CATABOLISM OF RAT GROWTH HORMONE-RELEASING FACTOR(1-29) AMIDE IN RAT SERUM AND LIVER

Clinical and veterinary uses of growth hormone-releasing factor [GRF(1-29)NH2] require the design of analogs that are resistant to proteolysis by serum and liver degrading enzymes. This study investigated rat GRF(1-29)NH2 processing in serum and fiver homogenate by means of high pressure liquid chromatography (HPLC). Synthetic rGRF(1-29)NH, (30-mu-M was incubated (0-120 min, 37-degrees-C) in serum (49 +/- 8 mg prot./ml). The rGRF(1-29)NH2 (10-mu-M) was also incubated (0-120 min, 37-degrees-C) with liver homogenate (200 +/- 6-mu-g prot./ml). Time course studies of rGRF(1-29)NH2 disappearance showed apparent half-lives of 18 +/- 4 min and 13 +/- 3 min in serum and liver homogenate, respectively. This was accompanied by the appearance of degradation products that were all less hydrophobic than the native peptide. In the serum, two major metabolites were detected and isolated by preparative HPLC. Combined results of amino acid analysis, sequencing, and chromatography with synthetic homologs revealed the presence of rGRF(1-20)OH and (3-20)OH. A small amount of rGRF(12-29)NH2, coeluting with rGRF(3-20)OH, was also found by sequencing. In the liver, rGRF(1-18)OH, (3-18)OH, and (I-10)OH were identified. The peptide bond Ala2-Asp3 (DPP IV cleavage site) was hydrolyzed in both serum and liver. Other tissue-specific cleavage sites were Arg11-Arg12 and Arg20-Lys21 (trypsin-like cleavage site) in the serum, and Tyr10-Arg11 and Tyr18-Ala19; (chymotrypsin-like cleavage site) in the liver. The enzyme responsible for the cleavage of Ala-Asp3 was sensitive to diprotin A, while those responsible for the cleavage of Tyr18-Ala19 and Arg20-Lys21 were sensitive to leupeptin. The low affinity of fragments (1-20)NH2 and (12-29)NH2 for pituitary GRF binding sites suggests that hydrolysis of Arg11-Arg12, Tyr18-Ala19, and Arg20-Lys21 bonds inactivates rGRF(1-29)NH2 in this target tissue.