Avian myeloblastosis virus reverse transcriptase

Effect of glycerol on its hydrodynamic properties

Thy Hou Lin, Tom Quinn, Mary Walsh, Duane Grandgenett, James Lee

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

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, M̄w, of (1.52 ± 0.05) × 105. Since the enzyme consists of an α and β subunit of equal molar ratio with M̄w of 6.3 × 104 and 9.4 × 104, respectively, it was concluded that the enzyme exists as an αβ 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 80 20,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 (αβ)2 is favored at 20°C with the αβ form predominating at 4°C. The secondary structure of the reverse transcriptase was measured by circular dichroism. Results showed that the enzyme consists of (16 ± 2)% α-helix, (24 ± 2)% β-sheet, (24 ± 2)% β-turn, and (36 ± 4)% undefined structures.

Original languageEnglish (US)
Pages (from-to)1635-1640
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number3
StatePublished - Jan 25 1991
Externally publishedYes

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Avian Myeloblastosis Virus
RNA-Directed DNA Polymerase
Hydrodynamics
Viruses
Glycerol
Enzymes
Sedimentation
Buffers
Tromethamine
Biochemistry
Dimerization
Circular Dichroism
Gel Chromatography
Proteins
Physical properties
Molecular Weight
Gels
Molecular weight

ASJC Scopus subject areas

  • Biochemistry

Cite this

Lin, T. H., Quinn, T., Walsh, M., Grandgenett, D., & Lee, J. (1991). Avian myeloblastosis virus reverse transcriptase: Effect of glycerol on its hydrodynamic properties. Journal of Biological Chemistry, 266(3), 1635-1640.

Avian myeloblastosis virus reverse transcriptase : Effect of glycerol on its hydrodynamic properties. / Lin, Thy Hou; Quinn, Tom; Walsh, Mary; Grandgenett, Duane; Lee, James.

In: Journal of Biological Chemistry, Vol. 266, No. 3, 25.01.1991, p. 1635-1640.

Research output: Contribution to journalArticle

Lin, TH, Quinn, T, Walsh, M, Grandgenett, D & Lee, J 1991, 'Avian myeloblastosis virus reverse transcriptase: Effect of glycerol on its hydrodynamic properties', Journal of Biological Chemistry, vol. 266, no. 3, pp. 1635-1640.
Lin, Thy Hou ; Quinn, Tom ; Walsh, Mary ; Grandgenett, Duane ; Lee, James. / Avian myeloblastosis virus reverse transcriptase : Effect of glycerol on its hydrodynamic properties. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 3. pp. 1635-1640.
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abstract = "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, M̄w, of (1.52 ± 0.05) × 105. Since the enzyme consists of an α and β subunit of equal molar ratio with M̄w of 6.3 × 104 and 9.4 × 104, respectively, it was concluded that the enzyme exists as an αβ 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 80 20,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 (αβ)2 is favored at 20°C with the αβ form predominating at 4°C. The secondary structure of the reverse transcriptase was measured by circular dichroism. Results showed that the enzyme consists of (16 ± 2){\%} α-helix, (24 ± 2){\%} β-sheet, (24 ± 2){\%} β-turn, and (36 ± 4){\%} undefined structures.",
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