectrons coming from each light pulse are integrated. The result, after conversion to digital form enters the computer; a high precision is thus reached: 10(-5) absorbance unit (A.U.) under very favorable conditions, and 10(-4) A.U. for conventional sedimentation studies. On the other hand, during its motion along the image, the precise slit position is permanently known by the system: the sector image is thus segmented into precisely defined zones, inside which the average absorbancies are determined, punched, plotted and printed. New and very useful utilizations and improvements of analytical ultracentrifugation are now possible: the fixed radius mode, permits precise s determination by following the radial dilution; s can be precisely measured by time difference curves even when the solution absorbancy is comparable to the base line variations (0.01 A.U.); round trip analyses permit very long analyses without the absorbancies distributions distorsions due to very long one-way scans during sedimentations runs; reductions of the effect of the stray light by a factor of ten. This system has been in routine use for four years.

Autoanalysis↗

PubMed1976

Volume of reaction by the Archibald ultracentrifuge method (lobster hemocyanin).

Samples of lobster hemocyanin (Homarus americanus) under conditions of reversible reaction between whole (25 S) and half (17 S) molecules have been subjected to accurately known nitrogen pressures in analytical ultracentrifuge cells. A modified pressurization chamber of the type developed by Schumaker and colleagues has been constructed for this purpose. The molecular weight was then determined at the top (liquid-gas) meniscus, by means of the Archibald method. The logarithmic dependence upon pressure of the derived equilibrium constant then gave directly the volume of reaction. Experiments were performed in veronal-citrate buffers at pH 8, where the molar volume of formation of whole (dodecameric) molecules from half molecules appears to be negative, and at pH 8.46 in veronal-citrate buffer in the presence of 0.003 molar free calcium ion, where the molar volume of formation was estimated to be + 390 cm3/mole. In glycine-sodium hydroxide buffer at pH 9.6 containing 0.0047 molar free calcium, the molar volume of formation of whole molecules was estimated to be +120 +/- 70 cm3, corresponding to an estimated difference in partial specific volume between whole molecules and half molecules of only 1.3 (10)-4cm3/gram. The correctness of the sign of this value in glycine buffer has been verified by pressure-jump light-scattering experiments.

Animals↗

PubMed1979

Ultracentrifugation evidence for a somatostatin-binding protein in serum.

Using a technique of high speed centrifugation of serum and a well validated immunoassay for the measurement of serum somatostatin-like immunoreactivity, we have demonstrated that somatostatin, unlike other peptide hormones, appears to sediment with large molecular weight proteins. When synthetic somatostatin of increasing concentration was incubated with serum prior to ultracentrifugation, a linear plot of concentration of somatostatin added against concentration sedimenting (or apparently bound to protein) revealed an association curve. These data provide further evidence for the existence of a serum-binding protein for somatostatin.

Adult↗

PubMed1976

The molecular weight of aspermatogenic antigen as determined by equilibrium ultracentrifugation.

The extraction of antigen from testis and sperm is described. This antigen, when administered in microgram amounts in a single injection, induces aspermatogenesis in 100% of guinea pigs so treated. The deletion of the seminiferous cell line is specific (no cross-reactions) and provides a tool for investigating inhibition of cell replication as well as reproductive control. The antigen is a polypeptide-polysaccharide complex and has been determined to have a molecular weight of 10,520, using the equilibrium ultracentrifugation method.

Animals↗

PubMed1978

Comparative studies of the ethynyl estrogens used in oral contraceptives. VII. Effects with and without progestational agents on ultracentrifugally fractionated plasma lipoproteins in humans, baboons, and beagles.

