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Biomedical subjects

D Korn

Publications and source records attributed to D Korn.

At least 73 records · Page 4Linked to original sources
PubMed1978

Starvation---an interesting model for the study of the renin-angiotensin-aldosterone system.

Five obese subjects were studied during prolonged starvation. Then renin aldosterone system and urinary aldosterone excretion were studied during prolonged starvation and refeeding in five obese subjects. An uniform increase in aldosterone urinary excretion was observed in all subjects studied. A progressive increase in renin plasma activity was found in all patients along the starvation period with a striking additional increase after refeeding. The possible factors involved are discussed.

Adolescent↗
PubMed1977

In vitro replication of mitochondrial DNA. Elongation of the endogenous primer sequence in D loop mitochondrial DNA by human DNA polymerase beta.

Whe incubated in the presence of Mn2+ as the divalent metal activator, highly purified human DNA polymerase beta performs a selective and limited replication of KB cell closed circular mtDNA. On the basis of biochemical and electron microscopic analyses of the reaction product, we demonstrate that the polymerase specifically recognizes and elongates the 9 S primer sequence in D loop mtDNA and then proceeds to copy the displaced strand. The point at which the enzyme switches template strands is most likely that at which all negative superhelical turns have been removed and an energetically unfavorable introduction of positive superhelical turns would be required for further synthesis on the initial parental template strand. The product of the reaction is an enlarged D loop that has been converted to a duplex structure. This is the first description of the capacity of a pure eukaryotic DNA polymerase to replicate a naturally occurring, specifically initiated duplex DNA molecule. Our results suggest that this system may be particularly useful in developing an in vitro duplex circular DNA replication system with purified eukaryotic components.

Cell Line↗
PubMed1977

DNA polymerase-alpha. Purification and structural characterization of the near homogeneous enzyme from human KB cells.

In this report we describe the purification and structural characterization of a near homogeneous preparation of DNA polymerase-alpha that we have obtained from cultured human KB cells. When analyzed by nondenaturing gel electrophoresis, velocity gradient centrifugation, and isoelectric focusing, the enzyme activity demonstrates a constancy of gel mobility, sedimentation coefficient, and isoelectric point during the final three chromatographic steps of the purification. Native gel electrophoresis of the penultimate polymerase fraction at seven concentrations of acrylamide reveals co-migration of the enzyme activity with a single discernible protein band, at constant specific activity, and indicates that the polymerase protein is electrophoretically homogeneous at this stage. The purified enzyme has a sedimentation coefficient of 7.1 to 7.2 S at high or low ionic strength, a molecular weight gel filtration of 149,000, and an isoelectric point of pH 5.0 to 5.2. Sodium dodecyl sulfate gel analyses demonstrate that the polymerase-alpha molecule is a dimer comprised of two dissimilar subunits of 76,000 and 66,000 daltons that are present in equimolar ratio.

Cells, Cultured↗
PubMed1977

Effect of calcium on the recovery and distribution of DNA polymerase alpha from cultured human cells.

It was recently reported (Lynch, W. E., Surrey, S., and Lieberman, I. (1975) J. Biol. Chem. 250, 8179-8183) that the extraction of regenerating rat liver in solutions of isotonic sucrose containing 4 mM CaCl2 leads to almost quantitative recovery of DNA polymerase alpha (Weissbach, A., Baltimore, D., Bollum, F., Gallo, R., and Korn, D. (1975) Science 190, 401--402) activity in the purified nuclear compartment. Our application of this method to the isolation of the DNA polymerase activities activities from cultured human epithelial and lymphoblastoid cells has led to substantially different results. We have observed that the inclusion of Ca2+ in either isotonic sucrose or hypotonic aqueous extraction media leads to the irreversible inactivation of the majority, cytoplasmic fraction of DNA polymerase alpha activity and is without quantitative effect on the recovery of the nuclear fraction of this activity.

Calcium↗
PubMed1977

Purification and partial characterization of the principal deoxyribonucleic acid polymerase from Mycoplasmatales.

