Scand. J. Immunol., Vol. 6, 1977.
A High Molecular Weight Serum Protein Is the Carrier for Amyloid-Related Protein SAA B. SKOGEN, J. B. NATVIG, T. E. MICHAELSEN & R. F. ANDERS Institute of Immunology and Rheumatology, Rikshospitalet University Hospital, National Institute of Pubhc Health, Oslo, Norway, and Department of Clinical Immunology, Karolinska Institute, Huddingc Hospital, Stockholm, Sweden
Skogen, B.. Natvig, J. B., Michaelsen, T. E. & Atidtrs, R. F. A High Molecular Weight Serum Protein Is the Carrier tor Amyloid-Related Protein SAA. Scand. } . Immunol. 6. 1365-1368, 1977. In the present study evidence is presented that SAA in serum complexes to a carrier protein with a molecular weight of 100,000-200.000 daltons. with mobility in the a-reglon on electrophoresis, and with a rather low normal scrum concentration. The carrier protein is apparently not albumin. SAA isolated from the carrier protein has a molecular weight of 14,000 daltons and does not complex to any considerable extent with itself under neutrsi conditions. B. Skogen. Institute of Immunology Norway
and Rheumatology,
F. Qiamsgl.
1, Oslo 1,
The xinique protein AA (8, 13) has been iden- under neutral and dissociating conditions, retified as the main fibrillar protein for amyloid spectively. These studies showed that SAA In deposits in secondary atnyloidosis. In serum serum complexes to a high molecular weight from such patients the related protein SAA protein of approximately 100,000-200,000 can usually be detected in elevated concentra- daltons and has a mobility in the «-region on tions before and during the developtncnt of electrophoresis. amyloidosis (5, 7, 15). This protein shows antigenic identity with protein AA when te.sted by double immunodiffusion against anti-protein AA antiserum. Protein SAA has the same N- MATERIALS AND METHODS tenninal sequence as protein AA (1) but has a Induction and isolation of protein SAA. subunit molecular weight of about 14,000 dal- SAA was induced in rabbits by subcutaneous tons (1), with small variations in several spe- injection of LPS, in saline (lipopolysaccharide cies (2), and is thus about "yO^/t- larger than B. Escherichia coli, .rtrain 0.26:B6; Difco Labprotein AA, which usually has a molecular oratories, Michigan), 2 mg/lvg body weight as weight of about 9000. previously described for mink (6, 11). The Although isolated protein SAA has an ap- rabbits were anesthetized and exsanguinated by parent molecular weight of 14,000 daltons cardiac puncture 24 h after treatment with under dissociating conditions, it is present in LPS. serum as a complex with molecular weight of The SAA-positive serutn was dialyzed over100,000-200,000 daltons (5, 16). The aim of night against water and spun at 15,000 g for this investigation was to study the mechanism 30 min, and formic acid was added to a final that causes protein SAA to occur as high concentration of 10%. SAA was isolated by molecular and low molecular weight protein gel filtration in 1 0 ^ formic acid on Bio-Gel
1364 B. Skogen, J. B. Satvig, T. E. Michaelsen 6 R. f. Anders Fig. IA. Gel filtration of SAA-positive rabbit serum on a 96 X 1.5 cm Si'phadex G-150 column, with a void volume of 18 ml. Elution rate, A ml/h. Fraction volume, ^.1 ml. Elution buffer. O.O^M Tris-HCi in O.JM NaCI and 0.2mM EDTA containing 0.2'7, NaN3. pH 7.6. Eluted at room temperature. ( • - • ) raJial immunodiffusion quantitation profile of SAA after thirtyfold concentration by lyophilization of the fractions. When normal rabbit serum was gel filtered on the same column, the associating activity was found as indicated by the bar. B. Association of normal rabbit serum and purified SAA. Gel filtration of normal rabbit serum after dissolving of different amounts of purifieii SAA. ( ..) - 0.2% mg/ml; \ ) = O.-SO and ( • - • ) mg/ml. Column conditions as in
