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PubMed1981

Plasma lipoprotein changes resulting from immunologically blocked lipolysis.

The role of lipoprotein lipase (LPL) in the generation of low density lipoprotein (LDL) and high density lipoprotein (HDL) was investigated. Intravenous injections of high titer goat antiserum against highly purified chicken LPL into fasted roosters quantitatively blocks the removal of plasma VLDL triglyceride (1976. J. Lipid Res. 17: 498-505). Analyses of the chemical components of lipoproteins after 8 hr of LPL inhibition showed that the very low density lipoprotein (VLDL) concentration increased over 10-fold, while LDL and HDL concentrations decreased by 5-fold and 48%, respectively. LDL and HDL cholesterol levels decreased logarithmically over the 8-hr period, with half-lives of 2.4 and 6 hr, respectively. The composition of these lipoprotein fractions on a percent weight basis changed significantly. Experimental LDL contained 37% less phospholipid, 64% less cholesterol, and 2.3-fold more triglyceride than control LDL. Experimental HDL contained 3.1-fold more triglyceride and 50% less unesterified cholesterol than control HDL. The Stokes' radii of HDL were determined by gel filtration on Biogel A5M and Ultrogel AcA 22: the radius of experimental HDL (44.9 A) was smaller than that of control HDL (55.4 A). These measurements were confirmed by electron microscopy (43 and 54 A, respectively). After rate zonal ultracentrifugations of plasma samples, control LDL was clearly resolved, while no LDL could be detected in the experimental samples. Rate zonal ultracentrifugations of plasma samples also indicated that control HDL had a higher flotation rate than experimental HDL. Equilibrium zonal ultracentrifugation showed experimental HDL to be more dense than control HDL with hydrated densities of 1.118 and 1.113 g/ml, respectively. These experiments provide in vivo evidence that LDL is a direct metabolic product of VLDL and that LPL plays a role in the transfer of surface constituents from VLDL to HDL.-Behr, S. R., J. R. Patsch, T. Forte, and A. Bensadoun. Plasma lipoprotein changes resulting from immunologically blocked lipolysis.

Animals↗

PubMed1981

Multilaboratory evaluation of an ultrafiltration procedure for high density lipoprotein cholesterol quantification in turbid heparin-manganese supernates.

High density lipoprotein (HDL) can be quantitated by measurement of cholesterol in supernates after precipitation of low and very low density lipoprotein (LDL and VLDL) with heparin and Mn(2+). Supernatant turbidity, often observed with hypertriglyceridemic specimens, indicates incomplete sedimentation of LDL/VLDL and precludes accurate quantitation of HDL. Ten Lipid Research Clinic Laboratories compared an ultrafiltration technique for clearing turbid heparin-Mn(2+) supernates to current methods involving repeat precipitation of either the original specimen after dilution or the d > 1.006 g/ml fraction after removal of VLDL from the initial specimen by ultracentrifugation. Results for ultrafiltration of 429 turbid supernates averaged only slightly higher (1.0-1.1 mg/dl) than results by the dilution or ultracentrifugation methods on the same specimens, but this difference was found to be significant (P < 0.005). The agreement of the ultrafiltration method with the other two methods is indicated by the following linear regression equations: a), ultrafiltration = (0.964 x ultracentrifugation) + 2.4 mg/dl, and correlation coefficient = 0.926; and b), ultrafiltration = (0.936 x dilution) + 3.3 mg/dl, and correlation coefficient = 0.933. We conclude that ultrafiltration of turbid heparin-Mn(2+) supernates is a convenient alternative to precipitation after either dilution or removal of VLDL.-Warnick, G. R., J. J. Albers, P. Bachorik, J. Turner, C. Garcia, C. Breckinridge, K. Kuba, S. McNeely, G. Hillerman, P. King, R. Muesing, B. Most, and K. Lippel. Multi-laboratory evaluation of an ultrafiltration procedure for high density lipoprotein cholesterol quantification in turbid heparin-manganese supernates.

Chemical Phenomena↗

PubMed1995

F-actin-myosin-subfragment-1 (S1) interactions. Identification of the refractory state of S1 with the S1 dimer.

