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S C Rall

Publications and source records attributed to S C Rall.

At least 73 records · Page 4Linked to original sources
PubMed1984

The lipid transport system in the mouse, Mus musculus: isolation and characterization of apolipoproteins B, A-I, A-II, and C-III.

Four of the principle apolipoproteins of murine serum have been isolated and characterized. On the basis of their physicochemical properties, they are homologous with the human and rat apoA-I, A-II, B, and C-III. The group of apolipoproteins of middle to low molecular weight, i.e., A-I, A-II and C-III, were separated from the protein moiety of high density lipoproteins (HDL) by gel filtration chromatography, followed by electrophoresis in alkaline-urea polyacrylamide gel with electrophoretic elution. Murine apoA-I, the major protein of HDL (60-80%) displayed an Mr of approximately 27,000, and was polymorphic (four prominent isoproteins with isoelectric points in the range of pH 5.5-5.7). The amino acid profiles of mouse, rat, and human apoA-I generally resembled each other, the former being distinguished by a content of one isoleucine residue per mole. Amino terminal sequence analysis revealed marked homology between the mouse, rat, dog, and human proteins; mouse and rat apoA-I differed at residues 9 and 18 with potential dissimilarities at residues 5 and 15, while the murine and canine sequences were distinct at residues 6, 9, 13, 15, and 30. Apolipoprotein A-II was a monomer, exhibiting an Mr approximately 11,000 in SDS gels; in addition, it was polymorphic (three apparent isoproteins with pI in the pH range 5.05-5.2), and resembled its human and rat counterparts in amino acid composition. ApoC-III, an acidic peptide of pI 4.74 and of Mr approximately 9,600, possessed an amino acid composition very like that of the homologous human and rat proteins. The homology of mouse apoC-III with the human protein was confirmed by NH2-terminal sequence analysis, which revealed identical amino acids in six positions (1, 2, 4, 8, 9, and 13). As shown earlier (Camus et al. 1983. J. Lipid Res. 24: 1210-1228), two forms of immunologically reacting apoB predominated in mouse VLDL and LDL. After isolation of these lipoproteins in the presence of 1 mM PMSF, the apparent sizes of the high and low Mr forms, apoBH and apoBL, were in the ranges approximately 400,000-530,000 and approximately 250,000-280,000, respectively, according to the SDS gel system. We observed that inclusion of 1 mM PMSF was essential to retard degradation of the high Mr form apoBH. The murine B proteins were isolated from apoVLDL and apoLDL by gel filtration chromatography on Sephadex G150 in anionic detergent, and displayed apparent Mr values of 460,000 (apoBH) and 250,000 (apoBL) in 3% SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗
PubMed1983

The receptor-binding domain of human apolipoprotein E. Binding of apolipoprotein E fragments.

To identify the domain of apolipoprotein E (apo-E) involved in binding to low density lipoprotein (LDL) receptors on cultured human fibroblasts, apo-E was cleaved and the fragments were tested for receptor binding activity. Two large thrombolytic peptides (residues 1-191 and 216-299) of normal apo-E3 were combined with the phospholipid dimyristoylphosphatidylcholine (DMPC) and tested for their ability to compete with 125I-LDL for binding to the LDL (apo-B,E) receptors on human fibroblasts. The NH2-terminal two-thirds (residues 1-191) of apo-E3 was as active as intact apo-E3 . DMPC, while the smaller peptide (residues 216-299) was devoid of receptor-binding activity. When apo-E3 was digested with cyanogen bromide (CNBr) and the four largest CNBr fragments were combined with DMPC and tested, only one fragment competed with 125I-LDL for binding to cultured human fibroblasts (CNBr II, residues 126-218). This fragment possessed binding activity similar to that of human LDL. The 125I-labeled CNBr II . DMPC complex also demonstrated high affinity, calcium-dependent saturable binding to solubilized bovine adrenal membranes. The binding of CNBr II . DMPC was inhibited by 1,2-cyclohexanedione modification of arginyl residues or diketene modification of lysyl residues. In addition, the CNBr II had to be combined with DMPC before it demonstrated any receptor-binding activity. Pronase treatment of the membranes abolished the ability of this fragment to bind to the apo-B,E receptors. This same basic region in the center of the molecule has been implicated as the apo-B,E receptor-binding domain not only by this study but also by other studies showing that 1) natural mutants of apo-E that display defective binding have single amino acid substitutions at residues 145, 146, or 158; and 2) the apo-E epitope of the monoclonal antibody 1D7, which inhibits apo-E binding, is centered around residues 139-146.

Adrenal Cortex↗
PubMed1983

Tangier disease: defective recombination of a specific Tangier apolipoprotein A-I isoform (pro-apo A-i) with high density lipoproteins.

