scholarly journals Isolation and identification of metallothionein isoforms (MT-1 and MT-2) in the rat testis

1998 ◽  
Vol 334 (3) ◽  
pp. 695-701 ◽  
Author(s):  
Junko S. SUZUKI ◽  
Naomi KODAMA ◽  
Andrei MOLOTKOV ◽  
Etsuko AOKI ◽  
Chiharu TOHYAMA
Keyword(s):  
Metal Binding ◽  
Binding Proteins ◽  
Gel Filtration ◽  
Specific Protein ◽  
Composition Analysis ◽  
Isolation And Identification ◽  
Rat Testis ◽  
Heavy Metal Binding ◽  
Protein Fragmentation ◽  
Male Genital

It has been a long-lasting controversial issue as to whether or not the male genital organs, such as the testis and prostate, contain metallothioneins (MTs), a group of cysteine-rich heavy-metal-binding proteins that play a role in detoxifying heavy metals such as cadmium (Cd). Earlier studies reported that the rodent testis lacks MTs and concluded that this is why the testis is very susceptible to Cd, although other indirect experimental evidence suggests that MTs are present in this organ. A deficiency of MTs in the testis was originally suspected on the basis of amino acid composition analysis, since MT-like proteins isolated as Cd-binding proteins did not have a characteristic MT structure. In the present study, we demonstrate that the rat testis indeed expresses Cd-binding proteins with sequences identical to those previously described for MT-1 and MT-2, the major isoforms. To confirm that MT-1 and MT-2 are present in the rat testis, we purified and isolated Cd-binding proteins by homogenization using Cd-containing buffer, followed by sequential purification using Sephadex G-75 gel filtration chromatography and anion HPLC column chromatography, which yielded Cd-binding protein-1 (Cd-BP-1) and -2 (Cd-BP-2). After pyridylethylation, Cd-BP-1 and Cd-BP-2 were subjected to specific protein fragmentation by acids and endopeptidases, which revealed that these Cd-binding proteins have the same primary structures as MT-1 and MT-2 respectively. Thus we believe that the present results clearly resolve the long-standing debate about the presence of MTs in the testis, at least in the rodent.

scholarly journals Cadmium-binding proteins of rat testes. Characterization of a low-molecular-mass protein that lacks identity with metallothionein

1984 ◽  
Vol 220 (3) ◽  
pp. 811-818 ◽  
Author(s):  
M P Waalkes ◽  
S B Chernoff ◽  
C D Klaassen
Keyword(s):  
Metal Binding ◽  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Or Groups

Cadmium-binding proteins in the cytosol of testes from untreated rats were separated by Sephadex G-75 gel filtration. Three major testicular metal-binding proteins (TMBP), or groups of proteins, with relative elution volumes of approx. 1.0 (TMBP-1), 1.7 (TMBP-2) and 2.4 (TMBP-3) were separated. Elution of Zn-binding proteins exhibited a similar pattern. TMBP-3 has previously been thought to be metallothionein (MT), and hence this protein was further characterized and compared with hepatic MT isolated from Cd-treated rats. Estimation of Mr by gel filtration indicated a slight difference between MT (Mr 10000) and TMBP-3 (Mr 8000). Two major forms of MT (MT-I and MT-II) and TMBP-3 (TMBP-3 form I and TMBP-3 form II) were obtained after DEAE-Sephadex A-25 anion-exchange chromatography, with the corresponding subfractions being eluted at similar conductances. Non-denaturing polyacrylamide-gel electrophoresis on 7% acrylamide gels indicated that the subfractions of TMBP-3 had similar mobilities to those of the corresponding subfractions of MT. However, SDS (sodium dodecyl sulphate)/12% (w/v)-polyacrylamide-gel electrophoresis resulted in marked differences in migration of the two corresponding forms of MT and TMBP-3. Co-electrophoresis of MT-II and TMBP-3 form II by SDS/polyacrylamide-gel electrophoresis revealed two distinct proteins. Amino acid analysis indicated much lower content of cysteine in the testicular than in the hepatic proteins. TMBP-3 also contained significant amounts of tyrosine, phenylalanine and histidine, whereas MT did not. U.v.-spectral analysis of TMBP-3 showed a much lower A250/A280 ratio than for MT. Thus this major metal-binding protein in testes, which has been assumed to be MT is, in fact, a quite different protein.

Insulin-like growth factors and their binding proteins in pleural fluid

1997 ◽  
pp. 467-473 ◽  
Author(s):  
Y Le Bouc ◽  
A Bellocq ◽  
C Philippe ◽  
L Perin ◽  
M Garabedian ◽  
...  
Keyword(s):  
Growth Factors ◽  
Pleural Fluid ◽  
Binding Proteins ◽  
Specific Binding ◽  
Gel Filtration ◽  
Specific Protein ◽  
Hydroxyvitamin D ◽  
Igf I ◽  
Acid Gel ◽  
Insulin Like Growth Factors

