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A partial cDNA sequence of the boar 28, 29-kDa proacrosin-binding protein was determined. The desired sequence was amplified and obtained by RT-PCR followed by nested PCR. The oligonucleotide primers for RT-PCR were designed by deduction from the internal amino acid sequence of the boar proacrosin-binding protein. An approximately 1-kbp DNA fragment was analysed by agarose gel electrophoresis, in which the boar testicular total RNA or poly(A)+ RNA was used as a template for RT-PCR followed by nested PCR. This PCR product was inserted into a TA vector. The DNA sequence of the inserted PCR product was then determined by the chain termination method.

Boar proacrosin binding protein has been purified and the partial peptide sequence of the CNBr-digested proacrosin binding protein has been determined. Proacrosin binding protein was purified as a proacrosin and proacrosin binding protein complex from the acid extracts of boar spermatozoa through gel filtration. After the proacrosin binding protein was dissociated from proacrosin by freeze-thaw method, the proacrosin binding protein was purified through gel filtration. Fractions containing the proacrosin binding protein were pooled and were concentrated by lyophilization and then subjected to CNBr digestion. Four major CNBr-digested peptides were subjected to N-terminal peptide sequencing. All four showed the same N-terminus sequence., (Copyright 1999 Wiley-Liss, Inc.)

Bioelectrical impedance for measurement of total body fat and computed tomography for visceral and subcutaneous fat at umbilicus levels were performed in 34 obese and 10 lean men. Insulin secretion in response to an oral glucose tolerance test (OGTT) and a GH stimulation test by L-dopa, growth hormone-binding protein (GHBP) and IGF-I were measured. Obese subjects were divided into three groups according to the OGTT. The obese type II diabetes mellitus group had the highest GHBP levels and the most visceral fat. GHBP levels were most strongly correlated with the ratio of visceral fat area to body weight (VWR) above any other parameters (r = 0.725, P<0.001). The insulin and free fatty acid (FFA) areas under curves (AUC) during the OGTT, and the IGF-I level, were also positively correlated with GHBP levels (r = 0.474, P<0.005; r = 0.572, P<0.005; r = 0.453. P<0.005). GH-AUC to the L-dopa stimulation test was negatively correlated with GHBP levels (r = -0.432. P<0.005). Stepwise multiple linear regression analysis showed that VWR, FFA-AUC and insulin-AUC significantly contributed to the variability of GHBP (r2 = 0.58). In conclusion, we demonstrated that: (i) visceral fat amount mainly determined GHBP levels in obese men with varying glucose tolerance: (ii) hyperglycemia per se did not influence the GHBP level, whereas insulin and FFA could play a role in regulation of GHBP: and (iii) although GH was not the main regulator of GHBP, the unchanged IGF-I level despite GH hyposecretion suggests that increased GHBP levels reflect GH hypersensitivity in order to compensate for decreased GH secretion in obesity.