Subunit interaction of rabbit muscle phosphofructokinase

Effects of purification procedures

Michael A. Luther, Lyndal K. Hesterberg, James Lee

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Structural, physical, and kinetic properties of rabbit muscle phosphofructokinase (PFK) purified by three different procedures were monitored in order to determine the effect of various purification procedures on the dynamics of subunit interaction. PFK was purified by three commonly used procedures: (1) differential heat precipitation [Kemp, R. G. (1972) Methods Enzymol. 42, 71-77], (2) differential heat and alcohol precipitation [Ling, R. H., Marcus, F., & Lardy, H. A. (1965) J. Biol. Chem. 240, 1893-1899], and (3) differential salt fractionation [Hesterberg, L. K., & Lee, J. C. (1980) Biochemistry 19, 2030-2039]. The physical, kinetic, and structural properties of these three preparations show that these proteins are not identical. Sedimentation velocity studies show that PFK purified by method 3 self-associates rapidly and that the system is thermodynamically homogeneous. The presence of an inactive or noninteracting component is not observed within an 8-h time limit. In contrast, PFK purified by method 1 or 2 is heterogeneous. In these preparations, a slowly sedimenting, noninteracting, inactive form of PFK is present. The remaining active protein is not stable but continuously converts to an inactive form. Active PFK can be fractionated from this inactive form by sedimentation. This active fraction behaves as a thermodynamically homogeneous system, and the subunits undergo rapid association-dissociation in a manner similar to PFK purified by method 3. Kinetic studies on these three preparations show that the inclusion of a heat and/or alcohol step in the purification procedure yields an enzyme that is less stable, has a lower specific activity, requires DTT for full activation, and is more susceptible to inhibition by ATP. PFK purified by method 2 has been demonstrated to have a lower subunit molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mapping. This study demonstrates unequivocally the detrimental effects of including heat and alcohol precipitation steps in the purification procedure for PFK. It also shows that the various oligomeric states of native PFK subunits are in a dynamic, rapid equilibrium and that the kinetic parameters of active PFK are dependent on protein concentration; thus, these results demonstrate that the self-assembly of PFK subunits must play a role in the regulation of PFK activity.

Original languageEnglish (US)
Pages (from-to)2463-2470
Number of pages8
JournalBiochemistry
Volume24
Issue number10
StatePublished - 1985
Externally publishedYes

Fingerprint

Phosphofructokinases
Purification
Muscle
Rabbits
Muscles
Hot Temperature
Alcohols
Sedimentation
Kinetics
Biochemistry
Proteins
Peptide Mapping
Fractionation
Electrophoresis
Kinetic parameters
Sodium Dodecyl Sulfate
Self assembly
Structural properties
Polyacrylamide Gel Electrophoresis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Luther, M. A., Hesterberg, L. K., & Lee, J. (1985). Subunit interaction of rabbit muscle phosphofructokinase: Effects of purification procedures. Biochemistry, 24(10), 2463-2470.

Subunit interaction of rabbit muscle phosphofructokinase : Effects of purification procedures. / Luther, Michael A.; Hesterberg, Lyndal K.; Lee, James.

In: Biochemistry, Vol. 24, No. 10, 1985, p. 2463-2470.

Research output: Contribution to journalArticle

Luther, MA, Hesterberg, LK & Lee, J 1985, 'Subunit interaction of rabbit muscle phosphofructokinase: Effects of purification procedures', Biochemistry, vol. 24, no. 10, pp. 2463-2470.
Luther, Michael A. ; Hesterberg, Lyndal K. ; Lee, James. / Subunit interaction of rabbit muscle phosphofructokinase : Effects of purification procedures. In: Biochemistry. 1985 ; Vol. 24, No. 10. pp. 2463-2470.
@article{1447a6912d254e80bb84cdc739356001,
title = "Subunit interaction of rabbit muscle phosphofructokinase: Effects of purification procedures",
abstract = "Structural, physical, and kinetic properties of rabbit muscle phosphofructokinase (PFK) purified by three different procedures were monitored in order to determine the effect of various purification procedures on the dynamics of subunit interaction. PFK was purified by three commonly used procedures: (1) differential heat precipitation [Kemp, R. G. (1972) Methods Enzymol. 42, 71-77], (2) differential heat and alcohol precipitation [Ling, R. H., Marcus, F., & Lardy, H. A. (1965) J. Biol. Chem. 240, 1893-1899], and (3) differential salt fractionation [Hesterberg, L. K., & Lee, J. C. (1980) Biochemistry 19, 2030-2039]. The physical, kinetic, and structural properties of these three preparations show that these proteins are not identical. Sedimentation velocity studies show that PFK purified by method 3 self-associates rapidly and that the system is thermodynamically homogeneous. The presence of an inactive or noninteracting component is not observed within an 8-h time limit. In contrast, PFK purified by method 1 or 2 is heterogeneous. In these preparations, a slowly sedimenting, noninteracting, inactive form of PFK is present. The remaining active protein is not stable but continuously converts to an inactive form. Active PFK can be fractionated from this inactive form by sedimentation. This active fraction behaves as a thermodynamically homogeneous system, and the subunits undergo rapid association-dissociation in a manner similar to PFK purified by method 3. Kinetic studies on these three preparations show that the inclusion of a heat and/or alcohol step in the purification procedure yields an enzyme that is less stable, has a lower specific activity, requires DTT for full activation, and is more susceptible to inhibition by ATP. PFK purified by method 2 has been demonstrated to have a lower subunit molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mapping. This study demonstrates unequivocally the detrimental effects of including heat and alcohol precipitation steps in the purification procedure for PFK. It also shows that the various oligomeric states of native PFK subunits are in a dynamic, rapid equilibrium and that the kinetic parameters of active PFK are dependent on protein concentration; thus, these results demonstrate that the self-assembly of PFK subunits must play a role in the regulation of PFK activity.",
author = "Luther, {Michael A.} and Hesterberg, {Lyndal K.} and James Lee",
year = "1985",
language = "English (US)",
volume = "24",
pages = "2463--2470",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "10",