In this report we present the first description of the isolation and partial characterization of the deoxyribonucleic acid (DNA) polymerase activity from two species of Mycoplasmatales, Mycoplasma orale type 1 and M. hyorhinis. We have identified only a single DNA polymerase species in the mycoplasma crude extracts, and the enzymes from the two organisms are very similar in their structural and enzymatic properties. The purified polymerase from each source has a specific activity of greater than 50,000 U/mg of protein, a sedimentation coefficient of 5.6s, and an estimated molecular weight by gel filtration of 130,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the most highly purified M. orale fraction contains a single major protein band of 130,000 daltons, which we believe may represent the polymerase protein. The enzymes are most reactive with gapped (activated) DNA and show a marked preference for this primer template over oligodeoxyribonucleotide-initiated homoribo- or homodeoxyribo-polymers. The most purified preparations are devoid of contaminating endonuclease activity and also appear to lack associated 5' leads to 3'- or 3' leads to 5'-exonuclease activities, as determined by highly sensitive assays. The absence of the 3' leads to 5'-exonuclease is particularly remarkable in that this activity is essentially ubiquitous among the DNA polymerases that have thus far been characterized from procaryotes.

Bacterial Proteins↗
PubMed1977

Role of calcium in bone maturation arrest after thyroparathyroidectomy in the rat.

Thyroparathyroidectomy in the rat results in decreased plasma calcium and magnesium and increased phosphorus. The associated bone changes are decreased calcium, hydroxyproline, carbonate, and wholebone density. Bone magnesium, sodium, mineral density, and percent crystallinity are increased. The delayed matrix formation and mineralization previously identified by histologic techniques are herein characterized by direct measurement as arrest of the normal increase of hydroxyproline/matrix and percent mineral. The bone mineral present is of high density and x-ray-diffraction crystallinity, suggesting a decrease in the mineralization front high in the amorphous phase and/or small nondiffracting crystalloids. The chemical studies reveal that in the absence of available Ca, Mg and Na are substituted, and CO3 is decreased. The restoration of these plasma and bone abnormalities to normal by a diet high in CaCO3 adds further emphasis to the essential role of Ca in bone cell function.

Animals↗
PubMed1977

Influence of acute hypophysectomy on the natriuresis of dogs submitted to expansion of the extracellular fluid volume.

Three days after creating an access to the turcica on the sphenoid bone, twelve anesthetized mongrel dogs, after control collections, were submitted to extracellular volume expansion with saline. Thereafter, maintained constant the rate of saline infusion, the dura-mater covering the pituitary gland was incised in all dogs, the hypohysis being removed in seven and remaining untouched in the other five dogs. Pharmacological doses of pitressin, glycocorticoids and mineralocorticoids were administered to all dogs. It was shown, for the same filtered sodium load(p is greater than 0.05), that hypophysectomy produces a significant reduction (p is less than 0.05) in urinary sodium excretion previously elevated by the extracellular volume expansion, while durostomy only, without removal of th hypophysis, did not change (p is greater than 0.05) the rate of sodium excretion. Thus, the pituitary gland could be considered as contributing to natriuresis during extracellular volume expansion, all other factors related to this expansion remaining constant in both, hypophysectomized and sham operated dogs.

Animals↗
PubMed1977

14C cyanate as a tag for red cell survival in normal and uremic man.

14C cyanate was employed as a red cell tag. Incubated cells were washed before reinjection to remove free cyanate. Neither early or late elution was observed, and the mean red cell life span of 10 normal subjects was 115+/-12 days. In nine uremic subjects the mean life span corrected for blood loss was 73 days with a mean red cell turnover of about 0.79 times basal. Survival curves in normal subjects were similar to those observed with DF32P and in uremic subjects to those with 51Cr corrected for elution. These observatios show the reliability of 14C cyanate as a nonelutable red cell tag.

Carbon Radioisotopes↗
PubMed1975

"Cytoplasmic" deoxyribonucleic acid polymerase. Structure and properties of the highly purified enzyme from human KB cells.

