Affinity chromatography of viral DNA polymerases on pyran-sepharose.

Abstract

Pyran covaiently linked to cyanogen bromide-activated Sepharose has been shown to be an effective affinity matrix for several viral DNA polymerases. Differential salt elution of viral compared with cellular polymerases, as well as substrate elution, suggests the affinity nature for the matrix. Unlike some other affinity systems described, pyran-Sepharose is totally resistant to nuclease digestion and is stable at 4°for several months. DNA polymerases isolated from several viruses by detergent treatment were recovered in good yield. Analysis of iodinated proteins by sodium dodecyl sulfate-gel electro-.phoresis revealed that the DNA polymerase of avian myeloblastosis virus found in crude preparations. of the virus could be purified nearly to homogeneity by a single passage through the column. These results suggest that pyran-Sepharose is an effective affinity column that is potentially adaptable as part of a general purification procedure for viral DNA polymerases.

Evidence against the Postulated Identity of e-Antigen with DNA Polymerase Associated with the Hepatitis B Candidate Virus

“…Although the columns adsorbed between 5 and 13% of the total protein applied and increases of ionic strength of the elution buffer resulted in detachment of the proteins in agreement with earlier results [9], eAg was detected only in the first fraction containing SB. Similar results were obtained with preparations of eAg dialyzed against SB before application to the column, with a starting buffer described for chromatography on pyran-Sepharose [8], and with eAg purified about 200-fold by affinity chromatography [11], eAg failed to become attached to immobilized calf thymus DNA or to pyran-Sepharose under conditions appropriate for complete retention of nucleic acid polymerases of diverse origin [5][6][7][8]. This is an observation in dicating that eAg is not a DNA polymerase.…”

Mechanism of release of active alpha subunit from dimeric alpha beta avian myeloblastosis virus DNA polymerase

“…As shown in Fig. 4, a progressive decrease in the weight of the (3 subunit (97,000 daltons) was observed with increases in the a subunit (63,000 daltons). Although several polypeptides were released, it should be pointed out that the three major polypeptides observed upon glycerol treatment (Fig.…”

“…The product of the reaction was alkali and RNase stable but sensitive to DNase. The AMV RELEASE OF a SUBUNIT FROM AMV DNA POLYMERASE 905 DNA polymerase was purified either by conventional methods or by affinity chromatography (3,9) and subjected to a glycerol gradient centrifugation as a final step.…”

Structural requirements for binding of bovine tRNATrpwith avian myeloblastosis virus DNA polymerase

“…AMV 1DA polymerase in contrast, was reported to bind to intact tTrP but not to fragments that resulted fran nuclease digestion (10,11). Our interest has been in studies dealing with the enzymology of AMV DNA polymerase and its interaction with tRATrP (6,(12)(13)(14). In this paper we report the isolation of a bovine tRNATrP fragment that binds to AMV D1…”