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  • Author: Katsumi Wakabayashi x
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Masa-aki Hattori, Kazunori Ozawa and Katsumi Wakabayashi

Abstract. The present study was performed to evaluate the different carbohydrate structure of rat LH isoelectric components related to their intrinsic biological activities. Terminal sialic acid residues were essential to the formation of multiple LH components observed in the isoelectric focussing profile, which was proved by their interaction with Ricinus communis agglutinin-120 following neuraminidase treatment, and the conversion of component F (pI, 10.0) to less alkaline components after incubation with liver Golgi membrane fraction in the presence of CMP-NeuNAc. The affinity studies using lentil lectin indicated that component F was not an asialo form of component A (pI 8.4). The serial removal of sialic acid residues from these components led to increases in the steroidogenic activity, owing to increases in the activation of the receptor-adenylate cyclase system. The enhancement of the steroidogenic activity by desialylation was very great in component A'(pI, 8.0) (751% increase), and decreased with increasing pI. It can be concluded that the different biological potencies of intact LH components are attributable principally to terminal sialic acid residues. However, the peripheral chains of asialo oligosaccharides of less alkaline components (pI, 8.0, 8.4) seem to prevent the maximal cellular responses, since their desialylated forms did not attain the maximum activity.

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Masa-aki Hattori, Kazunori Ozawa and Katsumi Wakabayashi

Abstract. Biosynthesis and secretion of LH were investigated using primary cultures of rat gonadotropes following exposure to swainsonine, an inhibitor of N-linked oligosaccharide processing. The inhibition of terminal glycosylation by swainsonine was assessed by the increase in incorporation of [3H]mannose and the decrease of incorporation of [3H]galactose into the trichloroacetic acid-insoluble and anti-ovine LHβ immunoprecipitable fractions. This inhibitor led to an increase in cellular LH with high affinity for concanavalin A and a decrease of LH with less affinity for the lectin, although the cellular LH was not decreased. The swainsonine-treated cells accumulated highly basic LH (pI, 9.6, 10.0) in a time-dependent manner. Although the treated cells showed a decrease in the responsiveness to hormonal stimulation, there were no differenes in the rate and extent of LH secretion induced by maximal stimulation with 100 nmol/I GnRH between the control and treated cells over 24 h. The accumulation of highly basic LH molecules did not result from a decrease in their secretion, since they were secreted in the presence and absence of GnRH. It is concluded that significant changes in N-linked oligosaccharide structure of LH have no effect upon its secretion.

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Masahide Shodono, Takao Nakamura, Yuichi Tanabe and Katsumi Wakabayashi

ABSTRACT

Simultaneous determinations of plasma levels of oestradiol-17β, progesterone and luteinizing hormone (LH) during the ovulatory cycles of hens were performed using radioimmunoassay techniques without chromatography. Blood samples were collected from the wing vein of 7 hens with regular ovulatory cycle at intervals of 1, 2 or 4 h throughout 36 h starting 24–25 h before the second egg (C2) ovulation of the clutch and ending 11–12 h after C2 ovulation. The oestradiol-17β level of the plasma during the ovulatory cycle varied from 50 pg/ml to 250 pg/ml. The value was high 24 h before ovulation and decreased until 10 h before ovulation, then increased and reached a peak 4–5 h before ovulation, when it started to decrease again until 12 h after ovulation. The plasma levels of progesterone varied from 1 ng/ml to 6 ng/ml, and reached a peak 4–6 h before ovulation. The plasma LH value varied from 1 ng/ml to 4 ng/ml, and the changes in the levels corresponded well with that of progesterone, also showing a coincident peak.

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Masatomo Mori, Kihachi Ohshima, Hitoshi Fukuda, Isao Kobayashi and Katsumi Wakabayashi

Abstract. Heterogeneity of pituitary TSH was investigated in rats following thyroidectomy. Adult male rats were sacrificed at varying periods (2–28 days) after thyroidectomy. In another experiment, thyroidectomized rats were injected daily with various doses of l-T4 (0.3–7.5 μg/100 g body weight, ip) and sacrificed 2 weeks later. The homogenate of the pituitaries was applied on an isoelectric focusing column or a Sephacryl S-200 column.

The normal rat pituitary contained 5 major components of immunoreactive (IR) TSH in isoelectric focusing, in which the isoelectric point (pi) ranged from 6.6 to 8.8. The multiple components of IR-TSGβ were observed almost in the same areas as those of IR-TSH. Following thyroidectomy IR-TSH components with more acidic pI, associated with IR-TSHβ, were evident. A large amount of IR-TSHβ in the pituitaries of thyroidectomized rats appeared near the void volume in gel filtration, suggesting the presence of big TSHβ Supplement of l-T4 minimized these thyroidectomy-induced changes in isoelectric focusing and gel filtration. Furthermore, big IR-TSHβ was little affected by ultracentrifugation and was relatively stable after treatment with 6 m guanidine hydrochloride.

We demonstrated that the rat pituitary gland contained multiple components of IR-TSH and IR-TSHβ, both of which became variegated after thyroidectomy. It is suggested that a discernible degree of heterogeneity of TSH, particularly of TSHβ, is dependent upon the increased rate of TSH biosynthesis at the pituitary level.