The distribution of carnosine and related dipeptides in rat and human tissues.

Carnosine and anserine are present in high concentrations in most skeletal muscles. In addition, carnosine and homocarnosine have been detected in brain and cardiac muscle. Other tissues have been found to be devoid of these histidine-containing dipeptides. However, Flancbaum et al. reported that carnosine was present in every rodent and human tissue analyzed. These authors postulated that carnosine serves as a non-mast cell reservoir for histidine which becomes available for histamine synthesis during periods of physiological stress. We have analyzed many rat and human tissues using an immunohistochemical procedure. Carnosine and related dipeptides were detected in skeletal muscle, cardiac muscle and brain, but not in kidney, liver, lung or several other organs. These negative results seem valid since the immunoassay gave positive staining in the tissues generally known to contain carnosine.

Localization of a novel pathway for the liberation of GABA in the human CNS.

Serum carnosinase is a dipeptidase, which is synthesized in human brain, where it hydrolyzes homocarnosine to release free GABA. Immunohistochemical procedures were used to demonstrate the presence of this enzyme in several layers of the retina and in certain neuronal tracts of the cerebral cortex, cerebellar cortex, olfactory bulb, hippocampus, and in disseminated tracts presumably from the internal capsule, interspersed among the basal ganglia. The enzyme was also present in the epithelial cells of the choroid plexus and in corpora amylacea, which were seen in many regions of the CNS. Homocarnosine was localized either in the same tracts or in nearby neurons. For example, the Purkinje cells of the cerebellar cortex contained homocarnosine, whereas serum carnosinase was localized in adjacent neuronal projections apparently originating from outside the cerebellar cortex and having probable synaptic contact with the Purkinje cells. These findings suggest that in addition to glutamate decarboxylation, a second metabolic reaction for the formation of free GABA exists in specific neuronal tracts of the human CNS where GABA is released from homocarnosine by the action of serum carnosinase.

Homocarnosinosis patients and great apes have a serum protein that cross-reacts with human serum carnosinase.

A specific polyclonal antiserum to human serum carnosinase was raised in rabbits and was used to prepare an agarose-protein A-antibody matrix. An antigen capture procedure showed that sera from homocarnosinosis patients, which lack carnosinase activity, contain an immunoreactive protein (M(r) 75,000) indistinguishable from the carnosinase band from normal serum. Other higher primates have active serum carnosinase and a similar immunoreactive M(r) 75,000 protein. The immunoaffinity matrix was used in a facile procedure to isolate pure carnosinase from human plasma with a yield of 69%. The antiserum inhibited human serum carnosinase strongly, but the maximum inhibition attained averaged only 71%. The antiserum inhibited human and chimpanzee serum carnosinases more effectively than gorilla or other higher primate serum carnosinases.

Purification and properties of human serum carnosinase.

Carnosinase from human plasma was purified 18,000-fold to apparent homogeneity in a four step procedure. The dipeptidase was partially inactivated during DEAE-cellulose chromatography; however, it reactivated slowly when concentrated and stored at 4 degrees C. In the second purification step, hydroxylapatite column chromatography, two forms of the enzyme were separated from one another. Human serum carnosinase was found to be a glycoprotein with a pI of 4.4 and a subunit Mr of 75,000; the active enzyme was a dimer, the two subunits being connected by one or more disulfide bonds. The enzyme was especially active in hydrolyzing carnosine and anserine, preferring dipeptides with histidine in the C-terminal position. In most human tissues, the concentration of serum carnosinase was proportional to the percentage of trapped blood in the sample. However, the brain contained about 9 times more enzyme than expected, based on the amount of trapped blood present. The physiological function of this enzyme seems to be the hydrolysis of homocarnosine in the brain and the splitting of carnosine and anserine in the blood stream. Six higher primates were found to have serum carnosinase. Twelve nonprimate mammals were tested; all were lacking the serum enzyme except for the Golden hamster, which had very high concentrations of a carnosinase having somewhat different properties than the higher primate enzyme.

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".

Human cytosolic carnosinase: evidence of identity with prolinase, a non-specific dipeptidase.

In separate papers published in 1985, human cytosolic carnosinase and prolinase were purified and characterized for the first time. Prolinase had activity against many dipeptides not containing proline; carnosinase also had broad specificity. The present paper reports that carnosinase and prolinase activities were not separated from one another during chromatography on columns of DEAE-cellulose, AGMP-1, gel filtration media, hydroxylapatite or butyl-agarose. Both activities had identical pH-stability curves at 50 degrees C, being stabilized by manganese ions and dithiothreitol. Prolinase substrates competitively inhibited carnosinase activity and carnosinase substrates inhibited prolinase activity. Bestatin was a potent inhibitor of both activities, while cilastatin inhibited neither. It was concluded that prolinase and carnosinase activities reside in the same enzyme. High performance anion-exchange chromatography of extracts from kidney, liver or brain separated the enzyme into two forms having isoelectric points of 5.6 and 5.1. Because of the broad specificity of this dipeptidase, it is recommended that it be termed "human cytosolic non-specific dipeptidase".

Inborn errors of carnosine and homocarnosine metabolism.

