The Presence of Two Low Molecular Mass Proteins Immunologically Related to 14 Kilodalton Serum Amyloid A in the Lipoprotein Fraction and Their Decreased Serum Concentrations in Calves with Experimentally Induced Pneumonia

Abstract

ABSTRACT. A 14 kilodalton (kDa) serum amyloid A (apoSAA) protein was purified from cow serum. Rabbit antiserum to the 14 kDa apoSAA recognized, in addition to the 14 kDa protein, a 7.5-9.0 kDa protein and a protein having a molecular mass of less than 6.5 kDa (<6.5 kDa protein). The possibility that the two proteins were contaminants was excluded by results showing that the two proteins detected in early stages of purification procedures were not found in the purified 14 kDa apoSAA fraction, as revealed by immunoblot analysis. As in the 14 kDa apoSAA, the 7.5-9.0 kDa protein was localized in the high-density lipoprotein fraction, while the <6.5 kDa protein was in the low-density lipoprotein fraction. In calves with pneumonia induced by inoculation to the lungs of Pasteurella haemolitica, the serum concentration of the 14 kDa apoSAA was increased, whereas those of the 7.5-9.0 kDa and the <6.5 kDa proteins were conversely decreased. The time-course study indicated that the increase in concentration of the 14 kDa apoSAA and decrease in that of the <6.5 kDa protein occurred almost simultaneously. These results suggest that the 14 kDa apoSAA and the immunologically related 7.5-9.0 kDa and <6.5 kDa proteins act as positive and negative acute phase reactants, respectively, and also that concentrations of the three proteins are regulated in concert in acute phase plasma. fraction and, moreover, the serum concentration of the 14 kDa apoSAA was increased whereas those of the two low molecular mass proteins were conversely decreased in sera from calves with pneumonia induced by inoculation of Pasteurella haemolitica. MATERIALS AND METHODS Purification of apoSAA:ApoSAA was purified from cow serum during the course of purification of apoC-III [26]. Briefly, the total lipoprotein fraction [d<1.21, containing chylomicrons (CM) to HDL] prepared from sera of Holstein cows during midlactation was treated with an equal volume of acetone to extract low molecular mass apolipoproteins such as apoC-III and apoSAA. The supernatant obtained by centrifugation at 140 × g for 10 min was lyophilized and thereafter delipidated by mixing with 20 volumes of 2-propanol. Apolipoproteins pelleted by centrifugation at 1,300 × g for 10 min, were dissolved in 6 M urea and 50 mM Tris-HCl (pH 8.6), and applied to a column (2.5 × 90 cm) of Sephadex G-75 (Pharmacia Biotech, Uppsala, Sweden). Fractions rich in low molecular mass apolipoproteins were next applied to a DEAE-Sephacel (Pharmacia Biotech, Uppsala, Sweden) column (2 × 25 cm), and bound proteins were eluted by use of a linear gradient of NaCl (0-0.125 M, total 1 liter). Fractions containing apoSAA were dialyzed against 10 mM ammonium hydrogen carbonate, using cellulose dialyzer tubing with a 3,500 molecular weight cutoff, after which the dialysate was

Decreased Apolipoprotein C-III Concentration in the High-Density Lipoprotein Fraction from Calves Inoculated with Pasteurella haemolytica and Bovine Herpes Virus-1

“…Pneumonia was experimentally induced by inoculations of Pasteurella haemolytica and BHV-1 into calf lung lobes, as described previously [7,8,10,17]. Briefly, a suspension of Pasteurella haemolytica (serotype 1, I29 strain; 1 × 10 9 colony forming units) was administered to right lung lobes of 10 Holstein male calves (2 to 3 months old) using a fiberoptic bronchoscope.…”

Reduced Serum Lecithin:Cholesterol Acyltransferase Activity and Cholesteryl Ester Concentration in Calves Experimentally Inoculated with Pasteurella haemolytica and Bovine Herpes Virus-1

“…We have recently shown that Hp [8] and apoSAA [20] are induced in calves with pneumonic pasteurellosis. However, the induction was not specific to the inoculation, because the two proteins were also detected in sera from the control calves.…”

“…We recently reported that the induction of Hp [8] and apoSAA [20] in calves with pneumonic pasteurellosis was not specific to the bacterial inoculation. Appearance of the two acute-phase proteins in sera from the control calves were again shown by the present study.…”

“…Twenty 2-to 3-monthold male Holstein calves weighing 72 to 108 kg were used to induce experimental pneumonic pasteurellosis [9,20]. Calves were housed individually in pens, and were acclimated for 2 weeks.…”

“…Haptoglobin (Hp, [21]) and serum amyloid A (apoSAA, [20]) were purified from cow serum, and their antisera were raised in rabbits. Immunoblot analysis of Hp and apoSAA was done as described [20]. Serum concentrations of triglycerides (TG), total cholesterol (TC), FC and PL were measured enzymatically.…”