Several conflicting experiments have been carried out concerning the existence of a refractory state for myosin subfragment 1 (S1; i.e. unable to bind to F-actin), in the presence of Mg-nucleotide compounds, at low temperature. Contradictory experiments have been published on the existence of a S1 dimer, under the same conditions. By taking into account some elementary, but crucially important, precautions in the preparation of myosin and S1, it is possible to maintain intact the head-to-head sites of dimerisation on both myosin and S1. Moreover, it has been shown that Mg2+ alone, at low temperature, is able to induce a reversible S1-S1 dimerisation [Morel, J. E. & Garrigos, M. (1982) Biochemistry 21, 2679-2686). Here, we have studied the intrinsic viscosity of S1, in the presence of 2 mM MgCl2, versus temperature, between 0.5-20 degrees C. At approximately 0.5-2.0 degrees C, the intrinsic viscosity of S1 was found to be 19.8 cm3/g. Above 2.5-3.0 degrees C, a steep decrease in the intrinsic viscosity was observed. Between 7 degrees C and 20 degrees C, a constant intrinsic viscosity of 6.7 cm3/g was measured. Therefore, there is a dramatic temperature transition between approximately 2.5-7.0 degrees C (width, 4.5 degrees C; midpoint, 5 degrees C): below 2.5 degrees C we observed the presence of a S1 dimer (confirmed by analytical ultracentrifugation performed at 1 degree C) and above 7-8 degrees C we observed the presence of a S1 monomer (confirmed by analytical ultracentrifugation performed at 8 degrees C). We have also studied the interactions of F-actin with S1, under the same conditions of temperature, and in the presence of 2 mM MgCl2, by using preparative ultracentrifugation. At 1-2 degrees C, S1, which is in the dimeric form, is unable to bind to F-actin. At 6-20 degrees C, F-actin and S1 bind stoichiometrically (1S1/1actin), with an equilibrium constant of 0.5 microM-1, under our experimental conditions. We also performed binding experiments at 3-5 degrees C. Although we were unable to clearly reach the asymptote corresponding to stoichiometry, by assuming a 1:1 ratio, we found a temperature transition in the equilibrium constants, between 3-5 degrees C (width, 2 degrees C; midpoint, 4 degrees C). These phenomena are comparable to those observed for the dimer. Thus, we have shown that there is also a temperature transition in the F-actin-S1 binding process. We conclude that the dimeric and the refractory states of S1 are identical.

Actins↗

PubMed1993

Dietary fish oil modification of cynomolgus monkey low density lipoproteins results in decreased binding and cholesteryl ester accumulation by cultured fibroblasts.

Dietary fish oil (FO) has been reported to increase low density lipoprotein (LDL) receptor function resulting in lower plasma LDL concentrations in the rat (Ventura et al. J. Clin. Invest. 84: 528-537, 1989). The purpose of this study was to determine whether dietary FO, as compared to lard, affected the receptor-mediated uptake of LDL by cultured skin fibroblasts. Plasma LDL was isolated by combined ultracentrifugation and column chromatography from cynomolgus monkeys fed diets enriched in FO or lard and the effect of these two dietary fats on the binding of LDL and esterified cholesterol (EC) accumulation by cultured fibroblasts was determined. There was no difference in total plasma or LDL cholesterol concentrations between diet groups. The monkeys fed FO had significantly smaller LDL which, on average, contained less protein, phospholipid (PL), and free and esterified cholesterol compared to the LDL from monkeys fed the lard diet. FO LDL were less effective than lard LDL in competing for binding, internalization, and degradation of a standard 125I-labeled LDL by fibroblasts (11.0 +/- 2.4 vs. 3.0 +/- 0.8 micrograms LDL protein/ml for 50% displacement of binding, respectively; P = 0.013). FO versus lard LDL also resulted in less accumulation of cellular EC after a 24-h incubation with fibroblasts (7.7 +/- 0.2 vs. 13.0 +/- 0.4 micrograms EC/mg protein, respectively; P = 0.0001). In general, cellular EC accumulation was proportional to LDL particle size and LDL apoE/B molar ratio; however, LDL from the lard group resulted in greater EC accumulation even when LDL particle size and apoE content were nearly equivalent between diet groups. When LDL were isolated from the same animals by sequential ultracentrifugation, the lard LDL apoE was reduced 22% compared to column isolated LDL and this resulted in a 32% decrease in cellular EC accumulation. However, for FO LDL, apoE content was reduced 34% by sequential ultracentrifugation but this only resulted in a 10% decrease in EC accumulation. These results suggested that lard LDL contained more receptor-active apoE than FO LDL. We conclude that isocaloric substitution of fish oil for lard in the diet of cynomolgus monkeys results in LDL particles that bind less avidly to LDL receptors and in less EC accumulation in fibroblasts. The decreased binding of LDL from the FO group appears related to their decreased size and CE content as well as the decreased content of receptor-active apoE relative to the lard group.