Ethynyestradiol and mestranol, in doses ranging from 50 to 100 microgram/day, were given to women in 21-day cycles; baboons and beagle dogs received 1 and 4 microgram/kg/day in a similar regimen. After a number of such cycles, megestrol acetate, norethindrone acetate, or dl-norgestrel was given concomitantly. Protein, cholesterol, triglyceride, and phospholipid levels were determined in total plasma and in ultracentrifugally separated lipoprotein fractions. Over the dosage range studied, the effects of the two kinds of estrogen were indistinguishable. Except for human total plasma triglyceride, no dose-related differences were observed. The lowering of serum protein and the increase in cholesterol induced by estrogen were more pronounced in baboons and beagles than in human subjects. The cholesterol-depressing effect of progestational compounds observed in humans was very pronounced in baboons but absent in beagles. In all three species, estrogen increased the lipoprotein fraction cholesterol, except for human low-density lipoprotein cholesterol, which was decreased. Human plasma triglyceride and phospholipid increased on estrogen administration and were decreased by the progestins; in the two animal species, triglyceride is normally very low and the estrogen-induced changes were negligible; the phospholipid rose with estrogen but was unaffected by progestins. In sum, the two animal species show many similarities to, as well as important differences from, the human response of plasma lipids to various contraceptive steroids.

Animals↗

PubMed1976

Regulatory properties of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa. Active enzyme ultracentrifugation studies.

Active enzyme ultracentrifugation studies of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa (EC 1.6.1.1.) show that the enzymatic reaction is catalyzed by a molecular species characterized by an S20,W value of about 34 S, whatever the reduced substrate may be (tri- or diphosphopyridine nucleotide). The filamentous aggregated form of the enzyme (S20,W = 121 S and higher), identified by previous investigations (Cohen, P. T., and Kaplan, N. O. (1970), J. Biol. Chem. 245, 2825-2836; Louie, D. D., Kaplan, N. O., and Mc Lean, J. D. (1972), J. Mol. Biol. 70, 651-664), appears, therefore, to be an inactive species. The physiological implications of the enzyme are discussed. Several lines of evidence lead to the conclusion that the transhydrogenase might act as an essential link between carbohydrate catabolism and the respiratory chain.

Macromolecular Substances↗

PubMed1977

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.

Hemoglobins↗

PubMed1976

[Electronmicroscopic characterisation of human plasma lipoproteins after isolation by zonal ultracentrifugation (author's transl)].

Human plasma proteins were isolated by zonal ultracentrifugation and the fractions obtained examined under the electron microscope. HDL2 had a high degree of uniformity in normal persons, characterised by numerous two-dimensional, hexagonal arrangements. Optical diffraction analysis of HDL2 gave a centre distance of 11.3 nm, individual HDL2 particles being spherical with a diameter of 9.8 nm. HDL3 represented a somewhat less uniform lipoprotein population with a particle diameter between 6.5 and 9.5 nm. LDL had a diameter of 20-25 nm. In patients with hyperlipoproteinaemia type III a third peak was isolated, in addition to the two normal lipoprotein peaks VLDL and LDL. It lies between them and has been called Lp III. VLDL from type III plasma consisted of spherical particles, diameter 31-81 nm, morphologically indistinguishable from normal VLDL. Lp III consisted of a relatively homogeneous spherical lipoprotein population with a diameter of 25-30 nm. LDL consisted of spherical particles whose size was highly homogeneous (18-23 nm diameter), indistinguishable from normal LDL.

Adult↗

PubMed1979

The use of equilibrium density-gradient ultracentrifugation in the isolation and characterisation of glycoproteins with blood group P1 activity from sheep hydatid-cyst fluid.

Equilibrium density-gradient ultracentrifugation in caesium choride and caesium sulphate has been used in the isolation and fractionation of the glycoproteins specific for blood-group P1 from hydatid cyst fluids. The fractions obtained have distinct and systematic differences specifically related to their buoyant densities, chemical compositions and specific-activities for group P1. High levels of specific-activity were maintained over a large range of chemical compositions. The peptide content varied systematically from 2.5% for the densest fraction to 37% for the least dense fraction. The amino acid composition was essentially constant over all fractions. The proportion of glucosamine decreased and the proportions of galactosamine, mannose and glucose increased with increasing peptide content of the fractions. The data presented suggest the present of oligosaccharide side-chains of various lengths and compositions and/or the presence of oligosaccharide side-chains with very different chemical compositions, of which only some are associated with the specificity for group P1. The properties of the glycoproteins from hydatid cyst fluids have been compared with those of the glycoproteins from human ovarian cysts. Although some similarities have been demonstrated there are significant differences.

