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The First Component (Cln) of the Complement System of the Nurse Shark (Ginglymostoma Cirratum)

II. Purification of the First Component by Ultracentrifugation and Studies of Its Physicochemical Properties¹

From the Department of Microbiology, University of Miami School of Medicine, P. O. Box 875, Biscayne Annex, Miami, Florida 33152

Abstract

The sedimentation coefficient of Cln varied inversely with the ionic strength in sucrose gradient ultracentrifugation. Natural antibody contamination was removed from impure Cln preparations by zonal ultracentrifugation in a high ionic strength (µ = 0.70) sucrose gradient, in which the Cln was 13.9S. The purified Cln gave a single precipitin line in Ouchterlony and immunoelectrophoresis with anti-whole nurse shark serum. Rabbit antibody against this preparation agglutinated cell-bound Cln, gave a single precipitin line of identity with purified Cln and whole nurse shark serum, and single precipitin arcs immunoelectrophoretically. At 0°C, 0.04 M EDTA inactivated Cln slowly and did not prevent Cln-site formation. EDTA inactivation was irreversible. In the presence of EDTA, immunoelectrophoresis of purified Cln gave three precipitin arcs. Esterase activity, present in the partially purified (urea-precipitated) Cln preparations, was absent in purified Cln with N-acetyl-L-tyrosine ethyl ester, p-tosyl-L-arginine methyl ester, or N-carbobenzoxy-l-tyrosine p-nitrophenyl ester as substrates.

Footnotes

¹ This report represents in part a doctoral dissertation (G. D. R.) submitted to the Graduate School of the University of Miami as partial fulfillment of the requirements for the Ph.D. degree; it was supported by United States Public Health Service Grant AI 08569 from the National Institute of Allergy and Infectious Diseases. G. D. R. was a recipient of a National Defense Education Act Title IV Fellowship while in Graduate School.

² Present address: National Jewish Hospital, Department of Allergy and Clinical Immunology, 3800 E. Colfax Avenue, Denver, Colorado 80206.

³ Requests for reprints should be directed to this author.

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				J. Jensen, E Festa, D. Smith, and M Cayer The complement system of the nurse shark: hemolytic and comparative characteristics Science, October 30, 1981; 214(4520): 566 - 569. [Abstract] [PDF]