Animals↗

PubMed1998

Quantitation of cholesterol-carrying particles in human gallbladder bile.

The inter-mixed micellar/vesicular (non-phospholipid-associated) bile salt concentration (IMC) can be rapidly measured in model biles by centrifugal ultrafiltration, thus allowing reliable separation of vesicular and micellar cholesterol carriers by gel filtration with an elution buffer containing bile salts at the correct IMC (Donovan, J. M., and A. A. Jackson. 1993. J. Lipid Res. 34: 1121-1129). We adapted this method to the more complex human gallbladder bile and examined the relationship between cholesterol solubilization and crystallization in gallbladder biles from 10 cholesterol gallstone patients. The IMC (mean +/- SEM) was 9.67 +/- 1.97 (range 3.56-35.02) mM with significant enrichment with hydrophilic bile salt species. Upon gel filtration of these biles with an eluant buffer containing 10 major bile salts at concentrations according to their IMC, cholesterol was found to be solubilized mainly in mixed micelles. Vesicles were detected in all 10 biles after separation by KBr density gradient ultracentrifugation but in only 5 of these biles with the IMC method. Biles without vesicles had a lower CSI (1.15 +/- 0.12 vs. 1.90 +/- 0.28, P < 0.05), a higher total lipid concentration (11.9 +/- 2.3 vs. 5.9 +/- 1.1, P < 0.05), and a higher bile salt/ (bile salt + phospholipid) ratio (0.83 +/- 0.01 vs. 0.74 +/- 0.04, P = 0.07). For both IMC and ultracentrifugation methods, vesicular cholesterol concentration showed a negative correlation with crystal observation time and a positive correlation with cumulative crystal score during 21 days. Our data indicate that methods such as density gradient ultracentrifugation overestimate vesicular cholesterol solubilization in human biles.

Bile↗

PubMed1965

CARP MYOGENS OF WHITE AND RED MUSCLES. GENERAL COMPOSITION AND ISOLATION OF LOW-MOLECULAR-WEIGHT COMPONENTS OF ABNORMAL AMINO ACID COMPOSITION.

1. The general composition of the carp myogens of white and red muscles was examined by electrophoresis and ultracentrifugation. 2. Eight and nine peaks were found in the electrophoretic analysis at pH7.3 and I0.075 of white and red muscle respectively. Lowering of the pH to 5 or 6 did not increase the number of peaks. The electrophoretic pattern of white-muscle myogen was remarkably different from that of red-muscle myogen, though ultracentrifugal analyses of the both types of myogen gave similar diagrams, in which about one-third of the total myogen sedimented slowly. 3. The pH-mobility curves of the myogen of white muscle indicated that the net charges of the components 2, 3 and 5 vary only slightly within the pH range 7.3-5.4, suggesting that their histidine content is very low. 4. The slow-sedimenting fraction of white-muscle myogen was isolated in fairly good yield by ammonium sulphate fractionation, by taking advantage of their high salting-out range, and the fraction was shown to be composed mainly of components 2, 3 and 5. 5. The same method of fractionation was applied to red-muscle myogen and the absence of the three components was confirmed. These results bring to light a new difference between the two types of fish muscle.

