α2-µ-Globulin fragment (a2-f) from kidneys of male rats (original) (raw)
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α2-μ-Globulin Fragment (A2-f) from Kidneys of Male Rats forms Multimers in Vitro
International Journal of Bioscience, Biochemistry and Bioinformatics, 2014
2-µ-Globulin fragment (A2-f) functions as a fatty acid binding protein in kidneys of male rats. It has 100% sequence homology with amino acids 10-160 of 2-µ-Globulin (A2), an 18.6-kDa protein that is synthesized in male rat liver and is present in urine. A2-f is produced from A2 by the removal of 3 residues from the N-terminus and 9 amino acids residues from the C-terminus. This project aimed to isolate and purify A2-f (to >90% purity) for NMR analysis since the solution state structure of A2-f is still unknown. The purification failed due to formation of multimers in vitro but it did reveal heterogeneity of this protein as isolated from tissue. Index Terms-2--Globulin fragment (A2-f), 2--Globulin (A2), kidney fatty acid binding protein, Lipocalins, protein aggregation, male rat urinary proteins.
Hepatic α2μ-globulin localizes to the cytosol of rat proximal tubule cells
Kidney International, 2000
Hepatic ␣ 2-globulin localizes to the cytosol of rat proximal ported in membrane-bound vesicles to lysosomes are tubule cells. degraded, and their constituent amino acids are recycled Background. ␣ 2-Globulin (A 2), an 18.6 kD protein of he-[1-3]. To date, accumulation of an exogenous, physiologpatic origin, accumulates in the proximal tubule as an abundant, ically relevant protein in the cytosol of the renal epithe-15.5 kD cleavage product termed "A 2-fragment" (A 2-f). A 2-f lial cell (REC) has not been reported. Using standard facilitates proximal tubule fatty acid oxidation, presumably by binding hydrophobic ligands. This requires some A 2-f to enter techniques for isolating low molecular weight molecules the cytosol of the renal epithelial cell (REC). The localization that bind hydrophobic ligands, we and others purified a of A 2 /A 2-f in the proximal tubule cell was evaluated in this soluble 15.5 kD protein from the male rat kidney [4-6]. study. This abundant protein has an identical primary sequence Methods. Immunoblot analysis of renal cortical homogeto amino acids 10 to 160 of hepatic ␣ 2-globulin (named nates separated by differential centrifugation and quantitative immunoelectron microscopy (IEM) was performed to localize "A 2 " in [7]). Although A 2 mRNA is present in the liver, A 2 /A 2-f using an affinity-purified antibody that detects both it cannot be detected in the kidney by polymerase chain proteins. To evaluate A 2 as a physiologically relevant ligand, reaction [8]. A comparison of the primary structure of the accumulation of A 2-f in the female rat kidney (normally these two proteins suggests that A 2 is converted to A 2devoid of A 2-f) was examined after the induction of hepatic fragment (A 2-f) by limited proteolysis, most likely within A 2 synthesis. Ligand binding, uptake, and degradation assays were used to assess A 2 processing by RECs in vitro. the renal parenchyma [9-11]. Results. Although A 2 and A 2-f were detected in the "lyso-A 2-f, a 15.5 kD protein, is unusually abundant in the somal" fraction, only A 2-f was found in the soluble protein male kidney, where it represents as much as 5% of total fraction. IEM confirmed the presence of significant signal in soluble protein [4, 5, 7]. A 2-f binds hydrophobic ligands, the vesicular and lysosomal as well as the cytosolic compartincluding long-chain fatty acids [4], and has been localments. In contrast, both  2 globulin (B 2) and cathepsin B were restricted to endosomes. In the female rat, induction of hepatic ized predominantly to the proximal tubule [5, 6, 8]. Al-A 2 production resulted in A 2-f accumulation in the renal cortex. though the function of A 2-f in the kidney is unclear, In RECs in culture, uptake of A 2 and B 2 demonstrated nonsaturecent studies using isolated proximal tubule segments rable, nondisplacable surface binding and similar uptake rates. in suspension suggested that A 2 fragment facilitates  Compared with B 2 , A 2 was markedly resistant to degradation. oxidation of long-chain fatty acids [8]. These findings Conclusions. A fraction of A 2 escapes lysosomal degradation, permitting A 2-f to accumulate in the cytosol of the proxisuggest that A 2-f accumulates in the epithelial cell of the mal tubule epithelial cell. A 2 may represent an unusual example Key words: fatty acid binding protein, renal epithelial cell, protein transport, long-chain fatty acid. nificant number of immunogold-labeled A 2 /A 2-f localized to the cytosol of the proximal tubule epithelial cell.
Properties and differential regulation of two fatty acid binding proteins in the rat kidney
The Journal of biological chemistry, 1988
A protein from rat kidney was characterized that had several properties common to a multigene family of fatty acid binding proteins identified in other tissues. The putative kidney fatty acid binding protein (FABP) was purified from the soluble fraction of kidney homogenates using gel filtration and ion exchange chromatography. It was relatively abundant, had an apparent molecular mass of 15.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, bound equimolar amounts of oleic acid, and could be distinguished from other FABPs on the basis of size, amino acid composition, and tissue distribution. Polyclonal antibodies to kidney FABP were obtained and used to show that only kidney contained the 15.5-kDa protein, although the antibodies also recognized a slightly larger and less abundant protein in kidney that also was present in bladder. Rat kidney also contained heart FABP, and the properties of both FABPs in rat kidney were compared. The distribution of bo...
