Hydrolysis of carnosine and related compounds by mammalian carnosinases

Comparative study of hydrolysis of carnosine and a number of its natural derivatives by human serum and rat kidney carnosinase was carried out. The rate of carnosine hydrolysis was 3–4-fold higher then for anserine and ophidine. The rate of homocarnosine, N-acetylcarnosine and carcinine hydrolysis was negligible by either of the enzymes used. Our data show that methylation, decarboxylation or acetylation of carnosine increases resistance of the molecule toward enzymatic hydrolysis. Thus, metabolic modification of carnosine may increase its half-life in the tissues.     

Carnosine is a quencher of 4-hydroxy-nonenal: through what mechanism of reaction?

The aim of this study was to understand the mechanism of action through which carnosine (beta-alanyl-L-histidine) acts as a quencher of cytotoxic alpha,beta-unsaturated aldehydes, using 4-hydroxy-trans-2,3-nonenal (HNE) as a model aldehyde. In phosphate buffer solution (pH 7.4), carnosine was 10 times more active as an HNE quencher than L-histidine and N-acetyl-carnosine while beta-alanine was totally inactive; this indicates that the two constitutive amino acids act synergistically when incorporated as a dipeptide and that the beta-alanyl residue catalyzes the addition reaction of the histidine moiety to HNE. Two reaction products of carnosine were identified, in a pH-dependent equilibrium: (a) the Michael adduct, stabilized as a 5-member cyclic hemi-acetal and (b) an imine macrocyclic derivative. The adduction chemistry of carnosine to HNE thus appears to start with the formation of a reversible alpha,beta-unsaturated imine, followed by ring closure through an intra-molecular Michael addition. The biological role of carnosine as a quencher of alpha,beta-unsaturated aldehydes was verified by detecting carnosine-HNE reaction adducts in oxidized rat skeletal muscle homogenate.     

Biochemical properties of new synthetic carnosine analogues containing the residue of 2,3-diaminopropionic acid: the effect of N-acetylation

Summary. Three novel carnosine analogues 7–9 containing the residue of L(+)2,3-diaminopropionic acid with different degree of N-acetylation instead of -alanine have been synthesized and characterized. Comparative analysis of hydrolysis by carnosinase revealed that the mono- and bis-acetylated compounds 8 and 9 are resistant to enzymatic hydrolysis and act as competitive inhibitors of this enzyme. The hydroxyl radical scavenging potential of the three analogues was evaluated by their ability to inhibit iron/H₂O₂-induced degradation of deoxyribose. The second-order rate constants of the reaction of compounds 7–9 with hydroxyl radical were almost identical to that of carnosine. These compounds were also found to act as protective agents against peroxynitrite-dependent damage as assessed by their ability to prevent nitration of free tyrosine induced by this species.     

The antineoplastic effect of carnosine is accompanied by induction of PDK4 and can be mimicked by l-histidine

Carnosine (β-alanyl-l-histidine) is a naturally occurring dipeptide that shows antineoplastic effects in cell culture as well as in animal experiments. Since its mode of action and the targets at the molecular level have not yet been elucidated, we performed qRT-PCR experiments with RNA isolated from glioblastoma cell lines treated with carnosine, β-alanine, l-alanine, l-histidine and the dipeptide l-alanine-l-histidine. The experiments identified a strong induction of expression of the gene encoding pyruvate dehydrogenase 4 (PDK4) under the influence of carnosine and l-histidine, but not by the other substances employed. In addition, inhibition of cell viability was only detected in cells treated with carnosine and l-histidine, with the latter showing a significantly stronger effect than carnosine. Since the tumor cells expressed the tissue form of carnosinase (CN2) but almost no serum carnosinase (CN1), we conclude that cleavage by CN2 is a prerequisite for the antineoplastic effect of carnosine. In addition, enhanced expression of PDK4 under the influence of carnosine/l-histidine opens a new perspective for the interpretation of the ergogenic potential of dietary β-alanine supplementation and adds a new contribution to a growing body of evidence that single amino acids can regulate key metabolic pathways important in health and disease.     

