The effects of interstitial cell-stimulating hormone, its subunits, and recombinants on isolated rat Leydig cells.

The stimulation of cyclic AMP accumulation and testosterone synthesis in isolated rat Leydig cells by ovine and bovine ICSH, their subunits prepared by a new, mild procedure (dissociation of subunits at pH 3 and salt fractionation) and the recombined hormones have been studied. Whereas the isolated subunits exhibit less than 0.2% of the potency of the native hormones, recombination of the subunits results in full restoration of the biological activity. In contrast to this, recombination of the subunits prepared by a countercurrent distribution method resulted in only partial regeneration of the biological activity. The ovine hormone was found to be twice as active as the bovine ICSH. Both heterologous hybrids of the subunits of the ovine and bovine hormones were significantly more active than the bovine hormone. The utility of the isolated rat Leydig cell system as a rapid, sensitive bioassay for ICSH is also discussed.     

The primary structure of ovine interstitial cell-stimulating hormone III: Disulfide bridges of the α-subunit

The disulfide linkage of the ten half-cystine residues in the α-subunit of ovine interstitial cell-stimulating hormone has been investigated by partial acid and enzymic hydrolysis of the intact molecule. Results indicate that the disulfide bridges are formed by residues 14–86, 11–64, 35–63, 32–89, and 36–91.     

The amino terminal sequence of cholera toxin subunits

The $\text{N}$-terminal amino acid sequence of Cholera toxin, molecular weight 84,000 daltons, has been established. A high sensitivity sequencing procedure, employing ³⁵S-labelled phenylisothiocyanate as the coupling reagent in the automated Edman degradation was used. The toxin was found to consist of two polypeptide chains in the approximate molar ratio of 4:1. The amino-terminal twenty residues of each subunit will be reported here.     

A simple procedure for separating the subunits of ovine and bovine pituitary interstitial cell stimulating hormone

A simple, effective procedure for separating ovine or bovine interstitial cell stimulating hormone into their subunits has been described with a yield of 70–80%. The β subunit isolated by the new procedure behaves differently from that obtained by the countercurrent distribution method, and is in a more native state. In addition, full biological activity is consistently restored upon recombination of the subunits isolated by the new procedure.     

Rearrangement of terminal amino acid residues in peptides by protease-catalyzed intramolecular transpeptidation

Protease-catalyzed rearrangements of amino acid residues in peptides are observed during enzymatic digestion of proteins. When two enzyme-specific cleavage sites are within one or two residues of each other in the protein sequence, only one of the two sites usually is hydrolyzed by the protease, resulting in a peptide that contains an extra cleavage site near one of its termini. It is observed that in this type of peptide, the residues between the two cleavage sites often rearrange from one terminus of the peptide to the other terminus, catalyzed by the protease that created the peptide. It is proposed that the rearrangement is caused by protease-catalyzed intramolecular transpeptidation through a cyclic peptide intermediate. Several cases of this type of rearrangement were observed for different peptides generated by different proteases, indicating that this type of rearrangement is a general phenomenon occurring during enzymatic digestion of proteins.     

The kinetics of dissociation and reassociation of ovine pituitary interstitial cell stimulating hormone

The dissociation of ovine interstitial cell stimulating hormone and the reassociation of its subunits have been followed by circular dichroism and sedimentation velocity measurements. The dissociation was found to be a rapid, first-order reaction accompanied by the exposure of previously buried tyrosyl groups. The reassociation reaction begins as a rapid, probably second-order formation of a complex between the α and β subunits. This is followed by a much slower, first-order rearrangement of the conformation resulting in the reburying of at least two tyrosyl groups, one contributed by each subunit.