Primary amino acid sequence of follicle-stimulating hormone from human pituitary glands. I. alpha subunit.

Follicle-stimulating hormone of a high state of physicochemical and biological purity was isolated from acetone-preserved human pituitary glands. The follicle-stimulating hormone was dissociated into alpha and beta subunits by treatment with 8 M urea and the subunits were separated by ion exchange chromatography on DEAE-Sephadex A-25. The subunits were freed of undissociated or reassociated follicle-stimulating hormone by gel filtration on Sephadex G-100. For the establishment of the primary amino acid sequence, the alpha subunit was reduced and either carboxyamidomethylated or S-aminoethylated prior to a thermolytic or a tryptic digestion. Each digest was gel filtered on a column of Sephadex G-50 to separate the glycopeptides from the peptides. The glycopeptides and the peptides were purified further by sequential gel filtration on Sephadex G-25, G-15, and Bio-Gel-P-2 and were isolated by high voltage electrophoresis at pH 6, 3.5, and 2. The purity of the isolated peptides was ascertained further by amino acid analysis. The amino acid sequences of the peptides were determined by Edman degradation followed by subtractive amino acid analysis. COOH-terminal sequences were established by digestion with carboxypeptidases A and B. The primary amino acid sequence of human follicle-stimulating hormone-alpha is identical to that of human chorionic gonadotropin-alpha and differs from that of human luteinizing hormone-alpha in having the tripeptide Ala-Pro-Asx- at the NH2-terminal end.

Follicle-stimulating hormone of a high state ofphysicochemical and biological purity was isolated from acetone-preserved human pituitary glands. The follicle-stimulating hormone was dissociated into Q and fi subunits by treatment with 8 M urea and the subunits were separated by ion exchange rhromatography on DEAE-Sephadex A-25. The subunits were freed of undissociated or reassociated follicle-stimulating hormone by gel filtration on Sephadex G-100. For the establishment of the primary amino acid sequence, the Q subunit was reduced and either carbaxyamidomethylated or S-aminoethylated prior to a thermolytic or a tryptic digestion. Each digest was gel filtered on a column of Sephadex G-50 to separate the glycopeptides from the peptides. The glycopeptides and the peptides were purified further by sequential gel filtration on Sephadex G-25: G-13, and Rio-Gel-P-2 and were isolated by high volt.age electrophoresis at pH 6, 35, and 2. The purity of the isolated peptides was ascertained further by amino acid analysis. The amino acid sequences of the peptides were determined by Edman degradation followed by subtractive amino acid analysis. COOH-terminal sequences were established by digestion with carbnxypeptidases A and H. The primary amino acid sequence of human follicle-stimulating hormone-a is identical to that of human chorionic gonadotropin-cu and differs from that of human luteinizing hormone-a in having the tripeptide Ala-Pro-Asx-at the NH,-terminal end.
Highly purified FSH' from human pituitary glands, suitable for structural studies, has been prepared by various investigators (l-6). The insufficient yields of FSH. and particularly of its subunits, however, have handicapped the determination of the primary structure. The availability of human pituitary glands from the Kational Pituitary Agency (Baltimore, Md.) has permitted the development of procedures for the isolation of anterior pituitary hormones of high physicochemical purity in quantities sufficient for structural studies. FSH, similar to LH (Y-11), TSH (12-U), and HCG (18, 13, has been shown to consist of a hormone-nonspecific cy subunit and a hormone-specific fi subunit (20)(21)(22) (27), TSH (28), GH (29). and ACTH (30), and PRL activity (31), as reported earlier (4). (1 x 100 cm) ofsephadex G-25 and G-15, and a column (I x 30 cm) of Bio~GeI~P~Z.

