Isolation and characterization of the major androgen-dependent glycoprotein of canine prostatic fluid.

W B Isaacs,J H Shaper

doi:10.1016/s0021-9258(18)32456-6

Abstract

Canine prostatic fluid, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, is characterized by the presence of a single diffuse band (Mr approximately 30,000) which accounts for over 90% of the total protein. The biosynthesis of this protein is under androgen control. Castration results in the disappearance of this protein, whereas its presence in the prostate can be maintained in the castrated animal with exogenous androgen. Analysis of the native protein by isoelectric focusing revealed the presence of 10-13 charged variants with pI values in the range of 6.5 to 8.4. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed that each isoform is constructed of two dissimilar polypeptide subunits covalently linked through disulfide bonds. One subunit has a molecular weight of 15,000 (H chain); the second subunit (L chain) has a variable molecular weight in the 12,000-14,000 range. The H and L subunits have been purified by preparative isoelectric focusing and chemically characterized. Based on tryptic peptide mapping, NH2-terminal analysis, amino acid and carbohydrate composition, the H and L subunits are structurally unrelated and consequently appear to be unique gene products. Furthermore, the L subunit is glycosylated which potentially accounts for its size heterogeneity. Quantitative NH2-terminal analysis indicated that the H and L subunits are present in the native molecule in a ratio of 2:1, suggesting that the native molecule is a trimer with an apparent molecular weight of 43,000. Based on electrophoretic data, the glycoprotein also constitutes the major fraction of the soluble protein in canine prostatic tissue; its presence is organ specific. This glycoprotein should prove useful as a marker for prostatic function under varying hormonal and environmental conditions.

Highlights

- reducing conditions, is characterized by the presence of a single diffuse band (Mr 30,000)which accounts for over 90% of the total protein
Starting Material-The secretions analyzed in the following experiments, consisting of canine ejaculate minus the sperm component, can be considered prostatic fluid since the dog has no seminal vesicles or Cowpers glandand contributions from other sources are minimal [27].This assumption is supported by our observations that theprotein profiles as analyzed by SDS or IEF electrophoresis were identical regardless of whether the ejaculate was obtained from intact dogs or dogs whose testes and epididymides had beenremoved and had beengiven exogenous testosterone
Analysis of Prostatic Fluid anTdissue by SDS-Polyacrylamide Gel Electrophoresis-To examine the proteins present in canine prostatic fluid and tissue, samples of each were analyzed on SDS-polyacrylamidegels

Summary

Introduction

- reducing conditions, is characterized by the presence of a single diffuse band (Mr 30,000)which accounts for over 90% of the total protein. The biosynthesis of this protein is under androgen control. The glycoprotein constitutes themajor fraction of the soluble protein in canine prostatic tissue; its presence is organ specific. This glycoprotein should prove useful as a marker for prostatic function under varying hormonal and environmental conditions. Little data exists, with the notable exception of the rat[10,11,12,13] regarding the chemical nature andfunction of the proteins synthesized and secreted by the prostate

Methods

Results

Conclusion