Abstract
Cleavage of parathyroid hormone by cathepsin D was studied. Four primary products were detected and separated by high performance liquid chromatography. Two of the fragments are fluorescent and therefore contain residue 23 (tryptophan). These fragments are NH2-terminal in origin. The other two cross-react with antisera directed against COOH-terminal portions of the hormone; they are the complementary COOH-terminal fragments. Microsequencing and amino acid analysis showed that the two COOH-terminal fragments are 35-84 and 38-84 bovine parathyroid hormone. By CNBr cleavage and amino acid analysis, the two NH2-terminal fragments were shown to be the complementary 1-37 and 1-34 fragments. The 1-37 fragment is transitory and is rapidly hydrolyzed to 1-34, so that only relatively small amounts are detected at any one time. However, 34-84 was not converted to 38-84, although cleavage at other sites in the COOH-terminal fragments was observed with more exhaustive digestion. The 1-34 fragment appears to be the final product of the action of cathepsin D on parathyroid hormone. Both enzymatically produced NH2-terminal fragments were fully active in the renal membrane adenylyl cyclase assay system.
Highlights
During studies of PTH receptors in bovine kidney, we found an enzyme generating PTH fragments very similar to those seen in viuo [16,17,18]
This system separates 4 peptides from the native hormone.Peaks 2-5 originate from PTH, but control experiments showed that peak 1 is not derived from the hormone and probably represents asmall “self-digestion” fragment from cathepsin D
Analysis of the breakthrough peak showed the presence of only small amounts of poorly defined peptides, and cathepsin D eluted in the washout following the native hormone
Summary
Microsequencing and amino acid analysis showed that the two COOH-terminal fragments are 35-84 and 38-84 bovine parathyroid hormone. Hamilton et al [21] obtained similar results in parathyroid gland tissue andfurther studied the nature of the PTH fragments generated by cathepsin D. They found thatthe enzyme cleaves PTH primarily at one position, producing fragments 1-34 and 35-84 as the dominant final products. Separation of CNBr fragments was by HPLC, using acetonitrile/ water/trifluoroacetic acid solvent mixtures as described in the figure legends. The methods for membrane preparation and bioassay were exactly as described earlier except that CAMP was determined by radioimmunoassay [26, 27]
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