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PubMed1975

A computer analysis of electrophoresis and ultracentrifugation patterns.

A program DIANA is designed to fit the scanning curves of electrophoresis and ultracentrifugation patterns with 10-15 overlapping peaks by a sum of Gaussian-like distribitions. The parameters of the distributions are adjusted by minimizing x, using a problem oriented minimizing procedure. The number of molecular components as well as starting values of the positions and the widths of the peaks must be choosen as input for each type of pattern. Programs are written in FORTRAN IV.

Computers↗

PubMed1984

Computation of the sedimentation coefficient, S0(20),w, from the analytical ultracentrifuge run by an interactive program on the Apple II microcomputer.

We have developed a computer program for the calculation of the sedimentation coefficient of a macromolecule from the raw data collected from an analytical ultracentrifuge run. The program is written for the Apple II microcomputer and is capable of calculating the sedimentation coefficient at an infinite dilution from the data collected at varying concentrations.

Computers↗

PubMed1990

Ultracentrifugal analysis of the quaternary structure of the raf repressor from Escherichia coli.

The raf repressor from Escherichia coli regulates the expression of the plasmid-borne raf operon by switching between active and inactive conformational states. Ultracentrifugal analysis of the largely purified repressor proves the DNA-free protein to undergo concentration-dependent dissociation-association. High-speed sedimentation equilibria show that the 72 kDa dimer prevails under meniscus depletion conditions. At intracellular concentrations the 144 kDa dimer-of-dimers is the dominating species. It is suggested that the tetrameric structure of the raf repressor is involved in the recognition of the 18-basepair operator DNA.

Base Composition↗

PubMedDate not supplied

Properties of two pig low density lipoproteins prepared by zonal ultracentrifugation.

Pig plasma lipoproteins were separted into four density classes (very low density, two low density and high density lipoproteins, VLDL, LDL1, LDL2 and HDL respectively) from 670 ml plasma by ultracentrifugation in a continuous density gradient using the Spinco Ti15 zonal rotor. LDL1 and LDL2 were partly characterised. LDL1 and LDL2 are beta-migrating lipoproteins of different size and hydrated density; they are similar to human LDL2 and LDL3 respectively. Pig plasma contains about twice as much LDL1 as LDL2. LDL1 migrates at Sf 4.9 (modal value), and has a mean diameter of 217 A and a modal density of 1.035 g/ml (range 1.03-1.04 g/ml). LDL2 migrates at Sf 1.8 and has a mean diameter of 195 A and a density of 1.050 g/ml. Both lipoproteins are precipitated by heparin and Mn++ or by dextran sulphate and Ca++. The apoproteins of LDL1 and LDL2 are both largely insoluble in 8 M urea solution. When dissolved in 1% sodium dodecyl sulphate solution and electrophoresed on polyacrylamide gel at pH 7.0, the apoproteins of LDL1 and LDL2 formed a pattern of multiple bands of high molecular weight similar to that obtained from the apoprotein of human LDL. Both LDL1 and LDL2 share a major antigen with each other and with VLDL; in this respect again they resemble human LDL. The amino acid compositions of LDL1 and LDL2 are very similar. We concluded that the apoprotein moieties of pig plasma LDL1 and LDL2 are probably identical, and similar to apoprotein B in human serum. Zonal ultracentifugation has proved to be a rapid and effective method for isolating large quantities of these two lipoprotein classes for further metabolic studies. This method allows rapid bulk preparation of lipoproteins, and provides a record of their distribution and quantity in a continuous density gradient.

Amino Acids↗

PubMed1977

Chemical composition of ultracentrifugal fractions in different patterns of human atheroslcerosis.

