Mean Sedimentation Coefficient Research Articles

Applying analytical ultracentrifugation to nanocrystal suspensions

While applied frequently in physical biochemistry to the study of protein complexes, thequantitative use of analytical ultracentrifugation (AUC) for nanocrystal analysisis relatively rare. Its application in nanoscience is potentially very powerful asit provides a measure of nanocrystal density, size and structure directly in thesolution phase. Towards that end, this paper examines the best practices forapplying data collection and analysis methods for AUC, geared towards the study ofbiomolecules, to the unique problems of nanoparticle analysis. Using uniformnanocrystals of cadmium selenide, we compared several schemes for analyzing rawsedimentation data. Comparable values of the mean sedimentation coefficients (s-value) were found using several popular analytical approaches; however, the distribution in samples-values is best captured using the van Holde–Weischt algorithm. Measureds-values could be reproducibly collected if sample temperature and concentrationwere controlled; under these circumstances, the variability for averagesedimentation values was typically 5%. The full shape of the distribution ins-values, however, is not easily subjected to quantitative interpretation. Moreover, theselection of the appropriate sedimentation speed is crucial for AUC of nanocrystals as thedensity of inorganic nanocrystals is much larger than that of solvents. Quantitative analysisof sedimentation properties will allow for better agreement between experimentaland theoretical models of nanocrystal solution behavior, as well as providingdeeper insight into the hydrodynamic size and solution properties of nanomaterials.

Expression of Sporophytic Storage Proteins in the Corm of the Quillwort (Isoetes echinospora Dur.).

Parenchyma cells from the corm tissue of the aquatic lycopod Isoetes echinospora Dur. were shown by electron microscopy to be packed with amyloplasts, lipid bodies, and protein bodies. The protein bodies are morphologically similar to those identified in seeds and certain vegetative tissues of higher plants. Globoid-containing protein bodies (1-10 [mu]m) isolated in a sucrose gradient possessed a buoyant density of 1.28 g/mL and contained globulin (salt-soluble) proteins. Sucrose gradient centrifugation of crude globulins revealed only two components with mean sedimentation coefficients of approximately 2S and 11S. The 2S component, designated VSP-IsA, was composed of a 15.7-kD polypeptide. The 11S component, designated VSP-IsB, had a molecular mass of 215 kD as estimated by gel filtration and was composed of 39- to 42-kD polypeptides. Two-dimensional gel electrophoresis showed constituent polypeptides distinguished by differences in net charge and molecular mass. Affinity-purified antibodies against VSP-IsA and VSP-IsB prepared and used as probes on immunoblots cross-react only with their specific antigens, suggesting that the proteins are not immunologically related. Indirect immunolocalization studies confirmed that VSP-IsB is deposited in protein bodies. These globulin proteins, like those from some seeds, form the principal storage reserves of the corm tissue.

Multimeric vitronectin. Identification and characterization of conformation-dependent self-association of the adhesive protein.

The adhesive glycoprotein vitronectin (VN) shows a high degree of conformational flexibility implicating that different molecular forms of the molecular may exist. Conformation-dependent monoclonal antibodies 13H1 or 16A7 that, per se, did not react with plasma VN bound to VN treated with heparin, chaotropes, detergents, pH below 6, or by heating at 56 degrees C. Dependent on the stimulus, recognition of VN by these antibodies varied and preceded heparin binding and self-association of VN resulting in the formation of noncovalently linked multimeric species of the protein. Both monoclonal antibodies also reacted with VN in serum or in platelet releasates as well as with VN in extracellular matrices of endothelial cells and inhibited cell adhesion on immobilized VN. Critical VN levels were needed for concentration-dependent multimerization indicating a nonlinear type of polymerization process. The nature of VN multimers was judged by nondenaturing gel electrophoresis, gel filtration, and sucrose gradient ultracentrifugation and revealed the formation of 3- to 16-mer multimeric species within an M(r) range of 200-1200 kDa representing a mean sedimentation coefficient of 9.6 S. In electron microscopy, multimeric VN occurred as globular specimens with an average diameter of 15-28 nm (monomeric plasma VN, 6-8 nm). In contrast to plasma VN, VN multimers were efficiently stabilized by covalent inter-molecular bonds following chemical or transglutaminase-induced cross-linking. A synthetic peptide comprising the central heparin binding region of VN (residues 348-361) not only bound to plasma VN but induced its multimerization also in plasma. During plasmin proteolysis of VN, fragments were generated that lacked the heparin binding region and that lost the ability to multimerize following urea or detergent treatment, implicating that the highly basic region is essential for multimer formation. These data suggest that non-plasma forms of VN, which are abundant in platelets and subendothelium, represent the prototype conformer of the reactive heparin binding form of VN. Our findings implicate that conformationally altered forms of VN enable the adhesive protein to multimerize in a characteristic fashion and thereby endow extracellular matrix sites with unique multivalent properties.

