文献 Human intestinal diamine oxidase_ substrate specificity and comparative inhibitor study

108 Histamine MetabolismHuman Intestinal Diamine Oxidase: Substrate Specificity and Comparative Inhibitor Studyby T. BIEGANSKI1, J. KUSCHE, K.-D. FEU13NER, R. HESTERBERG, H. RICHTER and W. LORENZ2Polish Academy of Sciences, Institute of Pharmacology Cracow, Department of Biogenic Amines, Narutowicza Str. 60, 90136 Lodz, Poland; Division of Experimental Surgery 5- ethylhistamine, iVa,5-dimethylhistamine (Prof. Dr W. Schun- ack, University of Mainz); aliphatic diamines and monoamines (Fluka).Determination of enzymic activityThe DAO activity was determined by two methods. (1) With putrescine as substrate mainly the modified isotope assay was used 2, 3. (2) The substrate specificity was tested by the coupled optic test 4. For testing the pH optimum and the influence of ionic strength in presence of putrescine both methods were applied concomitantly. Monoamine oxidase activity was assayed by the isotope test with 14C-tryptamine as substrate 5. All test procedures and reagents were composed as described in the corresponding original publications.Results1. Properties of human intestinal DAOThe pH optimum of the purified enzyme with histamine (104final concentration) was reached at pH6.7, with putrescine (10-3 M) at pH 7.0. Comparing substrate deamination rates the ratio putrescine/histamine was ca. 2/1 at their corresponding pH and substrate optimum.The optimum molarity of phosphate buffer in presenceHistamine Metabolism 109of putrescine (10“3 M) was 0.15 M. At a 0.5 M buffer concentration the enzyme was inhibited by 50%.The ability to purify intestinal DAO by affinity chromatography on Con-A Seph has already shown the presence of a glycoprotein group in the human intestinal DAO. The addition of Con-A Seph to an enzyme solution and the subsequent centrifugation removed all DAO activity from the supernatant. The resuspended pellet in which the enzyme was bound to Con-A Seph contained the same DAO activity as the original preparation. This shows that the glycoprotein group occupied by binding to Con-A Seph was not involved in the active site of human intestinal DAO.2. Substrate specificityAmong the aliphatic diamines putrescine and cada- verine at 10-3 M final concentrations were deaminated with nearly equal velocity (ratio 1/1.2). 1,3-Diaminopropane, however, at the concentration of 5 x 10-3 M was metabolized 1.5-fold more rapidly than putrescine (10-3 M). Diamines with longer aliphatic chains than cadaverine (tested up to 1,9-diaminononane) were deaminated with decreasing reaction velocities.As already mentioned above, histamine was deaminated about half as fast as putrescine (53% at pH 7.0). However, mong the histamine derivatives ring-methylated compounds were deaminated with considerable higher velocity than histamine itself (at 10一 4 M -methyl- histamine 1.8-fold, 2-methylhistamine 2.0-fold faster). Side chain methylated histamine derivatives, however, such as A-methylhistamine and iVa,iVa-climethylhistamine .were no substrates of human intestinal DAO.3. InhibitorsThe well-known inhibitors of DAO, semicarbazide and aminoguanidine, produced a 50% inhibition of human intestinal DAO at concentrations of 2 x 106 M and 1.1 x 108 M respectively, showing that in this aspect the human intestinal enzyme was behaving like a classical DAO.However, a comparative study (Table 1) of the human intestinal enzyme with DAO from pea seedlings concerning the effects of H2-receptor antagonists, the H2-receptor agonist dimaprit and the inhibitor of lysyloxidase, -amino- propionitrile, revealed some differences suggesting that even classical DAO might represent not one but several