The chemical composition of ultracentrifugal fractions of VLDL (d less than 1006), LDL (d 1006-1063) and HDL (d less than 1063) has been studied in males affected by atherosclerosis of different vascular beds. Thirty-seven subjects affected by post-infarction cardiopathy (M.I.) showed significantly higher values of total-C, VLDL-C and LDL-C when compared to 52 controls. Twenty-three patients affected by non-occlusive ischaemic heart disease (I.H.D.) showed higher values than controls of total-C, VLCL-C, LDL-C, total TG, VLDL-TG, and GDL-TG. Twenty-three patients with atherosclerosis of the inferior limbs (P.A.) were characterized by increased levels of total-TG, VLDL-TG, VLDL-C, HDL-C. A group of patients who had suffered a stroke from cerebro-vascular disease (C.V.D.) did not show any significant difference from controls. In the M.I. group, 56% of the patients had a high level of C-VLDL. Patients with I.H.D. were characterized mostly by an increase in C-LDL, Patients with P.A. showed the highest values of total -TG, VLDL-TG and LDL-TG. Some of the observed differences are probably due to different metabolic backgrounds. Some other differences may be due to variations in dietary habits after heart infarction. Patients with levels of plasma cholesterol and triglyceride beyond the 90th percentile of the normal group showed many abnormalities in the chemical composition of their lipoproteins. It is noteworthy that increased amounts of cholesterol may collect in lipoprotein classes different from LDL while increased amounts of triglyceride may collect in classes different from VLDL.

Adult↗

PubMed1978

Purification of human thyroglobulin for radioimmunoassay and testing by ultracentrifugal analysis and immunoelectrophoresis.

The development of sensitive radioimmunoassays for the measurement of thyroglobulin (Tg) in human serum has demanded a high degree of purity of the Tg preparation. A procedure for purification of Tg including immunological methods for both purification and control of purity was therefore used. Extract of human thyroid glandular tissue from a patient with Grave's disease was chromatographed on Sepharose CL6B and subsequently on an affinity column containing antibody to whole human serum. Control of both purification steps was by fused rocket electrophoresis of fractions and crossed immunoelectrophoresis of the concentrated solutions. It could thus be shown that traces of contaminating serum proteins, present after column chromatography, were removed by affinity chromatography. Ultracentrifugation of 125I-labelled Tg indicated that it had a sedimentation rate corresponding to 19 S.

Humans↗

PubMed1979

Quantitation of the membrane attack complex of complement in an air-driven ultracentrifuge.

A sensitive assay of complement (C) activation via either the classical or alternative pathway was developed by evaluating assembly of the terminal complexes (C5b-9)2 or SC5b-9. Activation of serum containing [125I]C7 resulted in the formation of a stable, radiolabeled complex which was separable from its precursors by sedimentation in an air-driven ultracentrifuge. The radioactivity in the sediment was directly proportional to the amount of complex formed and assembly of the complex could be detected after C activation by aggregated IgG in concentrations as low as 10 micrograms/ml. Mild detergents such as Triton X-100 could be included in the reaction mixture, because they affected neither the assembly nor the integrity of the complexes. The assay, which detects both assembly of the membrane attack complex (MAC or (C5b-9)2) on target membranes and formation of SC5b-9 in fluid phase, measures the potential of certain substances to trigger the cytolytic phase of C regardless of whether the classical or alternative pathway was activated. However, by using serum depleted of either factor B or C1q, activation of either pathway can be assessed individually.

Cell Membrane↗

PubMed1990

Ultracentrifugation study on interactions among calmodulin, cardiac troponin and tropomyosin-actin complex.

In an effort to clarify the regulation of contractions in cardiac muscle, we performed ultracentrifugation studies on the interactions between cardiac troponin and tropomyosin-actin complex in the presence of Ca2+ or Sr2+. When troponin C and troponin I were centrifuged with tropomyosin-actin complex, troponin I was not removed from tropomyosin-actin complex in the presence of bivalent-cation. Troponin C was observed to bind very weakly to troponin T-tropomyosin-actin complex in either the presence of absence of bivalent-cation. When troponin C was replaced by calmodulin, troponin I was not removed from tropomyosin-actin complex in the presence of bivalent-cation. Calmodulin bound to the troponin I-troponin T-tropomyosin-actin complex only in the presence of bivalent-cation. These results suggest that the inhibitory action of troponin I is neutralized by troponin C or calmodulin upon binding of bivalent-cation while troponin I binds to tropomyosin-actin complex in cardiac muscle. Therefore cardiac muscle seems to differ from skeletal muscle in regard to regulation of its contraction.