Journal of the American Society of Nephrology : JASN, 1999
Recently, poly alpha2,8 deaminoneuraminic acid (poly alpha2,8 KDN) was demonstrated in various embryonic and adult mammalian tissues. This study reports the purification and characterization of the single poly alpha2,8 KDN-bearing glycoprotein from rat kidney. Amino acid sequences of proteolytic fragments shared homology with megalin, a member of the LDL receptor family. Immunochemical analysis supported this finding, since immunoprecipitated poly alpha2,8 KDN-bearing glycoprotein was immunoreactive with anti-megalin antibodies in Western blotting and conversely immunoprecipitated megalin was immunoreactive with the monoclonal anti-poly alpha2,8 KDN antibody. Furthermore, receptor-associated protein affinity-purified megalin reacted with the anti-poly alpha2,8 KDN antibody. By immunoelectron microscopy, labeling for both poly alpha2,8 KDN and megalin coincided in the brush border, endocytic invaginations and vesicles, and apical dense tubules of proximal convoluted tubules. Immunore...
Journal of the American Society of Nephrology, 1999
Recently, poly ␣2,8 deaminoneuraminic acid (poly ␣2,8 KDN) was demonstrated in various embryonic and adult mammalian tissues. This study reports the purification and characterization of the single poly ␣2,8 KDN-bearing glycoprotein from rat kidney. Amino acid sequences of proteolytic fragments shared homology with megalin, a member of the LDL receptor family. Immunochemical analysis supported this finding, since immunoprecipitated poly ␣2,8 KDN-bearing glycoprotein was immunoreactive with anti-megalin antibodies in Western blotting and conversely immunoprecipitated megalin was immunoreactive with the monoclonal anti-poly ␣2,8 KDN antibody. Furthermore, receptor-associated protein affinity-purified megalin reacted with the anti-poly ␣2,8 KDN
α2U-Globulin: measurement in rat kidney and relationship to hyaline droplets
Clinica Chimica Acta, 1986
The concentration of renal quglobulin increased in a dose-dependent manner in adult male but not female rats which received a single dose of 2,2,4_trimethylpentane (TMP). After administration of a single dose of 12 mmol TMP/kg to adult male rats, the renal concentration of (Y ,,-globulin reached a peak at 48 hours and returned to near background level after 7 days. These changes in renal a,,-globulin concentration were closely paralleled by changes in renal hyaline droplet formation. Renal CY ,,-globulin and hyaline droplets were absent in normal pre-puberty male rats, and neither could be stimulated by a single dose of TMP. a,,-Globulin was localised in the renal cortex of adult male rats, in particular the S2 segment of the proximal tubule. A greater staining intensity due to a,,-globulin was seen in the S2 and adjacent segments after a single dose of TMP. A strong association is suggested between the presence of renal hyaline droplets and the occurrence of a,"-globulin.
Tissue-specific expression of the rat alpha 2u globulin gene family
Molecular and cellular biology, 1986
The rat alpha 2u globulin gene family encodes approximately 20 low-molecular-weight (20,000) proteins with pIs ranging from 4.5 to 7.9. alpha 2u globulin protein isoforms were detected in the liver and in the submaxillary, lachrymal, preputial, and mammary glands of Sprague-Dawley rats. The hormonal and developmental regulation of alpha 2u globulin synthesis in each of these tissues was unique, and it appears that different alpha 2u gene sets were transcribed in the various tissues.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1988
Hepatic synthesis of a~-globulin in the male rat begins at puberty (about 40 days), reaches a peak level at about 80 days, and ceases at about 750-800 days of age. The age-dependent changes in a2u-globulin synthesis are correlated with both the steady-state level of the hepatic mRNA for this protein and the rate of transcription of the a~-giobulin gene family. Transcriptional activation of the a2~-giobulin gene family at puberty and cessation of transcription at senescence correlate with the association and dissociation of this gene domain with the nuclear matrix. Unlike the aTe-globulin gene, the albumin gene in the liver shows preferential association with the nuclear matrix throughout the life. From these results we conclude that the age-dependent changes in a2u-globulin synthesis are due to the alteration in the rate of transcription of the a2u-globulin gene, and that the association of this gene domain to the nuclear matrix is a prerequisite to its transcriptional activation.
Progress in Growth Factor Research, 1989
The peptide amylin (previously termed Diabetes Associated Peptide) has recently been isolated and characterisedjirom the amyloid of the pancreatic islets of Langerhans from human type 2 diabetics [I]. Amylin shows about 46% identity in amino acid sequence on comparison with the calcitonin gene-related peptides (CGRPs) and also shows some similarity to insulin [I]. Recent studies have also shown that both amylin and CGRP are potent inhibitors of insulin-stimulatedglycogen synthesis in skeletal muscle in vitro [2,3]. Hormones may be arranged into families, therefore a degree of order exists even though hormone-mediatedeflects are complex [4]. Thepolypeptides insulin, insulinlike growth factors (IGFs) and relaxins have been grouped into such a family with similarities both at the protein-structural and genetic levels [4.5]. We now demonstrate that this insulin-related family, along with amylin and the CGRPs. are members of a peptide superfamily defined by structural similarity in the region corresponding to the Achain of insulin. In order to distinguish this grouping of small biologically active peptides from the previous one, we have designated it the amylin superfamily. All the members of the previously a'ejined insulin family have a region homologous to the insulin B-chain. Insulin, the IGFs. the relaxins, the CGRPs and amylin are all involved in carbohydrate metabolism and therefore these peptides are functionally as well as structurally related. This grouping of peptides may have important implications for the study of human metabolic disease.