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Biomedical subjects

D Korn

Publications and source records attributed to D Korn.

At least 55 records · Page 3Linked to original sources
PubMed1982

Preparation and preliminary characterization of monoclonal antibodies against human DNA polymerase alpha.

We report the successful establishment of 16 stable murine hybridoma monoclones that produce homogeneous antibodies against KB cell DNA polymerase alpha. All of the antibodies exhibit specific binding of polymerase alpha activity, and 3 of them possess anti-polymerase alpha neutralizing activity. None of the antibodies interacts detectably with KB cell DNA polymerases beta or gamma. All of the 5 antibodies so far examined demonstrate linear Scatchard binding plots and very high binding affinities, with equilibrium dissociation constants (Kd) ranging between 3.2 x 10(-9) and 3.4 x 10(-10) M. These monoclonal antibodies comprise a set of powerful and specific reagents that should facilitate the development and application of novel approaches to the complex biochemical mechanisms of mammalian DNA replication.

Animals↗
PubMed1982

DNA polymerase-alpha. Common polypeptide core structure of three enzyme forms from human KB cells.

Aqueous extracts of exponentially growing human KB cells contain three quantitatively significant forms of DNA polymerase-alpha activity. The predominant form (about 70% of total cellular activity), which is recovered from the cytoplasm, has been previously purified to near-homogeneity and structurally characterized (Fisher, P. A., and Korn, D. (1977) J. Biol. Chem. 252, 6528-6535). The other two polymerase forms include a minor cytoplasmic activity (about 20% of total cellular activity), DNA polymerase-alpha'; and a species that is tightly associated with detergent-purified nuclei (about 10% of total cellular activity), nuclear DNA polymerase-alpha. We have now purified both minor enzyme species to near-homogeneity and show that they are physically and enzymologically similar to the predominant cytoplasmic species. All three purified forms of KB polymerase-alpha behave as physically homogeneous 7 S species with minimum protomer molecular weights of about 140,000. By examining in vitro 14C-labeled enzyme fractions by high resolution sodium dodecyl sulfate-polyacrylamide gel fluorography, we demonstrate that the three purified species display a similar subunit structure composed of a quartet of polypeptides with molecular masses of 70,000, 65,000, 59,000, and 55,000 daltons, respectively. Extended fluorographic exposures of the gels fail to reveal the presence of any polypeptides larger than 70,000 daltons. It is our present interpretation, based on these and our previous observations, that the four polypeptides reflect microheterogeneity involving two primary species, and we do not yet know how they may associate to form the catalytically active protomer of 140,000 molecular weight. From arithmetical considerations, it is plausible that one or more pairwise combinations of them might suffice to generate the 7 S polymerase activity. These results thus corroborate the principal conclusions of our previous study of the structure of the KB cell cytoplasmic polymerase. In addition, they provide the first comparative analysis of the structure of multiple, operationally distinguishable polymerase-alpha forms obtained from a single tissue, as well as the first description of the purification and structural characterization of a nuclear DNA polymerase-alpha species from any source.

Carcinoma↗
PubMed1981

Ordered sequential mechanism of substrate recognition and binding by KB cell DNA polymerase alpha.

