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The Reaction of Zymosan with the Properdin System: Isolation of Purified Factor D from Guinea Pig Serum and Study of Its Reaction Characteristics¹

From the Department of Microbiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Abstract

On incubation with serum, zymosan (Z) combines with several serum components, collectively designated X, to form an intermediate, ZX, which activates the complement system by cleavage of components C3 and C5. The enzymes, responsible for cleaving C3 and C5 differ from , and ,,, and are collectively designated "properdin enzymes." In our earlier studies it was shown that these are multi-unit complexes comprising factor B and complement fragment C3b. The present experiments extend our work on the C3-cleaving properdin enzyme with respect to a third component, designated factor D. On extended incubation at 37°C and long storage at 0°C, ZX changes to a severely decayed precursor, designated ZX^d2, due to loss of factors B and D. if fresh factor B is supplied, ZX^d2 can serve as a reagent for the detection and measurement of D. This method is far more sensitive than assaying with factor B and the cobra venom factor. Rigorous evidence is presented in the present work that the factor detected by each of these assay systems is one and the same despite the large difference in sensitivity. A procedure is described for isolation of D from guinea pig serum in a highly purified state as judged by disc electrophoresis on polyacrylamide gel. The final product was found to have a molecular weight of about 22,000 daltons, and a frictional coefficient, f/fo, of about 1.1, as estimated from its sedimentation rate of ca. 2.6S, its diffusion coefficient of ca. 10.5 x 10^-7 cm²/sec, and an assumed partial specific volume of 0.725. The isoelectric point was 9.35. Adsorption of D on ZX^d2 is favored by low pH and low ionic strength and proceeds in the absence of divalent cations. The rate of uptake of D on ZX^d2 is not influenced greatly by temperature and, therefore, does not appear to involve enzymatic cleavage.

Footnotes

¹ This work was supported in part by United States Public Health Service Training Grant 5 TO1 AI00282-10, United States Public Health Service Research Grant 5 RO1 AI02566-15, and National Science Foundation Grant GB7406X3.

² Supported by a grant from Deutsche Forschungsgemeinschaft.

³ Supported by a grant from Stetler Research Fund for Women Physicians.