Abstract
Acute-phase serum proteins were induced by administrating a chicken with turpentine oil. One of these proteins was a new protein that appeared in front of albumin in polyacrylamide disc gel electrophoresis using a 4.5-16% gel. To purify this protein, turpentine-administrated chicken serum was fractionated by ammonium sulfate precipitation at 50% saturation, and the supernatant fraction was chromatographed on a DEAE-Toyopearl 650S column. The purified protein is a mannose-glycoprotein, and its N-terminal sequence, determined by the Edoman method, is not homologous from that of other reported acute-phase proteins. An analysis of physiological function with two different test systems, chemiluminescence measurement and electron spin resonance spectroscopy, showed that the purified protein has antioxidant activity and inhibits superoxide (O(2)) mediated by activation of the receptor. In support of these results, the complete amino acid sequence of 18-B is homologous to the scavenger receptor cysteine-rich (SRCR) family of proteins that participate in the regulation of leukocyte function. 18-B is composed of four SRCR domains, which is different from the previously characterized SRCR family of proteins such as Spalpha, CD6, and CD163. These findings indicate that turpentine-induced 18-B, a new member of scavenger receptor cysteine-rich family, may be implicated in regulation of cell function in a manner of inhibition of the overproduction of the reactive oxygen species.
Highlights
Several acute-phase proteins in serum, such as C-reactive protein, hemopexin, fibrin, fibrinogen, transferrin, ␣1-acid glycoprotein, and ␣2-macroglobulin, have been reported in chicken [1,2,3,4,5,6,7]
Urban et al [11] reported that a new acute-phase ␣-1 protein in rat was induced by turpentine administration and that its molecular mass based on SDS-polyacrylamide disc gel electrophoresis (PAGE) was 68 kDa. 18-B in chicken is similar in electrophoretic characteristics and molecular size to Urban’s protein in rat, but it is not clear whether both these proteins are identical or not
Identification—In the disc electrophoresis of chicken serum obtained after turpentine administration, two protein bands appeared in front of the albumin band (Fig. 1B)
Summary
Chicken Serum—An adult female Leghorn chicken was intramuscularly administrated with turpentine (3 ml/kg of body weight) and bled 48 h later. Proteins were eluted from the column by the linear gradient method with 0 – 0.5 M NaCl in borax-phosphate buffer at a flow rate of 1 ml/min. The N-terminal amino acid sequence was determined by Edoman method with a model 477A protein sequencer on-line with a model 120A phenylthiohydantoin-derivative analyzer (PerkinElmer Life Sciences). ESR settings were as follows: modulation frequency, 100 kHz; modulation amplitude, 0.1 millitesla; scanning field, 334.6 Ϯ 15 mT; receiver gain, 5 ϫ 103 for sOZ or 2.5 ϫ 10 3 for PMA; time constant, 0.3 s; sweep time, 8 min; microwave power, 12 milliwatts; microwave frequency, 9.415 GHz. RNA Isolation, cDNA Synthesis, and PCR Amplification—Total RNA was isolated from chicken tissues with TriZOL reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. Nested PCR was carried out using the primer P3 and AUAP, and the PCR products were subcloned and sequenced as described above
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