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A High Molecular Weight Serum Protein Is the Carrier for Amyloid-Related Protein SAA B. SKOGEN, J. B. NATVIG, T. E. MICHAELSEN & R. F. ANDERS Institute of Immunology and Rheumatology, Rikshospitalet University Hospital, National Institute of Pubhc Health, Oslo, Norway, and Department of Clinical Immunology, Karolinska Institute, Huddingc Hospital, Stockholm, Sweden
Skogen, B.. Natvig, J. B., Michaelsen, T. E. & Atidtrs, R. F. A High Molecular Weight Serum Protein Is the Carrier tor Amyloid-Related Protein SAA. Scand. } . Immunol. 6. 1365-1368, 1977. In the present study evidence is presented that SAA in serum complexes to a carrier protein with a molecular weight of 100,000-200.000 daltons. with mobility in the a-reglon on electrophoresis, and with a rather low normal scrum concentration. The carrier protein is apparently not albumin. SAA isolated from the carrier protein has a molecular weight of 14,000 daltons and does not complex to any considerable extent with itself under neutrsi conditions. B. Skogen. Institute of Immunology Norway
and Rheumatology,
F. Qiamsgl.
1, Oslo 1,
The xinique protein AA (8, 13) has been iden- under neutral and dissociating conditions, retified as the main fibrillar protein for amyloid spectively. These studies showed that SAA In deposits in secondary atnyloidosis. In serum serum complexes to a high molecular weight from such patients the related protein SAA protein of approximately 100,000-200,000 can usually be detected in elevated concentra- daltons and has a mobility in the «-region on tions before and during the developtncnt of electrophoresis. amyloidosis (5, 7, 15). This protein shows antigenic identity with protein AA when te.sted by double immunodiffusion against anti-protein AA antiserum. Protein SAA has the same N- MATERIALS AND METHODS tenninal sequence as protein AA (1) but has a Induction and isolation of protein SAA. subunit molecular weight of about 14,000 dal- SAA was induced in rabbits by subcutaneous tons (1), with small variations in several spe- injection of LPS, in saline (lipopolysaccharide cies (2), and is thus about "yO^/t- larger than B. Escherichia coli, .rtrain 0.26:B6; Difco Labprotein AA, which usually has a molecular oratories, Michigan), 2 mg/lvg body weight as weight of about 9000. previously described for mink (6, 11). The Although isolated protein SAA has an ap- rabbits were anesthetized and exsanguinated by parent molecular weight of 14,000 daltons cardiac puncture 24 h after treatment with under dissociating conditions, it is present in LPS. serum as a complex with molecular weight of The SAA-positive serutn was dialyzed over100,000-200,000 daltons (5, 16). The aim of night against water and spun at 15,000 g for this investigation was to study the mechanism 30 min, and formic acid was added to a final that causes protein SAA to occur as high concentration of 10%. SAA was isolated by molecular and low molecular weight protein gel filtration in 1 0 ^ formic acid on Bio-Gel
1364 B. Skogen, J. B. Satvig, T. E. Michaelsen 6 R. f. Anders Fig. IA. Gel filtration of SAA-positive rabbit serum on a 96 X 1.5 cm Si'phadex G-150 column, with a void volume of 18 ml. Elution rate, A ml/h. Fraction volume, ^.1 ml. Elution buffer. O.O^M Tris-HCi in O.JM NaCI and 0.2mM EDTA containing 0.2'7, NaN3. pH 7.6. Eluted at room temperature. ( • - • ) raJial immunodiffusion quantitation profile of SAA after thirtyfold concentration by lyophilization of the fractions. When normal rabbit serum was gel filtered on the same column, the associating activity was found as indicated by the bar. B. Association of normal rabbit serum and purified SAA. Gel filtration of normal rabbit serum after dissolving of different amounts of purifieii SAA. ( ..) - 0.2% mg/ml; \ ) = O.-SO and ( • - • ) mg/ml. Column conditions as in
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A
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