|
|
Hokkaido University Collection of Scholarly and Academic Papers >
Theses >
博士 （獣医学） >
ウシの鉄結合タンパク質フェリチンに関する生化学的研究
| Title: | ウシの鉄結合タンパク質フェリチンに関する生化学的研究 |
| Other Titles: | Biochemical Studies on Bovine Ferritin |
| Authors: | 折野, 宏一1 Browse this author |
| Authors(alt): | Orino, Kouichi1 |
| Issue Date: | 25-Mar-1998 |
| Publisher: | Hokkaido University |
| Abstract: | Ferritin (Ft) is an iron-storage protein with a molecular weight of 480,000. Ft is an intracellular protein but also present in serum. Serum Ft concentration reflects the amount of storage iron in the body, and increases in inflammatory and malignant diseases. Fetal serum Ft level increases as gestation progresses, but there has been no information about the fetal serum Ft in the animals other than humans. It has been suggested that serum or plasma contains Ft-binding proteins which can inhibit immunoassay of Ft. The aims of this study are to clarify the molecular structure of bovine Ft and to assess the pathophysiological significance of the serum Ft. For this, first, I studied the molecular properties of bovine tissue Ft. Next, I investigated inhibitory effects of serum and plasma of bovine and horse on ELISA of their Fts. Finally, I measured the serum Ft level of bovine with various diseases and also of bovine fetus. The results are summarized as follows: 1) In order to clarify what causes the reversed mobility of bovine Ft subunits compared to other mammalian Ft subunits by SDS-PAGE, the fulllength cDNA clones for bovine Ft H(eart)- and L(iver)-type subunits were isolated from a bovine spleen λ gt11 cDNA library using anti-Ft subunit antibodies and sequ enced. The bovi ne H and L chains were compbsed of 180 and 174 amino acid residues with calculated molecular weights of 20,920 and 19,856, respectively, based on amino acid sequences of the H and L chains deduced from their cDNA s equences. These molecular weights of the H and L chains of bovine Ft contrast with those of the H and L chains of the Ft determined by SDS-PAGE (H: 18kDa; L: 21kDa). The H and L chains have 54% amino acid sequence homology. On the other hand, amino acid sequence of the H chain shows 91, 92, and 93% homologies with those of human, rat, and mouse H chains, respectively. The bovine L chain consists of the same amino acid residue number as horse, human, and rabbit L chains, and amino acid sequence of the L chain also displays high homologies with those of horse, human, and rabbit L chains (86, 84, and 87%, respectively), with 24, 28, and 23 amino acid substitutions, respectively. These sequence comparisons showed that there were neither insertion of amino acid(s) or peptide(s)in the bovine L nor deletion, On lectin blotting, bovine Ft subunits did not react with concanavalin A, suggesting that these subunits do not contain carbohydrate chain, When bovine Ft H and L chains were expressed in Spodoptera frugiperda cells using a baculovirus expression system, the expressed H and L chains showed the almost same mobilities as the respective chains of bovine spleen Ft on SDS-PAGE. These results suggest that the much slower mobility of bovine L chain compared to other mammalian L chains on SDS-PAGE may result from significant differences in the binding affinity of SDS to these L chains. 2) To confirm the effects of horse serum on immunoassay of horse Ft, two sandwich ELISA systems were developed. In System A, affinity-purified antibody to horse spleen Ft and its conjugate with alkaline phosphatase were used as the first and the second antibodies, respectively. In System B, whole antisemm and its conjugate with the enzyme were used. The recoveries of horse spleen Ft added to horse sera were very low in both systems (50-71% in System A; 42-79%in System B). However, heat treatment of the sera at 75℃ for 15 min improved the recoveries (90-96%) in System A, although the recoveries in System B were not improved sufficiently by the same treatment (75-83%). The apparent Ft concentrations of adult and newborn horse sera increased by heat treatment of the samples. These results indicate that horse serum contains a heat-unstable substance(s) which inhibits the immunoassay of horse Ft. 3) Ft concentrations of horse plasma determined by System A were 10-50% lower than those of the sera. However, Ft concentrations of plasma and serum were elevated to the almost same levels by heat treatment. The apparent Ft concentrations in horse sera were markedly decreased by adding horse fibrinogen to the sera. It was also found that horse fibrinogen bound to spleen Ft and inhibited the immunoassay of this protein. From these results, it is concluded that horse fibrinogen is a plasma-specific Ft-binding protein which inhibits the assay. 4) A highly sensitive ELISA for bovine serum Ft was developed using avidin-biotin complex technique. Estimated Ft concentrations of bovine sera were increased when the serum samples were diluted with an ELISA buffer containing 0.5 M ammonium sulfate, whereas those of bovine plasma were not affected by ammonium sulfate, being lower than those of serum samples. The recovehes of bovine spleen Ft added to bovine sera were low (39-57%), but were improved by the addition of ammonium sulfate to the sera (91-109%). However, in plasma, the recoveries were still low even after the addition of ammonium sulfate (73-87%). These results indicate that bovine serum and plasma contain Ft-binding proteins which disturb the immunoassay of bovine Ft. 5) Serum Ft concentrations of cows with leukemia (n=14), inflammatory diseases (n=7), and theileriasis (n=13) were significantly higher (149±130, 553±530, and 415±337 ng/ml, respectively) than non-pregnant cows over 2 years old (46±28 ng/ml, n=69). These results suggest that bovine serum Ft may be inflammation and malignant markers and that high Ft levels of theileriasis caused by Theileria sergenti result from the activation of reticuloendothelial system. 6) To elucidate physiological roles of serum Ft in bovine fetal circulation, the levels of Ft and its iron in fetal bovine sera were estimated. In 13 lots of commercial fetal bovine sera, the Ft levels ranged between 800 and 6,000 ng/ml. The serum Ft iron concentration measured by a quantitative immunoprecipitation technique ranged from 0.16 to 0.96μg/ml, and the iron content of Ft was about 20% regardless of serum Ft concentration. The percentage of Ft iron to total serum iron ranged from 8.8 to 28.5%, and correlated significantly with Ft concentration (r=0.9368, p<0.0001). No significant proportion of the Ft in fetal serum bound to concanavalin A. Immunoblotting showed that the molecular sizes of H- and L-type subunits of fetal serum Ft were identical to those of H and L subunits of adult bovine spleen Ft (H: 18 kDa, L: 21 kDa), respectively, and that the L subunit predominated. Serum transferrin level was relatively constant (1.8-2.2 mg/ml), whereas transferrin saturation varied from 54.8 to 91.7%. There was a significant correlation between serum Ft concentration and transferrin saturation (r=0.8864, p<0.001). These findings demonstrate that bovine fetus has the elevated iron stores and that serum Ft m ay contribute to the iron transport in the fetal circulation. |
| Conffering University: | 北海道大学 |
| Degree Report Number: | 乙第5324号 |
| Degree Level: | 博士 |
| Degree Discipline: | 獣医学 |
| Type: | theses (doctoral) |
| URI: | http://hdl.handle.net/2115/51517 |
| Appears in Collections: | 学位論文 (Theses) > 博士 （獣医学）
|
Submitter: 折野 宏一
|