Homogeneous reconstituted oligonucleosomes, evidence for salt-dependent folding in the absence of histone H1.

Using the method of salt dialysis, we have reconstituted histone octamers onto DNA templates consisting of 12 tandem repeats, each containing a fragment of the sea urchin 5S rRNA gene [Simpson, R.T., Thoma, F., & Brubaker, J.M. (1985) Cell 42, 799-808]. In these templates, each sea urchin repeat contains a sequence for preferred nucleosome positioning. Sedimentation velocity and sedimentation equilibrium studies in the analytical ultracentrifuge indicate that at molar histone/DNA ratios of 1.0-1.1 extremely homogeneous preparations of fully loaded oligonucleosomes (12 nucleosomes/template) can be regularly obtained. Digestion of the oligonucleosomes with micrococcal nuclease, followed by restriction mapping of purified nucleosome-bound DNA sequences, yields a complicated but consistent pattern of nucleosome positioning. Roughly 50% of the nucleosomes appear to be phased at positions 1-146 of each repeat, while the remainder of the nucleosomes occupy a number of other minor discrete positions along the template that differ by multiples of 10 bp. From sedimentation velocity studies of the oligonucleosomes in 0-0.2 M NaCl, we observe a reversible increase in mean sedimentation coefficient by almost 30%, accompanied by development of heterogeneity in sedimentation. These results, in combination with theoretical predictions, indicate that linear stretches of chromatin in the absence of lysine-rich histones exist in solution in a salt-dependent equilibrium between an extended (low salt) conformation and one or more folded (high salt) structures. In addition, by 100 mM NaCl, salt-dependent dissociation of histone octamers from these linear oligonucleosomes is observed.

The isolation and partial characterization of chymotrypsinogen from the pancreas of the ostrich ( Struthio camelus)

1. 1. Cationic chymotrypsinogen from the pancreas of the ostrich was purified to homogeneity by sulfuric acid extraction of pancrei, (NH 4) 2SO 4 fractionation and SP-Sephadex C-50 and Sephadex G-100 chromatography. 2. 2. The final preparation was homogeneous when subjected to SDS-PAGE, isoelectric focusing and sedimentation equilibrium centrifugation. The M min value obtained from amino acid analysis was 25,572 Da. A mean sedimentation coefficient of 2.575 S was obtained by sedimentation velocity centrifugation. 3. 3. N-tenninal analysis by dansylation showed an Ala residue which is the N-terminal of a neo-chymotrypsinogen. 4. 4. The effects of pH, temperature and inhibitors (LBTI, PMSF, TPCK and DFP) on the chymotryptic activity were examined. A K m -value for ATEE as substrate was found to be 0.57 mM.

The effect of sodium valproate on the subcellular particles of the rat liver

Sodium valproate, 200 mg/kg, i.p., for three weeks produced an increase in the mean sedimentation coefficient of liver mitochondria, while no changes were observed in lysosomes and peroxisomes. As the mean density of the mitochondria, the lysosomes and the peroxisomes of the control and treated animals were not different, it was concluded that the size of the mitochondria had increased in the liver of the treated rats. The mean sedimentation coefficient of the mitochondria returned to normal after the administration of the drug was interrupted for 8 days, indicating a reversible phenomenon. The mechanical and osmotical fragility of the mitochondria and the peroxisomes were not affected by sodium valproate. The osmotic stability of the lysosomes was increased in the in vivo experimental conditions.

