Effect of selective nitration of ovine lutropin on the subunit association and biological activity of the hormone.

Abstract

Different nitrated lutropin (LH) subunits were tested for their abilities to recombine with their counterpart subunits. (A detailed description of the preparations tested is presented in the preceding paper (Burleigh, B. D., Liu, W.K., and Ward, D.N. (1976) J. Biol. Chem. 251, 308-315).) The results indicated that the fully nitrated beta subunit of ovine LH (oLHBeta) (diatyrosyl oHbeta) can combine with the alpha subunit of ovine LH (oLHalpha) as wen which tyrosine residues alpha21, alpha41, alpha93 are modified) recombined partially with oLHbeta yielding about 40% as recombined product under specified recombination conditions. The fully nitrated oLHalpha (pentaatyrosyl oLH) combined poorly with oLHbeta and only 20 to 25% was obtained as the recombined product. The various nitrated LH and recombined LH derivatives containing separately nitrated subunits were tested for their potency in an in vitro radioligand assay and their in vivo biological potency in the ovarian ascorbic acid depletion assay. In the radioligand assay, the monoatyrosyl oLH and the oLHalpha + diatyrosyl oLHbeta were the most active derivatives and had about the same competitive inhibtion curves as that of oLHalpha + oLHbeta. Diatyrosyl oLH had intermediate native potency and the triatyrosyl oLH, tetraatyrosyl oLH + OLHbeta, and pentaatyrosyl oLHalpha + oLHbeta were the least active derivatives in this series. No tetranitromethane-modified LH derivative in our study was completely devoid of the specific binding activity in the radioligand assay. At a dosage of 1000-fold excess over the 125I-labeled oH, all the preparations tested in this series completely inhibited the specific binding of 125I-labeled oLH to the hormone receptors in rat testis preparations. In the in vivo ovarian ascorbic acid depletion assay, both the monoatyrosyl oLH and oLHalpha + diatyrosyl oLHbeta retained about 50% of its original activity; the diatyrosyl oLH and triatyrosyl oLH had about 10% of the activity and the recombined products of tetraatyrosyl oHalpha + oLHbeta and pentaatyrosyl oLHalpha + oLHbeta showed virtually no activity. Based on the results obtained from this paper and the preceding paper, the role of the individual tyrosine residues in oLH is assessed.