Amino Acids↗

PubMed1979

Cholesterol in high-density lipoproteins: a comparison between dextran sulfate-magnesium chloride precipitation and preparative ultracentrifugation.

The cholesterol in high density lipoproteins (HDL) has been determined in 140 serum samples after dextran sulfate-magnesium chloride precipitation of the apolipoprotein B-containing lipoproteins. The values correlated well with simultaneously measured values from preparative ultracentrifugation (slope 0.98, r = 0.93). Furthermore, there was a significant correlation in 60 specimen between the dextran sulfate-MgCl2 and heparin-MnCl2-precipitation method.

Chemical Precipitation↗

PubMed1979

Isolation of stable aggregates of IgG by zonal ultracentrifugation in sucrose gradients containing albumin.

Soluble heat aggregates of [125I]IgG (A-IgG) were prepared and separated by gel filtration or by zonal ultracentrifugation, and fractions containing different size aggregates were then analyzed by reultracentrifugation. A-IgG formed single narrow peaks with constant S rates when isolated, stored and analyzed in solution containing 0.5% serum albumin. Thus, stable homogeneous aggregates of IgG of known size can be simply prepared and should prove useful both as a model for immune complexes of specified sizes and as a standard for immune complex assays.

Animals↗

PubMed1976

Determination of physical properties of bovine serum lipoproteins by analytical ultracentrifugation.

Pure samples of low density lipoprotein-one (d 1.019 to 1.039 g/ml), low density lipoprotein-two (d 1.039 to 1.060 g/ml), and high density lipoprotein (d 1.060 to 1.210 g/ml) were isolated from bovine serum and their properties studied in the analytical ultracentrifuge. Approach-to-equilibrium experiments indicated that the lipoprotein classes were homogeneous. Molecular weights of the lipoproteins given by this method (low density lipoprotein-one, 1.14 x 10(6); low density lipoprotein-two, 2.37 x 10(6); high density lipoprotein, .576 x 10(6)) agreed well with those obtained by a high-speed-equilibrium method (1.03 x 10(6), 2.15 x 10(6), and .567 x 10(6)). Linear plots of flotation rate (corrected for viscosity) against the density of the medium were obtained in sedimentation velocity experiments and on extrapolation gave values for the hydrated density of 1.032 g/ml for low density lipoprotein-one, 1.044 g/ml for low density lipoprotein-two, and 1.071 g/ml for high density lipoprotein. The density of the material which would have to be added to high density lipoprotein to give it the physical properties of low density lipoprotein one was less than 1 g/ml, which suggested that it was predominantly lipid.

Animals↗

PubMed1976

Plasma high-density lipoprotein cholesterol concentrations determined after removal of other lipoproteins by heparin/manganese precipitation or by ultracentrifugation.

The widely used heparin/MnCl2 precipitation procedure for determination of plasma high-density lipoprotein cholesterol has been re-examined in light of recent reports that isolated preparations of the lipoprotein are only partly precipitated under the test conditions. In the present study, the procedure as applied to plasma tolerated rather wide variations in heparin and MnCl2 concentrations without significant effects on the assayed values in several plasma pools tested. The procedure was further tested on 129 individual samples by comparison with an ultracentrifugal method in which high-density lipoprotein-cholesterol is assumed to be represented by the cholesterol content of the plasma fraction of relative (to water) density greater than 1.063. Our results indicate that high-density lipoprotein is not precipitated under the test conditions when applied to unfractionated plasma.

Cholesterol↗

PubMed1978

Polymorphism of apolipoprotein E. I. Methodological aspects and diagnosis of hyperlipoproteinemia type III without ultracentrifugation.