We have used a steady-state kinetic approach in conjunction with direct velocity gradient sedimentation binding studies to examine the detailed steps that are involved in the recognition of DNA primer-template and dNTPs by near-homogeneous human DNA polymerase alpha. We demonstrate that the interaction of the polymerase with its substrates obeys a rigidly ordered sequential terreactant mechanism, with template as the first substrate, followed by primer as the second substrate and dNTP as the third. Although the binding of primer is prerequisite to the kinetically significant binding of dNTP, specification of which of the four dNTPs can then add to the enzyme is absolutely determined by base sequence of the template (the first substrate). The critical element in the proof of the ordered mechanism is the demonstration of the phenomenon of induced substrate inhibition; the presence of a dideoxy-terminated primer (dead-end inhibitor) induces substrate inhibition by dNTP which is absolutely restricted to the dNTP complementary to the template to which the blocked primer is annealed. This inhibition is kinetically com the demonstration of the phenomenon of induced substrate inhibition; the presence of a dideoxy-terminated primer (dead-end inhibitor) induces substrate inhibition by dNTP which is absolutely restricted to the dNTP complementary to the template to which the blocked primer is annealed. This inhibition is kinetically com the demonstration of the phenomenon of induced substrate inhibition; the presence of a dideoxy-terminated primer (dead-end inhibitor) induces substrate inhibition by dNTP which is absolutely restricted to the dNTP complementary to the template to which the blocked primer is annealed. This inhibition is kinetically competitive with 3' -hydroxyl-terminated (unblocked) primer and approaches 100% at saturating levels of the complementary dNTP. Direct binding studies document the specific and exclusive ability of complementary dNTPs to drive the polymerase into a stable dead-end complex with the proposed structure, enzyme.template.dideoxy primer.dNTP, thus corroborating the kinetic observations. Attempts to elucidate the order of product release from the enzyme by product inhibition studies have shown the polymerization reaction to be essentially irreversible and have thus been unsuccessful. On the basis the known processivity of KB cell DNA polymerase alpha, a preliminary model involving initial release of pyrophosphate is reasonable; however, the relationship between product release and the process of polymerase translocation remains obscure. All of the kinetic and sedimentation binding studies were performed on a variety of homopolymeric and natural heteropolymeric DNA substrates, and the consistency of the results establishes absolutely the qualitative identity of the general mechanism by which human DNA polymerase alpha recognizes and replicated polydeoxynucleotide primer-templates, regardless of their precise physicochemical nature.

Binding Sites↗
PubMed1980

Structural and enzymological characterization of a deoxyribonucleic acid dependent adenosine triphosphatase from KB cell nuclei.

We have purified to near homogeneity the single DNA-dependent ATPase activity that we have identified in extracts of KB cell nuclei. The protein structure of the enzyme was defined by sodium dodecyl sulfate gel electrophoresis, which revealed a single protein band of 75000 daltons that was coincident with the profile of ATPase activity resolved by the final step of agarose-ATP chromatography or by isoelectric focusing. The enzyme has a pI of 8.5, a Stokes' radius by gel filtration of 3.8 nm, and a sedimentation coefficient in high salt of 5.3 S. At low ionic strength the enzyme activity sediments at 7.0 S, suggesting that it may dimerize under these conditions. The purified enzyme has a specific activity of 5.9 X 10(5) nmol of ATP hydrolyzed per h per mg of protein and is devoid of endonuclease, exonuclease, RNA or DNA polymerase, nicking-closing, and gyrase activities at exclusion limits of 10(-6)-10(-8) of the ATPase activity. The enzyme can hydrolyze only ATP or dATP, to generate ADP or dADP plus Pi, but the other NTPs and dNTPs are competitive inhibitors of the enzyme with respect to ATP. A divalent cation (Mg2+ greater than Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Single-stranded DNA or deoxyhomopolymers are most effective, but blunt-ended linear and nicked circular duplex DNA molecules are also used at Vmax values approximately 20% of that obtained with single-stranded DNA. Intact duplex DNA and polyribonucleotides are unable to support ATP hydrolysis. Velocity gradient sedimentation studies corroborate the interpretations of the kinetic analyses and demonstrate enzyme binding to single-stranded DNA and nicked duplex DNA but not to intact duplex DNA. Although we have not succeeded directly in demonstrating DNA unwinding by this protein, preliminary results suggest that in the presence of ATP, the ATPase can stimulate the reactivity of homogeneous human DNA polymerases alpha and beta on nicked duplex DNA substrates.

Adenosine Triphosphatases↗
PubMed1979

Structural and enzymological characterization of the homogeneous deoxyribonucleic acid polymerase from Mycoplasma orale.

