Purification, partial characterization, and radioimmunoassay of prolactin and growth hormone from the Bennett's wallaby

Abstract

Bennett's wallaby prolactin (wPRL) and growth hormone (wGH) were purified from an aqueous extract of pituitary glands. The extract from 202 glands (6.5 g wet wt) was processed by gel filtration on Sephadex G-100, gel filtration on Sephadex G-100 SF, and then anion-exchange chromatography on DEAE-Sepharose CL-6B. The yields of wPRL and wGH were 5.2 and 15.7 mg, respectively. Since recovery of wPRL from the anion exchange column was 10%, anion exchange was performed in the presence of 20% acetonitrile in a subsequent purification. Recovery from this column was markedly increased to 42%. The purified hormones each gave a single vand on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight under reducing conditions of 21,000 and 23,000 for GH and PRL, respectively. Each hormone was positively identified by its N-terminal amino acid sequence, which showed high sequence identity with the equivalent eutherian hormone. Semianalytical gel filtration of purified hormone was used to demonstrate that each hormone remained as a monomer in aqueous solution. Each purified hormone was tested in the heterologous PRL radioimmunoassay (RIA) which has been used in many earlier studies to measure marsupial PRL. Highly purified wPRL was less potent than ovine prolactin (5.3 compared with 1.5 ng/ml at 50% displacement) and the cross-reaction of wGH was <0.01%. Antibodies were raised against wPRL and wGH and a homologous RIA was developed for each hormone. The sensitivity of the wPRL assay was 0.8 ng/ml which is similar to that of the heterologous PRL assay. Cross-reaction with a number of eutherian pituitary hormones or wGH was <0.07%. The wGH assay detected 0.8 ng/ml, cross-reacted with GH from several eutherian species, and showed low cross-reaction with wPRL (<0.5). In both the wPRL and wGH assays, pituitary homogenates from several species of marsupial diluted in parallel with the wallaby standard, suggesting that these assays will be of use in studies of a number of marsupial species.