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.     

Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells

We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.     

Purification and partial characterization of a calmodulin-stimulated nucleoside triphosphatase from pea nuclei

A nucleoside triphosphatase/deoxynucleoside triphosphatase associated with the chromatin fraction from a highly purified preparation of pea nuclei has been isolated and characterized. The purified enzyme has a molecular weight of 47,000 as checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it has an isoelectric point of 6.6. In the presence of divalent cations (Mg2+ = Mn2+ greater than Ca2+), this enzyme hydrolyzes nucleoside triphosphates or deoxynucleoside triphosphates. Hydrolysis is optimal at pH 7.5 and is significantly inhibited by relatively low concentrations of quercetin, but is not sensitive to vanadate, nitrate, or oligomycin. The enzyme has a rather broad nucleotide substrate specificity and has a Km for MgATP2- of 0.6 mM. The enzyme activity is stimulated over 3-fold by Ca2+ and calmodulin, and the stimulation is blocked by the Ca2+ chelator EGTA and by the calmodulin antagonists compound 48/80 and chlorpromazine.     

Monodispersal and deoxyribonucleic acid analysis of prostatic cell nuclei.

We collected prostatic glands from 50 unselected autopsies at the Pathology Institute and compared their histologic sections with cytologic preparations and with results of photometric measurements of isolated prostatic cells and isolated nuclei. The results obtained with single cell photometry and flow-through cytophotometry proved to be comparable with one another and with the results of the cytologic and histologic studies. With these methods of cytophotometry we could differentiate inflammatory conditions, microcarcinomas and frank carcinomas from normal and hyperplastic prostatic tissue. We had difficulties, however, preparing adequate suspensions of cell nuclei from chronic fibrosing prostatitis. Our results indicate that it should be possible for diagnostic purposes to combine the technique of fine needle biopsy of the prostate with that of flow-through cytophotometry and to use the combined techniques for studying diseases of other organs.     

Partial purification and characterization of dynein adenosine triphosphatase from bovine sperm

Outer dynein arm polypeptides that possess Mg+2-adenosine triphosphatase (ATPase) activity have been extracted from the flagellar axonemes of demembranated bovine sperm. Electron microscopy of intact and salt-extracted sperm demonstrates a relatively selective removal of the outer dynein arms. The salt extract contains a specific ATPase activity of 55 nmoles inorganic phosphate (Pi)/min/mg protein. Sucrose density gradient centrifugation of this extract results in a 6-fold increase in specific activity of ATPase (333 nmole/Pi/min/mg protein), which sediments as a single 13S peak. Concomitant with the increase in specific activity, there is enrichment of three high molecular weight polypeptides (Mr greater than 300,000) characteristic of dynein heavy chains. ATPase activities in the initial extract and in the 13S peak are inhibited by concentrations of vanadate and erythro-9-[3-2-(hydroxynonyl)]adenine similar to those that inhibit ATPase activity in sea urchin sperm dynein. These findings indicate that outer arm dynein ATPase can be extracted and partially purified from bovine sperm.     

Isolation and characterization of adenosine triphosphatase from Salicornia pacifica var. utahensis

Membrane-bound ATPase associated with plasma membrane and solubleATPase associated with the cytoplasm were prepared from shootsof Salicornia pacifica var. utahensis by sucrose density gradientcentrifugation. The isolated ATPases were tolerant to high concentrationsof NaCl. The Km for membrane-bound ATPase was 1.75 mM and forsoluble ATPase, it was 1.4 mM. The relative effectiveness ofdivalent cations for stimulation of membrane-bound ATPase wasMg²⁺>Fe²⁺>Mn²⁺>Co²⁺>Cu²⁺. Soluble ATPase activitywas stimulated by Ba²⁺>Ca²⁺>Mg²⁺ and was inhibited byCu²⁺, Zn²⁺, Co²⁺ and Fe²⁺. The compounds N,N¹-dicyclohexylcarbodiimide,NaF and ADP, did inhibit the ATPases but ouabain, triphenyltinhydroxide, sodium azide, indoleacetic acid and abscisic aciddid not inhibit the ATPases from s. pacifica var. utahensis. ¹ Present address: Department of Biology, Kong-Ju National College,Kong-Ju, Korea. (Received April 1, 1980; )     

The activity of deoxyribonucleic acid polymerase and deoxyribonucleic acid synthesis in nuclei from brain fractionated by zonal centrifugation

1. The DNA polymerase (EC 2.7.7.7) activity in purified intact brain nuclei from infant rats was investigated. The effects of pH, Mg(2+), glycerol, sonication and storage of the nuclei under different conditions were examined and a suitable assay system was established. 2. The nuclei from infant brain cells were fractionated by zonal centrifugation in a discontinuous sucrose gradient into five zones: zone (I) contained neuronal nuclei (59%) and astrocytic nuclei (41%); zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (19%); zone (III) contained astrocytic nuclei (82%) and oligodendrocytic nuclei (18%); zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained oligodendrocytic nuclei (100%). 3. The content of DNA, RNA and protein for each fraction was measured. 4. The distribution of DNA polymerase activity in the fractionated infant and adult rat brain nuclei was determined. The highest activity was found in the neuronal nuclei from zone (I) and the following zones exhibited a progressive decline. In contrast with the nuclei from infant rats those from adults had a much higher activity and expressed a preference for native DNA as template. 5. The deoxyribonuclease activity in all classes of nuclei was measured with [(3)H]DNA as substrate. A general correspondence in the pattern of the relative activities in the nuclear fractions with the distribution of DNA polymerase was found. 6. The incorporation of [(3)H]thymidine into nuclear DNA in infant and adult rat brain was investigated. The specific radioactivity of the DNA in the 10-day-old rats was highest in zone (V) whereas in the nuclei of adult rats, which exhibited a comparatively low incorporation, the highest specific radioactivity was associated with zones (I) and (V).     

Adenovirus deoxyribonucleic acid replication. II. Synthesis of viral deoxyribonucleic acid in vitro by a nuclear membrane fraction from infected KB cells.

A cell-free system for the study of viral DNA replications was developed by the isolation of a nuclear membrane fraction "DNA replication complex" from adenovirus 2-infected human KB cells late after infection. This complex which possesses both DNA polymerase activity and a virus-specific endonuclease synthesizes exclusively virus-specific DNA sequences in vitro by a semiconservative mechanism. Analysis by rate zonal sedimentation in alkaline sucrose gradients showed that the products of the reaction are small DNA chains approximately 6 to 9 S, presumably "Okazaki intermediates," that are not sealed under our in vitro conditions. Analysis by rate zonal sedimentation in neutral sucrose gradients showed that labeled viral DNA fragments are hydrogen bonded to viral 18 S DNA segments, 0.25 the size of the linear, viral 31 S DNA genome. The 18 S DNA is probably a specific cleavage product of the viral endonuclease found in the replication complex and could represent intermediates in viral DNA replication or degradation products.