The occurrence of nicked human chorionic gonadotropin (hCG) by a thermolytic endoprotease.
A nicked form of human chorionic gonadotropin (nicked hCG), in which only one peptide bond between residues 47 and 48 (-Gly-Val-) of beta-subunit is cleaved, has been found in the urine and blood of pregnant women. In this study, we investigated the occurrence of nicked hCG and the localization of the nicking enzyme for hCG. First, to determine what type of protease nicks hCG, an in vitro proteolytic study using various proteases was performed. Amino-terminal amino acid sequence analysis of the beta-subunit purified from protease-treated hCG indicated that thermolysin actively nicks hCG. Secondly, to determine which tissues are related to the formation of nicked hCG, the distribution of radioactivity in various tissues after i.v. administration of radiolabeled hCG to female rats was examined. The radioactivity accumulated predominantly in the kidney (17%), liver (9.3%) and ovary (0.9%) after 30 min of injection. Analysis of molecular species of beta-radiolabeled hCG in various tissues and body fluids, using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by autoradiography, indicated that a nicked hCG-like molecule was found in the kidney, predominantly, as well as in the serum and urine. To examine the role of the kidney in producing nicked hCG, hCG was incubated with rat kidney particulate fraction (KPF). Immunoblot analysis of KPF-treated hCG indicated that KPF produced a nicked hCG-like molecule. Furthermore, the possibility that placental trophoblast cells produce nicked hCG was also examined using the choriocarcinoma cell BeWo.(ABSTRACT TRUNCATED AT 250 WORDS)
Kagimoto
A
A
Department of Biochemistry, School of Clinical Pharmaceutical Science, Nagasaki University, Japan.
Sakakibara
R
R
Fukushima
N
N
Ikeda
N
N
Ishiguro
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002822:Choriocarcinoma / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000097:blood / Q000378:metabolism* / Q000652:urine; D015342:DNA Probes; D005260:Female; D005821:Genetic Techniques; D006801:Humans; D015151:Immunoblotting; D007457:Iodine Radioisotopes; D007672:Kidney Cortex / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar; D013552:Swine; D014407:Tumor Cells, Cultured
D006063:Chorionic Gonadotropin; D015342:DNA Probes; D007457:Iodine Radioisotopes; D015260:Neprilysin