Oxidative decarboxylation of free and peptide-linked amino acids in phagocytizing guinea pig granulocytes. S K Adeniyi-Jones, M L Karnovsky J Clin Invest. 1981;68(2):365-373. https://doi.org/10.1172/JCI110264. Research Article The oxidative decarboxylation of amino acids by a system consisting of myeloperoxidase-hydrogen peroxide-chloride has been demonstrated previously by others and the process has been considered to be part of the microbicidal armamentarium of some phagocytic leukocytes. We were able to translate these earlier observations, made on model systems, to intact guinea pig granulocytes. We could demonstrate differences in the cellular handling of peptide-linked amino acids as particles, compared with free amino acids. Specific inhibitors were used to explore two routes of oxidative decarboxylation: (a) the myeloperoxidase-catalyzed direct decarboxylation-deamination reaction, and (b) oxidation of alpha-keto acids after transamination of amino acids. These inhibitors were cyanide, azide, and tapazole for the former pathway, and amino-oxyacetate for the latter. Amino-oxyacetate profoundly inhibited the decarboxylation of free 14C- amino acids (alanine and aspartate) in both resting and stimulated cells, but had only a minimal effect on 14CO2 production from ingested insoluble 14C-protein. On the other hand, the peroxidase inhibitors cyanide, azide, and tapazole dramatically inhibited the production of 14CO2 from ingested particulate 14C-protein, but had only small effects on the decarboxylation of free amino acid. Soluble, uniformly labeled 14C-protein was not significantly converted to 14CO2 even in the presence of phagocytizable polystyrene beads. These observation suggest that the amino acids taken up by phagocytosis (e.g., as denatured protein particles) are oxidatively decarboxylated and deaminated in […] Find the latest version: https://jci.me/110264/pdf Oxidative Decarboxylation of Free and Peptide-linked Amino Acids in Phagocytizing Guinea Pig Granulocytes S. K. ADENIYI-JONES and MANFRED L. KARNOVSKY, Departments of Biological Chemistry and Pediatrics, Harvard Medical School; and Children's Hospital Medical Center, Boston, Massachusetts 02115 A B S T R A C T The oxidative decarboxylation ofamino INTRODUCTION acids by a system consisting of myeloperoxidase-hy- drogen peroxide-chloride has been demonstrated It is now well accepted that the combination of myelo- previously by others and the process has been con- peroxidase (MPO),1 H2O2 and a halide constitutes a sidered to be part of the microbicidal armamentarium potent antimicrobial and antitumor system, but the of some phagocytic leukocytes. We were able to trans- precise mechanism of action remains obscure (1-4). late these earlier observations, made on model sys- Starting in 1968, interest in one possible mechanism tems, to intact guinea pig granulocytes. We could was aroused through the demonstration by Zgliczynski demonstrate differences in the cellular handling of pep- et al. (5) that MPO, prepared from leukocytes isolated tide-linked amino acids as particles, compared with from a patient with chronic granulocytic leukemia, free amino acids. Specific inhibitors were used to ex- could deaminate and decarboxylate amino acids in the plore two routes of oxidative decarboxylation: (a) the presence of H202 and Cl-. The products were alde- myeloperoxidase-catalyzed direct decarboxylation- hydes with one carbon less than the original amino deamination reaction, and (b) oxidation of a-keto acids acids. This was later confirmed by Strauss et al. (6) and after transamination of amino acids. These inhibitors Jacobs et al. (7) in a system in vitro that employed were cyanide, azide, and tapazole for the former path- neutrophil granules as the source of MPO. These work- way, and amino-oxyacetate for the latter. Amino-oxy- ers showed a relation between bactericidal activity and acetate profoundly inhibited the decarboxylation of decarboxylation of L-alanine, and suggested that the al- free "4C-amino acids (alanine and aspartate) in both rest- dehyde formed may itself be the bactericidal agent. ing and stimulated cells, but had only a minimal effect Aldehydes are known to have such activity (8). It was on "4CO2 production from ingested insoluble "4C- demonstrated in these studies that the reaction was protein. On the other hand, the peroxidase inhibitors rather specific for MPO, since horseradish peroxidase cyanide, azide, and tapazole dramatically inhibited the failed to manifest appreciable activity under similar production of "4CO2 from ingested particulate "4C- conditions. The same authors (9) demonstrated, with in- protein, but had only small effects on the decarboxyla- tact guinea pig leukocytes, a twofold increase in the de- tion of free amino