The localization of hippurate synthesis in the matrix of rat liver mitochondria.

In many species benzoate is conjugated in the liver with glycine to form hippurate, after prior formation of benzoyl-CoA, which then reacts with glycine in a transacylation catalysed by glycine N-acylase (EC 2.3.1.13) (Bridges et al., 1970). This enzyme has been prepared from mitochondria (Schachter & Taggart, 1954; Bartlett & Gompertz, 1974), although there is no information about its submitochondrial localization. Conversion of benzoate into benzoyl-CoA is presumably catalysed by butyryl-CoA synthetase (EC 6.2.1.2) (Mahler et al., 1953), which is found in the mitochondrial matrix (Garland et al., 1970). If glycine N-acylase also occurs in the matrix both benzoate and glycine would have to enter this compartment, and the hippurate formed would have to leave. If it is outside the permeability barrier of the inner mitochondria1 membrane, activated benzoyl groups would have to be exported as benzoylcarnitine. We therefore investigated the submitochondrial localization of hippurate synthesis. The synthesis of hippurate by intact and by sonicated preparations of mitochondria was measured. Mitochondria were prepared as described by Osmundsen & Sherratt (1975) and incubated at 20°C in 120m~-KC1/2m~-MgSO~/5m~-Hepes[2-(N-2hydroxyethylpiperazine4"'yl)sulphonic acid], adjusted to pH7.2 with solid KOH, with 50jm-[l-14C]benzoate and appropriately supplemented with lOmM-ATP, 0.5 ~M-COA and lorn-glycine. Antimycin (1 pg/ml) and oligomycin (I pg/ml) were also added to block ATP synthesis and hydrolysis respectively. Incubations were quenched with HClO, to give a final concentration of 3% (v/v). Samples were then chromatographed on Whatman SG 81 paper with diethyl ether/80% (v/v) formic acid (lO:l, v/v) (Osmundsen, 1973) and the separated benzoic acid, hippuric acid and benzoyl-CoA were detected with U.V. light (Rp values 0,0.80 and 0.95 respectively). These compounds were determined from measurements of the radioactivity of appropriate areas of the chromatograms. Benzoyl-CoA was synthesized by standard methods (Stadtman, 1957). More hippurate was synthesized by sonicated than by intact mitochondria (10 and 1.4nmol/mg of protein during a 1 h incubation, respectively). Added CoA was required for high rates of hippurate synthesis by sonicated mitochondria. Benzoyl-CoA synthetase activity was latent in intact mitochondria and was released by sonication, confirming that it is located in the matrix. Mitochondria treated with digitonin as described by Schnaitman & Greenwalt (1968) to remove the outer membrane and the contents of the intermembrane space, and suspended in lWmM-Tris/HCI, pH8.2 at 20°C, still synthesized hippurate (3.7nmol/mg of protein during a 1 h incubation). These observations indicate that glycine N-acylase occurs in the matrix, where it will have access to benzoyl-CoA generated in this compartment. Suspensions of sonicated mitochondria incubated with 5Ophi-benzoate attained a nearly constant concentration of benzoyl-CoA after lOmin (about 2 5 ~ ) . This fell when glycine was added, with the synthesis of more than an equivalent amount of hippurate. The concentration of benzoate also fell. The failure to convert allthe benzoateadded into benzoyl-CoA in the absence of glycine suggests that there is hydrolysis of benzoyl-CoA. Indeed, benzoyl-CoA hydrolase activity with an apparent K,,, of low was found in the supernatant fraction obtained after centrifugation of a suspension of sonicated mitochondria for 1 h at 150000g.,., although this activity was unstable. Further, short-chain acyl-CoA hydrolase activity occurs in the matrix of liver mitochondria (Osmundsen & Sherratt, 1975). When intact mitochondria were incubated with 5Op~-benzoate, the maximum extent of acylation of their CoA was only about 0.5nmol/mg of protein. Rat liver mitochondria contain about 2-3nmol of CoA/mg of protein (Holland & Sherratt, 1973), of which about 50% was acylated by deca-2,4,6,8-tetraenoate in the steady state