Ascorbic acid transport by 3T6 fibroblasts. Regulation by and purification of human serum complement factor

Area of Science:

• Biochemistry
• Immunology
• Cell Biology

Background:

• Serum complement activation by endotoxins or immunocomplexes previously shown to inhibit ascorbate transport in fibroblasts.
• Understanding the mechanism and nature of the inhibitor is crucial for comprehending cellular nutrient regulation during immune responses.

Purpose of the Study:

• To elucidate the mechanism by which complement activation inhibits ascorbate transport.
• To identify the nature and origin of the inhibitor generated during complement activation.

Main Methods:

• Kinetic analysis of ascorbate transport in 3T6 fibroblasts.
• Assessing heat stability and reversibility of the inhibitor.
• Complement component consumption assays (C3, C5, Factor B).
• Purification of the inhibitor from human serum and molecular mass determination.
• Inhibition assays using antiserum against C3a.

Main Results:

• The inhibitor increased the Km for ascorbate without altering Vmax, indicating reduced transporter affinity.
• The inhibitor was heat-stable and reversible, suggesting a proteinaceous nature.
• Complement component C3 consumption was observed, but not C5 or Factor B.
• A 9000-dalton inhibitor was purified, and its activity was neutralized by anti-C3a antiserum.

Conclusions:

• The inhibitor of ascorbate transport is generated during the C3 step of complement activation.
• The inhibitor is likely a small protein fragment related to C3a.
• Complement activation during infection or autoimmune conditions may impair tissue ascorbate supply.