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.

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Bestatin is a dipeptide containing a unique ß-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 K^ of O.SnM. 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 ß-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. Hemmung menschlicher Gewebs-Carnosinase, einer unspezifischen cytosolischen Dipeptidase, durch BestatinZusammenfassung: Bestatin ist ein Dipeptid mit einer nur hier gefundenen ß-Aminosäure. Es findet gewöhnlich als Aminopeptidase-Inhibitor Verwendung. Unlängst hat sich das Interesse jedoch auf seine immunstimulierende Aktivität konzentriert, und es konnte in mehreren klinischen Untersuchungen gezeigt werden, daß es bei bestimmten Krebsformen eine wirkungsvolle Ergänzung zur Bestrahlung und Chemotherapie darstellt. Wir fanden, daß Bestatin gegenüber menschlicher Gewebs-Carnosinase wesentlich stärker wirksam ist als gegenüber Aminopeptidasen. Die Hemmung erwies sich als kompetitiv, mit einem ^-Wert von O.SnM. Die Bildung des Enzym-Inhibitor-Komplexes erfolgte schnell, Bestatin wurde von Carnosinase nicht hydrolysiert. Eine Dipeptidase aus Schweineniere hatte eine ähnliche Empfindlichkeit gegenüber diesem Inhibitor wie die Carnosinase aus menschlichem Gewebe. Das Rückgrat des Bestatinmoleküls ist identisch mit dem des Carnosins; nach unseren Ergebnissen ist die Hemmaktivität jedoch in erster Linie den Seitenketten des ß-Aminosäure-Anteils zuzuschreiben. Die menschliche Ge-

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