Characterization of two carnosine‐degrading enzymes from rat brain

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 CL4B 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 K, value (0.02 mM) towards this substrate. Carnosinase also degrades anserine but not homocarnosine or homoanserine. The other carnosine-degrading enzyme hydrolyzes PAla-Arg considerably faster than carnosine and, therefore, has been tentatively designated PAla-Arg hydrolase. This enzyme exhibits high K , values with carnosine ( K , = 25 mM) and PAla-Arg ( K , = 2 mM). Homocarnosine and y-aminobutyryl-arginine are not degraded by PAlaArg 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. Carnosinase is also not inhibited by bestatin but PAla-Arg hydrolase, although not an aminopeptidase, is strongly inhibited by this aminopeptidase inhibitor (IC50 = 50 nM). While carnosinase is strongly inhibited by thiolreducing agents such as dithioerythritol and 2-mercaptoethanol, PAla-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 MnZ+ as well as by heavy metal ions. In contrast, BAla-Arg hydrolase requires MnZ + 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.As early as 1900, carnosine had been isolated from Liebig's meat extract [l] and subsequently identified as P-alanylhistidine [2, 31. Since then, various w-aminoacyl amino acids such as anserine (j-alanyl-1 -methylhistidine), homocarnosine (y-aminobutyryl-histidine), /I-alanylornithine, P-alanyl-lysine etc. have been isolated from excitable tissues, brain and muscle (for reviews see [4] and [5]).Biochemical studies with cell-free tissue extracts strongly indicated that all w-aminoacyl amino acids are synthesized by only one enzyme, carnosine synthetase [L-histidine : p-alanine ligase (AMP forming), EC 6.3.2.111, which is present in brain and muscle [6,7]. In agreement with these results, we recently demonstrated that carnosine and related dipeptides are actively synthesized by glial cells in culture [8]. In order to understand the mechanisms regulating the metabolism of w-aminoacyl amino acids, we were interested in studying the degradation of these peptides by brain enzymes. Here we describe the id...

Renoprotective Effects of l-Carnosine on Ischemia/Reperfusion-Induced Renal Injury in Rats

“…l-Carnosine is known to be hydrolyzed by a carnosinedegrading enzyme, carnosinase, which is present in several tissues and serum of humans or animals (Lenney et al, 1985;Kunze et al, 1986;Jackson et al, 1991;Nagai et al, 2003). These findings raise a possibility that enzymatic hydrolysis of exogenous l-carnosine by carnosinase may be involved in l-carnosine actions on ischemic ARF.…”

“…However, a possibility that l-carnosine improves the I/R-induced renal injury, at least in part, via its antioxidative activity cannot be ruled out, because oxidative stress is definitely involved in the pathogenesis of ischemic ARF (Chatterjee et al, 2000;Takaoka et al, 2002). l-Carnosine is decomposed to ␤-alanine and l-histidine by a carnosine-degrading enzyme, carnosinase, which is present in serum and several tissues (Lenney et al, 1985;Kunze et al, 1986;Jackson et al, 1991;Nagai et al, 2003). In rat brain, two types of carnosine-degrading enzymes are present: one enzyme is carnosinase, which preferentially hydrolyzes carnosine, and the other enzyme hydrolyzes ␤-alanyl-l-arginine considerably faster than carnosine, both of which do not degrade acetyl-l-carnosine (Kunze et al, 1986).…”

“…These findings raise a possibility that enzymatic hydrolysis of exogenous l-carnosine by carnosinase may be involved in l-carnosine actions on ischemic ARF. Therefore, we investigated the effect of N-␣-acetyl-l-carnosine (N-acetyl-␤-alanyll-histidine, acetyl-l-carnosine) resistant to enzymatic hydrolysis by carnosinase (Kunze et al, 1986;Babizhayev et al, 1996;Pegova et al, 2000) or l-histidine cleaved from l-carnosine by carnosinase on renal injuries induced by I/R, and the findings were compared with those observed by the lcarnosine treatment. Furthermore, we examined whether renoprotective effects of l-carnosine on ischemic ARF would be affected by a histamine H 3 receptor antagonist, thioperamide, since Yamano et al (2001) reported that the suppressive effect of l-carnosine on hyperglycemia induced by i.c.v.…”

Extracellular Arginine Aminopeptidase from Streptococcus gordonii FSS2

“…Both Lactococcus lactis and L. helveticus express biochemically identical dipeptidases, PepDA (459 amino acids) and cytosol nonspecific dipeptidase EC 3.4.13.18 (474 amino acids), which are approximately 30% identical and 47% similar to RAP (11). These enzymes are biochemically distinct from RAP, are specific for dipeptides of variable composition, and have been isolated from the cytoplasm of prokaryotic cells (34,35).…”

Hormone-brain-aging relationships, broadly reactive with imidazole-containing dipeptides: targeting of telomere attrition as an aging biomarker and dynamic telomerase activity flirting

“…The level of carnosine in tissues is controlled by a number of enzymes transforming carnosine into other carnosine related compounds, such as carcinine, N-acetylcarnosine, anserine, or ophidine (by decarboxylation, acetylation, or methylation, respectively) or its cleavage into the amino acids histidine and β-alanine. Hydrolysis is mainly due to tissue carnosinase (EC 3.4.13.3), which is widely distributed among different subjects [133][134][135], or serum carnosinase (EC 3.4.13.20), which is present in the brain and blood plasma of primates and humans [135][136][137]. Carnosine has been proven to scavenge ROS as well as α-β unsaturated aldehydes formed from peroxidation of cell membrane fatty acids during oxidative stress [138,139].…”