The isolation and partial characterization of trypsin from the pancreas of the ostrich Struthio camelus

1. 1. Cationic trypsin was isolated and purified from the pancreas of the ostrich ( Struthio camelus) by affinity chromatography on a Trasylol-Sepharose column. 2. 2. External activation of trypsinogen was required before trypsin could be isolated. 3. 3. The final preparation was homogeneous by SDS-PAGE and by sedimentation equilibrium centrifugation studies, resulting in M r values of 24,547 and 22,091, respectively. The M min value obtained from amino acid analysis was 22,450. A mean sedimentation coefficient of 2 S was obtained by sedimentation velocity centrifugation. Results obtained from N-terminal and amino acid analyses were similar to those from trypsins of other species. 4. 4. The effects of pH, temperature and inhibitors (LBTI, KBPTI and PMSF) on the tryptic activity were examined. The effect of calcium ions and enzyme concentration on the rate of self-digestion of ostrich trypsin was also investigated.

The sedimentation coefficients of human jejunal gut hormone storage granules

Centrifugation techniques have been applied to human jejunal homogenates to determine the sedimentation coefficients of the peptide hormone storage granules and mitochondria. The manually computed mean values from three experiments were: motilin (3020), gastrin (3300), enteroglucagon (4880), gastric inhibitory poly-peptide (7240), somatostatin (7580). The mean sedimentation coefficient of the mitochondria was 12600. These results provide independent correlates of granule size determined morphologically and provide the basis for determination of changes in disease states affecting the jejunum.

Poly(A)- and oligo(A)-adjacent sequences in RNA from the large hnRNP complexes of Rat liver

The present report describes the isolation of hnRNP complexes with sedimentation coefficients greater than 80 S from rat liver nuclei without the use of ribonuclease inhibitors. The RNA moiety from these complexes is heterogeneous in size, with a mean sedimentation coefficient of 16 S when assayed with polyacrylamide-formamide gel electrophoresis. About 3% of this RNA binds to oligo(dT)-cellulose, the size of the bound fraction being somewhat smaller than the bulk of the hnRNP-RNA. This poly(A) RNA contains two adenylate tracts, one with about 30 and the second with about 200 adenylate residues. Reverse transcription of the poly(A)-containing RNA and hybridization of the cDNA with their respective templates shows two well-defined frequency populations, the first one separated from the second by almost 2.5 logs in the R 0 t curve. The same distribution was found, whether the hybrids were analysed with S1 nuclease or hydroxyapatite. Both separated frequency classes hybridize to total genomic DNA, the abundant one at C 0 t values typical for repetitive and unique sequences, the scarce one only at C 0 t values typical for unique sequences. The two populations are also able to hybridize with polysomal polyadenylated mRNA, the dilution of both frequencies being very similar in the cytoplasm.

Analysis of mammalian DNA for the presence of carcinogen-induced phosphotriesters: Application of the technique of difference sedimentation

The concentration of alkyl phosphotriesters induced in mammalian DNA by many carcinogens can be determined by measuring the mean sedimentation coefficient of the single strands before and after hydrolysis of the triesters in 0.5 m NaOH at 37°C for 1 h. Experiments show that the difference between hydrolyzed and unhydrolyzed DNA containing methyl phosphotriesters can be quantitatively determined using the technique of difference sedimentation. Theoretical analysis indicates that there is no requirement for the accurate matching of the meniscus positions but that differences in DNA concentration between the two solutions have to be avoided. Practical procedures for the analysis and the calculation of the results are discussed.

Lipoproteins in human atherosclerotic vessels: II. Biochemical properties of the major apolipoproteins of arterial low density and very low density lipoproteins

