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Two proteins with structural characteristics similar to peptide sequences identified in the inhibin alpha-subunit precursor sequence have been isolated from bovine follicular fluid. A side-fraction from the purification of bovine follicular fluid inhibin with high levels of inhibin immunoactivity relative to its inhibin bioactivity was fractionated through a sequence of procedures which included triazine dye affinity and phenyl-Sepharose chromatography, gel permeation chromatography on Sephadex G-100, reverse phase HPLC, and preparative polyacrylamide gel electrophoresis. The first of the two proteins identified had a molecular mass of 25-26K under reducing and nonreducing conditions and a NH2-terminal sequence identical to that of 43K inhibin alpha-subunit and showed minimal activity (less than 2% activity) compared with bovine 31K inhibin in either the inhibin in vitro bioassay or the RIA. These data suggest that this protein is the alpha 1-166 sequence of the bovine inhibin alpha-subunit (designated alpha N-subunit), most likely released after processing of either the inhibin alpha-subunit precursor or the 43K alpha-subunit involved in the conversion of 58K to 31K inhibin. The other protein identified (designated pro-alpha C-subunit) has a molecular mass of 27K under nonreducing conditions and 20K and 6K under reducing conditions. It is inactive in the in vitro bioassay, although highly reactive in the inhibin RIA, and has NH2-termini identical to the pro sequence of the inhibin alpha-subunit precursor and the 20K alpha-subunit sequence. These results suggest that pro-alpha C is a disulfide-linked structure and may represent an intermediate in the dimerisation of alpha- and beta-subunits to form inhibin while the alpha N-subunit is probably a proteolytic product of either the alpha-subunit precursor or 58K inhibin.

Reaction of the hydrobromide salt of 1,3,5-tris(1,4,7-triazacyclonon-1-ylmethyl)benzene, Lؒ9HBr, with copper() nitrate followed by cation-exchange chromatographic purification affordedThe ESR and magnetic susceptibility data indicated that the complex consists of three identical non-interacting copper() centres. Reaction of 1 with a phosphate source produces {[Cu 3 L(µ-OH)(µ 3 -HPO 4 )(H 2 O)][PF 6 ] 3 ؒ3H 2 O} n 2 the polymeric lattice of which contains trinuclear copper() sites with structural similarities to laccase (Lc) and ascorbate oxidase (AO). These trinuclear sites consist of two type 3 copper() centres, at a separation of 3.557(4) Å, linked by an hydroxo bridge and two phosphate oxygens while another phosphate oxygen links these two centres to the further removed type 2 copper() centre, establishing separations of 4.561(4) and 5.474(4) Å. The magnetic properties of 2 were investigated in the temperature range 4.2-300 K and they revealed an S = ¹ 2 molecular ground state arising from antiferromagnetic coupling. A number of models have been employed in order quantitatively to fit the µ eff versus temperature data including those applicable to the resting oxidised state of laccase and ascorbate oxidase, vis-à-vis dimer plus uncoupled monomer, but the best fits were obtained using a symmetrical trinuclear approximation with J 12 for the doubly bridged [CuL(µ-OH)(µ-HPO 4 )Cu] moiety of Ϫ53 or ca. Ϫ80 cm Ϫ1 combined respectively with J for the two equal three-atom phosphato bridges of Ϫ90 or Ϫ77 to Ϫ90 cm Ϫ1 . The 77 K solid-state ESR spectrum of 2 is unusual for S = ¹ 2 ground-state systems and shows six components of a probable seven-line copper hyperfine multiplet between 2500 and 3100 G and a strong x, y resonance at ca. 3200 G, most probably due to weak copper() pair interactions or the superimposition of S = ¹ 2 signals from both molecular doublets. The 77 K solution ESR spectrum (dimethylformamide, water-glycol 1 : 1) for 2 is typical of a monomeric copper() centre and closely resembles the ESR spectrum of the type 2 site in Lc and AO. The ESR, electrospray mass spectrometric and NMR data indicate that 2 dissociates in solution to give a trinuclear unit consisting of two type 3 copper() centres (ESR silent), which are linked by the hydroxo and phosphate group (phosphate is released from the complex only on addition of acid), and an isolated type 2 copper() centre which is probably responsible for the ESR features.

The primary amino acid structures of the 43-kDa (A) and 15-kDa (B) subunits of the 58-kDa form of the hormone inhibin have been elucidated by cloning and analysis of cDNA species derived from bovine granulosa cell mRNA.The A subunit (Mr = 32,298) is a protein of 300 amino acids with two potential N-glycosylation sites and two potential proteolytic processing sites and'has a pre-pro region of 60 amino acids. The mature B sublinit (Mr = 12,977) is a protein of 116 amino acids synthesized from a separate mRNA. These data establish that a 31-kDa forn of inhibin also isolated from bovine follicular fluid, with subunits qf 20 kJa (Ac) and 15 kDa (B), is derived from the 58-kDa form by proteolytic processing of the A subunit.Considerable evidence has now accumulated to support the concept that the gonads produce a protein termed inhibin (1) that selectively suppresses the pituitary secretion of folliclestimulating hormone (2), a key hormone in controlling folliculogenesis and spermatogenesis. Controversy still exists concerning the nature of inhibin (3) partly because of the fact that some of the bioassays used in its characterization do not monitor follicle-stimulating hormone directly (4). For instance, inhibin-like activity has been detected in seminal plasma; the proteins associated with this activity have been purified and characterized but subsequently they were shown not to be of gonadal origin (5)(6)(7)(8)(9).We have isolated inhibin from a gonadal source, bovine follicular fluid, with an apparent molecular mass of 58 kDa, composed of two subunits now designated A and B, of 43 kDa and 15 kDa, respectively, linked together by disulfide bonds (10). Subsequently, 32-kDa inhibin was isolated by others from porcine follicular fluid, with two subunits of 18/20 kDa and 13/14 kDa (11,12). We have also shown that a 31-kDa form of bFF inhibin, composed of 20-kDa and 15-kDa subunits, is generated during a pH precipitation step in the purification procedure (13) but the precise relationship between the different forms has been unknown. However, the biological activity of the 31-kDa form is neutralized in vitro by an antiserum raised against the 58-kDa form (13), suggesting that the 31-kDa inhibin is a processed form of the 58-kDa inhibin.This article describes the amino acid sequences of the two subunits of the 58-kDa bovine inhibin as determined fromn cDNA sequencing and reports that the 20-kDa subunit of the 31-kDa inhibin is derived from the 43-kDa subunit of the 58-kDa inhibin, thus clarifying the relationship between these different forms.MATERIALS AND MIETIODS NH2-Terminal Amino Acid Sequencing of the 58-kDa Inhibin. The 58-kDa inhibin was isolated from bFF (10).Briefly, this involved a four-step' purjfication procedure: (i) gel permeation chromatography on Sephacryl 3200 in 0.05 M ammonium acetate, (it) gel permeation chromatography on Sephadex G100 in 4 M acetic acid, (iii) reversed-phase HPLC on an Ultrapore RPSC column-(Beckman) using a 0.1% trifluoroacetic acid-acetonitrile-H2O gradient, and (iv) prepara...