Studies on the Biochemical Properties of Highly Purified HCG

Abstract

By a combination of Sephadex gel filtration, CM-C and DEAE-C chromatography, 2 highly purified HCG fractions (dA: 18,000 IU/mg, dB: 20,000 IU/mg) were obtained from a crude preparation (2130 IU/mg) which was extracted from the urine of normal pregnant women in the first trimester. They were characterized by histological response of the ovary of hypophysectomized immature rats, bioassay (the Steelman-Pohley method and the OAAD method), amino acid analysis and determination of carbohydrate content, and their high homogeneity was proved by polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. The components confirmed were 17 amino acids, galactose, mannose, hexosamine and sialic acid. LH-like activity was dominant in fraction dA, and this fraction was richer in proline and valine than fraction dB and poorer in aspartic acid, glutamic acid, glycine, alanine, lysine and histidine. Fraction dB had dominant FSH-like activity in its biological character and was richer in acidic amino acids and sialic acid. Biological potency was particularly well correlated between LH-like activity of dA and hexose, and between FSH-like activity of dB and acidic amino acids and sialic acid. These results suggested that HCG is composed of 2 components. One has LH-like activity and the other has FSH-like activity. The ratio of these activities in HCG preparations differs according to the stage of pregnancy and biological and physicochemical properties of this hormone change throughout pregnancy. (Endocrinology 87: 233, 1970)

In vitro biosynthesis of human chorionic follicle stimulating hormone.

“…It was formerly demonstrated that hCG with LH-like activity and hCFSH with FSHlike activity were isolated separately from lyophilized powder of trophoblastic tissue (Ashitaka, 1970;Tojo et al, 1973). Recently the biosynthesis in vitro of hCG from placenta was also confirmed using 3H-proline as a radioactive precursor .…”

“…Up to this time, however, there has been no report which detected the presence of radioimmunological FSH activity in the medium of tissue culture of chorionic tissue although some workers have tried to assay chorionic FSH activity by radioimmunoassay for pituitary FSH (Benagiano et al, 1972). The chorionic FSH activity in the cultured medium should be detected by anti-hCFSH serum since hCFSH did not cross-react with anti-FSH serum of pituitary origin in Ouchterlony immunodiffusion (Ashitaka et al, 1970).…”

“…It has already been clarified and reported that human chorionic gonadotropin (hCG) with LH-like activity and human chorionic FSH (hCFSH) with FSH-like activity could be isolated separately from the chorionic tissue (Tojo, 1968;Ashitaka, 1970;Tojo et al, 1973). Protein hormone synthesis by placenta is not well understood yet although there have been several reports available on the biosynthesis of hCG (Benagiano et al, 1972;Patrito et al, 1973;Tojo et al, 1974;Maruo et al, 1974) and on human placental lactogen (Friesen et al, 1968 These findings prompted us to study the incorporation in vitro of labeled amino acid into hCFSH.…”

BIOSYNTHESIS OF HUMAN CHORIONIC GONADOTROPHIN IN VITRO: INCORPORATION OF [14C]l-LEUCINE

“…The recent demonstration (Ashitaka, 1970; Ashitaka, Tokura, Tane, Mochizuki & Tojo, 1970;Hamashige, Alexander, Abravanel & Astor, 1971;Ashitaka, Mochizuki & Tojo, 1972) that HCG extracted from placental tissue and urine has different bio¬ chemical characteristics, prompted us to study the conditions and the rate of incor¬ poration in vitro of [14C]L-leucine into human placental HCG with the purpose of investigating the biosynthesis and release of this hormone using such techniques as absorption with polymerized antisera, ion exchange chromatography, polyacrylamide gel electrophoresis and radioimmunoassay.…”

Localizations and Biological Activities of HCG Fractions in Rat Organs

“…Isojima et al(1969) and Koyama (1971) also prepared two different fractions from HCG preparation and proved that one of them (Ag-1) showed biological activity and antigenicity of HCG, but the other (Ag-2) only showed the antigenicity of HCG. Later Ashitaka et al(1970) *:The antigenic activity of HCG was assayed by radioimmunoassay (Isojima et al, 1968) and expressed as IU HCG-equivalent by bioassay. **: Borate buffered saline (5 parts of 0 .1M borate buffer (pH8.6) mixed with 95 parts of 0.9% saline).…”