A Novel SH‐Type Carboxypeptidase in the Inner Membrane of Rat‐Liver Mitochondria

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Abstract
A carboxypeptidase from rat liver mitochondria was partially purified by discontinuous sucrose gradient centrifugation, washes with NaCl/KBr/Tris buffer, and solubilization with 2 M NaCl in the presence of soybean trypsin inhibitor bound to CM-cellulose. By means of dodecylsulfate/polyacrylamide gel electrophoresis a molecular weight of 34500 was determined; a value of 38000 was estimated by Sephadex G-100 gel filtration. The carboxypeptidase was completely inhibited by 3 mM Hg2+. In the presence of 3 mM Cu2+ 50% of the catalytic activity was inhibited. Among several peptides tested Cbz-Ala-Phe, Cbz-Leu-Phe, Cbz-Phe-Leu, and Cbz-Phe-Phe, were good substrates. The enzyme activity exhibited a pH optimum of around 9 with Cbz-Ala-Phe as a substrate. After submitochondrial fractionation it was found that the carboxypeptidase is located in the inner mitochondrial membrane.