enzymes. /ff-Aminopropionitrile was a very strong inhibitor of theTable 1Inhibitor concentrations causing 50% inhibition of DAO from human intestine and pea seedling.Inhibitors Inhibitor concentration MHumanintestinal DAOPea seedling DAOyff-Aminopropionitrile 2.0 x 10-4 8.0 x 10_6Burimamide 2.2 x 10-5 (io-3)Metiamide 5.0 x 10_4 (103)Cimetidine 4.2 x 104 (10_3)Dimaprit 3.0 x 10_4 1.0 x 10_3Each value represents x from 3 determinations by isotope assay, ( 10-3) means that at this concentration less than 20% inhibition were observed.enzyme from pea seedlings but by two orders of magnitude less effective against the DAO from human intestine. The reverse situation was observed with burimamide which strongly inhibited the intestinal enzyme but was really ineffective on pea seedling DAO.DiscussionIn many aspects the human intestinal DAO resembles other mammalian diamine oxidases such as in its substrate and inhibitor specificity. However, some special properties of this enzyme are (1) the relatively low pH optimum for enzyme activity using putrescine and histamine as substrate as compared with the intestinal DAO of rabbits and dogs 6, 7 and (2) the high reaction velocity of Nx- and 2-methyl- histamine. These substrates were metabolized nearly as fast as putrescine. This might indicate that the product of histamine methylation by histamine methyltransferase (HMT) 8 in human intestinal mucosa is further catabolized by DAO. This metabolic pathway was already suggested by GRANERUS et al. 9 for human subjects and for mice by SCHAYER et al. 10.A rather interesting finding was the different extent of inhibition of the DAO from human intestine and pea seedlings by /ff-aminopropionitrile and burimamide. Interpreting these results we should call in mind that the function of lysyloxidase is strongly connected with the formation of connective tissue 11 which surely is a process of growing and regeneration. Therefore, it seems difficult to accept that the inhibitor of lysyloxidase, -aminopropionitrile is only by chance so effective on the pea seedling enzyme which is originating during germination, i.e. during growth.On the other hand burimamide was highly effective on the intestinal DAO but nearly without effect on pea seedling DAO. From several experiments (liberation of DAO during anaphylaxis 12, protective function of DAO during intestinal ischemia 13, 14 and beneficial and detrimental actions of and H2-receptor antagonists in the circulatory shock 15) we can assume a detoxicating function of this enzyme by histamine catabolism in the mammalian organism. Moreover, one should also account the ratio of histamine/putrescine catabolism which for the pea seedling enzyme was about 1/20 4, but for the human intestinal enzyme 1/2. At least we should remember that -methyl- histamine is a strong inhibitor of HMT 16. Thus the human intestinal DAO might influence the histamine degradation (1) by removing the product inhibition of HMT and (2) by deamination of histamine itself.Taking together results and hypotheses one could argue that KAPELLER-ADLER 17 was not completely wrong when differentiating between histaminase and diamine oxidase which means in the present view that the histaminase is involved in the detoxication of histamine and diamine oxidase in the regulation of di- and polyamine levels.Received 18 September 1979References1 W. LORENZ, H.-J. REIMANN, H. BARTH, J. KUSCHE, R. MEYER, A. DOENICKE and M. HUTZEL, A Sensitive and Specific Method for Determination of Histamine in Human Whole Blood and Plasma, Hoppe-Seylers Z. Physiol. Chem. 353,911 (1972).2 T. OKUYAMA and Y. KOBAYASHI, Determination of Diamine Oxidase Activity by Liquid Scintillation Counting, Arch. Biochem. Biophys. 