The effectiveness of inhibitors of soluble prolyl hydroxylase against the enzyme in the cisternae of isolated bone microsomes

Abstract

Inhibitors of purified, soluble prolyl hydroxylase ( K. Majamaa et al. (1984) Eur. J. Biochem. 138, 239–245; K. Majamaa et al. (1986) J. Biol. Chem. 261, 7819–7823 ) were tested against isolated chick embryo bone microsomes containing intracisternal prolyl hydroxylase and its radiolabeled, unhydroxylated procollagen substrate. Two groups of inhibitors were used which consisted of pyridine-2-carboxylate and 1,2-dihydroxybenzene (catechol) derivatives. The 2,4- and 2,5-pyridine dicarboxylic acids, which are potent inhibitors of the soluble enzyme ( K i values 2 and 0.8 μ m, respectively), were effective in the same concentration range against intracisternal prolyl hydroxylase, although their relative affinities were reversed. Inhibition by pyridine-2,4-dicarboxylate in the microsomal system was reversed by increasing the concentration of 2-oxoglutarate. Pyridine-2,4-dicarboxylic acid did not inhibit the uptake of 2-[ 14C]oxoglutarate into microsomes, so it appears likely that the inhibitor must traverse the microsomal membrane and act directly at the enzyme level. Pyridine-2-carboxylic acid was ineffective in the microsomal system at 1 m m whereas it is a relatively potent inhibitor of the soluble enzyme with a K i of 25 μ m. This finding suggests that the second carboxyl group of the pyridine carboxylate derivatives may be required for their transport into the microsomal lumen. In the soluble system, 3,4-dihydroxybenzoic acid and 1,2-dihydroxybenzene had been found to be competitive inhibitors with relatively low K i values of 5 and 25 μ m, respectively. In the microsomal system, half-maximal inhibition was obtained at approximately 50–100 μ m and inhibition was not reversed by increasing the concentrations of either 2-oxoglutarate or ascorbate, alone or together. These results imply that in situ these compounds do not inhibit prolyl hydroxylase directly. Thus, the microsomal system can assess the accessibility of the intracisternal enzyme to potential inhibitors and offers an insight into the in cellulo potential of such compounds.