Abstract：
:A rapid, three-step purification of DNA alpha-polymerase from calf thymus is described. The key feature is immunoaffinity chromatography using a column of immobilized monoclonal immunoglobulin G (IgG) developed against human KB cell alpha-polymerase. This step is followed by preparative sucrose gradient sedimentation. The highly purified polymerase has a specific activity of 35 000 nmol of nucleotide incorporated per hour per milligram. Its molecular weight is 404 000. This molecular weight is higher than observed in some earlier purifications, possibly because salt concentrations are kept at nearly physiological levels. Also, the rapidity of purification in the presence of multiple protease inhibitors minimizes degradation. The purified enzyme is inhibited by aphidicolin, N-ethylmaleimide, and the specific monoclonal IgG, thereby identifying it as DNA alpha-polymerase. ATP at 4 mM concentration stimulates enzymatic activity up to 4-fold on calf thymus DNA templates. The enzyme is also capable of priming single-stranded DNA with RNA. The procedure represents a significant advance from purifying alpha-polymerase from calf by conventional means, since it avoids ion-exchange chromatography and harsh conditions. It also minimizes the time required to produce sufficient quantities of purified high molecular weight polymerase for analysis.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Wahl AF,Kowalski SP,Harwell LW,Lord EM,Bambara RAdoi
10.1021/bi00304a001subject
Has Abstractpub_date
1984-04-24 00:00:00pages
1895-9issue
9eissn
0006-2960issn
1520-4995journal_volume
23pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract：:Kinetics studies of dNTP analogues having pyrophosphate-mimicking β,γ-pCXYp leaving groups with variable X and Y substitution reveal striking differences in the chemical transition-state energy for DNA polymerase β that depend on all aspects of base-pairing configurations, including whether the incoming dNTP is a puri...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi500101z
更新日期：2014-03-25 00:00:00
abstract：:Two enantiomeric analogues of farnesyl pyrophosphate (1) were tested as inhibitors and anomalous substrates of trichodiene synthase, which catalyzes the cyclization of trans,trans-farnesyl pyrophosphate (1) to the sesquiterpene hydrocarbon trichodiene (2). The reaction has been shown to involve preliminary isomerizati...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00475a011
更新日期：1990-06-12 00:00:00
abstract：:Electron spin resonance (ESR) spectroscopy using spin-labeled ATP was used to study nucleotide binding to and structural transitions within the multidrug resistance P-glycoprotein, P-gp. Spin-labeled ATP (SL-ATP) with the spin label attached to the ribose, was observed to be an excellent substrate analogue for P-gp. S...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi0512445
更新日期：2005-10-25 00:00:00
abstract：:The complete amino acid sequence of human liver NADPH-cytochrome P-450 reductase has been determined by microsequence analysis and mass spectrometry. The total sequence consists of 676 amino acids initiated by an amino-terminal acetyl group. There is no evidence for posttranslational modifications, including Asn-linke...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00447a054
更新日期：1989-10-17 00:00:00
abstract：:As part of our studies on the molecular mechanism of mutation [Chambers, R. W. (1982) in Molecular and Cellular Mechanisms of Mutagenesis (Lemontt, J. F., & Generoso, W. M., Eds.) pp 121-145, Plenum, New York and London], we wanted to prepare specific oligonucleotides carrying O2- or O4-alkylthymidine residues. Since ...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00430a007
更新日期：1989-02-21 00:00:00
abstract：:The photolyase/cryptochrome family is a large family of flavoproteins that encompasses DNA repair proteins, photolyases, and cryptochromes that regulate blue-light-dependent growth and development in plants, and light-dependent and light-independent circadian clock setting in animals. Phylogenetic analysis has reveale...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi801085a
更新日期：2008-09-30 00:00:00
abstract：:A preparation consisting of isolated dimeric peripheral antenna complexes from green plant photosystem I (light-harvesting complex I or LHCI) has been characterized by means of (polarized) steady-state absorption and fluorescence spectroscopy at low temperatures. We show that this preparation can be described reasonab...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi0007369
更新日期：2000-07-25 00:00:00
