Journal of Endocrinology and Reproduction

Taruna Arora ¹, G. Arundhati ¹, K. Muralidhar ¹

Affiliations
1 Hormone Research Laboratory, Department of Zoology, University of Delhi, Delhi-110007, IN

Abstract

Presence of free alpha subunit has been noticed in a side fraction obtained during the processing of buffalo pituitary glands for thyrotropin and gonadotropins using standard isolation and fractionation procedures. In addition to the gel chromatographic data (Ve/Vo), when the material was analyzed with different antisera, it clearly reacted with alpha subunit directed antibodies. The NaCI precipitation method of Sairam for the separation of the free subunits from each other was applied to the material. The yield of the alpha subunit by the NaCI precipitation method with respect to the immunogenicity and the content was higher than what was obtained by the counter current distribution.

The sugar content of the free alpha subunit was less (70 μg/mg) compared to the sugar content of the alpha subunit isolated by the dissociation from the intact hormone (204 μg/mg). Most of the free alpha subunit eluted as the unbound fraction from a Concanavalin A sepharose column thus supporting the idea that there was an alteration in carbohydrate structure in these naturally occurring free subunits.

Keywords

Alpha Subunit, Free Subunit, Glycoprotein Hormones, Thyrotropin.

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Taruna Arora ¹, K. Muralidhar ¹

Affiliations
1 Hormone Research Laboratory, Department of Zoology, University of Delhi, Delhi-110007, IN

Abstract

Microheterogeneity has been observed in the thyrotropin isolated from buffalo pituitary glands using the standard isolation and fractionation procedures. Crude thyrotropin was passed through CM-Sephadex column for enrichment, followed by sizing through Sephadex G-200 and ultimately DEAE-Sephacel was used for the segregation of TSH from LH. The buTSH obtained had 72% TSH immunoreactivity and a specific bioactivity of 71U/mg. It had only about 10% LH content in it. N-terminal analysis of buTSH revealed heterogeneity where the two N-termini were determined as Phe and Gly. The carbohydrate analysis of the buTSH fraction revealed the presence of three microheterogenous forms, out of which the form containing the biantennary carbohydrate structure had the highest immunoreactivity. However, buffalo TSH was found to be different from sheep TSH. The latter appears to have a high mannose/hybrid carbohydrate structure in the most immunoreactive fraction.

Keywords

Buffalo TSH, Glycoprotein Hormones, Heterogeneity, Pituitary, Sheep TSH, Thyrotropin.

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Kapil Maithal ¹, H. G. Krishnamurty ¹, K. Muralidhar ²

Affiliations
1 Department of Chemistry, University of Delhi, Delhi - 110 007, IN
2 Department of Zoology, University of Delhi, Delhi - 110 007, IN

Abstract

Three different extraction protocols were applied to freshly frozen buffalo pituitaries collected from a local abattoir with a view to obtain a highly immunopotent preparation of buffalo GH. The 'enriched GH' obtained from each protocol was assessed on the basis of immunopotency and hormonal homogeneity. It was observed that, use of Ellis and Spitsberg protocols in tandem yielded the highest amount of immunoreactive prolactin free GH (ECS). Further, it was found to be relatively less contaminated with other protein impurities. Application of various chromatographic procedures on this enriched GH preparation (ECS) showed that Sephadex G-200 chromatography yielded most immunopotent GH preparation in high amounts. Further, the purification protocol was most convenient and reproducible procedure for obtaining bulk quantities of pure growth hormone from buffalo pituitaries.

Keywords

Buffalo, Growth Hormone, Purification, Immunopotency.

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