Actins↗

PubMed1985

Instability of brain synaptosomal membrane preparations to repeated ultracentrifugation in isoosmotic density gradients.

Synaptosomal membranes from rat brain were found to be remarkably unstable, from the criterion of buoyant density, to repeated density gradient ultracentrifugation. These membranes underwent progressive changes in buoyant density through at least three cycles of fractionation on density gradients generating large proportions of material of both higher and lower buoyant density than the original fraction. The observed density changes were not due to osmotic effects of the various gradient density zones, clumping due to the influence of divalent cations, age of the membrane preparation, length of centrifugation time, intermediate processing steps or a variety of other factors. In addition, the method of homogenization including duration and rate of homogenization had only minimal effects on this property of buoyant density instability.

Animals↗

PubMed1982

The plasma lipoproteins as risk factors: comparison of electrophoretic and ultracentrifugation results.

Epidemiologic data from 1,859 men aged 45-65 were examined to explore which approach to analyzing plasma lipids and lipoproteins would be most useful for clinical medicine. In addition to analyzing fasting plasma lipids (cholesterol and triglyceride), we used two chemical techniques to measure the three major classes of lipoproteins: ultracentrifugation with heparin/manganese precipitation which yielded values for HDL-, LDL- and VLDL-cholesterol, and quantitative electrophoresis which yielded values for alpha-, beta- and pre-beta-lipoprotein. Both measures of the cholesterol-rich lipoprotein class (LDL-cholesterol and beta-lipoprotein) were related to CHD prevalence, but neither appeared to be superior to total serum cholesterol in the strength--approximately twofold--of the association. Both measures of the protein-rich lipoprotein class (HDL-cholesterol and alpha-lipoprotein) showed a two-fold inverse association with CHD, although the latter did not attain statistical significance. No member of the triglyceride-rich class (pre-beta-lipoprotein, VLDL-cholesterol, or serum triglyceride itself) was independently associated with CHD. We conclude that plasma total cholesterol remains a good single test for most clinical situations because of the prognostic information it contains, its widespread availability and its role in treatment decisions; also that either the plasma alpha-lipoprotein or HDL-cholesterol level can provide additional information on the likelihood of disease.

Aged↗

PubMed1991

Improved detection of HSV by electron microscopy in clinical specimens using ultracentrifugation and colloidal gold immunoelectron microscopy: comparison with viral culture and cytodiagnosis.

Three tests were compared to diagnose herpes virus infection: electron microscopy (EM), viral culture (VC) and cytodiagnosis (Tzanck smear). The study comprised 67 patients with skin or mucous membrane lesions suggestive of herpes simplex virus (HSV) infection. The sensitivity of EM increased 25% after virus concentration by ultracentrifugation. Herpes virus infection was confirmed in 55 of the 67 cases by EM or VC or both. EM detected 53 herpes virus-positive lesion samples of which 14 were not detected by VC; only two lesion samples that were herpes virus-positive in VC were not detected by EM. The sensitivities of EM, VC, and Tzanck smear for the group of 55 herpes virus-positive cases were 96%, 75% and 76%, respectively. The specificity of the Tzanck smear was 83% (prevalence 82%). Colloidal gold immuno-EM was used to rapidly type HSV-1, HSV-2 and varicella zoster virus (VZV) present in skin and mucous membrane lesions in less than 4 h. Immuno-EM was able to detect antiviral antibodies on viral envelopes and viral cores in lesion samples with negative VC. Antiviral antibodies do not interfere with typing of herpes viruses by immuno-EM. It is suggested that formation of viral immune complexes and inactivation of virus particles by antibodies may have caused a negative VC. Improved EM is discussed for its applicability to special cases that cannot rely on VC and cytodiagnosis or when rapid diagnosis is required.

Adolescent↗

PubMed1988

Action of virginiamycin M on the stability of different ribosomal complexes to ultracentrifugation.