abstract：:We consider the effects that different lipid surfaces have upon the denaturation and subsequent formation of amyloid fibrils of bovine insulin. The adsorption and unfolding kinetics of insulin being adsorbed onto the different lipid surfaces under denaturing conditions are studied using FTIR ATR spectroscopy and are c...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi020525z
更新日期：2002-12-31 00:00:00
abstract：:The binding of horse ferricytochrome c to yeast cytochrome c peroxidase at pH 6.0 in 8.7 mM phosphate buffer (0.0100 M ionic strength) is characterized by a small, unfavorable enthalpy change (+1.91 +/- 0.16 kcal mol-1) and a large, positive entropy change (+37 +/- 1 eu). The free energy of binding depends strongly up...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00026a022
更新日期：1995-07-04 00:00:00
abstract：:To elucidate early stages in protein folding, we have adopted a fragment reconstitution method for small proteins. This approach is expected to provide nuclei for protein folding and to allow us to investigate folding mechanisms. In previous work [Kobayashi, N., et al. (1995) FEBS Lett. 366, 99-103.] we demonstrated t...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi982271g
更新日期：1999-01-26 00:00:00
abstract：:A new fluorescence method to measure viscosity in cell cytosol [Fushimi, K., & Verkman, A. S. (1991) J. Cell Biol. 112, 719-725] has been applied to determine fluid-phase viscosity in sea urchin eggs. Freshly harvested eggs from Lytechinus pictus were loaded with the dyes 2,7-bis(2-carboxyethyl)-5-(and-6-)carboxyfluor...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00115a600
更新日期：1991-12-24 00:00:00
abstract：:The importance of a specific hydrogen bond between thermolysin and a phosphonamidate inhibitor, Z-NHCH2-PO(O-)-Leu-Leu (1) [Bartlett, P. A., & Marlowe, C. K. (1987) Science (Washington D.C.) 235, 569-571], has been reevaluated. We have determined the inhibition constants (binding free energies) for thermolysin of phos...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00438a006
更新日期：1989-06-13 00:00:00
abstract：:The proposal that microtubule dynamic instability results from stabilization of microtubule ends by tubulin-GDP-Pi subunits (where Pi is inorganic phosphate) [Melki et al. (1996) Biochemistry 35, 12038] was based on studies of GTP hydrolysis and microtubule assembly that showed that tubulin-GDP-Pi subunits can transie...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi972602+
更新日期：1998-09-15 00:00:00
abstract：:The carbon monoxide binding curve to human hemoglobin A0 has been measured to high precision in experimental conditions of 600 microM heme, 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid, 0.1 M NaCl, 10 mM inositol hexaphosphate, 1 mM disodium ethylenediaminetetraacetic acid, pH 6.94, and 25 degrees C. Co...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00394a031
更新日期：1987-10-06 00:00:00
abstract：:The symmetric, tandem GU mismatch motifs, and , which only differ in the mismatch order, have an average difference in thermodynamic stability of 2 kcal/mol at 37 degrees C. Thermodynamic studies of duplexes containing these motifs indicate the effect is largely localized to the mismatches and adjacent base pairs. The...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi970122c
更新日期：1997-07-01 00:00:00
abstract：:Base excision repair (BER) plays a vital role in maintaining genomic integrity in mammalian cells. DNA polymerase λ (Pol λ) is believed to play a backup role to DNA polymerase β (Pol β) in base excision repair. Two oxidized abasic lesions that are produced by a variety of DNA-damaging agents, including several antitum...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi301592x
更新日期：2013-02-05 00:00:00
abstract：:Filamin A (FLNA) is a ubiquitously expressed actin cross-linking protein and a scaffold of numerous binding partners to regulate cell proliferation, migration, and survival. FLNA is a homodimer, and each subunit has an N-terminal actin-binding domain followed by 24 immunoglobulin-like repeats (R). FLNA mediates mechan...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/acs.biochem.9b00100
更新日期：2019-11-26 00:00:00
abstract：:The electric organ of the ray, Torpedo ocellata, can serve as a source for both the acetylcholine (ACh) receptor and its ionic channel. The two entities were identified by their specific binding of [3H]ACh and [3H]perhydrohistrionicotoxin ([3H]H12-HTX), respectively. Binding of [3H]H12-HTX was inhibited by certain dru...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00618a023
更新日期：1978-12-12 00:00:00
abstract：:The high-frequency resonance Raman spectra of FeIII yeast native cytochrome c peroxidase (CCP) and five of its mutants [CCP(MI), Phe-51, Leu-48, Lys-48, Asn-235, and Phe-191] were recorded in phosphate buffer, pH 7.0, and in glycerol/phosphate mixtures at 295 and 10 K. Glycerol induces heme coordination changes in som...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00438a024