The freshly prepared crude cytoplasmic fraction of aqueously extracted KB cells contains a single major species of DNA polymerase activity (DNA polymerase C) that sediments homogeneously in low ionic strength sucrose gradients with a peak at 10.8 S. The enzyme activity from frozen crude extracts sediments heterogeneously under these conditions with peaks at 8.4 and 10 S. In 0.45 M salt-containing gradients all of the polymerase activity is recovered as a single 6.4 S species. When purified to a specific activity of 7,300, DNA polymerase C sediments in low ionic strength gradients as a single species of 6.5 S. From combined sedimentation and gel filtration analysis, we estimate the molecular weight of the active protomeric species of the polymerase to be about 170,000. Under no conditions of ionic strength does the enzyme disaggregate to active species smaller than 6.4 to 6.5 S. Sodium dodecyl sulfate-polyacrylamide gel analysis of the most highly purified enzyme fractions reveals two major protein bands of 87,000 and 175,000 daltons, respectively. These data suggest that DNA polymerase C contains an 87,000-dalton component and permit the interpretation that the active protomer of Mr equal 170,000 may be a dimer. The purified enzyme shows maximal activity with gapped duplex DNA and has an absolute requirement for 3'-hydroxyl termini. It utilizes initiated polydeoxynucleotide templates poorly and initiated polyribonucleotide templates not at all. Although the polymerase is inhibited by PPi it has only minimal ability to promote PPi exchange (0.8% of the polymerase activity). The purified enzyme is free of endonuclease and exonuclease activities (less than or equal to 0.003% of the polymerase activity) and demonstrates no primer-template-dependent conversion of substrate dNTP to free dNMP during the polymerization reaction. Finally, DNA polymerase C does not excise misparied primer termini from a synthetic homopolymer primer-template but can utilize such termini as initiation sites, although at a very slow rate.

Cell Line↗
PubMed1975

Nuclear deoxyribonucleic acid polymerase. Further observations on the structure and properties of the enzyme from human KB cells.

At low ionic strength KB cell DNA polymerase N1 forms large aggregates of a size comparable to those of DNA polymerase C. However, in contrast to polymerase C, the polymerase N1 aggregate: (a) retains the distinctive features of the polymerase N1 monomer, specifically its relative insensitivity to salt and to p-hydroxymercuribenzoate, and its pI of 9.3; and (b) is quantitatively converted to the polymerase N1 monomer form at appropriate ionic strength. It is important to recognize that since both polymerase N1 and polymerase C undergo salt-dependent association-dissociation reactions, attempts to distinguish these clearly indedependent polymerase species on the basis of size criteria can be very misleading. This is particularly true in relatively impure enzyme fractions that are generally isolated from eukaryotic tissue sources in low ionic strength buffers. We had earlier reported (Wang, T. S.-F., Sedwick, W. D., and Korn, D. (1974) J. Biol. Chem. 249,841-850; Sedwick, W. D., Wang, T. S.-F., and Korn, D. (1972) J. Biol. Chem. 247,5026-5033; Sedwick, W. D., Wang, T. S.-F., and Korn, D. (1974) Methods Enzymol. 29, 89-102) that DNA polymerase N1 could not utilize homoribopolymer templates. We have re-examined this question with a modified and more stringent method of product assay, and we show here that a greater than or equal 95% homogeneous preparation of polymerase N1 can copy the primer-template (A)n-(dT)-/16 at about one-half the rate that it copies activated DNA under optimum incubation conditions.

Cell Line↗
PubMed1975

Identification of a new DNA polymerase activity in human KB cells.

We have isolated and partially characterized a new DNA polymerase activity from the purified nuclear fraction of cultured human KB cells. The physical and chemical properties of this enzyme which we have designated DNA polymerase N3 define it as a unique entity that can be distinguished from the several classes of eukaryotic D-DNA and R-DNA polymerase activity that have been recognized to date.

Cell Line↗