Purification and characterization of alpha-macroglobulin and ovomacroglobulin of the green turtle (Chelonia mydas japonica).
The plasma alpha-macroglobulin and egg white ovomacroglobulin were purified from the sea turtle, Chelonia mydas japonica, and their structural and functional properties were studied with the aim of clarifying the degree of evolutional divergence of two homologous proteins specific to different tissues of the same animal. The concentration of alpha-macroglobulin in green turtle plasma was about 4 mg/ml. The protein was purified from the plasma by precipitation with polyethylene glycol 6000, followed by zinc chelate chromatography and gel chromatography on Sepharose CL-6B. The concentration of ovomacroglobulin in green turtle egg white was about 0.4 mg/ml. Ovomacroglobulin was purified by gel chromatography on Sepharose CL-6B. The two proteins had similar molecular weights and amino acid compositions, and both inhibited proteinases such as trypsin, chymotrypsin, papain, and thermolysin. The amino terminal sequences of the two proteins were homologous to each other but higher homologies were found between the ovomacroglobulin of turtle and chicken, and between the serum macroglobulins of the same animals. The functional difference between turtle alpha-macroglobulin and ovomacroglobulin became clear when they were treated with methylamine, which is known to destroy the inhibitory activity of human alpha 2-macroglobulin by splitting internal thiolester bonds. The inhibitory activity of the turtle plasma protein was completely destroyed by methylamine but that of ovomacroglobulin was only partially affected. The number of sulfhydryl groups as titrated with 5,5'-dithiobis(2-nitrobenzoate) before and after treatment with proteinases or methylamine was different for the two proteins. The amount of radioactive methylamine that was incorporated was also different between the two proteins. The two proteins purified in this study had no immunological cross-reactivity.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004527:Egg Proteins / Q000032:analysis / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D008259:Macroglobulins / Q000032:analysis / Q000302:isolation & purification*; D008744:Methylamines / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000032:analysis; D014361:Trypsin Inhibitors; D014426:Turtles / Q000097:blood / Q000378:metabolism*; D014461:Ultracentrifugation; D000511:alpha-Macroglobulins / Q000032:analysis / Q000302:isolation & purification*
D000596:Amino Acids; D004527:Egg Proteins; D008259:Macroglobulins; D008744:Methylamines; D013438:Sulfhydryl Compounds; D014361:Trypsin Inhibitors; D000511:alpha-Macroglobulins; C036828:ovomacroglobulin; C027451:methylamine; D010447:Peptide Hydrolases