95, 242 (1961).110 Histamine Metabolism3 J. KUSCHE，H. RICHTER, R. HESTERBERG, J. SCHMIDT and W. LORENZ, Comparison of the 14C-Putrescine Assay with the NADH Test for the Determination of Diamine Oxidase: Description of a Standard Procedure with a High Precision and Improved Accuracy, Agents and Actions 3, 148 (1974).4 W. LORENZ, J. KUSCHE and E. WERLE，Uber eine neue Methode zur Bestimmung der Diaminoxy- daseaktivitat, Hoppe-Seylers Z. Physiol. Chem. 348, 561 (1967).5 RJ. WURTMANN and J. AXELROD, A Sensitive and Specific Assay for the Estimation of Monoamine Oxidase， Biochem. Pharmacol. 12, 1439 (1963).6 J. KUSCHE, H. RICHTER, J. SCHMIDT, R. HESTERBERG, A. FRIEDRICH and W. LORENZ, Diamine Oxidase in Rabbit Small Intestine: Separation from a Soluble Monoamine Oxidase， Properties and Pathophysiological Significance in Intestinal Ischemia, Agents and Actions 5,431 (1975).7 J. KUSCHE, W. LORENZ and J. SCHMIDT, Oxidative Deamination of Biogenic Amines by Intestinal Amine Oxidases: Histamine is Specifically Inactivated by Diamine Oxidase， Hoppe-Seylers Z. Physiol. Chem. 356, 1485 (1975).8 H. BARTH, M. CROMBACH, W. SCHUNACK and W. LORENZ, Gastric Histamine Methyltransferase has a Less Hish Acceptor Substrate Specificity， Fed. Proc. 37, 392 (1978).9 G. GRANERUS，H. WETTERQUIST and T. WHITE, Histamine Metabolism in Healthy Subjects Before and During Treatment with Aminoguanidine， Scand. J. Clin. Lab. Invest. 22, 39 (1968).10 M.A. REILLY and R.W. SCHAYER, In Vivo Studies onHistamine Catabolism and its Inhibition, Br. J. Pharmac. 38, 478(1970).11 P.J. SHERIDAN, L.G. KOZAR and S.C. BENSON, Increased Lysyl Oxidase Activity in Aortas of Hypertensive Rats and Effect of pAminopropionitrile, Exp. Mol. Pathol. JO, 315 (1979).12 W. SCHMUTZLER, F. HAHN, G. SESEKE and W. BERNAUER, Ober die Herkunft der Plasmahistaminase Im anaphylaktischen Schock des Meerschweinchens, Naunyn-Schmiedebergs Arch. Pharmacol. 252, 332 (1966).13 J. KUSCHE, C.-D. STAHLKNECHT, W. LORENZ, G. REICHERT and H. RICHTER, Diamine Oxidase and Histamine Release in Dogs Following Acute Mesenteric Artery Occlusion， Agents and Actions 7, 81 (1977).14 J. KUSCHE, C.-D. STAHLKNECHT, W. LORENZ, G. REICHERT and W. DIETZ, Comparison of Alterations in the Histamine-Diamine Oxidase System During Acute Intestinal Ischemia in Pigs, Dogs and Rabbits: Evidence for a Uniform Pathophysiological Mechanism? Agents and Actions P, 49 (1979).15 B.M. ALTURA and S. HALEVY, Beneficial and Detrimental Actions of Histamine Hx- and H2-Receptor Antagonists in Circulatory Shock, Proc. Natl. Acad. Sci. USA 75, 2941 (1978).16 H. BARTH, W. LORENZ and I. NIEMEYER, Inhibition and Activation of Histamine Methyltransferase by Methylated Histamines， Hoppe-Seylers Z. Physiol. Chem. 354, 1021 (1973).17 R. KAPELLER-ADLER and H. MCFARLANE, Purification and Identification of Hog-Kidney Histaminase, Biochim. Biophys. Acta 57, 542 (1963).The Influence of Carcinoma Growth on Diamine Oxidase Activity in Human Gastrointestinal Tractby J. KUSCHE, T. BIEGANSKI1, R. HESTERBERG, C.-D. STAHLKNECHT, K.-D. FEUI3NER, I. STAHLENBERG and W. LORENZ2 Department of Experimental Surgery and Pathological Biochemistry and Surgery Clinic of the University of Marburg, Robert-Koch-Str. 8, D-3550 Marburg, Federal Republic of GermanyAbstractThe distribution of diamine oxidase (DAO) activity was studied in patients having no carcinoma disease. Besides in gut DAO occurred in high activity only in kidney and mesenteric lymph nodes.In patients with adenocarcinoma of the large bowel or of the stomach the enzymic activity was reduced in the tumour tissue itself as compared with the adjacent mucosa in which part the highest activities were observed. In stomac