Two methods for phenotyping apolipoprotein E are compared. One is based on preparation of VLDL by conventional ultracentrifugation, whereas the other uses heparin/Mg precipitation of VLDG. In principle, the same results were obtained by both methods. However, the group limits for the three different phenotypes Apo E-N, Apo E-ND and Apo E-D were slightly different by the two methods. Phenotype Apo E-D - the phenotype characterizing type III dyslipoproteinemia - is clearly definable by both methods. Hence the precipitation-I.E.F. method for Apo E phenotyping provides a simple tool for genetic and population genetic studies and also for the routine diagnosis of hyperlipoproteinemia type III, based on the only specific marker known for this disease.

Apolipoproteins↗

PubMed1979

Lipoprotein abnormalities in cholestasis. I. Electrophoretic and ultracentrifugal analyses.

The alterations of lipid composition in sera of patients with liver diseases, particularly intrahepatic cholestasis and biliary obstruction, were studied by ultracentrifugation and polyacrylamide-gel disc-electrophoresis of lipoproteins and apoproteins. The elevation of serum cholesterol in intrahepatic cholestasis was greater than in biliary obstruction. The appearance of lipoprotein X in obstructive disease accounted for most of the increased cholesterol. The level of non-lipoprotein X cholesterol in intrahepatic cholestasis was significantly elevated, this being in part ascribed to the appearance of a new class of cholestatic lipoprotein, Slow-migrating HDL. The electrophoretic pattern of lipoprotein in cholestasis was generally characterized by a decrease in alpha band intensity and, in some types of cholestasis, by the appearance of Slow-migrating HDL. In addition, other abnormal lipoproteins exhibiting the characteristics of triglyceride-rich LDL (LP-Y), LP-X-like HDL and LDL-like HDL were found in some cases of intrahepatic cholestasis and biliary obstruction.

Apolipoproteins↗

PubMed1978

Characterization of microtubule protein oligomers by analytical ultracentrifugation.

Samples of microtubule protein prepared by repeated cycles of assembly-disassembly were examined at low temperatures by sedimentation velocity ultracentrifugation. Sedimenting boundaries corresponding to the 6 S tubulin subunit as well as to two oligomeric species with s020,w values of 18.6 S and 30.6 S were observed. The 30 S to 6 S mass ratio varied with total protein concentration, suggesting that a concentration-dependent equilibrium exists between 6 S and oligomeric species of tubulin. A study of the effects of pH on the mass distribution among the species demonstrated that the 6 S species was favored at low pH values (5.8 to 6.5), the 30 S oligomer was favored at moderate pH values (6.5 to 7.4), and the 18 S oligomer was formed in increasing proportions at higher pH values (7.4 to 8.2). Incubation of purified microtubule protein solutions with increasing concentrations of NaCl initially favored conversion of the 30 S oligomer to the 18 S species with further increases in salt concentration resulting in the dissolution of both the 30 S and 18 S oligomers. The depolymerizing effects of high salt concentrations were substantially reversible, providing further evidence for 6 S-oligomer equilibria. The manipulation of the solution variables of pH and ionic strength in a systematic fashion led to the construction of a "phase diagram" for the microtubule protein species which provided a relatively complete description of the mass distribution among the 6 S, 18 S, and 30 S species over a range of physiological and near-physiological solution conditions.

Animals↗

PubMed1977

[Isolation of flagellin antigens of salmonellae by the method of ultracentrifugation and their use for preparation of erythrocytic H-diagnostic agents].

Flagellar preparations were obtained from 6 salmonellae strains by differential ultracentrifugation; they were characterized by morphological, immunochemical, and serological tests. The results of investigations showed that the preparations possessed high serological activity; somatic antigen admixture was insignificant. Flagellins extracted from the flagellae were used to prepare erythrocytic H-diagnostic agents. The results of their use in the examination of sera of healthy persons, and of those suffering from typhoid fever and salmonellosis indicated the expediency of using passive H-hemagglutination for diagnostic purposes.

Antigens, Bacterial↗