Ovine inhibin

• US 5,015,729 A
• Filed: 06/23/1988
• Issued: 05/14/1991
• Est. Priority Date: 06/24/1986
• Status: Expired due to Term

• Current Assignee: Salk Institute for Biological Studies
• Original Assignee: Salk Institute for Biological Studies
• Inventors: Spiess, Joachim, Bardin, C. Wayne, Vale, Wylie W. Jr., Rivier, Jean E. F.
• Primary Examiner(s): Doll, John
• Assistant Examiner(s): WESSENDORF, TERESA D
• Application Number:
  US07/210,683
• Time in Patent Office: 1,055 Days
• Field of Search: 530/350, 530/324, 530/398, 530/397, 530/387, 435/68.1, 514/12, 514/21
• US Class Current: 530/350
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/575 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695
• CPC Class Codes: C07K 16/26 against hormones ; against hormone releasing or inhibiting factors

Abstract

A dimeric protein with inhibin activity is isolated from ram reta testis fluid using reverse-phase high-performance liquid chromatography and gel filtration. The isolated molecule is composed of two chains having apparent molecular weights of about 18,000 and about 16,500 to 14,500 Daltons, as measured by gel electrophoresis, which are bound together by disulfide bonding and the longer of which is likely glycosylated. Microsequencing revealed the NH₂ -terminal portion of the longer chain to be Ser- Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Gln-Arg-Pro-Pro- Glu-Glu-Pro-Ala-Ala-His-Ala-Asp-Cys and of the shorther chain to by Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe. This dimeric protein specifically inhibits basal secretion of FSH, but not of LH, in a rat anterior pituitary monolayer culture system, exhibiting a half-maximal effective dose of about 0.1 to 0.3 ng/ml. Furthermore, antibodies raised against synthetic replicates of the N-terminal six residues of the longer chain, as well as against the N-terminal 25 residues, are effective to reduce the activity of highly purified intact ovine inhibin and might be used to exert a profertility effect in rams, ewes and other mammals.

Claims

1. A substantially pure ovine inhibin protein consisting essentially of greater than 90% by weight of total protein,

said inhibin protein consisting of a dimer of a first polypeptide chain having an apparent molecular weight of about 18,000 Daltons, as measured by gel electrophoresis, and a second polypeptide chain having a molecular weight of about 16,500 Daltons, as measured by gel electrophoresis, said first chain being linked to said second chain through disulfide bonding,

said dimer, when subjected to reverse phase HPLC elutes from a C₈ column of 5 micron particle size and 300 angstrom pore size, at between about 27 and about 28 minutes following the instigation of a straight line gradient flow of Buffers A and B which varies from 25% Buffer B to 90% Buffer B over a period of 45 minutes, at a flow rate of 0.7 ml per minute and a back pressure of about 890 psi, with Buffer A being 0.1% trifluoroacetic acid (TFA) and with Buffer B being 80% acetonitrile in 0.1% TFA:

said first chain having an amino-terminal sequence beginning with Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His-Ala-Asp-Cys-His-Arg -Ala and said second chain having the formula;

H-Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-Tyr-Val-Ser-Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-Ile-Ile-Ala-Pro-Ser-Gly -Tyr-His-Ala-Asn-Tyr-Cys-Glu-Gly-Glu-Cys-Pro-Ser-His-Ile-Ala-Gly-Thr-Ser-Gly-Ser-Ser-Leu-Ser-Phe-His-Ser-Thr-Val-Ile-Asn-His-Tyr-Arg-Met-Arg -Gly-His-Ser-Pro-Phe-Ala-Asn-Leu-Lys-Ser-Cys-Cys-Val-Pro-Thr-Lys-Leu-Arg-Pro-Met-Ser-Met-Leu-Tyr-Tyr-Asp-Asp-Gly-Gln-Asn-Ile-Ile-Lys-Lys-Asp -Ile-Gln-Asn-Met-Ile-Val-Glu-Glu-Cys-Gly-Cys-Ser-OH, said protein specifically inhibiting basal secretion of follicle-stimulating hormone while not inhibiting basal secretion of luteinizing hormone.

2. A protein according to claim 1 having a relative amino acid composition as determined by hydrolysis followed by amino acid analysis in the approximate amounts as follows:

Asx 22, Thr 11, Ser 17, Glx 22, Gly 16, Ala 16, Val 11, Met 5, Ile 8, Leu 22, Tyr 8, Phe 10, His 8, Trp 4, Lys 10, Arg 10, Cys 12, and Pro 23.

3. A protein according to claim 1 wherein said first chain is glycosylated.

4. A protein subunit of ovine inhibin having a purity of greater than 90% by weight based upon total protein selected from the group consisting of (a) a first polypeptide having an apparent molecular weight of about 18 kD, as measured by gel electrophoresis, having about 135 amino acid residues, and having an amino terminal sequence beginning with Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-His-Ala-Asp-Cys -His-Arg-Ala, and (b) a second polypeptide having an apparent molecular weight of about 16.5 kD, as measured by gel electrophoresis, having the sequence Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-Tyr-Val-Ser-Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-Ile-Ile-Ala-Pro -Ser-Gly-Tyr-His-Ala-Asn-Tyr-Cys-Glu-Gly-Glu-Cys-Pro-Ser-His-Ile-Ala-Gly-Thr-Ser-Gly-Ser-Ser-Leu-Ser-Phe-His-Ser-Thr-Val-Ile-Asn-His-Tyr-Arg -Met-Arg-Gly-His-Ser-Pro-Phe-Ala-Asn-Leu-Lys-Ser-Cys-Cys-Val-Pro-Thr-Lys-Leu-Arg-Pro-Met-Ser-Met-Leu-Tyr-Tyr-Asp-Asp-Gly-Gln-Asn-Ile-Ile-Lys -Lys-Asp-Ile-Gln-Asn-Met-Ile-Val-Glu-Glu-Cys-Gly-Cys-Ser, wherein the first polypeptide is combined through disulfide bonding to the second polypeptide create a dimer which dimer, when subjected to reverse phase HPLC, elutes from a C₈ column of 5 micron particle size and 300 angstrom pore size, at between about 27 and about 28 minutes following the instigation of a straight line gradient flow of Buffers A and B which varies from 25% Buffer B to 90% Buffer B over a period of 45 minutes, at a flow rate of 0.7 ml per minute and a back pressure of about 890 psi, with Buffer A being 0.1% trifluoroacetic acid (TFA) and with Buffer B being 80% acetonitrile in 0.1% TFA.