Glycerol Gradient Ultracentrifugation Research Articles

Immunoaffinity purification of DNA polymerase δ1

A monoclonal antibody against human DNA polymerase δ (pol δ) was isolated with properties suitable for its utilization for immunoaffinity chromatography. The antibody was immobilized after periodate oxidation and coupled to a hydrazide-activated support. Starting from a partially purified preparation, calf thymus pol δ was purified about 200-fold in a single step. Further purification on ssDNA-cellulose resulted in isolation of a homogeneous preparation. The amount of enzyme isolated, ca. 0.3 mg of pure pol δ from 0.75 kg of calf thymus, is about 15-fold greater than can be achieved by conventional procedures. This procedure provides a significant advance in the isolation of pol δ in allowing its facile isolation from tissues in good yield. The isolated enzyme consisted of two subunits of 125 and 50 kDa. Characterization of the enzyme showed that these two subunits remained associated on glycerol gradient ultracentrifugation even in the presence of 2.8 m urea.

Tyrosine phosphorylated proteins in chronic myelogenous leukemia.

An aberrantly expressed and highly active abl tyrosine kinase (p210bcr-abl) appears critical for the development and pathogenesis of chronic myelogenous-leukemia (CML). CML cells and cell lines each displayed a similar spectrum of phosphotyrosyl proteins. Analysis of these proteins by glycerol-gradient ultracentrifugation showed that many apparently existed as multimeric complexes. Confirming this, several of these proteins co-immunoprecipitated, along with the p210bcr-abl, with antibody to abl. Included were co-precipitating proteins identified as the p120 ras GTPase-activating protein (GAP) and the p62 protein that binds both to GAP and to a number of other tyrosine-phosphorylated proteins having peptide regions homologous to the second domain of src. Because p62, ras GAP and ras are involved in growth-factor and oncogene activation of cells, this pathway may also play an important role in CML.

Single-stranded DNA binding protein from calf thymus. Purification, properties, and stimulation of the homologous DNA-polymerase-alpha-primase complex.

A binding protein for single-stranded DNA (ssDNA) was purified from calf thymus to near homogeneity by chromatography on DEAE-cellulose, blue-Sepharose, ssDNA-cellulose and FPLC Mono Q. The most purified fraction consisted of four polypeptides with molecular masses of 70, 55, 30, and 11 kDa. The polypeptide with the molecular mass of 55 kDa is most likely a degraded form of the largest polypeptide. The complex migrated as a whole on both glycerol gradient ultracentrifugation (s = 5.1 S) and gel filtration (Stokes' radius approximately 5.1 nm). Combining these data indicates a native molecular mass of about 110 kDa, which is in accord with a 1:1:1 stoichiometry for the 70 + 55/30/11-kDa complex. The ssDNA binding protein (SSB) covered approximately 20-25 nucleotides on M13mp8 ssDNA, as revealed from both band shift experiments and DNase I digestion studies. The homologous DNA-polymerase-alpha-primase complex was stimulated by the ssDNA binding protein 1.2-fold on poly(dA).(dT)14 and 10-13-fold on singly primed M13mp8 DNA. Stimulation was mainly due to facilitated DNA synthesis through stable secondary structures, as demonstrated by the vanishing of many, but not all, pausing sites. Processivity of polymerase-primase was not affected on poly(dA).(dT)14; with poly(dT).(rA)10 an approximately twofold increase in product lengths was observed when SSB was present. The increase was attributed to a facilitated rebinding of polymerase alpha to an already finished DNA fragment rather than to an enhancement of the intrinsic processivity of the polymerase. Similarly, products 300-600 nucleotides long were formed on singly primed M13 DNA in the presence of SSB, in contrast to 20-120 nucleotides when SSB was absent. DNA-primase-initiated DNA replication on M13 DNA was inhibited by SSB in a concentration-dependent manner. However, with less sites available to begin with RNA priming, more homogeneous products were formed.

