Purification of calcitonin-like peptides from rat brain and pituitary.

Accumulating evidence supports the existence of nonthyroidal calcitonin (CT)-like peptides, more similar to fish CTs, which may act as endogenous regulators of CT receptors in brain and other tissues. In this study, we have carried out large-scale extractions from Sprague-Dawley rat brain diencephalon and pituitary, and purified a novel, biologically active, CT-like peptide from pituitary. Monitoring of the calcitonin-like activity of the peptides from rat brain and pituitary required different detection systems. While the brain CT cross-reacted with C-terminally directed salmon CT-specific antisera, the pituitary CT did not. However, the pituitary CT was biologically active, exhibiting specific interaction with CT receptors to activate adenylate cyclase. Conventional chromatographic techniques were employed to purify the CT-like peptides. Although the brain CT was not purified to homogeneity, size exclusion chromatography revealed the presence of multiple molecular weight forms of immunoreactive CT. Of these, only the lowest molecular weight form was biologically active. Purification from the pituitary resulted in the isolation of a biologically active peptide with a mass of 3267 Da. This mass differs from the mass of both salmon and thyroid-derived rat CT. Initial amino acid sequencing of the pituitary CT indicated that it was N-terminally blocked. Following aminopeptidase digestion, a unique six amino acid sequence, EKSQSP, was identified. Elucidation of the amino acid composition provided supporting evidence that the peptide was novel and was consistent with a full length peptide of approximately 30 amino acids. These data support the existence of novel, nonthyroidal, CTs which are potential regulators of CT receptor-mediated functions.

Structure/function relationships of calcitonin analogues as agonists, antagonists, or inverse agonists in a constitutively activated receptor cell system.

The structure/function relationship of salmon calcitonin (sCT) analogues was investigated in heterologous calcitonin receptor (CTR) expression systems. sCT analogues with progressive amino-terminal truncations intermediate of sCT-(1-32) to sCT-(8-32) were examined for their ability to act as agonists, antagonists, or inverse agonists. Two CTR cell clones, B8-H10 and G12-E12, which express approximately 5 million and 25,000 C1b receptors/cell, respectively, were used for this study. The B8-H10 clone has an approximately 80-fold increase in basal levels of intracellular cAMP due to constitutive activation of the overexpressed receptor. In whole-cell competition binding studies, sCT-(1-32) was more potent than any of its amino-terminally truncated analogues in competition for 125I-sCT binding. In cAMP accumulation studies, sCT-(1-32) and modified analogues sCT-(2-32) and sCT-(3-32) had agonist activities. SDZ-216-710, with an amino-terminal truncation of four amino acids, behaved as a partial agonist/antagonist, whereas amino-terminal truncations of six or seven amino acid residues produced a 16-fold reduction in basal cAMP levels and attenuated the response to the agonist sCT-(1-32) in the constitutively active CTR system. This inverse agonist effect was insensitive to pertussis toxin inhibition. In contrast, the inverse agonist activity of these peptides was not observed in the nonconstitutively active CTR system, in which sCT analogues with amino-terminal truncations of four or more amino acids behaved as neutral competitive antagonists. These results suggest that the inverse agonist activity is mediated by stabilization of the inactive state of the receptor, which does not couple to G protein, and attenuates basal signaling initiated by ligand-independent activation of the effector adenylyl cyclase.

Isolation and characterization of human LH isoforms.

Thirty-nine human LH (hLH) isoforms were chromatographically separated from human pituitary extracts using a mild purification procedure which consisted of preparative isoelectric focusing, high-performance ion-exchange chromatography and immobilized metal-affinity chromatography. Twenty of these hLH isoforms were characterized by LH radioreceptor assay, SDS-PAGE and amino acid analysis, and 17 were shown to be highly purified (> 90% pure). The specific activities of these hLH isoforms ranged from 1980 to 38,650 IU/mg protein in terms of the 2nd IS for human pituitary LH, based on protein content as determined by amino acid analysis. hFSH and hTSH content were < 0.5% and < 7.8% respectively. The purity was assessed by silver staining on SDS-PAGE. Under non-reducing conditions, a single band of apparent molecular mass 23.5-24.5 kDa was observed, whereas under reducing conditions the isoforms migrated as two distinct bands, 21.1-22.4 kDa and 18.0-20.5 kDa, probably corresponding to the alpha and beta subunits of hLH respectively. The remaining three less pure isoform preparations (70-90% pure) contained additional bands of 16 kDa and 26.3 kDa under non-reducing conditions. All isoforms showed a low molecular mass band(s) of 11-14 kDa which was < 7% of stained material as assessed by densitometry. Amino acid composition of the 17 hLH isoforms was similar to the published cDNA composition of hLH. Further fractionation of one hLH isoform (hLH IIc) on reversed-phase high-performance liquid chromatography yielded four peaks identified by N-terminal sequencing as two alpha and two beta hLH subunits identical to their cDNA-derived N-terminal sequences. No additional sequences indicative of internal clipping of hLH were observed. The two pairs of alpha and beta subunits probably represent two separate hLH isoforms in this preparation. It was concluded that a mild purification procedure with high recoveries for the isolation of intact hLH isoforms has been developed, and 17 isoforms of high purity suitable for further biological and physicochemical characterization have been isolated. These isoform preparations are free of other contaminating proteins, but may still contain multiple hLH-related species.