Platelet-derived growth factor. Isolation by a large-scale procedure and analysis of subunit composition

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Platelet-derived growth factor (PDGF) is a potent mitogen involved in cell growth and proliferation.
• Previous purification methods often involved SDS, potentially altering protein structure and activity.

Purpose of the Study:

• To develop a large-scale purification method for PDGF that avoids SDS.
• To characterize the biochemical and structural properties of purified PDGF.

Main Methods:

• Large-scale purification of PDGF from human platelets without SDS.
• Analytical gel electrophoresis in the presence of SDS.
• Sedimentation-equilibrium analysis in an ultracentrifuge.
• Amino acid analysis.
• SDS-PAGE under reducing and non-reducing conditions.

Main Results:

• Obtained 0.5 mg of highly pure PDGF from 3 x 10^13 platelets.
• Purified PDGF exhibited high specific activity in multiplication-stimulating assays.
• Amino acid analysis confirmed the presence of all common amino acids except tryptophan and the absence of hexosamine.
• Determined molecular weight of ~33,000 Da by sedimentation-equilibrium and non-reducing SDS-PAGE.
• SDS-PAGE under reducing conditions revealed two distinct lower molecular weight components (17,000 and 14,000 Da).

Conclusions:

• PDGF was successfully purified on a large scale without SDS, preserving its biological activity.
• The findings suggest PDGF is a heterodimer composed of two different polypeptide chains linked by disulfide bonds.