The biochemical behavior of the major apoproteins of low density lipoproteins (LDL) and very low density lipoproteins (VLDL) isolated from human atheroslerotic arteries was examined. On Sephadex G-150, delipidated arterial LDL behaved like delipidated serum LDL and eluted as a single peak (apoLDL) at the void volume. The apoLDL fractions migrated as a single band when examined by electrophoresis on cellulose acetate or acrylamide gel and had a mean sedimentation coefficient of 3.2. Upon double immunodiffusion arterial apoLDL formed immunopreciptin lines of complete identity with serum apoLDL, when reacted against antisera to either human serum apoLDL or LDL. However the amino acid composition of arterial and serum apoLDL was significantly different. When delipidated arterial VLDL was examined on Sephadex G-150, it separated into two major apoprotein fractions, SF-1 and SF-3, and into a minor SF-2 fraction. These fractions appeared to have similar elution volumes and immunochemical reactivity as the corresponding Sephadex fractions of delipidated serum VLDL. Arterial and serum SF-1, SF-2, and SF-3, when reacted respectively against apoLDL antisera, HDL antisera and apo C-III antisera, formed immunoprecipitin lines of complete identity. The sedimentation coefficients of arterial and serum SF-1 appeared similar, as were the coefficients of arterial and serum of SF-3. However the amino acid composition of arterial SF-1 and SF-3 was clearly distinguishable from that of their corresponding serum apoprotein fractions. When examined by acrylamide electrophoresis arterial and serum SF-3 showed a similar band pattern but with different electrophoretic mobility. SF-1 of arterial apoVLDL behaved much like apoLDL from arterial LDL as indicated by electrophoretic and immunochemical behavior and amino acid composition. Although the mean sedimentation coefficients of these apoprotein fractions were similar, the analytical pattern suggested SF-1 of arterial apoVLDL to be more polydisperse in its composition. The present studies indicate that major differences exist between the arterial and serum apoproteins especially in regard to their amino acid composition. They also suggest that arterial LDL and VLDL contain apoproteins which have some characteristics that are similar to the apoB and apoC proteins contained in serum LDL and VLDL.

The mitochondrial and cytoplasmic valyl tRNA synthetases in Tetrahymena are indistinguishable

The mitochondrial and cytoplasmic valyl tRNA synthetases from Tetrahymena pyriformis are indistinguishable. These synthetases cannot be differentiated through hydroxylapatite, DEAE-cellulose, or phosphocellulose column chromatography. Both enzymes show the same mean sedimentation coefficient of 5.9 S in sucrose gradient centrifugation analysis; when bound with tRNA, they are relatively stable and sediment at 7.8 S. The temperature optimum for aminoacylation reaction is 27.5 °C, the optimum Mg 2+ concentration is 4.4 m m, and substrate affinities ( K m values) for valine and ATP in aminoacylation are the same for both enzymes at 1.0 μ m and 2.5 m m, respectively. These enzymes show identical specificities for acylation of different tRNA species, i.e., Tetrahymena and rat liver tRNAs can be equally well recognized, but no significant acylation can be observed with Escherichia coli and Saccharomyces tRNAs. These observations suggest the probable molecular identity of mitochondrial and cytoplasmic valyl tRNA synthetases in Tetrahymena.

The rate of hydrolysis of methyl phosphotriesters in DNA under conditions used in alkaline sucrose gradients

Methyl phosphotriesters have been introduced into DNA, in vitro, by reaction with N-methyl- N-nitrosourea and the rate of degradation in alkali has been followed by measurements of the mean sedimentation coefficient using an analytical ultracentrifuge. In 0.3 M NaOH 0.7 M NaCl , a solution commonly used in alkaline sucrose gradient experiments, hydrolysis of the methyl phosphotriesters present is complete after 15 h at 20°C, or after 2–3 h at 37°C. In addition to breaks formed by the latter reaction there was a continuous background degradation of the DNA giving rise to 6.3 and 63 breaks 10 6 nucleotides per h at 20° and 37°C, respectively. The problem of obtaining quantitative data on phosphotriester concentrations from results of alkaline sucrose gradient experiments has been discussed.

Determination of sedimentation coefficient distributions for cartilage proteoglycans

Sedimentation coefficient distributions of widely polydisperse proteoglycan preparations were made using a previously developed transport sedimentation methodology. Boundary stability was improved by centrifuging samples in a preformed CsCl density gradient (0.016 g/cm 4). The results were compared with the distributions obtained with an interferometric analytical centrifugation method. When these two techniques were applied to analyze A1 and A1–D1 proteoglycan preparations, results were in substantial agreement with respect to the mean sedimentation coefficients of the peaks, average S value, sedimentation coefficient distribution, skewness, proportion of monomer and aggregates, and linearity of the plot ln( s) versus C extrapolations to zero concentration. The lower solute concentration compatible with the transport (velocity gradient) method makes this technique particularly suitable for studying the details of proteoglycan distribution of molecular sizes, especially for aggregates.

