Purification and characterization of relaxin from the tammar wallaby (Macropus eugenii): bioactivity and expression in the corpus luteum.
The objective of this study was to isolate and purify prorelaxin or mature relaxin from the tammar wallaby corpus luteum (CL), determine their structure and bioactivity, and test the hypothesis that enzymatic cleavage of prorelaxin occurs in late gestation. Tammar relaxin peptides were extracted from pooled corpora lutea of late pregnant tammars using a combination of HPLC methods, and they were identified using Western blotting with a human (H2) relaxin antisera and matrix-assisted laser desorption ionization time of flight mass spectrometry. Although no prorelaxin was identified, multiple 6-kDa peptides were detected, which corresponded to the predicted mature tammar relaxin amino acid sequence, with an A chain of 24 amino acids, and different B chain lengths of 28, 29, 30, and 32 amino acids. Tammar relaxin bound with high affinity to rat cortical relaxin receptors and stimulated cAMP production in the human monocytic cell line, THP-1, which expresses the relaxin receptor. Analysis of individual CL indicated that equivalent amounts of mature relaxin peptides were present throughout gestation and also in unmated tammars at equivalent stages of the luteal phase in the nonpregnant cycle. Immunoreactive relaxin was localized specifically to the luteal cells of the CL and the intensity of immunostaining did not vary between gestational stages. These data show that the CL of both pregnant and unmated tammar wallabies produces mature relaxin and suggests that relaxin expression in this species is not influenced by the conceptus. Moreover, the presence of mature relaxin throughout gestation implies that prohormone cleavage is not limited to the later stages of pregnancy
Bathgate
Ross A D
RA
Department of Zoology, Howard Florey Institute. University of Melbourne, Victoria 3010, Australia. r.bathgate@hfi.unimelb.edu.au
Siebel
Andrew L
AL
Tovote
Philip
P
Claasz
Antonia
A
Macris
Mary
M
Tregear
Geoffrey W
GW
Parry
Laura J
LJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001681:Biological Assay; D015153:Blotting, Western; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003338:Corpus Luteum / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007614:Macropodidae / Q000502:physiology*; D013058:Mass Spectrometry; D008970:Molecular Weight; D011247:Pregnancy; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide / Q000737:chemistry / Q000302:isolation & purification; D012065:Relaxin / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000596:Amino Acids; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; C033395:relaxin receptors; D012065:Relaxin