}

TY - JOUR

T1 - Subunit interaction of rabbit muscle phosphofructokinase

T2 - Effects of purification procedures

AU - Luther, Michael A.

AU - Hesterberg, Lyndal K.

AU - Lee, James

PY - 1985

Y1 - 1985

N2 - Structural, physical, and kinetic properties of rabbit muscle phosphofructokinase (PFK) purified by three different procedures were monitored in order to determine the effect of various purification procedures on the dynamics of subunit interaction. PFK was purified by three commonly used procedures: (1) differential heat precipitation [Kemp, R. G. (1972) Methods Enzymol. 42, 71-77], (2) differential heat and alcohol precipitation [Ling, R. H., Marcus, F., & Lardy, H. A. (1965) J. Biol. Chem. 240, 1893-1899], and (3) differential salt fractionation [Hesterberg, L. K., & Lee, J. C. (1980) Biochemistry 19, 2030-2039]. The physical, kinetic, and structural properties of these three preparations show that these proteins are not identical. Sedimentation velocity studies show that PFK purified by method 3 self-associates rapidly and that the system is thermodynamically homogeneous. The presence of an inactive or noninteracting component is not observed within an 8-h time limit. In contrast, PFK purified by method 1 or 2 is heterogeneous. In these preparations, a slowly sedimenting, noninteracting, inactive form of PFK is present. The remaining active protein is not stable but continuously converts to an inactive form. Active PFK can be fractionated from this inactive form by sedimentation. This active fraction behaves as a thermodynamically homogeneous system, and the subunits undergo rapid association-dissociation in a manner similar to PFK purified by method 3. Kinetic studies on these three preparations show that the inclusion of a heat and/or alcohol step in the purification procedure yields an enzyme that is less stable, has a lower specific activity, requires DTT for full activation, and is more susceptible to inhibition by ATP. PFK purified by method 2 has been demonstrated to have a lower subunit molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mapping. This study demonstrates unequivocally the detrimental effects of including heat and alcohol precipitation steps in the purification procedure for PFK. It also shows that the various oligomeric states of native PFK subunits are in a dynamic, rapid equilibrium and that the kinetic parameters of active PFK are dependent on protein concentration; thus, these results demonstrate that the self-assembly of PFK subunits must play a role in the regulation of PFK activity.

AB - Structural, physical, and kinetic properties of rabbit muscle phosphofructokinase (PFK) purified by three different procedures were monitored in order to determine the effect of various purification procedures on the dynamics of subunit interaction. PFK was purified by three commonly used procedures: (1) differential heat precipitation [Kemp, R. G. (1972) Methods Enzymol. 42, 71-77], (2) differential heat and alcohol precipitation [Ling, R. H., Marcus, F., & Lardy, H. A. (1965) J. Biol. Chem. 240, 1893-1899], and (3) differential salt fractionation [Hesterberg, L. K., & Lee, J. C. (1980) Biochemistry 19, 2030-2039]. The physical, kinetic, and structural properties of these three preparations show that these proteins are not identical. Sedimentation velocity studies show that PFK purified by method 3 self-associates rapidly and that the system is thermodynamically homogeneous. The presence of an inactive or noninteracting component is not observed within an 8-h time limit. In contrast, PFK purified by method 1 or 2 is heterogeneous. In these preparations, a slowly sedimenting, noninteracting, inactive form of PFK is present. The remaining active protein is not stable but continuously converts to an inactive form. Active PFK can be fractionated from this inactive form by sedimentation. This active fraction behaves as a thermodynamically homogeneous system, and the subunits undergo rapid association-dissociation in a manner similar to PFK purified by method 3. Kinetic studies on these three preparations show that the inclusion of a heat and/or alcohol step in the purification procedure yields an enzyme that is less stable, has a lower specific activity, requires DTT for full activation, and is more susceptible to inhibition by ATP. PFK purified by method 2 has been demonstrated to have a lower subunit molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mapping. This study demonstrates unequivocally the detrimental effects of including heat and alcohol precipitation steps in the purification procedure for PFK. It also shows that the various oligomeric states of native PFK subunits are in a dynamic, rapid equilibrium and that the kinetic parameters of active PFK are dependent on protein concentration; thus, these results demonstrate that the self-assembly of PFK subunits must play a role in the regulation of PFK activity.

UR - http://www.scopus.com/inward/record.url?scp=0021874989&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021874989&partnerID=8YFLogxK

M3 - Article

VL - 24

SP - 2463

EP - 2470

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 10

ER -