Isolation of single stranded transcription sites from human nuclear DNA

Single stranded DNA (ss-DNA) isolated from nuclear DNA of human rhabdomyosarcoma (RD line) cells amounts to 2% of total DNA. As compared with native double-stranded DNA (ds-DNA), ss-DNA has a lower mean sedimentation coefficient, higher buoyant density, contains fewer repeated DNA sequences and a greater proportion of it can be hybridized to human RNAs (up to 35% and less than 8% for ds-DNA). The hybridization kinetics determined by S 1 nuclease digestion and verified by other methods (hydroxylapatite chromatography, density gradient centrifugation and thermal melting) indicate that ss-DNA corresponds to a great number and variety of transcripts. These data are discussed as evidence for DNA strand dissociation in the course of gene activity.

Appearance of rapidly labeled, high molecular weight RNA in nuclear ribonucleoprotein. Release from chromatin and association with protein.

Chromatin and nuclear ribonucleoprotein (nRNP) have been prepared from a human carcinoma cell line. Following a 1-hour (3H)uridine pulse, 60 to 70% of the nuclear radioactivity, after removal of nucleoli, was found in the chromatin, the balance in nRNP. This was true whether the chromatin and nRNP were separated by velocity centrifugation or by isopycnic centrifugation on Metrizamide gradients. Radioactivity in chromatin and nRNP was found in high molecular weight RNA, with mean sedimentation coefficients of 20 S and 15 S, respectively, as determined on sodium dodecyl sulfate-sucrose gradients. Experiments on the kinetics of appearance of radioactivity in the RNA of the two fractions suggest that some of the chromatin-associated RNA is precursor to nRNP-RNA. The proteins of nRNP are complex as revealed by sodium dodecyl sulfate gel electrophoresis. The contamination by chromatin protein was estimated to be 5%. Experiments involving short pulses of (3H)tryptophan, and pulse-chase, suggested that the rapidly turning over proteins of nRNP were not complexed with RNA while still associated with chromatin. However, it was also shown that the radioactivity in nRNP following short pulses of (3H)tryptophan did not correspond to the major bands seen on stained sodium dodecyl sulfate gels. It is therefore concluded that the protein of nRNP consists of two classes: species present in large amounts, possibly common to all RNA in nRNP, which are relatively stable and may be complexed to RNA still associated with chromatin; and a large number of rapidly turning over species, each present in small amounts and associated with nRNP only after its release from chromatin.

Physical aspects and cytoplasmic distribution of messenger RNA in mouse kidney

As a prerequisite to examining mRNA metabolism in compensatory renal hypertrophy, polyadenylated RNA has been purified from normal mouse kidney polysomal RNA by selection on oligo(dT)-cellulose. Poly(A)-containing RNA dissociated from polysomes by treatment with 10 mM EDTA and sedimented heterogeneously in dodecyl sulfate-containing sucrose density gradients with a mean sedimentation coefficient of 20 S. Poly(A) derived from this RNA migrated at the rate of 6–7 S RNA in dodecyl sulfate-containing 10% poly-acrylamide gels. Coelectrophoresis of poly(A) labeled for 90 min with poly(A) labeled for 24 h indicated the long-term labeled poly(A) migrated faster than pulse-labeled material. Twenty percent of the cytoplasmic poly(A)-containing mRNA was not associated with the polysomes, but sedimented in the 40–80 S region (post-polysomal). Messenger RNA from the post-polysomal region had sedimentation properties similar to those of mRNA prepared from polysomes indicating post-polysomal mRNA was not degraded polysomal mRNA. Preliminary labeling experiments indicated a rapid equilibration of radioactivity between the polysomal and post-polysomal mRNA populations, suggesting the post-polysomal mRNA may consist of mRNA in transit to the polysomes.

Intermediates in the strand-separation transition of T7 DNA.