We have purified the DNA polymerase from Mycoplasma orale to homogeneity. The protein structure of the enzyme was declined by sodium dodecyl sulfate gel electrophoresis, which revealed a single band of 116 000 daltons that was coincident with the polymerase activity profile in the final step of DNA--cellulose chromatography, and by two-dimensional gel analysis, which demonstrated a single protein species at pI = 6.8 that was congruent with enzyme activity and contained the same 116 000 polypeptide. although severe enzyme aggregation occurs during nondenaturing gel electrophoresis, a monomer species can be resolved with a Mr of 140 000 by the Ferguson plot analysis. Gel filtration and velocity gradient centrifugation yield a Stokes radius of 4.8 nm and a sedimentation coefficient of 5.6 S, respectively, from which Mr values of 106 000--128 000 can be computed. The different size values suggest that the polymerase molecule is asymmetric. The purified enzyme has a specific activity of approximately 6 x 10(5) units/mg of protein and in completely devoid of exodeoxyribonuclease and endodeoxyribonuclease activities, at exclusion limits of 10(-4)--10(-6%) of the polymerase activity. The mechanism of polymerization is moderately processive, with an average of 14 +/- 4 nucleotides incorporated per binding event, and the "effective template length" on activated DNA is approximately 40 nucleotides.

DNA-Directed DNA Polymerase↗
PubMed1979

Enzymological characterization of DNA polymerase alpha. Basic catalytic properties processivity, and gap utilization of the homogeneous enzyme from human KB cells.

This report describes the results of our initial enzymological characterization of a homogeneous preparation of DNA polymerase alpha that we have purified from cultured human KB cells. Although the enzyme is most reactive with duplex DNA substrates that contain short gaps (optimally activated) in incubations that require Mg2+, the polymerase possesses the intrinsic capacity to copy the initiated ribohomopolymer template, (A)-n, (dT)-200, at low rates in the presence of Mn2+. Because of the preponderance of DNA polymerase alpha in actively multiplying vertebrate cells, it is probable that this low level of activity comprises the majority of the ribopolymer copying activity that can be detected in crude tissue extracts. The presence of contaminating or associated deoxyribonuclease activities can be excluded from the purified enzyme to levels of 10(-4) to 10(-7) of the polymerase activity. The mechanism of polymerization on activated DNA under optimum conditions is moderately processive, with 11 +/- 5 nucleotides incorporated per polymerization cycle. The polymerase is unable to work at nicks or at short gaps of approximately 20 to 30 nucleotides in length, and it measures a surprisingly invariant effective template length on optimally activated DNA and on DNA molecules that have been gapped to varying extents with Escherichia coli exonuclease III. In the "Appendix" we present an amplification of the theoretical formulation of Bambara et al. (Bambara, R. A., Uyemura, D., and Choi, T. (1978) J. Biol. Chem. 253, 413--423) that permits the use of DNA polymerases with significant associated 3' leads to 5'-exonuclease activities for the accurate measurement of average template lengths (gap sizes) and titration of usable 3'-hydroxyl primer termini in gapped, duplex DNA substrates.

Binding, Competitive↗
PubMed1979

Cardiac-pulmonary edema and low pulmonary capillary wedge pressure.

We describe a patient who presented with acute massive pulmonary edema, clinically and on chest roentgenogram. Two hours later the patient became hypotensive and was found to have a low pulmonary capillary wedge pressure (PCWP). The blood pressure returned to normal after administration of fluids. Acute pulmonary edema develops if PCWP rises higher than 25 to 30 mm Hg. In our patient, the elevated PCWP fell to low normal within two hours, when chest roentgenogram and clinical examination still suggested severe pulmonary edema. A phase lag existed between lowering of the pulmonary capillary wedge pressure and clearing of fluid from the alveolar and interstitial spaces in the lungs. At least three different pathogenetic mechanisms in patients with coronary artery disease can produce this phase lag. Transient global ischemia of the left ventricle was thought to be the responsible mechanism in our patient.

Blood Pressure↗