更新日期：1989-06-13 00:00:00
abstract：:The RecB and RecC subunits of the RecBCD enzyme from Escherichia coli were purified from cells containing plasmids overproducing these proteins [Boehmer, P.E., & Emmerson, P.T. (1991) Gene 102, 1-6]. RecB hydrolyzes ATP in the presence of either single- or double-stranded DNA. RecC stimulates ATP hydrolysis by RecB, p...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00069a024
更新日期：1993-05-11 00:00:00
abstract：:DNA-DNA interstrand cross-links are thought to be important for the cytotoxicity of many chemotherapeutic agents. To study this more definitively, adduct site-specific methods are used to construct a plasmid with a single nitrogen mustard interstrand cross-link (inter-HN2-pTZSV28). Replication efficiency (RE = [coloni...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi962778w
更新日期：1997-03-25 00:00:00
abstract：:The multimeric cytochromes described to date in sulfate- and sulfur-reducing bacteria are associated with diverse respiratory modes involving the use of elemental sulfur or oxidized sulfur compounds as terminal acceptors. They exhibit no structural similarity with the other cytochrome c classes and are characterized b...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi9707741
更新日期：1997-09-02 00:00:00
abstract：:The three-dimensional chalice-like crystal structure of initiation factor 2 IF2/eIF5B from Methanobacterium thermoautotrophicum represents a novel fold and domain architecture in which the N-terminal G domain and the C-terminal C domain are separated by an approximately 40 A alpha-helix. Homologous Thermus thermophilu...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi702295g
更新日期：2008-04-29 00:00:00
abstract：:A mutant form of the nitrogenase iron protein with a deletion of residue Leu 127, located in the switch II region of the nucleotide binding site, forms a tight, inactive complex with the nitrogenase molybdenum iron (MoFe) protein in the absence of nucleotide. The structure of this complex generated with proteins from ...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi001645e
更新日期：2001-01-23 00:00:00
abstract：:Site-specific mutagenesis has been used to create two mutant versions of aspartate transcarbamoylase. Arg-167 and Gln-231, both previously identified as interacting with the portion of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate (PALA) that corresponds to aspartate [Krause, K. L., Voltz, K. W., & Lipscomb...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00468a003
更新日期：1990-04-24 00:00:00
abstract：:The unfolding and dissociation of the tetrameric enzyme fructose-1,6-bisphosphatase from pig kidney by guanidine hydrochloride have been investigated at equilibrium by monitoring enzyme activity, ANS binding, intrinsic (tyrosine) protein fluorescence, exposure of thiol groups, fluorescence of extrinsic probes (AEDANS,...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi034203m
更新日期：2003-06-17 00:00:00
abstract：:Mg2+ is important for the RNase activity of the hammerhead ribozyme. To investigate the binding properties of Mg2+ to the hammerhead ribozyme, cleavage rates and CD spectra for substrates containing inosine or guanosine at the cleavage site were measured. The 2-amino group of this guanosine interfered with the rate of...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00235a005
更新日期：1991-05-28 00:00:00
abstract：:In type 2 diabetics, the hormone amylin misfolds into amyloid plaques implicated in the destruction of the pancreatic β-cells that make insulin and amylin. The aggregative misfolding of amylin is pH-dependent, and exposure of the hormone to acidic and basic environments could be physiologically important. Amylin has t...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi401164k
更新日期：2014-01-21 00:00:00
abstract：:Two thiol-activated endopeptidases with pH optima near pH 7.5 were isolated from the supernatant fraction of rabbit brain homogenates by DEAE-cellulose chromatography, gel filtration and isoelectrofocusing. Peptide bond hydrolysis was measured quantitatively by ion-exchange chromatography with an amino acid analyzer. ...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00654a026
更新日期：1976-05-04 00:00:00
abstract：:Fourier transform infrared (FTIR) spectroscopy was applied to the blue-light photoreceptor photoactive yellow protein (PYP) to investigate water structural changes possibly involved in the photocycle of PYP. Photointermediates were stabilized at low temperature, and difference IR spectra were obtained between intermed...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi000357f
更新日期：2000-07-11 00:00:00