Serum carnosinase deficiency with carnosinuria has been reported in 23 children with neurological signs and/or mental retardation. In adults four cases in one family had serum carnosinase deficiency, carnosinuria, and in addition elevated homocarnosine in CSF and in the brain. The mother was one of these cases but had no clinical symptoms; however her three children have spastic paraparesis, retinitis pigmentosa and mental retardation. Serum carnosinase deficiency alone is not the cause of the neurological symptoms. When two of the affected children consumed carnosine, anserine or homocarnosine, they metabolized these compounds much less rapidly than did two normal control individuals.

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.

Characterization of human tissue carnosinase.

Human tissue carnosinase (EC 3.4.13.3) had optimum activity at pH9.5 and was a cysteine peptidase, being activated by dithiothreitol and inhibited by p-hydroxymercuribenzoate. By optimizing assay conditions, the activity per g of tissue was increased 10-fold compared with values in the literature. The enzyme was present in every human tissue assayed and was entirely different from serum carnosinase. Highly purified tissue carnosinase had a broader specificity than hog kidney carnosinase. Although tissue carnosinase was very strongly inhibited by bestatin, it did not hydrolyse tripeptides, and thus appears to be a dipeptidase rather than an aminopeptidase. It had a relative molecular mass of 90 000, an isoelectric point of 5.6, and a Km value of 10 mM-carnosine. Two forms of kidney and brain carnosinase were separated by high-resolution anion-exchange chromatography, although only one form was detected by various electrophoretic methods. Homocarnosinase and Mn2+-independent carnosinase were not detected in human tissues, although these enzymes are present in rat and hog kidney.

Cathepsins J and K: high molecular weight cysteine proteinases from human tissues.

Human tissue extracts contained two high Mr proteinases active in hydrolyzing the fluorogenic substrate Cbz-phe-arg-aminomethylcoumarin. By gel filtration chromatography, cathepsins J and K had apparent molecular weights of 230,000 and 650,000, respectively. Both enzymes were cysteine proteinases with optimum activity at pH 6.2-6.8; neither had aminopeptidase activity. Human kidney, lung and spleen were rich sources of these enzymes, while liver contained moderate amounts. Cathepsins J and K were partially characterized and appeared to differ from the mammalian high Mr cysteine proteinases described in the literature. In rat liver and kidney and in mouse liver, cathepsin J was localized in the particulate fraction, whereas cathepsin K was not detected in these tissues.

Homocarnosinosis: lack of serum carnosinase is the defect probably responsible for elevated brain and CSF homocarnosine.

Patients afflicted with homocarnosinosis have elevated concentrations of homocarnosine in brain and CSF. It has been reported that they lack brain homocarnosinase. However, we have found that these patients are deficient in serum carnosinase, a dipeptidase which hydrolyzes homocarnosine about 5% as rapidly as it splits carnosine. Homocarnosinase could not be detected in normal human brain extracts after isoelectric focusing or DEAE-cellulose column chromatography. The ability of brain extracts to hydrolyze homocarnosine thus appears to be attributable solely to the serum carnosinase which is present because of serum trapped in the brain sample. Preliminary data indicate that homocarnosinase is probably not present in 13 other human tissues. Normal CSF contained serum carnosinase, whereas the CSF of a homocarnosinosis patient was lacking this enzyme. Thus it appears that the elevated concentrations of homocarnosine in the CSF of homocarnosinosis patients are attributable to serum carnosinase deficiency.

Human serum carnosinase: characterization, distinction from cellular carnosinase, and activation by cadmium.

Human serum carnosinase was assayed using a simple and sensitive fluorometric method. Under optimum conditions, the average adult serum hydrolyzed 42 mu mol of carnosine per ml per hour, about 17 times the average activity reported in the literature. Cadmium was twice as effective as manganese as an activator of this enzyme. Serum carnosinase was found to be different in many respects from cellular carnosinase. For example, the serum isozyme hydrolyzed homocarnosine, whereas the cellular carnosinase did not. The apparent molecular weight of serum carnosinase was 160 000, while that of the cellular isozyme was 90 000. Although it has been reported that serum contains two molecular forms of carnosinase, only one form was detected using several electrophoretic methods and two ion exchange chromatography procedures. The concentration of serum carnosinase varied greatly between individuals. Little or no enzyme was detected in children below 10 months in age. Thereafter, the average concentration of carnosinase increased gradually to reach the adult range at age 13-15.

Similarity of tuna N-acetylhistidine deacetylase and cod fish anserinase.

1. The brain and ocular fluid of skipjack tuna (Katsuwonus pelamis) contained high levels of N-acetylhistidine deacetylase. 2. This enzyme had a molecular weight of about 120,000 and was activated by zinc or cobaltous ions. 3. Cod (Gadus callarias) brain, ocular fluid and muscle contained a similar metal-activated thiol hydrolase, the muscle enzyme being known as anserinase. 4. The purified enzymes hydrolyzed N-acetylhistidine, carnosine, homocarnosine, anserine and certain other dipeptides. 5. Their specificity resembled that of hog kidney homocarnosinase. 6. In both fish, brain and ocular fluid were rich sources of this hydrolase, whereas muscle contained only trace amounts.