Production of acetyl-L-leucyl-L-argininal, inhibitor of dipeptidyl aminopeptidase III by bacteria.

Sir: As part of our continuous effort toward the discovery of novel enzyme inhibitors from microbial cultures1~3), we have searched for inhibitors against dipeptidyl aminopeptidases. We wish to report the discovery of acetyl-L-leucyl-L-argininal as a specific inhibitor against dipeptidyl aminopeptidase III (DAP-III, EC 3.4.14.4) from the culture filtrate of a bacterium (BMG520-yF2). Although this inhibitor is identical to a known analogue of leupeptin', '), we also wish to report its isolation and characterization because of the important inhibitory actions. DAP-III was partially purified from rat pancreas by ammonium sulfate fractionation according to the method of ELLIS and NUENKE6) The method of testing the enzyme inhibitory activity was as follows: a mixture consisting of 0.1 ml of 2 MM L-arginyl-L-arginine naphthylamide (purchased from Bachem.), 0.5 ml of 110 mm TrisHCI buffer (pH 9.0 containing with 5.6 mm 2mercaptoethanol), 0.3 ml of a mixture of water and the test solution and 0.1 ml of DAP-III enzyme solution (34 fig protein in 10 mm phosphate buffer, pH 7.0) was incubated at 37°C for 30 minutes. Then 10,0 of 40 mm NaIO4, was added and incubated at 37-C for 5 minutes. Finally 1.0m] of 3 mm fast garnet solution was added and the absorbance at 525 nm was determined after 15 minutes at room temperature. The concentration of the inhibitor required for 50% inhibition (IC50) was determined. The inhibitor was produced by shaken culture of BMG520-yF2 in a medium containing glycerol 2 %, dextrin 2 %, soy peptone 1 %, yeast extract 0.3%, (NH,)2SO40.2%, and CaCO, 0.2%, pH 7.4 with 5 N NaOH before sterilization. The maximum production was attained after 14-j 20 hours of culture at 27~C. The inhibitor was adsorbed onto activated carbon and eluted with 80% acetone solution. The active eluate was evaporated under reduced pressure to a brown powder (IC,, 18 fig/nil; 61 % yield). The crude powder was dissolved in water and applied onto a column of Amberlite XAD-7. After washing the column with water and 80% methanol, the inhibitor was eluted with 0.05 N HCI in 80 methanol. The active eluate was adjusted to pH 4.0 with Dowex WGR and concentrated under reduced pressure. The concentrate was rechromatographed by the same column using a linear gradient of 80% o methanol to 0.05 N HCI in 80 methanol. The active fraction gave a pale brown powder (IC,, 1.2 jig/ml; 16% yield). Further purification was achieved by column chromatography over Sephadex LH-20 developed with 80 methanol, giving a white powder (IC50 0.04 ug/ml; 13 % yield, mp 129 132°C). The inhibitor is soluble in water, methanol, ethanol and butanol and is insoluble in acetone, ethyl acetate and chloroform. It gives positive color reactions with red tetrazolium, RYDONSMITH's and SAKAGUCHI's reagents, but negative with ninhydrin. A thin-layer chromatogram of the inhibitor and leupeptin is shown in Fig. 1. The pure inhibitor was separated into two spots on TLC as did leupeptin. In the case of leupeptin this observation was rationalized by assuming an equilibrium mixture of the aldehyde, hydrate, and cyclic alkanolamine forms in aqueous solution7) The present inhibitor showed no absorption maximum between 210400 nm and [a]n -45.2° (c 0.5, MO). Amino acid analysis of the acid hydrolysate of the oxidized compound prepared