We investigated the expression and potential regulatory role of insulin-like growth factors (IGFs) and their specific binding proteins (BPs) in tuberculous and nontuberculous pleuritis. By using a radioimmunoassay after acid gel filtration chromatography, we found that mean concentrations of IGF-I were 211.9 +/- 20.2 microg/l and 203.2 +/- 31.1 microg/l in pleural fluid of 14 patients with tuberculous pleuritis and 9 patients with malignant pleuritis respectively. These values were near those in serum of the same patients (221.3 +/- 19.5 microg/l and 204.6 +/- 21.0 microg/l respectively). By using a specific protein-binding assay, we found that mean concentrations of IGF-II were 345.3 +/- 61.0 microg/l and 167.6 +/- 22.7 microg/l in tuberculous and malignant pleural effusions respectively. These values were significantly lower than those in serum of the same patients (628.3 +/- 79.0 microg/l, P<0.025 and 532.0 +/- 85.9 microg/l, P<0.025 respectively). Because bioavailability and bioactivity of IGFs may be regulated by their binding to IGFBPs, we studied IGFBP patterns in the pleural fluid of 6 patients with tuberculous pleuritis. As assessed by Western ligand blotting the levels of IGFBP-1 and IGFBP-2 were increased whereas those of IGFBP-3 were decreased in pleural fluid in comparison with serum. The decrease in IGFPB-3 levels reflected increased proteolysis, as assessed by Western immunoblotting. In spite of this presence of IGFBPs, IGFs could be responsible for the local biosynthesis of 1.25-dihydroxyvitamin D (1,25-(OH)2D) since pleural fluid levels of both IGF-I and IGF-II significantly correlated with those of 1,25-(OH)2D. These results indicate that IGFs are detectable in pleural fluid and may contribute to control the activity of 25-hydroxyvitamin D-1alpha hydroxylase in tuberculous pleuritis.

Heavy metal-binding proteins from metal-stimulated bacteria as a novel adsorbent for metal removal technology

2006 ◽  
Vol 53 (6) ◽  
pp. 221-226 ◽  
Author(s):  
D. Sano ◽  
K. Myojo ◽  
T. Omura
Keyword(s):  
Heavy Metals ◽  
Heavy Metal ◽  
Activated Sludge ◽  
Metal Binding ◽  
Binding Proteins ◽  
Metal Removal ◽  
Heavy Metal Removal ◽  
Copper Ion ◽  
Amino Acid Sequences ◽  
Heavy Metal Binding

Water pollution with toxic heavy metals is of growing concern because heavy metals could bring about serious problems for not only ecosystems in the water environment but also human health. Some metal removal technologies have been in practical use, but much energy and troublesome treatments for chemical wastes are required to operate these conventional technologies. In this study, heavy metal-binding proteins (HMBPs) were obtained from metal-stimulated activated sludge culture with affinity chromatography using copper ion as a ligand. Two-dimensional electrophoresis revealed that a number of proteins in activated sludge culture were recovered as HMBPs for copper ion. N-termini of five HMBPs were determined, and two of them were found to be newly discovered proteins for which no amino acid sequences in protein databases were retrieved at more than 80% identities. Metal-coordinating amino acids occupied 38% of residues in one of the N-terminal sequences of the newly discovered HMBPs. Since these HMBPs were expected to be stable under conditions of water and wastewater treatments, it would be possible to utilize HMBPs as novel adsorbents for heavy metal removal if mass volume of HMBPs can be obtained with protein cloning techniques.

scholarly journals Cadmium-binding proteins of rat testes. Apparent source of the protein of low molecular mass

1984 ◽  
Vol 220 (3) ◽  
pp. 819-824 ◽  
Author(s):  
M P Waalkes ◽  
S B Chernoff ◽  
C D Klaassen
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Amino Acid Analysis ◽  
Binding Proteins ◽  
Binding Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Breakdown Product ◽  
Heat Stable ◽  
Metal Binding Protein

Fractionation of rat testicular cytosolic proteins by gel filtration indicates three major metal-binding proteins, or groups of proteins, termed testicular metal-binding protein (TMBP) 1, 2 and 3 by order of elution. The major heat-stable, metal-binding proteins in testes is TMBP-2, which has an Mr of approx. 25000. In most tissues, metallothionein (MT) is the major heat-stable, metal-binding protein, but it has an Mr of 6000. This testicular protein (TMBP-2) is much larger than MT, and since polymeric forms of MT have been previously reported, further characterization of TMBP-2 was performed. TMBP-2 was separated into two forms by DEAE-Sephadex A-25 anion-exchange chromatography. Amino acid analysis of both forms of TMBP-2 revealed that they differed markedly from MT, having particularly low cysteine contents. However, amino acid analysis showed that TBMP-2 was strikingly similar to TMBP-3, with an approximate stoichiometric relationship of 4:1. Therefore, experiments were conducted to determine if TMBP-3 could be a breakdown product of TMBP-2. Heat treatment of testicular cytosol in room air before gel filtration resulted in a marked increase in TMBP-3 and loss of TMBP-2. Storing intact testes at −20 degrees C for 2 weeks before processing for gel filtration also resulted in an increase in TMBP-3 and a loss of TMBP-2. Addition of a reducing agent (dithiothreitol) or proteinase inhibitor (N-ethylmaleimide) in processing of samples before gel filtration inhibited the appearance of TMBP-3. Results suggest that the low-Mr Cd-binding protein (TMBP-3) of rat testes results from either proteolytic or oxidative breakdown of a higher-Mr species, or from a combination of such factors.

Isolation of heavy metal binding proteins from marine vertebrates

1974 ◽  
Vol 28 (2) ◽  
pp. 83-86 ◽  
Author(s):  
K. W. Olafson ◽  
J. A. J. Thompson
Keyword(s):  
Heavy Metal ◽  
Metal Binding ◽  
Binding Proteins ◽  
Heavy Metal Binding ◽  
Marine Vertebrates
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