Avian myeloblastosis virus reverse transcriptase. Effect of glycerol on its hydrodynamic properties.
Although reverse transcriptase has been the subject of intensive investigation, minimal information is available regarding the physical properties of the enzyme. The basic hydrodynamic properties of avian myeloblastosis virus reverse transcriptase in solution were measured by both sedimentation velocity and equilibrium measurements in two buffer systems. In a 0.3 M potassium phosphate buffer system, pH 7.8, the enzyme sedimented as a homogenous particle with a sedimentation coefficient of (7.1 +/- 0.3) S with a weight-average molecular weight, Mw, of (1.52 +/- 0.05) x 10(5). Since the enzyme consists of an alpha and beta subunit of equal molar ratio with Mw of 6.3 x 10(4) and 9.4 x 10(4), respectively, it was concluded that the enzyme exists as an alpha beta heterodimer in this buffer system. In a Tris buffer system, pH 7.9, containing 0.46 M NaCl and 4% glycerol, the native enzyme also sedimented as a homogeneous particle with an apparent sedimentation coefficient of (10.1 +/- 0.5) S, without considering the effect of glycerol on solvent-protein interaction. Based on the results of Gekko and Timasheff (Gekko, K., and Timasheff, S. N. (1981) Biochemistry 20, 4667-4676) and the polarity of the enzyme, it was estimated that there is significant solvent-protein interaction even at 4% glycerol leading to a value of -0.06 g/g in the preferential solvent interaction parameter. When the solvent effect was taken into consideration, the value for s020,w increased from 10.1 to 11.9 S, implying that the native enzyme dimerizes in the presence of 4% glycerol. The combined results of gel filtration and sedimentation velocity showed that the dimerization of the enzyme to form (alpha beta)2 is favored at 20 degrees C with the alpha beta form predominating at 4 degrees C. The secondary structure of the reverse transcriptase was measured by circular dichroism. Results showed that the enzyme consists of (16 +/- 2)% alpha-helix, (24 +/- 2)% beta-sheet, (24 +/- 2)% beta-turn, and (36 +/- 4)% undefined structures.
Lin
T H
TH
Institute for Molecular Virology, St. Louis University School of Medicine, Missouri 63104.
Quinn
T
T
Walsh
M
M
Grandgenett
D
D
Lee
J C
JC
D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D005990:Glycerol / Q000494:pharmacology; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D014461:Ultracentrifugation
D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D012194:RNA-Directed DNA Polymerase; D005990:Glycerol