SUMMARY: Several methods were used in a vain search for a possible specific, isolable complex of the enzymes involved in the conversion of mandelate into benzoate in and Previous cross-linking experiments done by other workers with were repeated and slightly extended. Extracts prepared from bacteria that had been treated with dimethylsuberimidate were analysed by ultracentrifugation and gel-filtration chromatography. In confirmation of the original observations, some of the mandelate enzymes were found to be linked to high- material. However, several control enzymes behaved in the same way and only a small proportion of the ‘soluble’ mandelate enzymes were found in exactly the same fraction as the membrane-bound L(+)-mandelate dehydrogenase. Similarly, no specific cross-linking was observed in experiments with , even when the bacteria were supplied with substrates and cofactors for the mandelate enzymes. There was no evidence for co-fractionation of the mandelate enzymes from extracts that had been prepared in various ways. Treatment of homogenates with antiserum that had been raised against phenylglyoxylate decarboxylase failed to co-precipitate the other mandelate enzymes. Incubation of homogenates with anti-D(---)-mandelate dehydrogenase did lead to the removal of some phenylglyoxylate decarboxylase from the supernatant fraction, but this is ascribed to the interaction of phenylglyoxylate decarboxylase with a membrane component other than D(---)-mandelate dehydrogenase. Control and cross-linked extracts were analysed by SDS-PAGE followed by immunoblotting with the three antisera. Although there was evidence for the formation of high- species, there was no sign of specific cross-linking of any of the three enzymes to each other. Experiments involving cross-linking with other dimethylimidates and with phenylene dimaleimide all failed to provide evidence for a mandelate complex.


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