Ovine Luteinizing Hormone. III. Relationships between the Charge Heterogeneity of Partially Purified Subunits and the Native Hormone1

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Abstract
Extracts of anterior pituitaries from wethers were prepared by homogenization and centrifugation at 100,000 .times. g. When chromatofocused on pH 10.5-7.0 gradients, eight peaks of immunoreactive ovine luteinizing hormone (oLH) were observed six exhibited apparent pIs in the range of 9.33-8.83, one eluted unbound (apparent pI > 9.8), and one was bound to the column (apparent pI .ltoreq. 7.0). A portion of the same extracts was subjected to gel filtration on Sephadex G-100 Superfine to resolve native oLH and its uncombined subunits. oLH, oLH.alpha., and oLH.beta. were present at concentrations of 0.907 .+-. 0.127, 0.089 .+-. 0.020, and 0.010 .+-. 0.023 .mu.g/mg tissue, respectively, which translated to oLH.alpha./oLH and oLH.beta./oLH molar ratios of .apprxeq.0.19 and .apprxeq.0.02. Fractions containing immunoreactive oLH or uncombined subunits (oLH.alpha. and oLH.beta.) were pooled, lyophilized, and chromatofocused. Native oLH resolved from uncombined subunits by gel filtration displayed a similar pattern of isohormones to those in crude extracts. In contrast, three purified oLH preparations exhibited distinct chromatofocusing patterns. Uncombined oLH.alpha. in pituitary extracts resolved from native oLH by gel filtration exhibited a higher percentage (.apprxeq.37%) of acidic components when chromatofocused, while more than 97% of purified oLH.alpha. focused as basic forms having pIs greater than 8.9. When uncombined oLH.beta. in pituitary extracts was chromatofocused, more than half of the immunoreactivity was bound to the column (apparent pI .ltoreq. 7.0); purified oLH.beta. displayed a nearly identical pattern. These results suggest that 1) native oLH resolved from uncombined subunits by gel filtration displays a similar chromatofocusing profile to that of oLH in crude pituitary extracts; 2) uncombined oLH.alpha. and oLH.beta. in pituitary extracts separated from native oLH by gel filtration focus predominantly as acidic forms; 3) uncombined LH subunits in pituitary extracts minimally contribute to the observed pattern of immunoreactive oLH isohormones; and 4) the charge characteristics of native oLH appear to reflect those of the alpha subunit, assuming that the acidic nature of uncombined alpha in pituitary extracts could be due to a high percentage of O-glycosylated forms.

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