Bestatin inhibition of human tissue carnosinase, a non-specific cytosolic dipeptidase

Bestatin is a dipeptide containing a unique beta-amino acid. It is usually referred to as an aminopeptidase inhibitor. Current interest has focused on the immunostimulating activity of bestatin and several clinical trials have demonstrated that it is an effective adjunct to radiation or chemotherapy in the treatment of certain types of cancer. We found that bestatin was much more effective against human tissue carnosinase than against aminopeptidases. Inhibition was competitive, with a Ki of 0.5nM. Carnosinase did not hydrolyse bestatin and the enzyme-inhibitor complex formed rapidly. A hog kidney dipeptidase similar to human tissue carnosinase was equally sensitive to this inhibitor. Bestatin has a backbone structure identical to that of carnosine; however, our results indicate that the inhibitory activity of this compound is primarily attributable to the side chains of the beta-amino-acid moiety. Human tissue carnosinase is a non-specific dipeptidase, actively hydrolysing many dipeptides, including prolinase substrates. Inhibition of this cytosolic enzyme is probably at least partially responsible for the intracellular accumulation of dipeptides which occurs following the in vivo administration of bestatin.     

Problems and perspectives in studying the biological role of carnosine

In describing carnosine among the constituents of muscle tissue in 1900, V. Gulevitsch opened the question of its real biological role. Investigation of carnosine-related phenomena occurred simultaneously with the study of its metabolic transformation within the cell. It has now been demonstrated that carnosine has the ability to protect cells against oxidative stress as well as to increase their resistance toward functional exhaustion and accumulation of senile features. Mechanisms of such protection are explained in terms of proton buffering, heavy metal chelating, as well as free radical and active sugar molecule scavenging, preventing modification of biomacromolecules and keeping their native functional activity under oxidative stress. Several carnosine derivatives are characterized by different rates of splitting by tissue carnosinase and by different biological efficiencies, thus the biological significance of enzymatic modification of carnosine during its tissue metabolism may be increased resistance of cells operating under unfavorable conditions.     

The Sod Like Activity of Copper: Arnosine,Copper: Anserine and Copper: Homocarnosine Complexes

Carnosine, anserine and homocarnosine are natural compounds which are present in high concentrations (2–20 mM) in skeletal muscles and brain of many vertebrates. We have demonstrated in a previous work that these compounds can act as antioxidants, a result of their ability to scavenge peroxyl radicals, singlet oxygen and hydroxyl radicals. Carnosine and its analogues have been shown to be efficient chelating agents for copper and other transition metals. Since human skeletal muscle contains one-third of the total copper in the body (20–47 mmol/kg) and the concentration of carnosine in this tissue is relatively high, the complex of carnosine:copper may be of biological importance. We have studied the ability of the coppenarnosine (and other carnosine derivatives) complexes to act as superoxide dismutasc. The results indicate that the complex of copper:carnosine can dismute superoxide radicals released by neutrophils treated with PMA in an analogous mechanism to other amino acids and copper complexes. Copper:anserine failed to dismute superoxide radicals and coppwhomocarnosine complex was efficient when the cells were treated with PMA or with histone-opsonized streptococci and cytochalasine B. The possible role of these compounds to act as physiological antioxidants that possess superoxide dismutase activity is discussed.     

Separation and characterization of two carnosine-splitting cytosolic dipeptidases from hog kidney (carnosinase and non-specific dipeptidase)