Several regions of human estrogen receptor are involved in the formation of receptor-heat shock protein 90 complexes

Several mutants of the human estrogen receptor (ER) were transiently expressed in Cos 7 cells in order to determine the regions involved in the formation of complexes with the heat shock protein Mr approximately 90,000 (hsp 90). The formation of the cytosol non-DNA binding 8-9 S complexes (8-9 S ER) was monitored by glycerol gradient ultracentrifugation. It was established that the N-terminal region of the receptor, including the two zinc fingers of the DNA binding domain (DBD), is not required for the formation of the 8-9 S ER complexes. Conversely, deletion of the entire ligand binding domain (LBD) produced truncated receptor mutants that are constitutive transcriptional activators and did not form 8-9 S ER complexes, confirming results obtained previously with the glucocorticosteroid receptor (Pratt, W.B., Jolly, D.J., Pratt, D.V., Hollenberg, S.M., Giguerre, V., Cadepond, F., Schweizer-Groyer, G., Catelli, M.G., Evans, R.M., and Baulieu, E.E. (1988) J. Biol. Chem. 263, 267-273). However, no limited subregion of the LBD was found to be uniquely involved in hsp 90 binding. A highly positively charged region situated at the C-terminal extremity of the DBD (between amino acids 251 and 271) also appeared to be implicated. Although not sufficient, this sequence is necessary for the formation of the 8-9 S ER; it also corresponds to the NL1 nuclear localization domain of steroid receptors. Taken together, these results suggest that the formation of complexes with hsp 90 involves several receptor regions, and they are consistent with the proposal that hsp 90 inhibits receptor function and can be released by hormone binding to the LBD.

Substructure of human von Willebrand factor. Proteolysis by V8 and characterization of two functional domains.

The effects of Staphylococcus aureus V8 protease (V8) on the multimeric structure of human von Willebrand factor (vWF) were studied to test and expand our model for the substructure of vWF. Electron microscopy of V8 digests of vWF revealed that the multimers were cleaved where the flexible rod (R) domains join the large elongated globular (G) domains. The resulting two major fragments, which were purified by affinity and hydrophobic interaction chromatography and by glycerol-gradient ultracentrifugation, are disulfide-linked homodimers of these domains (i.e. RR and GG) and are morphologically identical to the alternating RR and GG domains of intact vWF. The glycoprotein fragment GG (6.5 X 35 nm) has mass 343 kDa by sedimentation equilibrium and the amino-terminal sequence of intact plasma vWF. It contains the binding site for heparin within 300 residues of its amino terminus and a separate site for the platelet GPIb receptor responsible for platelet agglutination in the presence of ristocetin. With approximately 18% alpha-helix and approximately 15% beta-pleated sheet, fragment GG accounts for most of the ordered secondary structure present in whole vWF. The two thin flexible rod domains (1.8-2.0 X 30-34 nm) of fragment RR are joined at a small central nodule (approximately 5 nm diameter) and also have a small nodule at each free end. Fragment RR contains an extraordinarily high cystine content, lower than average amounts of other hydrophobic residues, and essentially no alpha-helix, as judged by circular dichroism. The amino-terminal sequence and amino acid composition of fragment RR corresponded to that of the COOH-terminal 685 residues of the intact vWF subunit (Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., and Fujikawa, K. (1986) Biochemistry 25, 3171-3184). This sequence analysis gives a mass of 180 kDa for glycosylated fragment RR, somewhat higher than the 130 kDa we obtained by sedimentation equilibrium. Our sequence analysis of a 110-kDa plasmic vWF peptide also permitted identification of a major plasmin cleavage site 705 residues from the COOH terminus and a half-cystine residue (1360) involved in maintaining the multimeric structure of plasmin-degraded vWF.(ABSTRACT TRUNCATED AT 400 WORDS)

A new retrovirus produced by tissue culture cell line from mammary tumor of a house musk shrew, Suncus murinus

A new type of retrovirus (Sm-MTV) released by cultured cells of a spontaneous mammary tumor from a house musk shrew, Suncus murinus, is described. The Sm-MTV is distinct morphologically from type C particles. In spite of certain morphological similarities to type B and type D retroviruses, the Sm-MTV is readily distinguishable. The extracellular virions had a spikeless envelope containing a centrally located nucleoid with a small electron-dense core surrounded by an inner membrane. The budding particles contained a doughnut-shaped nucleoid. Intracytoplasmic type A particles similar in profile to those associated with mouse mammary tumor cells were also found, and tended to form a small cluster of several particles in the cytoplasm. The virus banded at 1.169 g/cm 3 in isopycnic centrifugation and possessed constitutive Mg 2+-dependent reverse transcriptase. The viral RNA had a molecular size ranging from 50 to 70 S in its native form and 30 to 40 S in its denatured form by a glycerol gradient ultracentrifugation. Major viral polypeptides were 72K, 69K, 47K, 44K/43K, 27K, 20.5K, and 15K.

