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SUMMARY
Separation and partial purification of chicken pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH) has been obtained by methods which were effective in the purification of the corresponding human and horse hormones. Increases in chicken LH activity were observed after chromatography on DEAE-cellulose and on Amberlite IRC-50 suggesting removal of an LH inhibitor. The biological potencies of chicken FSH and LH preparations when assayed in mammals were very much lower than those of the corresponding mammalian fractions on a weight basis. A weak immunological cross-reaction between chicken and human pituitary LH was used to estimate LH in chicken pituitary fractions and the results were compared with bioassays of the same fractions.
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SUMMARY
The gonadotrophic activities of crude glycoprotein fractions from pituitaries of man, sheep, chicken and carp and of partially purified follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the three tetrapod species were examined in both sexes of the lizard Anolis carolinensis. The piscine material did not show activity in the lizard but all mammalian and avian preparations promoted spermatogenesis, ovarian growth, ovulation and steroidogenesis. FSH preparations were far more potent than LH, in fact the actions of the LH preparations may have been due largely to FSH contamination. These findings are consistent with earlier conclusions that FSH alone may be able to stimulate all types of gonadal activity in lizards, except that high doses of FSH block ovulation. Comparisons of the relative potencies of the avian and mammalian preparations provide evidence for the zoological specificity of vertebrate gonadotrophins. In general, the relative potency of chicken gonadotrophins in lizards was greater than that estimated from standard rodent bioassays: i.e. the lizard is relatively more sensitive to chicken gonadotrophin than the rodent.
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Search for other papers by ANNE STOCKELL HARTREE in
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SUMMARY
Univalent antibody fragments were isolated from a papain-digested antiserum to human chorionic gonadotrophin. The univalent antibody fragments were purified, iodinated with 125I and used in a sensitive immunoradiometric assay of human luteinizing hormone. Unlike intact divalent antibodies, the univalent radioactive antibody fragments cannot simultaneously bind to both soluble antigen and antigen-immunoadsorbent.
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Search for other papers by MARJORIE THOMAS in
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SUMMARY
Human growth hormone prepared by Raben's (1959) method was chromatographed on various ion exchangers and the resulting fractions were assayed for growth-promoting, luteotrophic and immunological activity. A critical quantitative study of the biological activities of the fractions showed that there were significant differences between their ratios of growth-promoting to luteotrophic potency. The luteotrophic activity of some of the preparations was slightly higher than that of the 2nd International Ovine Prolactin Standard.
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In a previous communication from this laboratory (Stockell Hartree, Thomas, Braikevitch, Bell, Christie, Spaull, Taylor & Pierce, 1971) it was reported that the sialic acid contents of the subunits of human luteinizing hormone (LH) were very much less than in the intact hormone. This finding was unexpected since the subunits were still capable of recombination with restoration of full biological activity. The demonstration that marked loss of biological activity occurred in human LH after removal of sialic acid by treatment of the intact hormone with neuraminidase (Braunstein, Reichert, Van Hall, Vaitukaitis & Ross, 1971) has prompted us to reinvestigate the sialic acid contents of the subunits. In addition, the tryptophan content of the subunits has been measured by the spectrophotometric method of Bencze & Schmid (1957).
Estimations were performed on subunits prepared by two different methods. In each case intact LH of potency 3–5 mg NIH-LH-S1/mg (Stockell Hartree, 1966) was
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SUMMARY
Ion-exchange chromatography was used to further purify a human pituitary fraction rich in thyrotrophic and luteinizing hormone activities. Approximately twofold concentration of both activities was obtained by chromatography on IRC-50 at pH 7·5, but the hormones were not separated. Subsequent chromatography on DEAE-cellulose at pH 9·5 led to a tenfold concentration of the luteinizing hormone in a fraction practically free of thyrotrophic activity and to a fourfold concentration of the thyrotrophic hormone in a fraction still exhibiting substantial luteinizing hormone activity.
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Because of a misunderstanding, the information on chicken pituitary fractions provided by one of us (A.S.H) was erroneously presented in a paper by two of us (Langslow & Hales, 1969). Figure 1 gives an accurate representation of the purification scheme that was employed together with further information on the pituitary fractions that were used.
The designation of certain fractions as 'GTN residue' and 'GTN supernate' (Langslow & Hales, 1969) is misleading since there is no evidence that either fraction contains significant amounts of glycoprotein or of gonadotrophic activity. The fraction designated GTN precipitate contains follicle-stimulating and luteinizing hormone activities (Stockell Hartree & Cunningham, 1969) and there is suggestive evidence that thyrotrophin is also present (Mitchell, 1967).
The fraction designated crude 'growth hormone' might be expected to contain growth-promoting activity by analogy with corresponding fractions from human and horse pituitaries (Stockell Hartree, 1966; Stockell Hartree, Mills, Welch & Thomas, 1968). However,
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SUMMARY
Highly purified human pituitary FSH was partially dissociated by treatment with 8 m-urea, and α- and β-subunits were isolated by ion-exchange chromatography and gel filtration. Tests of biological activity by in-vivo assays and in-vitro radioreceptor assays were in good agreement and showed that preparations of isolated α-subunit had less than 1%, and β-subunit from 2 to 10% of the FSH activity of the intact hormone. In contrast to results reported elsewhere, most of the subunit preparations reassociated with counterpart subunit to regain biological activity equal to that of intact FSH (around 160 mg NIH-FSH-S1/mg). The intact FSH recovered as a by-product after isolation of subunits was of high biological activity, and its LH contamination was reduced by more than 90% when compared with the purified FSH starting material. The subunits are relatively inactive in a radioimmunoassay specific for intact FSH. Sialic acid and tryptophan determinations indicated that both subunits contain sialic acid and that tryptophan is present only in the β-subunit.
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This report describes some of the properties of a clinical-grade preparation of human growth hormone (hGH) extracted from acetone-preserved autopsy human pituitary glands and used in Great Britain from 1967 to 1980. Gel filtration of this hGH on Sephadex G-100 yielded (on a weight basis) an average of 48% of a high molecular weight fraction, 10% of an intermediate fraction expected to contain dimeric forms of the hormone and 33% of a fraction considered to be the hGH monomer. The immunoassay potency of the monomer fraction was twice that of the clinical-grade preparation and the amino-acid composition of the monomer fraction agreed well with that obtained from published hGH sequence data. The results of polyacrylamide-gel electrophoresis (under reducing and dissociating conditions) and amino-acid analysis of the high molecular weight fraction suggest that it contains around 30% of aggregated hGH as well as other material not separated from hGH by the purification procedure.
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Procedures for purification of human luteinizing hormone (LH) and human folliclestimulating hormone (FSH) (Stockell Hartree, 1966; Roos, 1968) have been applied to acetone-preserved baboon pituitaries. After death of the baboons in Kenya pituitaries were removed and placed in excess cold acetone. The glands were transported to Ohio where an acetone-dried powder was prepared, the yield from 185 pituitaries was 10·1 g. This material was extracted with 6% ammonium acetate at pH 5·1 in 40% ethanol and protein was precipitated from the soluble extract by addition of ethanol to 80% (GTN ppt). This fraction was then chromatographed on a column of CM-cellulose as described previously (Stockell Hartree, 1966). The nonadsorbed fraction (CM-1) was pooled, concentrated by rotary evaporation and freeze-dried. The adsorbed fraction (CM-2) was eluted with 1 m-ammonium acetate, and was precipitated with 80% ethanol. Fraction CM-2 (44 mg) was chromatographed on a column of DEAE-cellulose (Whatman DE-23, 1