High performance anion-exchange chromatography was used to separate two carnosine-hydrolysing dipeptidases from hog kidney. Both enzymes (peaks I and II) were cytosolic and were activated and stabilized by Mn2+ and dithiothreitol. Peak I had a narrow specificity when assayed without added metal ions, but a broad specificity in the presence of Mn2+ or Co2+. Peak II was inactive unless both Mn2+ and dithiothreitol were present. Bestatin and leucine inhibited peak II, but not peak I. Peak I had a Km of 0.4 mM carnosine, a pI of 5.5 and a Mr of 57,000. Peak II had a Km of 5 mM carnosine, a pI of 5.0 and a Mr of 70,000. Hog and rat brain and liver carnosinase activity was completely inhibited by bestatin, indicating that these organs contained peak II, with little or no peak I enzyme. Hog kidney peak I contained the classical carnosinase of Hanson and Smith, who first described this enzyme. It also contained activity against homocarnosine ("homocarnosinase") and showed "manganese-independent carnosinase" activity. These three activities could not be separated using 8 different chromatographic procedures; it was concluded that they are attributable to one enzyme. It is recommended that the name carnosinase be retained for this enzyme and the names "homocarnosinase" and "manganese-independent carnosinase" be withdrawn. The properties of hog kidney peak II closely resembled those of human tissue carnosinase (also known as prolinase, a non-specific dipeptidase), mouse "manganese-dependent carnosinase" and a rat brain enzyme termed "beta-Ala-Arg hydrolase". Since these terms appear to represent closely related enzymes with broad specificity, the recommended name for each is "non-specific cytosolic dipeptidase".     

Carnosine derivatives: new multifunctional drug-like molecules

Carnosine (β-alanyl-L-histidine) is an endogenous dipeptide widely and abundantly distributed in the muscle and nervous tissues of several animal species. Many functions have been proposed for this compound because of its antioxidant and metal ion-chelator properties. Many potential therapeutic properties have been recognized especially related to the antioxidant activity, but the therapeutic uses are strongly limited by the mechanism governing its homeostasis. This fact has been the main reason for developing the synthesis of carnosine derivatives with interesting potentiality, but until now there have been very few applications. These derivatives could represent the future drugs for many pathologies related to oxidative stress and metal ion dyshomeostasis.     

In vitro demonstration of histamine biosynthesis from carnosine by kidneys of pregnant mice

Kidneys of pregnant mice synthesize histamine when incubated in the presence of carnosine, manganese, and pyridoxal phosphate. Intensity of biosynthesis increases linearly with the amount of enzyme and the incubation time. The reaction can only be catalysed by two enzymes that are located in kidneys and act in succession: carnosinase, which hydrolyzes carnosine into its two moieties, and histidine decarboxylase, which transforms histidine, a product of carnosine degradation, into histamine. The biosynthesis of histamine from carnosine seems to increase with the progress of pregnancy. In nonpregnant mice, kidneys do not effect this biosynthesis. The above results directly demonstrate that carnosine may be used for histamine synthesis when the activity of histidine decarboxylase is high, as in pregnant mouse kidney. Vertebrate carnosine, its role still enigmatic, might thus be mainly a potential histidine reservoir that would be mobilized any time there is a significant requirement for histidine, such as for histamine biosynthesis.     

Low plasma carnosinase activity promotes carnosinemia after carnosine ingestion in humans

A polymorphism in the carnosine dipeptidase-1 gene (CNDP1), resulting in decreased plasma carnosinase activity, is associated with a reduced risk for diabetic nephropathy. Because carnosine, a natural scavenger/suppressor of ROS, advanced glycation end products, and reactive aldehydes, is readily degraded in blood by the highly active carnosinase enzyme, it has been postulated that low serum carnosinase activity might be advantageous to reduce diabetic complications. The aim of this study was to examine whether low carnosinase activity promotes circulating carnosine levels after carnosine supplementation in humans. Blood and urine were sampled in 25 healthy subjects after acute supplementation with 60 mg/kg body wt carnosine. Precooled EDTA-containing tubes were used for blood withdrawal, and plasma samples were immediately deproteinized and analyzed for carnosine and β-alanine by HPLC. CNDP1 genotype, baseline plasma carnosinase activity, and protein content were assessed. Upon carnosine ingestion, 8 of the 25 subjects (responders) displayed a measurable increase in plasma carnosine up to 1 h after supplementation. Subjects with no measurable increment in plasma carnosine (nonresponders) had ～2-fold higher plasma carnosinase protein content and ～1.5-fold higher activity compared with responders. Urinary carnosine recovery was 2.6-fold higher in responders versus nonresponders and was negatively dependent on both the activity and protein content of the plasma carnosinase enzyme. In conclusion, low plasma carnosinase activity promotes the presence of circulating carnosine upon an oral challenge. These data may further clarify the link among CNDP1 genotype, carnosinase, and diabetic nephropathy.     