Isolation of a transcription complex for ribosomal 5S RNA.

Cloned 5S rRNA genes from Xenopus borealis oocytes can be used to assemble functional transcription complexes from cytoplasmic HeLa cell extracts as a source for polymerase III and all factors additionally required for faithful 5S RNA transcription. Such complexes can be isolated by glycerol gradient ultracentrifugation and non-denaturing gel electrophoresis. They contain less than 1% of the cellular protein and retain their fidelity to synthesize 5S rRNA. The assembly of the complex is unaffected by KCl concentrations up to 140 mM whereas the transcription of 5S rRNA by the isolated complex is significantly reduced at this ionic strength. This indicates that the latter process, involving re-initiation by RNA polymerase III, is more sensitive to elevated salt concentrations than is the assembly of the transcription complexes. Furthermore, we show that complex formation also takes place in the absence of exogenously added nucleoside triphosphates, although this results in a slight shift in the sedimentation position which can be reversed by addition of the initial nucleotides GTP and CTP. We have analyzed the isolated transcription complexes by the protein blotting technique in an attempt to characterize their DNA-binding components. The results show a single component, corresponding to a protein with a mol. wt. of approximately 45 kd, which binds selectively, but not exclusively to a DNA fragment containing the 5S gene. The possible relationship of this protein to transcription factor IIIA from Xenopus oocytes is discussed.

Porcine liver nuclear histone acetyltransferase. Partial purification and basic properties.

The major histone acetyltransferase activity from porcine liver nuclei has been isolated and partially purified by a simple, rapid, and reproducible method. Extraction of nuclei in buffered 30% saturated ammonium sulfate and subsequent ammonium sulfate fractionation, chromatography on DEAE-Sephacel and hydroxylapatite, and ultracentrifugation on linear 15-30% glycerol gradients provides an 8650-fold purification (over nuclei) in 42% yield. The molecular weight of the enzyme is approximately 94,000 as determined by glycerol gradient ultracentrifugation and gel filtration on Sephacryl S-200. The optimum pH for the reaction is 7.5 and the activity is inhibited by monovalent and divalent salts and by sulfhydryl blocking reagents. The enzyme activity is substantially protected from thermal denaturation at 37 degrees C by the addition of glycerol to the incubation medium. In the presence of the core histones, the enzyme catalyzes the acetylation reaction in the order H3 greater than H4 greater than H2B greater than H2A; the order for histones bound in nucleosome core particles is H4 greater than H2B greater than H3 greater than H2A. The high mobility group proteins 14 and 17 serve as substrates for the enzyme in vitro, suggesting a possible role for enzymatic high mobility group acetylation in chromatin function.

Cytotoxic activity of partially purified Escherichia coli alpha haemolysin.

Alpha haemolysin, produced by Escherichia coli, grown in a chemically defined medium, was purified 19-fold and the endotoxin content reduced 2176-fold by ultrafiltration and glycerol-gradient ultracentrifugation. Immunodiffusion of purified alpha haemolysin (PH) against antiserum to crude haemolysin (CH) revealed only one precipitation line. PH was cytotoxic in nanogram amounts for mouse-fibroblast 3T3 cells, and the cytotoxicity exhibited proportional dose-response and time-course kinetics. The cytotoxic and haemolytic activities of PH were neutralised by immunoglobulins to CH. A mutant, produced by treating the haemolytic wild type with mitomycin C, possessed all of the biochemical characteristics of the wild type with the exception that the extracellular products of the mutant were non-haemolytic and non-cytotoxic.