Characterization of two carnosine‐degrading enzymes from rat brain

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Abstract
From rat brain extracts, two carnosine‐degrading enzymes have been identified and partially purified by ion‐exchange chromatography, hydrophobic interaction chromatography on phenyl‐Sepharose CL‐4B and gel filtration. These enzymes, exhibit distinct differences in their chemical characteristics and substrate specificities.One enzyme, designated carnosinase, preferentially hydrolyzes carnosine and exhibits a low Km value (0.02mM) towards this substrate. Carnosinase also degrades anserine but not homocarnosine or homoanserine. The other carnosine‐degrading enzyme hydrolyzes βAla‐Arg considerably faster than carnosine and, therefore, has been tentatively designated βAla‐Arg hydrolase. This enzyme exhibits high Km values with carnosine (Km= 25 mM) and βAla‐Arg (Km= 2 mM). Homocarnosine and γ‐aminobutyryl‐arginine are not degraded by βAla‐Arg hydrolase.Neither enzyme is inhibited by agents reactive on activated hydroxyl groups, such as diisopropyl fluorophosphate, and also not by a variety of peptidase inhibitors of microbial origin or from other sources. Carno‐sinase is also not inhibited by bestatin but βAla‐Arg hydrolase, although not an aminopeptidase, is strongly inhibited by this aminopeptidase inhibitor (IC50= 50 nM). While carnosinase is strongly inhibited by thiol‐reducing agents such as dithioerythritol and 2‐mercaptoethanol, βAla‐Arg hydrolase is stabilized and activated by these substances. Both enzymes are strongly inhibited by metal‐chelating agents. Carnosinase, however, is not dependent on exogeneously added metal ions and is strongly inhibited by Mn2+ as well as by heavy metal ions. In contrast, βAla‐Arg hydrolase requires Mn2+ ions for full enzymatic activity.Based on these differences, selective incubation conditions could be evaluated in order to determine specifically both enzyme activities in crude tissue extracts. In rat, both enzymes are present in all tissues tested, except skeletal muscles, but considerable differences in their relative distribution among different tissues are also observed.

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