It was previously shown that virginiamycin M produces in vivo an accumulation of pressure-sensitive (60 S) ribosomes, and in vitro an inactivation of the donor and acceptor sites of peptidyl transferase. The latter action, however, is expected to cause the accumulation in vivo of ribosome complexes carrying acylated tRNA species: such complexes are usually endowed with pressure resistance. However, present data indicate that poly(U).ribosome complexes carrying Phe-tRNA, Ac-Phe-tRNA or Ac-Phe-Phe-tRNA at either the A or the P site become pressure-sensitive after exposure to virginiamycin M in vitro. It is known also that uncoupled EF-G GTPase is stimulated by P-site-bound unacylated tRNA, not by the acylated species. Our data show, however, a stimulation of EF-G GTPase, when ribosomal complexes carrying Ac-Phe-tRNA or Ac-Phe-Phe-tRNA at the P site are incubated with virginiamycin M. The interpretation proposed to account for all these findings is that complexes carrying A- and P-site-bound aminoacyl-tRNA derivatives, which undergo a stable interaction with the peptidyl transferase, are endowed with ultracentrifugal stability, whereas complexes with unacylated tRNA (which does not interact with the enzyme) are pressure-sensitive. By inactivating the donor and acceptor sites of peptidyltransferase, virginiamycin M causes aminoacyl-tRNA.ribosome complexes to mimic tRNA.ribosome complexes in their pressure-lability and competence in EF-G GTPase stimulation. This interpretation is supported by the finding that the ribosome-promoted protection of aminoacyl-tRNA against spontaneous hydrolysis is suppressed by virginiamycin M.

Binding Sites↗

PubMed1987

Binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase from bovine pancreas under pH conditions of maximum activity. Analysis by ultracentrifugation, fluorescence quenching and chemical modification.

The binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase (EC 6.1.1.2) from beef is examined by three approaches, under pH conditions of maximum activity (pH 8.0). (1) Analytical ultracentrifugation evidences the binding of a single mol of tRNATrp in a 2.5-10 microM concentration range. (2) tRNATrp quenches the fluorescence of the enzyme. The dependence of this fluorescence quenching on the tRNATrp concentration (0.1-4 microM) reflects also the binding of 1 mol of tRNA per mol of enzyme, with a Kd value of 0.19 +/- 0.02 microM. (3) tRNATrp protects the enzyme against derivatization by oxidized ATP. Out of the two fast-reacting lysine residues of the native enzyme, only one is prevented from reacting by tRNATrp in the 0.5-110 microM concentration range. This protection can be significantly analyzed only by assuming a one-to-one complex between the enzyme and tRNA. These results, obtained at pH 8.0 and 25 degrees C, are in contrast with the stoichiometry of 2 mol of tRNA to 1 mol of enzyme, previously observed at pH 6.0 and 4 degrees C.

Amino Acyl-tRNA Synthetases↗

PubMed1989

Estimation of cholesterol loading of the low-density lipoprotein fraction in diabetic subjects without ultracentrifugation.

We report here a new formula for estimating apolipoprotein (apo) B concentration in the low-density lipoprotein (LDL) fraction from measurements of plasma triglyceride and apoB. ApoB in plasma and in the triglyceride-rich lipoprotein fraction (VLDL, d less than 1.019) and plasma triglyceride were measured in 112 subjects, including 56 diabetics. There was a significant correlation between VLDL-apoB and plasma triglyceride (Y = 0.07X + 1, r = 0.73, P less than 0.001). We calculated LDL-apoB according to this formula: LDL-apoB = total apoB - (0.07 x total triglyceride + 1). We found an excellent relationship between LDL-apoB (total apoB - VLDL-apoB) and calculated LDL-apoB (Y = 1.0X + 1, r = 0.96, P less than 0.001). This new formula will enable us to estimate the apoB concentration in the LDL fraction without ultracentrifugation.

Apolipoproteins B↗

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↗

PubMed1987

A comparison by ultracentrifugation of the effects on DNA of ethidium bromide and of acridine orange at low ionic strength.

The application of scaled particle theory to the gels formed by DNA in the ultracentrifuge has provided values for the effective length and the effective radius of the DNA particle. Ethidium bromide has been shown to cause extensive lengthening of the DNA in dilute salt. Acridine orange interaction with DNA resulted in modest changes in DNA dimensions. These results are explained in terms of binding for acridine orange and of denaturation of DNA by ethidium bromide.

Acridine Orange↗