Amino Acids↗

PubMed1964

ACTIVITY OF DISSOCIATED AND REASSOCIATED 19S ANTI-GAMMA-GLOBULINS.

19S anti-gamma-globulins were isolated in a high state of purity from the sera of two patients with rheumatoid arthritis. Following reduction with ethyl mercaptan and alkylation by iodoacetamide, fragments were produced which retained the capacity to combine with 7S gamma-globulin. The fragments from one of the 19S anti-gamma-globulins agglutinated red cells coated with incomplete anti-Rh antibodies. This activity was shown by density gradient ultracentrifugation to be associated with low molecular weight fractions. The agglutination of the coated red cells by the fragments was strongly inhibited by normal and myeloma 7S gamma-globulins and showed a greater specificity than the parent 19S material. Analytical ultracentrifuge experiments demonstrated that the fragments from either of the 19S anti-gamma-globulins formed complexes with 7S gamma-globulin. Reassociation of the dissociated fragments through reformation of disulfide bonds resulted in the formation of fast sedimenting molecules having properties similar to those of the untreated 19S material in respect to precipitation with aggregated gamma-globulin and agglutination of coated red cells.

Antibodies, Anti-Idiotypic↗

PubMed1964

ESTIMATION OF THE LOW-DENSITY (BETA) LIPOPROTEINS OF SERUM IN HEALTH AND DISEASE USING LARGE MOLECULAR WEIGHT DEXTRAN SULPHATE.

Studies have been made of the factors affecting the specificity of the interaction between high molecular weight dextran sulphate and low-density lipoproteins, both in pure solution and in serum. The results have been used in the development of a simple assay method for the serum concentration of low-density lipoproteins in small volumes of serum. The results obtained by this assay procedure have been found to correlate acceptably with parallel estimations of low-density lipoproteins by an ultracentrifugal technique and by paper electrophoresis. The technique has been applied to a survey of serum levels of these proteins in a normal population. The results have been compared with data in the literature. Satisfactory agreement was found between mean levels, matched for age and sex, between the dextran sulphate method and those methods based ultimately on chemical estimation of one or more components of the isolated lipoproteins. A systematic difference was observed when the dextran sulphate method was compared with estimates based on analytical ultracentrifugation or turbidimetry using amylopectin sulphate.Some indication of the range of application of the dextran sulphate method in clinical chemistry is provided.

Aging↗

PubMed1996

Selective adsorption: a new method for purification of protein A-gold complexes.

Protein gold complexes are prepared by adding gold colloids to cytochemically active proteins in solution. The gold particles of the colloid form complexes with the protein spontaneously, but some of the protein remains uncomplexed. Currently, when protein A-gold complexes are prepared, the uncomplexed protein. A is separated from the complex by ultracentrifugation, which is a lengthy procedure and requires special equipment. This report describes a simple and rapid method for removing uncomplexed protein A from freshly-prepared "crude" protein A-gold at the laboratory bench. In this method, larger gold particles of 15-nm diameter are added to a crude protein A-gold preparation made with smaller particles (e.g.,6nm diameter). The 15-nm particles adsorb uncomplexed protein A preferentially, but do not form complexes with already-formed 6-nm protein A-gold. The adsorbed protein A, attached to the 15-nm particles, can then be sedimented in a bench centrifuge, leaving the purified 6-nm protein A-gold complexes in the supernatant. The stability, immunocytochemical activity, and degree of aggregation of the protein A-gold complexes prepared by this method are comparable to protein A-gold complexes prepared by ultracentrifugation. The method is simple to perform, avoids lengthy purification procedures, and yields complexes with reproducible labelling characteristics.

Centrifugation↗

PubMed1984

Self-associating IgG rheumatoid factors in MRL/l autoimmune mice.

