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

Abstract

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.

Properties of cadmium-binding proteins in rat testes. Characteristics unlike metallothionein

“…The two larger-M, cadmium-binding proteins (peaks I and II from the gel-filtration step) have also been characterized (Waalkes et al, 1984b). The amino acid compositions of these two groups of proteins are similar to those of the low-Mr ones discussed in the present work and in their other work (Waalkes et al, 1984a).…”

“…In other work from this same laboratory (Waalkes et al, 1984b), evidence was presented to indicate intercon-Vol. 231 Table 1.…”

Similarities between metallothionein and low molecular weight testicular cadmium-binding protein

“…The lower molecular weight peak had the same Ve/Vo ratio as the hepatic and renal MT and the high molecular weight peak had a molecular weight of at least 70,000 daltons. These results are in agreement with previously published reports from this laboratory 5, 6 and others2-4, 8, 9, [14][15][16] The immunoreactivities of the heat-stable proteins with the MT-antibody are superimposed over the 1~ distribution patterns in figure 1. MT is a heat-stable protein, therefore, in both liver and kidney the antibody showed clear reactivity with the fractions containing the l~ peak.…”

Role of metallothionein-like cadmium-binding protein (MTLCdBP) in the protective mechanism against cadmium toxicity in the testis

“…The MT-like Cd-binding protein (MTLCdBP) is also present in the testis9, 11, 12 ) . The heat-stable, metal-binding MTLCdBP is low in cysteine, in contrast to cysteine-rich MT, and is induced and synthesized in response to Cd accumulation in the testis9, 11,12,13,14,15,16,17,18,19,20 ) . In rodents, MT-I/-II has been localized in spermatogenic, Sertoli, and interstitial cells, although there are discrepancies in their precise cellular distribution, and their role in protein induction is not clear18, 20,21,22,23,24,25,26,27,28 ) .…”