AbstractSedimentation velocity runs as a function of temperature in the region of the alkaline helix‐coil transition have enabled us to demonstrate the existence of stable two‐stranded intermediates in the strand‐separation process for T7 DNA. The strand‐separation transition under these conditions has an intrinsic breadth of ∼1°C, and within this temperature range (Tm + 2°C < T < Tm + 3°C) the intermediate forms are progressively converted (as a function of temperature) to single‐stranded DNA. Parallel characterizations of the strand‐separation transition by viscosity and absorbance–renaturation studies in the alkaline solvent are entirely consistent with the sedimentation experiments. Comparison of the experimental mean sedimentation coefficient of the intermediate forms with theoretical predictions for branched structures suggests that in these molecules the two strands are connected at a single point, not centrally located with respect to the ends of the molecule.

Co-existence of non-histone messenger RNA species lacking and containing polyadenylic acid in sea urchin embryos

The non-histone messenger RNAs of sea urchin embryos have been separated on oligo(T)-cellulose into fractions containing poly(A) and those entirely lacking poly(A). Proof of the existence of the class of mRNA lacking poly(A) is afforded by the following demonstrations: (1) the pulse-labeled RNA analyzed is bound to polyribosomes and has no non-polysomal contamination, (ii) The methods of extraction, as tested by mixing experiments between cytoplasmic extracts of sea urchin embryo and mammalian tissue culture cells, preclude partial degradation of mRNA containing poly(A) to yield artifactual species lacking poly(A), (iii) The non-histone mRNA lacking poly(A) has a mean sedimentation coefficient of 22 S, as measured in a denaturing solvent. It is shown not to consist of molecular aggregates of the putative histone 9 S mRNA, and its base composition differs markedly from those of both 9 S and ribosomal RNA, but resembles closely that of poly(A)-containing mRNA. (iv) Although the non-histone mRNAs lacking and containing poly(A) have similar base compositions and sizes (approximately 22 S), they differ widely in their nucleotide sequences. Complementary DNA, prepared with reverse transcriptase instructed by poly(A)-containing mRNA, hybridized to a negligible extent with RNA lacking poly(A).

A study of the RNA associated with chromosomal proteins in chromatin from Pisum sativum L. embryos

1. RNA associated with chromosomal proteins was isolated by the method of Bonner and Widholm (Bonner, J. and Widholm, J (1967) Proc. Natl. Acad. Sci. U.S. 57, 1379) which was used for the preparation of a specific chromosomal RNA covalently bound to chromosomal proteins. Several properties of the isolated RNA (column chromatographic behaviour and behaviour on polyacryl amide gel electrophoresis) were in agreement with the data of Bonner and coworkers (Huang, R. and Bonner, J. (1965) Proc. Natl. Acad. Sci. U.S. 54, 960, Bonner, J. and Widholm, J. (1967) Proc. Natl. Acad. Sci. U.S. 57, 1379, Bekhor, I., Kung, G. and Bonner, J. (1969) J. Mol. Biol. 39, 351, Sivolap, Y. and Bonner, J. (1971) Proc. Natl. Acad. Sci. U.S. 68, 387 and Bonner, J. (1967) in Regulation of Nucleic Acid and Protein Biosynthesis (Koningsberger, V. and Bosch, L., eds), p. 211, Elsevier, Amsterdam). The base composition of the RNA was very similar to that of tRNA and rRNA isolated from Pisum sativum seeds and the hUMP content did not rise over 1%. 2. Analysis of the incubation of the protein pellicles (obtained by centrifugation of the chromatin in 4 M CsCl) with pronase showed that during this step a degradation of the RNA in the protein pellicle might occur. In view of this observation the pellicle RNA was isolated by a procedure in which the treatment with pronase was omitted. The total pellicle RNA was very heterogeneous and had a mean sedimentation coefficient of about 8 S. After purification of the total pellicle RNA by DEAE—Sephadex column chromatography, the base composition and hUMP content of the RNA were found to be similar to that of the isolated chromosomal RNA. The results suggested the pellicle RNA to be a mixture of degraded rRNA and tRNA. 3. By analysis of the centrifugation of chromatin in 4 M CsCl and by analytical gel electrophoresis no evidence was found that significant amounts of the RNA at the top of the 4 M CsCl gradient were covalently bound to chromosomal proteins. The same conclusion was drawn after analysis of the RNA associated with chromosomal proteins isolated by the procedure of Bekhor (Bekhor, I. (1973) Arch. Biochem. Biophys. 155, 39).