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

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.

Rat peritoneal mast cell carboxypeptidase: localization, purification, and enzymatic properties

“…The carboxypeptidase hydrolyzed an ester substrate, hippuryl-L&phenylactate [5], and a number of dipeptides with carboxyl terminal aromatic residues, e.g., Z-AlaPhe [Z]. High ionic strength was required for solublization and the enzyme was inhibited by o-phenanthroline [2,5] and mercurials [2]. Estimates of molecular weight ranged from 34 500 [2] to 43 000 [4].…”

Proteolytic enzymes of rat liver mitochondria. Evidence for a mast cell origin

“…Similarly, insoluble sediments exhibiting proteinase and carboxypeptidase activities were obtained after The preparation of the high density sediments is described in section 2. The proteinase activity was measured with N-acetyltyrosine-ethylester [2], the carboxypeptidase was tested with Cbz-Ala-Phe [3] sucrose step gradient centrifugation of mitochondrial fractions from rat heart and rat kidney. No high density sediment could be isolated from the mitochondrial fraction of rat brain.…”

“…The details of the sucrose step gradient centrifugation used to isolate the high density sediments which contained the proteinase and the carboxypeptidase have been reported [ 11. The assay conditions for the measurement of both enzyme activities have been described [2,3]. For the histochemical detection of mast cells small tissue blocks of rat liver, heart, kidney and brain were frozen in isopentane cooled with liquid nitrogen.…”

“…Evidence for the mast cell origin of the serine proteinase and the carboxypeptidase associated with the high density sediment obtained after step gradient centrifugation of digitonin-treated mitochondria came from the foilowing findings: (i) A > loo-fold higher proteinase activity/g wet wt was measured in the high density fraction obtained from a digiton~-treated mast cell mitochondrial fraction compared to liver (table 1). (ii) In addition to the proteinase the carboxypeptidase which was only recently discovered in liver [3] is also found in the high density fraction isolated from mast cells. (iii) The protein patterns after SDS-polyacrylamide gel electrophoresis are the same for solubilized high density fractions from liver and mast cells.…”

“…Both enzymes have been purified and their physicochemical properties were studied [2-41. After subcellular fractionation, digitonin treatment and step gradient centrifugation both enzymes were localized in mitochondria [ 1,3]. After submitochondrial fractionation both proteolytic enzymes were found in the inner mitochondrial membrane [ 1,3].…”