Isolation of Subunits of FSH
The peptide-containing fractions recovered from these columns were finally purified on Whatman No. 1 paper by two-dimensional high voltage electrophoresis at pH 2. and/or pH 3.5, and/or pH 6. Guide strips (0.5 cm) were cut and stained with 0.1% ninhydrin in acetone to locate the peptides. The peprides were &ted from the paper using 50% pyridine in water. Aliquots of the peptide fractions were hydrolyzed and subjected to amino acid analysis.   (Table II). The amino acid composition, however, showed a few differences from that of a 24-hour hydrolysate of an earlier preparation of FSH-cv (201, which was not purified by gel filtration on Sephadex G-100. The methionine content reported for the earlier preparation was higher due to the addition of a peak in the region of methionine sulfoxide to methionine. The amino acid composition of FSH-a and the recovery of 3 residues of methionine in sequence determinations indicates that the peak for methionine sulfoxide was an artifact and was erroneously added to the methionine in our earlier report (20). The values of serine, tyrosine, and cysteine in the earlier preparation were not extrapolated to zero time and were lower due to partial destruction during hydrolysis. b Calculated on the basis of total micrograms of residues recovered using a molecular weight of 10,326.
These differences were resolved by the performance of amino acid analysis after 24, 48, and 52 hours of hydrolysis of highly purified FSH-a. The amino acid analysis of FSH-a as reported by Reichert and Ward (21) has a lower proline, valine, and cysteine content and a higher tyrosine and glutamic acid content. NH,-and COOH-terminal Analyses-Alanine was found to be the major NH,-terminal amino acid present. A predominant alanine NH,-terminal peptide was also recovered from both digests of Ae-FSH-a confirming that FSH-a is similar to HCG-a (41) at the NH,-terminal.
The data obtained with the automatic Sequencer indicated the NH,-terminal sequence of FSH-cu to be Ala-Pro-Asp-Val-Glu-Asp-.
The COOH-terminal analyses (Table III) showed 1 mol of serine, lysine, and histidine,and 2 mol of tyrosine per mol of protein in 6 hours, which is consistent with the sequence of -Tyr-Tyr-His-Lys-Ser obtained from tryptic and thermolytic peptides of FSH-a (Fig. 7).
Amino Acid Sequence of FSH-a2-Purification and amino acid composition of the thermolytic and the tryptic peptides are given in Diagrams 1 and 2 and Tables IV and VI, respectively. The amino acid sequence of each peptide is given in Tables V and VII. The complete sequence of FSH-cu showing the overlap of peptides obtained from two enzymatic digests is given in Fig. 7. The experimental details are presented in Appendices A and B.
During the tryptic digestion of Ae-FSH-u, an uptake of 18  (Fig. 7) is represented by 1.2 residues of tyrosine.
Ala -Pro -Asp -Val -Glu -Asp -Cys -Pro -Th-1 mol of alkali/m01 of protein was obtained, which is in close agreement with the total of 17 tryptic peptides obtained (Table  VII).
The alignment of amino acid sequences of the tryptic and thermolytic peptides of FSH-cu did not permit overlaps between positions 28-29, 32-33, 60-61, and 67-68; however, the arrangement shown in Fig. 7 is almost certainly the correct one since it results in identical sequences of FSH-(Y and hCG-cu (411, and homologous sequences when FSH-ol is compared to human, ovine, and bovine LH-cu (12, 49-51) and bovine TSH-(Y (52). Moreover, the validity of this arrangement is supported by the observation that it would confirm the sequence given in the preliminary report of Shome and Parlow (50). Assignment of Carbohydrate Side Chains-Glycopeptides T-13 and T-8, following extensive proteolytic digestion and removal of the enzymes, peptides, and amino acids, showed only aspartic acid on amino acid analysis, indicating that the carbohydrate moiety is linked to the asparagine at positions 52 and 78. This is also suggested by glycopeptide Th-8, which contains only asparagine that can be linked to the carbohydrate moiety. Linkages of the carbohydrate moiety to asparagine, serine, or threonine are commonly found in glycoproteins The solid lines below represent peptides recovered and sequenced. (44). The sequences in the region of attachment of the carbohydrate moieties, uiz.
The primary amino acid sequence of FSH-cu is identical with that of HCG-a (41), and differs from that of human LH-cu and human FSH-a as reported by Shome and Parlow (50) and Inagami et al. (51) in having the tripeptide Ala-Pro-Asp at the NH, terminus.
The sequence at the NH, terminus also was confirmed by the results obtained from experiments using the automatic sequenator. The sequence at positions 84 and 85 as Cys-Ser of FSH-(u, however, is in agreement with Shome and Parlow (50) and differs from Ser-Cys at these positions reported by .
Recombination of subunits of glycoprotein hormones has indicated that the N subunits are interchangeable at both intra-and interspecies levels (58-61). It may be of interest to note that significantly higher FSH activity was recovered during recombination experiments between HCG-ol and FSH-P subunits, and between FSH-(Y and FSH-P subunits, than between human LH-cu and FSH-@ subunits (61). In addition to this, FSH-u generated a h' h 1g er HCG-like activity when incubated with HCG-P, as compared to human LH-o( (58). The recovery of 81% of FSH activity by Reichert (62) on incubation of human FSH (u, /3) in the presence of HCG-cu and the 93.fold increase in HCG-like activity, when human FSH (a, /3) was incubated in the presence of HCG-/3, further demonstrates that biologically active molecules can be formed between human FSH-/3 and HCG-cu, and between human FSH-cu and HCG-P. These data further support the identity of FSH-cu with HCG-cu and suggest an essential nature of the NH,-terminal tripeptide Ala-Pro-Asp.