Isoforms of apolipoprotein A-I (apo A-I) from subjects with Tangier disease were characterized, and their ability to recombine with normal high density lipoproteins (HDL) was studied. In contrast to normal serum, in which isoprotein 4 is the dominant species [79 +/- 1.8% (mean +/- SD)], the Tangier serum contained much less total apo A-I (approximately equal to 1% of that in normal serum), and isoproteins 2 and 4 were present in roughly equivalent amounts (35.3 +/- 2.5% and 42.7 +/- 3.6%, respectively). The Tangier isoprotein 2 was shown to correspond to pro-apo A-I, having a six-amino acid amino-terminal extension with the sequence: Arg-His-Phe-Trp-Gln-Gln-. The Tangier isoprotein 4 had the same amino-terminal sequence as normal circulating plasma apo A-I. Its association with normal HDL (70%) was similar to the association of normal apo A-I with HDL (80-90%) in recombination experiments. In marked contrast to this behavior, very little (less than 10%) of Tangier isoprotein 2 (pro-apo A-I) associated with HDL in recombination experiments. These results suggest that the underlying defect in Tangier disease may be a faulty conversion of pro-apo A-I to mature apo A-I, either due to a defect in the converting enzyme activity or to a further specific structural defect in Tangier apo A-I. The failure of Tangier pro-apo A-I to associate with HDL may be at least partially responsible for the HDL deficiency in Tangier subjects.

Amino Acid Sequence↗
PubMed1983

Identical structural and receptor binding defects in apolipoprotein E2 in hypo-, normo-, and hypercholesterolemic dysbetalipoproteinemia.

Apolipoprotein E (apoprotein E or apo-E) from type III hyperlipoproteinemic subjects with the E2/2 homozygous phenotype displays both structural and receptor binding heterogeneity. The apo-E from all subjects thus far studied, however, has been functionally defective, though to different degrees. Although nearly every type III hyperlipoproteinemic subject has the E2/2 phenotype, 95-99% of the people with this same phenotype do not display type III hyperlipoproteinemia, nor do they have elevated plasma cholesterol levels. Consequently, it became important to determine whether the apo-E2 from hypo- and normocholesterolemic individuals with the E2/2 phenotype is also functionally abnormal. To do this, apo-E2 was isolated from two hypo-, two normo- and two hypercholesterolemic homozygous E2/2 subjects. The apo-E2 was recombined with vesicles and tested for its ability to displace (125)I-low density lipoproteins (LDL) from apo-B,E (LDL) receptors on human fibroblasts. The apo-E2 from all six subjects was found to be severely defective in receptor binding (<2% of the binding activity of normal apo-E3). In all cases, the binding activity of the apo-E2 was increased 10- to 20-fold by treating the apoproteins with cysteamine, a reagent that converts cysteine residues to positively charged lysine analogues. The cysteine content of each apo-E was determined by monitoring the change in the isoelectric focusing position of the cysteamine-treated apo-E2. Using this method, it was found that the apo-E2 from each subject contained two cysteine residues per mole. A partial sequence analysis of the cysteine-containing regions of the apo-E from three of the six subjects indicated that the two cysteine residues were at residues 112 and 158 in the amino acid sequence. The cysteine at residue 158 has previously been implicated in the severe binding defect of the apo-E2 from a type III hyperlipoproteinemic subject. Since the apo-E2 of the hypo-, normo-, and hypercholesterolemic subjects in this study all displayed a severe functional abnormality, it is apparent that factors in addition to the defective receptor binding activity of the apo-E2 are necessary for the manifestation of type III hyperlipoproteinemia.

Amino Acid Sequence↗
PubMed1983

Identification of a new structural variant of human apolipoprotein E, E2(Lys146 leads to Gln), in a type III hyperlipoproteinemic subject with the E3/2 phenotype.

A type III hyperlipoproteinemic subject having the apolipoprotein E (apo E) phenotype E3/2 was identified. From isoelectric focusing experiments in conjunction with cysteamine treatment (a method that measures cysteine content in apo E), the E2 isoform of this subject was determined to have only one cysteine residue, in contrast to all previously studied E2 apoproteins, which had two cysteines. This single cysteine was shown to be at residue 112, the same site at which it occurs in apo E3. From amino acid and sequence analyses, it was determined that this apo E2 differed from apo E3 by the occurrence of glutamine rather than lysine at residue 146. When phospholipid X protein recombinants of the subject's isolated E3 and E2 isoforms were tested for their ability to bind to the human fibroblast apo-B,E receptor, it was found that the E3 bound normally (compared with an apo E3 control) but that the E2 had defective binding (approximately 40% of normal). Although they contained E3 as well as E2, the beta-very low density lipoproteins (beta-VLDL) from this subject were very similar in character to the beta-VLDL from an E2/2 type III hyperlipoproteinemic subject; similar subfractions could be obtained from each subject and were shown to have a similar ability to stimulate cholesteryl ester accumulation in mouse peritoneal macrophages. The new apo E2 variant has also been detected in a second type III hyperlipoproteinemic subject.

Aged↗
PubMed1982

Structural basis for receptor binding heterogeneity of apolipoprotein E from type III hyperlipoproteinemic subjects.

