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Title: Studies on homocarnosinosis and on human tissue carnosinase and its inhibition by bestatin and by endogenous inhibitors 
Author: Peppers, Steven Carl
Date: 1984
Abstract: Carnosine (β-alanyl-L-histidine) is a major constituent of human skeletal muscle. Its function in muscle is yet undetermined, but there is evidence that carnosine may serve as a buffer, an activator of myosin ATPase, or a source of supplementary histidine or histamine. Carnosine is also highly localized in the olfactory bulb of the mouse, where it is a putative neurotransmitter for olfactory chemoreceptor neurons. Tissue carnosinase may be responsible for its degradation and consequent inactivation at the synapse. Another histidine dipeptide, homocarnosine (γ-aminobutyryl-L-histidine), is almost exclusively localized in the CNS; little is yet understood about its physiological function. Patients afflicted with homocarnosinosis have elevated concentrations of homocarnosine in brain and cerebrospinal fluid. It has been reported that they lack brain homocarnosinase. However, in the present study it was found that these patients are deficient in serum carnosinase, a dipeptidase (mol.wt. 160,000) which hydrolyzes homocarnosine in addition to carnosine. When an extract of normal human brain was chromatographed on DEAE-cellulose, all homocarnosine-hydrolyzing activity was present in one peak and had a molecular weight of 160,000. Also, homocarnosinase (Mol.wt. 57,000) was not detected in brain extracts after isoelectric focusing. Homocarnosine-hydrolyzing activities and serum carnosinase activities of human CSF samples were significantly correlated (p<0.001). In homocarnosinosis patients, both of these activities were negligible in serum samples, a CSF sample, and a brain biopsy sample when compared to corresponding controls. Thus, the ability of brain extracts and CSF to hydrolyze homocarnosine appears to be attributable solely to serum carnosinase, which is deficient in patients with homocarnosinosis. The homocarnosine-hydrolyzing activity in 12 other tissues was related to the amount of trapped blood, but brain contained 15 times the activity expected from its blood content; hence, serum carnosinase may be synthesized in the brain. Human tissue carnosinase was found to be optimally active at pH 9.5. It was inhibited by p-hydroxymercuribenzoate and activated by 2 mM dithiothreitol: indicating it to be a cysteine peptidase. By optimizing assay conditions, tissue carnosinase activities per g of tissue were 5- to 15-fold greater than those previously reported. The enzyme was present in every human tissue assayed and was entirely different from plasma 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 hydrolyze tripeptides and thus appears to be a dipeptidase rather than an aminopeptidase. It was found to have a molecular weight of 90,000, an isoelectric point of 5.6, and a Km value of 10 mM carnosine under the conditions of assay. Two forms of kidney and brain carnosinase were separated by high resolution anion exchange chromatography, although only one form was detected using various electrophoretic methods. Two other enzymes, homocarnosinase and manganese-independent carnosinase, were not detected in human tissues, but were readily measured in rat and hog kidney. Crude extracts of human tissues contain endogenous tissue carnosinase inhibitors that were dialyzable and thermostable over a pH range of 1 to 11. Among 14 different tissues the amount of inhibition varied considerably, but no tissue was without inhibitor. An endogenous inhibitor from human liver was isolated and identified as L-leucine. It was found to be the most inhibitory (IC50 = 0.2 mM) of all the common amino acids, followed by cysteine and cystine. L-leucine inhibition was stereospecific, D-leucine being inactive, and was partially dependent on manganese; this indicates that it binds to the active site of tissue carnosinase. In contrast, cysteine probably inhibited carnosinase by chelating manganese; as manganese concentrations were increased above 0.02 mM, the inhibition decreased. Cystine inhibition was attributed to its reduction to cysteine by the dithiothreitol present in the digest. Since the concentrations of leucine and other amino acids in a liver extract accounted for only one fourth of its inhibitory activity, another inhibitor(s) must have been present. Inhibition by L-leu-L-leu was greater than by L-leucine, and plots of the reciprocal velocity vs. L-leucine concentration displayed upward curvature these results suggest that L-leucine binds to two sites on tissue carnosinase. L-leucine inhibition was mixed competitive and noncompetitive, being predominantly competitive at concentrations below 0.2 mM L-leucine and more noncompetitive at higher concentrations. Bestatin is a dipeptide that has potential clinical value as an immunostimulant and chemotherapeutic agent. Commonly known to inhibit leucine aminopeptidase and aminopeptidase B, bestatin was found to be an extremely potent inhibitor of human tissue carnosinase (Ki = 0.5 nM). The affinity of bestatin binding to tissue carnosinase was 18 to 40 times greater than to nonparticulate leucine aminopeptidase and 120 times greater than to aminopeptidase B. Bestatin was a competitive inhibitor and was most inhibitory at a manganese concentration close to the optimum for enzyme activity. The structures of bestatin and carnosine are similar; both contain an N-terminal β-amino connected to an a-amino acid having an R-group with a branch-point at the y-carbon. Therefore, it is proposed that bestatin binds with high affinity to tissue carnosinase because its backbone chain is identical to that of carnosine.
Description: Typescript. Thesis (Ph. D.)--University of Hawaii at Manoa, 1984. Bibliography: leaves 139-157. Photocopy. Microfilm. xii, 157 leaves, bound ill. 29 cm
URI: http://hdl.handle.net/10125/9411
Rights: All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Keywords: Tissues, Carnosine, Bestatin

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