Carnosine and anserine concentrations in the quadriceps femoris muscle of healthy humans

The content of anserine and carnosine in the lateral portion of the quadriceps femoris muscle of 50 healthy, human subjects has been studied. Anserine was undetectable in all muscle samples examined. Muscle carnosine values for the group conformed to a normal distribution with a mean (SD) value of 20.0 (4.7) mmol.kg-1 of dry muscle mass. The concentration of carnosine was significantly higher in the muscle of male subjects (21.3, 4.2 mmol.kg-1 dry mass) than in females of a similar age and training status (17.5, 4.8 mmol.kg-1 dry mass) (P less than 0.005). The test-retest reliability of measures was determined on a subgroup of 17 subjects. No significant difference in mean carnosine concentration was found between the two trials [21.5 (4.0) and 22.0 (5.2) mmol.kg-1 dry muscle mass; P greater than 0.05]. The importance of carnosine as a physicochemical buffer within human muscle was examined by calculating its buffering ability over the physiological pH range. From the range of carnosine concentrations observed (7.2-30.7 mmol.kg-1 dry muscle mass), it was estimated that the dipeptide could buffer between 2.4 and 10.1 mmol H+.kg-1 dry mass over the physiological pH range 7.1-6.5, contributing, on average, approximately 7% to the total muscle buffering. This suggests that in humans, in contrast to many other species, carnosine is of only limited importance in preventing the reduction in pH observed during high intensity exercise.     

Carnosine Release from Olfactory Bulb Synaptosomes Is Calcium-Dependent and Depolarization-Stimulated

Abstract: The dipeptide carnosine (β-alanyl-L-histidine) has been proposed as a neurotransmitter in the mammalian olfactory pathway. Therefore, the efflux of in vivo -synthesized [¹⁴C]carnosine from mouse olfactory bulb synaptosomes was investigated. Carnosine was found to be released from the olfactory bulb synaptosomes by two mechanisms. The first is a slow spontaneous process that is independent of depolarization. The rate of this release was doubled in the presence of 1 m M external carnosine. Release by the second mechanism was markedly stimulated in the presence of calcium by depolarization with either 60 m M K⁺ or 300 μ M veratridine. Omission of calcium abolished the stimulatory effect of both of these agents. Further, blockage of the veratridine-induced depolarization by tetrodotoxin also inhibited carnosine release. These results are consistent with the hypothesis that carnosine acts as a neurotransmitter in the mouse olfactory pathway.     

Mechanisms of carnosine-induced activation of neuronal cells

Abstract

Carnosine (β-Ala-l-His), an imidazole dipeptide, is known to have many functions. Recently, we demonstrated in a double-blind randomized controlled trial that carnosine is capable of preserving cognitive function in elderly people. In the current study, we assessed the ability of carnosine to activate the brain, and we tried to clarify the molecular mechanisms behind this activation. Our results demonstrate that carnosine permeates the blood brain barrier and activates glial cells within the brain, causing them to secrete neurotrophins, including BDNF and NGF. These results point to a novel mechanism of carnosine-induced neuronal activation. Our results suggest that carnosine should be recognized as a functional food factor that helps achieve anti-brain aging.     

Kidney and liver carnosinase activity and carnosine level in goose.

