Biosynthesis and processing of endogenous parathyroid hormone related peptide (PTHRP) by rat Leydig cell tumor H-500

• Published in: Biochemistry (Easton) Vol. 32; no. 18; pp. 4931 - 4937
• Main Authors: Rabbani, Shafaat A, Haq, Mahmudul, Goltzman, David
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 11.05.1993
• Subjects: Aminoacids, peptides. Hormones. Neuropeptides; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cycloheximide - pharmacology; Fundamental and applied biological sciences. Psychology; Leydig Cell Tumor - metabolism; Male; Neoplasm Proteins - biosynthesis; Neoplasm Proteins - immunology; Parathyroid Hormone - biosynthesis; Parathyroid Hormone - immunology; Parathyroid Hormone-Related Protein; Protein Biosynthesis; Protein Processing, Post-Translational; Proteins; Proteins - immunology; Proteins - isolation & purification; Rats; Time Factors; Tumor Cells, Cultured; Protein hormone; Parathormone; Vertebrata; Mammalia; Molecular processing; Rat; Peptide hormone; Rodentia; Biosynthesis
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00069a030

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.