Previous work has shown that the intermediate complexes isolated from the plasma of patients with rheumatoid arthritis are composed of self-associating IgG rheumatoid factors. Mice of the MRL/l strain develop spontaneous autoimmune disease with arthritis that is pathologically similar to human rheumatoid arthritis. Also, the sera of MRL/l mice contain autoantibodies to nuclear antigens as well as IgM and IgG rheumatoid factors. The present studies were done to determine if the IgG rheumatoid factors isolated from these mice undergo self-association. MRL/l mouse sera were categorized into groups A and B based on serum-serum precipitin interactions. Thirteen of 13 MRL/l mice sera examined contained intermediate complexes sedimenting between the 6.6S and 19S components of normal serum by sedimentation velocity ultracentrifugation. There were no differences in the level of intermediate complexes between groups A and B. IgG rheumatoid factors were isolated from the sera of 9 other mice. Upon sedimentation equilibrium ultracentrifugation, these rheumatoid factors underwent concentration-dependent self-association similar to that described for human self-associating IgG rheumatoid factors, although the precise stoichiometry of self-association could not be determined. The IgG rheumatoid factors from group B had higher energies of self-interaction than those from group A. These studies provide additional evidence that MRL/l mice may be the best available animal model for the study of human rheumatoid arthritis.

Animals↗

PubMed1997

Development of a lipoprotein profile using capillary electrophoresis and mass spectrometry.

A new program for lipoprotein characterization is outlined where capillary electrophoresis (CE) plays a central role in the analysis of intact lipoprotein serum components and the apoprotein domains. The first characterization step involves separation and particle density analysis of very low-, low-, and high-density lipoprotein fractions (VLDL, LDL, HDL) by ultracentrifugation and image analysis. VLDL, HDL, and LDL fractions are analyzed by capillary electrophoresis. Sodium dodecyl sulfate (SDS) at low concentrations in the background electrolyte used in the CE analysis is incorporated into the lipoprotein particle without appreciable delipidation, as determined by ultracentrifuge particle density analysis. Increasing the concentration of SDS results in extensive delipidation, resulting in the release of apoproteins (apo) which are detected as components of the electropherogram. Apo B-100 is detected in the delipidated VLDL and LDL fractions along with micelles of the lipids. Micelles from LDL delipidation have uniform charge densities. Apo A-I and A-II are detected in the HDL fraction. A new method for lipoprotein delipidation is introduced where the lipoprotein fraction is adsorbed on a reversed-phase hydrophobic cartridge. Delipidation and recovery of the apoprotein fractions is made by serial elutions with acetonitrile. CE of the lipid-free apoprotein mixture shows the presence of apoC-I,II,III and apoE in the VLDL fraction, and apoA-I,II apoC-I and apoE in the HDL fraction. Electrospray ionization mass spectrometry analysis gives the isoform distribution for each apoprotein. The identification of the apoproteins in the electropherograms is the first step in developing a CE-based quantitation method for measuring serum levels of these apoproteins and their distribution between the lipoprotein fractions. The assay described in this paper is being used as a level 2 and 3 cardiac risk profile analysis for individuals with normal lipid profiles who have a documented or family history of cardiovascular disease.

Apolipoproteins A↗

PubMed1997

Redefining the separation factor: a potential pathway to a unified separation science.

Understanding the separation process in capillary electrophoresis (CE) leads to the unification of the theories for separation science. While the separation of analytes is governed by equilibria in chromatography, and by (centrifugal) field in ultracentrifugation, the separation in CE is governed by both equilibria and (electric) field. Therefore, a comprehensive separation theory that describes the separation process of analytes in CE should be able to describe the separation processes in both chromatography and ultracentrifugation. In this paper, we propose that individual capacity factors for each analyte species be used to describe the migration behavior of an analyte. The effect of field on each analyte species, as well as the effect of equilibria are considered in deriving a generalized equation that is applicable for all separation techniques. The separation factor defined at present does not directly relate to the migration rates of the analytes, and therefore can not be used in a generalized theory. We propose that the ratio of the migration rates of a pair of analytes (gamma) should be used as the separation factor, instead of the ratio of the two capacity factors. When gamma is used to describe the separation of two closely migrating analytes, all separation techniques have the same resolution equation.

Chromatography↗

PubMed1979

Suppression of T cell-mediated immunity by tumor cells: immunogenicity versus immunosuppression and preliminary characterization of the suppressive factors.