Activity of kidney and liver carnosinase and concentration of carnosine in leg muscles were determined for 8 weeks in old geese of three races: Italian white, Bilgoraj and Lublin. significant differences were noted between the three races with respect to all parameters under study. the following correlations were found: 1. Between live goose weight and carnosine concentration in muscles (r= 0.5276). 2. Between weight of leg muscles and carnosine level in these muscles (r=0.4912). 3. Between liver weight and carnosine level in muscles (r= 0.3292). 4. Between kidney carnosinase activity and liver carnosinase activity (r= .2104). 5. Between liver carnosinase activity and carnosine level (r= 0.2280). 6. Between kidney carnosinase activity and carnosine level (r= -0.1675). 7. Between the ratio of kidney:liver carnosinase activity and carnosine level in muscles (r =0.1816).     

Enzymatic and immunological evidence for two forms of carnosinase in the mouse.

Carnosinase (aminoacyl-L-histidine hydrolase, EC 3.4.13.3) hydrolyzes the dipeptide carnosine (beta-alanyl-L-histidine), which is thought to play a role in cerebral and skeletal muscular function and has been implicated as a neuroaffector in the olfactory bulb. Carnosinase activity is present in many tissues of the mouse including heart, liver and lung, but it is most active in kidney, uterus and nasal olfactory mucosa. Kinetic measurements with 1H-NMR spectroscopy indicate that the enzyme is stereospecific and can hydrolyze L-but not D-carnosine. Anserine is a poorer substrate, while homocarnosine is essentially a non-substrate. However, these two dipeptides are effective inhibitors of the hydrolysis of L-carnosine. Carnosinase activity is unaffected when assayed in 2H2O at 99% isotopic purity. From considerations of the effect of Mn2+ on (1) substrate concentration velocity curves; (2) thermostability, and (3) inhibitor behavior, tissues with carnosinase can be divided into two groups. Kidney, uterus and olfactory mucosa represent one group, while central nervous system, muscle, spleen, etc. represent the second. The validity of this classification is confirmed by immunological evidence. Antiserum prepared against carnosinase purified from kidney cross-reacts with and inhibits the activity of olfactory mucosa, kidney and uterus but not that from central nervous system, heart or liver.     

Salmonella typhimurium peptidase active on carnosine.

Wild-type Salmonella typhimurium can use carnosine (beta-alanyl-L-histidine) as a source of histidine, but carnosine utilization is blocked in particular mutants defective in the constitutive enzyme peptidase D, the product of the pepD gene. Biochemical evidence for assigning carnosinase activity to peptidase D (a broad-specificity dipeptidase) includes: (i) coelution of carnosinase and dipeptidase activity from diethylaminoethyl-cellulose and Bio-Gel P-300 columns; (ii) coelectrophoresis of carnosinase and dipeptidase on polyacrylamide gels; and (iii) inactivation of carnosinase and dipeptidase activities at identical rates at both 4 and 42 degrees C. Genetic evidence indicates that mutations leading to loss of carnosinase activity map at pepD. Several independent pepD mutants have been isolated by different selection procedures, and the patterns of peptide utilization of strains carrying various pepD alleles have been studied. Many pepD mutations lead to the production of partially active peptidase D enzymes with substrate specificities that differ strikingly from those of the wild-type enzyme. The growth yields of carnosinase-deficient strains growing in Difco nutrient broth indicate that carnosine is the major utilizable source of histidine in this medium.     

Carnosine and anserine act as effective transglycating agents in decomposition of aldose-derived Schiff bases

There are numerous publications describing the positive effects of carnosine (beta-alanyl-histidine) and anserine (beta-alanyl-1-N-methyl-histidine) on cell and organ function. Of special interest to us is the fact that these dipeptides act to retard and (in one instance) reverse non-enzymatic glycation. To date, the primary explanation for these anti-glycating effects has been the fact that carnosine and anserine can serve as alternative and competitive glycation targets, thereby protecting proteins from this deleterious process. In this paper, we document another mechanism by which these two peptides can retard or reverse glycation. The process involves decomposition of the very first intermediates of the non-enzymatic glycation cascade (aldosamines a.k.a. Schiff bases) by nucleophilic attack of carnosine and/or anserine on the preformed aldosamine such as glucosyl-lysine. If future research shows this reaction is to be physiologically important, this mechanism could explain some of the beneficial effects of carnosine and anserine as anti-glycating agents.