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Watanabe, Kiyotaka; Hayashi, Kazuhiro; Miyamoto, Toshihiro; Tanaka, Mina; Okano, Shozo; Yamamoto, Shinji

doi:10.1023/a:1009267515715

Cited by 30 publications

(18 citation statements)

References 28 publications

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“…Blood samples and livers were collected 2 days after the final administration from the untreated and iron-loaded rats under pentobarbital-induced anesthesia prior to sacrifice. The serum samples and tissues were stored at -25°C until use.Ferritin was purified from rat livers as previously described [19]. Protein was determined according to the method of Lowry et al…”

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“…Ferritin iron in the liver extracts was determined essentially by an immunoprecipitation technique described for measuring ferritin iron in fetal bovine serum [9]. Ferritin in rat liver extracts and sera was determined using a sandwich enzyme-linked immunosorbent assay (ELISA) established previously [19].Serum ferritin iron was determined as follows. First, 5 µg of purified rabbit antibodies specific for rat liver ferritin was added to 1 ml of rat serum, and the mixture was incubated overnight at 4°C.…”

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“…Rabbit antisera to rat liver ferritin were prepared, and antibodies to the ferritin were purified from the antisera by affinity chromatography as described previously [19].…”

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“…Ferritin was purified from rat livers as previously described [19]. Protein was determined according to the method of Lowry et al [11] using bovine serum albumin as a protein standard.…”

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confidence: 99%

“…Protein was determined according to the method of Lowry et al [11] using bovine serum albumin as a protein standard. Rabbit antisera to rat liver ferritin were prepared, and antibodies to the ferritin were purified from the antisera by affinity chromatography as described previously [19].…”

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Iron Content of Rat Serum Ferritin.

Watanabe

Yamashita

Ohgawara

et al. 2001

The Journal of Veterinary Medical Science

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ABSTRACT. The serum ferritin concentration was significantly higher in female than in male rats, reflecting higher iron stores in females than in males. The mean iron/protein ratio of serum ferritin was 0.018 ± 0.008 (SD) (µg of Fe/µg of protein) in female rats and 0.011 ± 0.011 in male rats, being much lower than that of liver ferritin (0.233 ± 0.014 in females and 0.227 ± 0.020 in males). Iron loading of rats significantly increased serum ferritin concentration, but did not influence the iron content of serum ferritin. These results indicate that rat serum ferritin contains only a small amount of iron independent of body iron stores. KEY WORDS: ferritin iron, rat, serum ferritin.J. Vet. Med. Sci. 63(5): 587-589, 2001 Ferritin is a major iron-storage protein that is composed of 24 subunits of two types, termed H and L, the molecular masses of which are 21 kDa and 20 kDa, respectively [4,8,16]. Ferritin is present not only in cells but also in sera, and the serum ferritin level is positively correlated with body iron stores [1-3, 12, 15, 18]. The major function of tissue ferritin is to detoxify and store intracellular iron [8], and it contains a large amount of iron [5,14,17]. Serum ferritin has been found to have different amounts of iron in some mammals [6,9,13,20,22]. The physiological significance of serum ferritin and its iron remains unclear. Although rat serum ferritin was purified by Halliday et al. [7], its iron has yet to be determined. In the present study, the iron content of rat serum ferritin was measured, and compared with that of serum ferritin from other mammals.Female and male Wistar rats aged 9-12 weeks (Clea Japan, Tokyo, Japan) were used. Male rats were iron-loaded by intraperitoneal injection of iron dextran (100 mg iron/ml; Sigma, St. Louis, MO, U.S.A.) to increase body iron stores. Rats received single doses of 7 mg of iron three times on alternate days. Control rats were injected with an equivalent volume of 0.9% NaCl. Blood samples and livers were collected 2 days after the final administration from the untreated and iron-loaded rats under pentobarbital-induced anesthesia prior to sacrifice. The serum samples and tissues were stored at -25°C until use.Ferritin was purified from rat livers as previously described [19]. Protein was determined according to the method of Lowry et al.[11] using bovine serum albumin as a protein standard. Rabbit antisera to rat liver ferritin were prepared, and antibodies to the ferritin were purified from the antisera by affinity chromatography as described previously [19].Each rat liver was homogenized in a Waring blender for 3 min with 10 volumes of 10 mM Tris containing 0.2 mM Pefabloc SC (Merck, Darmstadt, Germany) as a serine proteinase inhibitor. Each homogenate was divided into two parts; one was used for measuring non-heme iron; and the other was centrifuged at 24,000 × g for 20 min at 4°C, then the supernatant (liver extract) was used for measuring ferritin protein and ferritin iron. Total non-heme iron in liver homogenates was measured as previo...

