Hormonotoxins. Preparation and characterization of ovine luteinizing hormone-gelonin conjugate.
With the aim of targeting toxins to selected cells in the gonad, we have prepared conjugates of ovine luteinizing hormone (oLH) with a single chain ribosome-inactivating protein called gelonin. The two proteins were thiolated by using N-succinimidyl-3-(2-pyridyldithio)propionate and subsequently reacted under appropriate conditions to form oLH-S-S-gelonin complex. A complete biochemical analysis of thiolated oLH and oLH-gelonin conjugates has been performed. The linkage of the hormone to the toxin probably occurred through a single amino group in the alpha-subunit, with the beta-subunit remaining free. Modification of a single amino group on the alpha-subunit reduced receptor binding and immunological reactivity of the thiolated oLH, but subsequent complexing with the toxin-gelonin did not seriously compromise these activities. oLH and gelonin were calculated to be present in a 1:1 ratio in the hormonotoxin preparation. The conjugate retained significant steroidogenic activity in rat granulosa cells. Upon reaction with mouse tumor Leydig cells (MA-10 cells), the toxin component of the complex became internalized to a sufficient degree to effectively inhibit protein synthesis. The studies provide a rational basis for the design and study of large hormonotoxins.
Singh
V
V
Reproduction Research Laboratory, Clinical Research Institute of Montreal, Quebec, Canada.
Sairam
M R
MR
Bhargavi
G N
GN
Akhras
R G
RG
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004151:Dipeptides / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects; D007984:Leydig Cell Tumor / Q000378:metabolism; D007986:Luteinizing Hormone / Q000032:analysis / Q000138:chemical synthesis* / Q000494:pharmacology; D051379:Mice; D010940:Plant Proteins / Q000032:analysis / Q000138:chemical synthesis* / Q000494:pharmacology; D011374:Progesterone / Q000096:biosynthesis / Q000378:metabolism; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors; D011863:Radioimmunoassay; D051381:Rats; D011974:Receptors, LH / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1; D012756:Sheep; D013388:Succinimides; D013438:Sulfhydryl Compounds
D000596:Amino Acids; D003432:Cross-Linking Reagents; D004151:Dipeptides; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D011974:Receptors, LH; D054789:Ribosome Inactivating Proteins, Type 1; D013388:Succinimides; D013438:Sulfhydryl Compounds; C058542:luteinizing hormone-gelonin conjugate protein, plant; C058543:chrysobactin; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D011374:Progesterone; C025674:GEL protein, Gelonium multiflorum; D007986:Luteinizing Hormone; D004229:Dithiothreitol