After extracting converting enzyme from a membrane fraction of homogenized human kidney, "enkephalinase" activity was solubilized with Triton X-100. Ion-exchange chromatography resolved two peaks of the "enkephalinase" activity, both of which cleaved Leu5-enkephalin at the Gly3-Phe4 bond. The major "enkephalinase" form was purified 1140-fold to homogeneity with a 14% yield. This homogeneous "enkephalinase" had a specific activity of 46 mumol min-1 mg-1 with Leu5-enkephalin as substrate. The purified enzyme, in addition to hydrolyzing Leu5-enkephalin, cleaved synthetic substrates with protected N- and C-terminal ends. On the basis of the specificity of the enzyme and its inhibition by chelating agents, human "enkephalinase" can be classified as a neutral metalloendopeptidase with a broad substrate specificity. The activity of this neutral endopeptidase with several biologically active peptides was compared to that of homogeneous human kidney converting enzyme. Both enzymes inactivated bradykinin by release of the C-terminal dipeptide but were inhibited differentially by specific inhibitors. Comparison of hydrolysis of bradykinin with that of its protected C-terminal peptide indicated that the neutral endopeptidase is more active toward the larger substrate than is converting enzyme. Although the neutral endopeptidase did not convert angiotensin I to II, it did hydrolyze angiotensin I at Pro7-Phe8 and inactivate angiotensin II by cleavage at the Tyr4-Ile5 bond.

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The particulate enzyme fraction from mung bean (Phaseolus aureus) seedlings catalyzes the incorporation of mannose from GDP-[14Clmannose into mannosyl-phosphoryl-dolichol and of N-acetylglucosamine from UDP-13HIN-acetylglucosamine into N-acetylglucosanine-pyrophosphoryl-polyisoprenol. Bacitracin inhibits the transfer of both of these sugars into the lipid-linked saccharides with 50% inhibition being observed at 5 mM bacitracin. This antibiotic did not inhibit the transfer of glucose from UDP-1'4Clglucose into steryl glucosides or the incorporation of glucose into a cell wall glucan. Bacitracin also inhibited the in vivo incorporation of I14Clmannose into mannosyl-phosphoryl-dolichol and into glycoprotein by carrot (Daucus carota) slices. While bacitracin also inhibited the incorporation of lysine into proteins by these slices, protein synthesis was less sensitive than glycosylation. Thus at 2 mM bacitracin glycosylation was inhibited 92%, while protein synthesis was inhibited only 50%.Bacitracin is a polypeptide antibiotic produced by Bacillus licheniformis, which has been shown to act on bacteria by interfering with the formation of lipid-linked sugars which are involved in peptidoglycan synthesis (10, 12). There is evidence that this interference is the result of the formation of a complex between bacitracin, the undecaprenyl-pyrophosphate, and a divalent cation (11). The undecaprenyl-pyrophosphate, thus bound, cannot be dephosphorylated to regenerate the undecaprenyl-monophosphate which is necessary to form lipid-sugar intermediates. This binding of phospholipids may also explain the observation that bacitracin disrupts the cell membrane (13).That lipid-linked sugars act as intermediates in the biosynthesis of polysaccharides and glycoproteins has been well established in plants and animals as well as bacteria (4,6,9,14). Figure I liquid scintillation spectrometer using scintillation fluid containing 500 ml of Triton X-100, 500 ml of toluene, 5 g of PPO and 0.2 g of POPOP.Plant Material. Mung bean seeds and carrot roots were purchased in local supermarkets and stored at 4 C. Seeds of Phaseolus aureus (mung beans) were sterilized for 5 min in commercial bleach diluted 1:10 (about 0.5% hypochlorite) and rinsed with distilled H20. They were then planted in moist Vermiculite and allowed to grow in the dark for 40 hr. The in vivo experiments were performed using thin discs (I mm in thickness and 9 mm in diameter) of carrot phloem parenchyma preincubated for 16 hr in water at 27 C.Preparation of Particulate Enzyme. Hypocotyls from mung bean sprouts were ground in cold buffer (5 ml/g containing 50 mM Tris [pH 7.5], 2 mm ,8-mercaptoethanol, and PVP [5 g/l]). The homogenate was filtered through cheesecloth and the particulate material was collected by centrifugation at 30,000g for 15 min. The pellet was resuspended in 50 mm Tris (I ml/g) (pH 7.5), containing 2 mmi f-mercaptoethanol.Formation and Analysis of Products. Assays for the incorporation of GlcNAc, mannose, or glucose into lipid in vitro, at differ...