Biosynthesis and processing of endogenous parathyroid hormone related peptide (PTHRP) by rat Leydig cell tumor H-500.
We have examined in vitro the biosynthesis and processing of endogenous PTHRP in cultured rat H-500 Leydig tumor cells. Cells were grown to confluence and pulse labeled with [3H]Ile, 50 microCi/mL, in Ile free culture medium for 2 min to 6 h. In some experiments incubations were carried out in culture medium alone in the presence of 0.3 mM cycloheximide or 20 micrograms/mL unlabeled Ile. Cell extracts and culture media were analyzed by affinity chromatography employing an antibody directed against the bioactive NH2-terminal region, PTHRP(1-34), followed by gel-permeation or reversed-phase high-performance liquid chromatography. Incorporation of [3H]Ile into PTHRP in cell extracts increased over 20 min during pulse labeling and then remained constant throughout the incubation period up to 6 h. In contrast, the release of [3H]PTHRP into culture medium increased progressively over 6 h. Addition of cycloheximide or unlabeled Ile almost completely blocked incorporation of [3H]Ile into newly synthesised PTHRP. Three molecular forms of PTHRP were seen which comigrated with PTHRP(1-36), PTHRP(1-86), and PTHRP(1-141) standards in both chromatographic systems employed. After 20 min these species comprised approximately 63%, 30%, and 7% of newly synthesized PTHRP, respectively. These three molecular forms of PTHRP were observed both intra- and extracellularly, and no further metabolism of these species was seen after release into conditioned medium. Pulse-chase studies demonstrated a rapid decrease of newly synthesized PTHRP forms within cells after 20 min; there was, however, a progressive increase in [3H]PTHRP in conditioned culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)
Rabbani
S A
SA
Department of Medicine, McGill University, Montreal, Canada.
Haq
M
M
Goltzman
D
D
D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D007984:Leydig Cell Tumor / Q000378:metabolism*; D008297:Male; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000276:immunology; D010281:Parathyroid Hormone / Q000096:biosynthesis* / Q000276:immunology; D044162:Parathyroid Hormone-Related Protein; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000276:immunology / Q000302:isolation & purification; D051381:Rats; D013997:Time Factors; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D003513:Cycloheximide