In studying the immunogenicity of spleen cells and tumor cells in the generation, of cytotoxic T lymphocyte (CTL) in the allogeneic mixed lymphocyte culture (MLC) or mixed lymphocyte tumor cell culture (MLTC) reactions, we have found that the tumor cells not only appear to be poorly immunogenic, but are also immunosuppressive. This was shown by the ability of the tumor cells or their cell-free extracts to suppress standard MLC reactions. This suppression was acting mainly at the induction phase of the cytotoxic response. It could not interfere with the killing activity of the fully generated CTLs. In a Friend virus-induced leukemia FBL-3 system, at least two major components could be attributed to the cause of immunosuppression; one was of viral origin and the other was of non-viral origin. The viral component was sensitive to UV-irradiation and could be pelleted after ultracentrifugation at 100,000 g. The non-viral component was UV-resistant and was retained in the supernatant fraction after ultracentrifugation. Friend virus and 12 commonly found murine viruses have been excluded as the possible candidates causing the immunosuppression. The immunosuppressive viruses are very likely of endogenous origin and are defective in replication as shown by electromicroscopy, and by the virus focus-inducing and reverse transcriptase assays. These findings indicate that probably all tumor cells possess the immunosuppressive factor(s) which may account for their apparent lack of immunogenicity and the lack of proper immune responses in the tumor-bearing hosts.

Animals↗

PubMed2006

Determination of bioaccessibility of beta-carotene in vegetables by in vitro methods.

The in vitro method in use for the determination of beta-carotene bioaccessibility involves simulated gastrointestinal digestion followed by ultracentrifugation to separate the micellar fraction containing bioaccessible beta-carotene and its quantitation. In this study, the suitability of two alternatives viz., membrane filtration and equilibrium dialysis were examined to separate the micellar fraction. Values of beta-carotene bioaccessibility obtained with the membrane filtration method were similar to those obtained by the ultracentrifugation method. Equilibrium dialysis was found not suitable for this purpose. Among the vegetables analyzed, fenugreek leaves had the highest content of beta-carotene (9.15 mg/100 g), followed by amaranth (8.17 mg/100 g), carrot (8.14 mg/100 g) and pumpkin (1.90 mg/100 g). Percent bioaccessibility of beta-carotene ranged from 6.7 in fenugreek leaves to 20.3 in carrot. Heat treatment of these vegetables by pressure cooking and stir-frying had a beneficial influence on the bioaccessibility of beta-carotene from these vegetables. The increase in the percent bioaccessibility of beta-carotene as a result of pressure-cooking was 100, 48 and 19% for fenugreek leaves, amaranth and carrot, respectively. Stir-frying in presence of a small quantity of oil led to an enormous increase in the bioaccessibility of beta-carotene from these vegetables, the increase being 263% (fenugreek leaves), 192% (amaranth leaves), 63% (carrot) and 53% (pumpkin).

Amaranthus↗

PubMed1994

Two domains of interaction with calcium binding proteins can be mapped using fragments of calponin.

Native calponin is able to bind 2 mol of calcium binding protein (CaBP) per mole calponin. This study extends this observation to define the 2 domains of interaction, one of which is near the actin binding site, and the other in the amino-terminal region of calponin. Also, the first evidence for a differentiation in the response of calponin to interaction with caltropin versus calmodulin is demonstrated. The binding of caltropin to cleavage and recombinant fragments of calponin was determined by 3 techniques: tryptophan fluorescence of the fragments, CD measurements to determine secondary structure changes, and analytical ultracentrifugation. In order to delineate the sites of interaction, 3 fragments of calponin have been studied. From a cyanogen bromide cleavage of calponin, residues 2-51 were isolated. This fragment is shown to bind to CaBPs and the affinity for caltropin is slightly higher than that for calmodulin. A carboxyl-terminal truncated mutant of calponin comprising residues 1-228 (CP 1-228) has been produced by recombinant techniques. Analytical ultracentrifugation has shown that CP 1-228, like the parent calponin, is able to bind 2 mol of caltropin per mol of 1-228 in a Ca(2+)-dependent fashion, indicating that there is a second site of interaction between residues 52-228. Temperature denaturation of the carboxyl-terminal truncated fragment compared with whole calponin show that the carboxyl-terminal region does not change the temperature at which calponin melts; however, there is greater residual secondary structure with whole calponin versus the fragment. A second mutant produced through recombinant techniques comprises residues 45-228 and is also able to bind caltropin, thus mapping the location of the second site of interaction to near the actin binding site.