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confidence: 99%

“…Rabbit antisera to rat liver ferritin were prepared, and antibodies to the ferritin were purified from the antisera by affinity chromatography as described previously [19].…”

mentioning

confidence: 99%

“…Ferritin was purified from rat livers as previously described [19]. Protein was determined according to the method of Lowry et al [11] using bovine serum albumin as a protein standard.…”

mentioning

confidence: 99%

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confidence: 99%

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Iron Content of Rat Serum Ferritin.

Watanabe

Yamashita

Ohgawara

et al. 2001

The Journal of Veterinary Medical Science

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Mass spectrometric characteristics and kinetics of iron release in visceral mass of Saccostrea cucullata

Zhu

Huang

2011

Rapid Comm Mass Spectrometry

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Ferritins with electrophoretic homogeneity were prepared from the visceral mass of Saccostrea cucullata in batch. The native PAGE approach showed similar electrophoretic mobility among pig pancreatic ferritin, liver ferritin of Dasyatis akajei, and visceral mass ferritin of Saccostrea cucullata. SDS-PAGE indicated that the Saccostrea cucullata visceral ferritin (SCVF) consisted of a single subunit type and had a molecular weight (MW) of approximately 20 kDa, suggesting that the protein shell in SCVF was composed of a single subunit. In addition, peptide mass fingerprinting and transmission electron microscopy were used to identify SCVF further, and to observe its molecular structure. We found that the molecular structure in SCVF was similar to that of most mammalian ferritins, which are composed of a protein shell and an iron core. The results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry under the assistance of an acidic matrix, sinapic acid, also showed that SCVF was composed of a single subunit type and its subunit MW was calculated to be 19871.042 Da in the absence of heme. Kinetics analysis revealed that the complete process of iron release fitted the law of a first-order reaction, which is similar to that of most ferritins in mammals. Similar to bacterial ferritin, studies indicated that the shell consisted of a single subunit type and showed similar kinetics of iron release, suggesting that this subunit plays two important roles in iron release and storage, and that it shows different stability and intensity of interaction in carrying out its physiological functions in SCVF.

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Purification and Characterization of Canine Serum Ferritin-binding Proteins

et al. 2006

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Ferritin-binding protein (FBP) is known to interact with circulating ferritins in mammals. Canine FBPs were purified from canine serum by affinity chromatography and were identified as IgM, IgG, and IgA by immunoblotting with alkaline phosphatase-labeled antibodies to canine IgM, IgG, and IgA heavy chains. Following further purification by application to a Sephacryl S-300 column, canine FBPs were separated into 81.3- and 27.7-kDa bands by sodium dodecyl sulfate-polyacryamide gel electrophoresis, and the 81.3-kDa band reacted with the anti-canine IgM heavy chain antibody. Purified canine FBP bound to canine liver ferritin, but not to canine albumin and transferrin. FBP showed greater binding to the expressed bovine ferritin H-chain homopolymer than to the expressed bovine ferritin L-chain homopolymer. The binding of FBP with canine liver ferritin was dose-dependently inhibited by anti-rat liver ferritin antibody, and the anti-ferritin antibody dissociated the bound FBP in a dose-dependent manner, even after binding FBP with liver ferritin. The canine ferritin H subunit peptide fragment with amino acid residues 148-155 (NH(2)-GDHVTNLR-COOH) in its C-terminal region was recognized by FBP. These results indicate that canine serum FBPs are autoantibodies to ferritin (IgM, IgG, and IgA) and that anti-ferritin autoantibody (IgM) recognizes the C-terminal region of ferritin H subunit.

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Cited by 30 publications

References 28 publications

Iron Content of Rat Serum Ferritin.

Iron Content of Rat Serum Ferritin.

Mass spectrometric characteristics and kinetics of iron release in visceral mass of Saccostrea cucullata

Purification and Characterization of Canine Serum Ferritin-binding Proteins

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