acid. Soluble, uniformly labeled carboxylation of L-alanine during phagocytosis of latex 4C-protein was not significantly converted to beads. Subsequently, using leukocyte granules they 14CO2 even in the presence of phagocytizable poly- demonstrated decarboxylation of [1,714C]diamino- styrene beads. These observations suggest that the pimelic acid of bacteria that had incorporated this sub- amino acids taken up by phagocytosis (e.g., as dena- stance (10). Zgliczynski et al. (11) with a similar granule tured protein particles) are oxidatively decarboxylated system showed the formation of an intermediate and deaminated in the phagosomes by the myelo- product, a chloramine, from amino acids. These un- peroxidase-hydrogen peroxide-chloride system; stable products decomposed spontaneously to yield soluble free amino acids that enter the cytoplasm NH3, CO2, and the corresponding aldehydes. by diffusion or transport are oxidatively decarboxylated Although the data obtained are suggestive and make after transamination by the normal cellular amino acid this system an attractive one in the context of granulo- oxidative pathway. I Abbreviations used in this paper: AOA, amino-oxyacetate; BSA, bovine serum albumen; KRP, Krebs-Ringer phosphate; MPO, myeloperoxidase; PLP, pyridoxal phosphate; PMA, Receivedfor publication 12 September 1980 and in revised phorbol-12-myristate 13-acetate; PMN, polymorphonuclear form 2 April 1981. neutrophilic leukocytes. J. Clin. Invest. © The American Society for Clinical Investigation, Inc. - 0021-9738/81/0810365/09 $1.00 365 Volume 68 August 1981 365-373 cyte function, there are some uncertainties regarding dehydrogenase pathway and the MPO-H202-halide- its relevance to intact granulocytes. All the studies mediated pathway. cited above, with one exception, were done with (c) Lack of information as to whether the actual cleav- soluble enzyme systems or isolated leukocyte granules. age of ingested protein proceeds by the MPO-catalyzed MPO-catalyzed decarboxylation-deamination of regu- reaction, or how much is due to released granule-bound lar, protein (peptide-linked) amino acids has not been proteases after fusion of the lysosomelike granules with demonstrated with intact cells. The one study that did the phagosome (12). employ whole cells (9) did not take account of the possi- (d) Questions as to whether aldehydes derived from bility that the decarboxylation of the free alanine used the relevant amino acids are importantly antibacterial as substrate occurred via the normal amino acid oxida- (13) at the concentrations that might result from the tive pathway after transamination. The pathways rele- MPO-catalyzed reaction, and what their fate might be. vant to the observations above, and the findings of this In the present study we have explored the release paper are summarized in Fig. 1. of 14CO2 from protein-bound amino acids, compared The uncertainties in the chain of evidence regarding with that from free amino acids, using intact leukocytes. the proposed MPO-catalyzed protein cleavage and de- We have tried to determine the degree to which this oc- amination-decarboxylation reactions as potentially im- curs via transamination followed by decarboxylation, portant in phagocytizing granulocytes may be sum- or via attack by the MPO-H202-halide system in each marized as follows: case, i.e., to cover points a and b above. Points c and d (a) Lack of information as to whether soluble free are mentioned only to indicate the scope of the amino acids and protein-bound amino acids (particu- problem. Inhibitors of transaminase (amino-oxyacetate larly of insoluble proteins accumulated by phagocyto- [AOA]) or of peroxidase (cyanide, azide, methimazole sis) are subject to the same reactions. This question [tapazole]) were employed to dissect the problem in concerns differences that might exist between reactions whole cells. in the cytoplasm (free amino acids brought into the cell by diffusion or transport) or in the phagosomes (amino METHODS acids of insoluble protein accumulated by phagocyto- Materials. All reagents used were of analytical grade. Uni- sis). The conditions (e.g., pH) and available enzymes formly labeled 14C-denatured algal (Chlorella) protein (58 are quite different in these two locations; mCi/m atom C) was obtained from the Radiochemical (b) Paucity of evidence concerning the extent of the Centre Ltd., Amersham, England. 14C-protein from Esche- relative contributions of the transaminase-a-keto acid richia coli (0.053 mCilmg) was obtained from New England Nuclear, Boston, Mass. The former organism was grown in an atmosphere of 14CO2, the latter on a medium with [14C]acetate. Free Amino Acids Carboxylic Acid Both protein preparations were delipidated and freed of R A R R nucleic acids, carbohydrate,