Actins↗

PubMed1998

Domain structure analysis of elongation factor-3 from Saccharomyces cerevisiae by limited proteolysis and differential scanning calorimetry.

Elongation-factor-3 (EF-3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF-3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultracentrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39 degrees C, and a minor transition at 58 degrees C. Ultracentrifugation shows that EF-3 is a monomer; thus, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF-3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF-3 with trypsin produced the first cleavage at the N-side of Gln775, generating a 90-kDa N-terminal fragment and a 33-kDa C-terminal fragment. The N-terminal fragment slowly undergoes further digestion generating two major bands, one at approximately 75 kDa and the other at approximately 55 kDa. The latter was unusually resistant to further tryptic digestion. The 33-kDa C-terminal fragment was highly sensitive to tryptic digestion. A 30-min tryptic digest showed that the N-terminal 60% of EF-3 was relatively inaccessible to trypsin, whereas the C-terminal 40% was readily digested. These results suggest a tight structure of the N-terminus, which may give rise to the 58 degrees C transition, and a loose structure of the C-terminus, giving rise to the 39 degrees C transition. Three potentially functional domains of the protein were relatively resistant to proteolysis: the supposed S5-homologous domain (Lys102-Ile368), the N-terminal ATP-binding cassette (Gly463-Lys622), and the aminoacyl-tRNA-synthase homologous domain (Glu820-Gly865). Both the basal and ribosome-stimulated ATPase activities were inactivated by trypsin, but the ribosome-stimulated activity was inactivated faster.

Amino Acid Sequence↗

PubMed2005

Investigation of molecular size of transcription factor TFIIE in solution.

Human general transcription factor IIE (TFIIE), a component of a transcription preinitiation complex associated with RNA polymerase II, was characterized by size-exclusion chromatography, mass spectrometry, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS). Recombinant human TFIIE was purified to homogeneity and shown to contain equimolar amounts of TFIIEalpha (50 kDa) and TFIIEbeta (35 kDa) by SDS-PAGE. In the analysis of size-exclusion chromatography of the purified sample, as already reported, TFIIE was shown to be a 170-kDa alpha(2)beta(2) heterotetramer. However, by using electrospray ionization mass spectrometry the purified sample gave the molecular mass of 84,152 +/- 5, indicating that TFIIE is an alphabeta heterodimer but not a heterotetramer. Analytical ultracentrifugation experiment of TFIIE provided that only a single component with the molecular mass of ca. 80,000 existed in solution, also suggesting an alphabeta heterodimer. In addition, its extraordinarily rod-like molecular shape was confirmed by SAXS. It is likely that the rod-like molecular shape of TFIIE has misled larger molecular size in size-exclusion chromatography, which was calibrated by globular proteins. It is demonstrated that TFIIE exists as a heterodimer under our present conditions in solution, although two molecules of heterodimer might be required for the formation of the preinitiation complex with RNA polymerase II for starting the transcription process.

Amino Acid Motifs↗

PubMed2005

Individualization of single-walled carbon nanotubes: is the solvent important?

The influence of different solvents on the individualization of single-walled carbon nanotubes (SWNTs) was investigated by several methods. A stable solution of individual SWNTs in o-dichlorobenzene was obtained by a combination of ultrasonication and ultracentrifugation. Atomic force microscopy height analysis confirmed that the final supernatant solution contained 85 % of individual tubes with a diameter equal to or less than 1.5 nm, which is consistent with the average diameter of arc-discharge tubes. Both the solvent and the ultracentrifugation speed are important parameters in determining the final degree of individualized SWNTs. Notably, the SWNTs were stable in o-dichlorobenzene for more than eight months. This technique for obtaining highly individualized SWNTs in an organic solvent is expected to have great potential in many applications, such as functional materials and nanoelectronics.

Acetamides↗