The three major isoforms of human apolipoprotein E (apo-E2, -E3, and -E4) are coded for by three alleles (epsilon 2, epsilon 3, and epsilon 4) which have a common genetic locus. Previously, we demonstrated that E2, E3, and E4 differ in primary structure from one another at two substitution sites, site A (residue 112) and site B (residue 158). At sites A/B, apo-E2, -E3, and -E4 contain cysteine/cysteine, cysteine/arginine, and arginine/arginine, respectively. We demonstrated that the substitution of cysteine for arginine at site B is at least partly responsible for the defective binding of apo-E2 to human fibroblast low density lipoprotein receptors, compared to the normal binding activity of apo-E3 or -E4. Subjects with the genetic disorder type III hyperlipoproteinemia are phenotypically homozygous for apo-E2, but the binding activity of apo-E to the fibroblast receptor differs considerably from one type III individual to another. We therefore undertook a partial comparative sequence analysis of apo-E2 from three type III subjects whose apo-E displayed this heterogeneity. The subject with the poorest binding apo-E2 was genotypically homozygous for an apo-E allele (epsilon 2); cysteine was found at sites A and B. The subject with the most active apo-E2 was genotypically homozygous for an apo-E allele (epsilon 2); cystine was found at site A and at a new site (site C, residue 145). The epsilon 2 allele specifies a protein that has arginine at site B (residue 158); the epsilon 2 allele specifies a protein that has arginine at site C (residue 145). Therefore, the two alleles differ from one another by cysteine/arginine interchanges at two positions, sites B and C. The third subject, whose apo-E2 displayed binding activity intermediate between the activities of the other two, was genotypically heterozygous, having one epsilon 2 allele and one epsilon 2 allele. The intermediate binding activity of apo-E2 from this subject resulted from having a mixture of severely defective apo-E (specified by epsilon 2) and slightly defective apo-E (specified by epsilon 2).

Alleles↗
PubMed1977

Histone composition of nucleosomes isolated from cultured Chinese hamster cells.

Nuclei isolated from cultured Chinese hamster cells were treated with micrococcal nuclease and lysed, and the resulting chromatin subunit classes (nucleosomes) were purified by sedimentation and resedimentation through isokinetic sucrose gradients. Nucleosomes isolated from [3H]thymidine-labeled cells were analyzed for DNA size using both polyacrylamide gel and electron microscopic techniques. Nucleosomes isolated from [14C]lysine-labeled cells were analyzed for protein content using a sodium dodecyl sulfate-polyacrylamide gel system. The results from monitoring the [14c]lysine in each protein indicate that, in the nucleosome classes (monomer through tetramer), the molar ratios of histones H2A, H2B, H3, and H4 are equivalent. Furthermore, in each population of the nucleosome classes monomer through tetramer, it was possible to demonstrate that this histone unit (H2A + H2B + H3 + H4) is present, on the average, in the amount of two for monomers, four for dimers, six for trimers, and eight for tetramers. This is direct experimental confirmation of the prediction of R.D. Kornberg [(1974) Science 184, 868] concerning the substructure of chromatin.

Cell Fractionation↗
PubMed1976

Induction of stable protein-deoxyribonucleic acid adducts in Chinese hamster cell chromatin by ultraviolet light.

Ultraviolet (uv)-light-mediated formation of protein-DNA adducts in Chinese hamster cell chromatin was investigated in an attempt to compare chromatin alterations induced in vitro with those observed in vivo. Three independent methods of analysis indicated stable protein-DNA associations: (1) a membrane filter assay which retained DNA on the filter in the presence of high salt-detergent; (2) a Sepharose 4B column assay in which protein eluted cincident with DNA; and (3) a CsCl density gradient equilibrium assay which showed both protein and DNA banding at densities other than their respective native densities. Treatment of the irradiated chromatin with DNase provided further evidence that protein-DNA and not protein-protein adducts were being observed in the column assay. There is a fluence-dependent response of protein-DNA adduct formation when the chromatin is irradiated at low ionic strength and is linear for protein over the range studied. When the chromatin is exposed to differing conditions of pH, ionic strength, or divalent metal ion concentration, the quantity of adduct formed upon uv irradiation varies. Susceptibility to adduct formation can be partially explained in terms of the condensation state of the chromatin and other factors such as rearrangement, denaturation, and dissociation of the chromatin components. Besides providing information on the biological significance of these types of uv-induced lesions, this technique may be useful as a probe of chromatin structure.

Binding Sites↗
PubMed1976

The so20,w of unsheared DNA from whole cell lysates of Escherichia coli.

We present measurements of the sedimentation coefficients of DNA present in whole cell lysates of E. coli. The method used is a preparative version of the band sedimentation experiment of Bruner and Vinograd. We show that in order to obtain reliable data on the time dependence of sedimentation, it is necessary to accelerate and decelerate the rotor over much longer times than the standard centrifuge allows. We describe the necessary modifications to the preparative centrifuge and use them to determine the So20,W of unsheared E. coli DNA. The value for the fastest moving components in the lysate is 220 S. The molecular weight of the DNA corresponding to this sedimentation coefficient is probably 1.7 X 10(9) g/mole. However, alternative values cannot be ruled out.

DNA, Bacterial↗