Specificity Determinants for Lysine Incorporation in Staphylococcus aureus Peptidoglycan as Revealed by the Structure of a MurE Enzyme Ternary Complex

Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.     

d-Glucosone and l-Sorbosone, Putative Intermediates of l-Ascorbic Acid Biosynthesis in Detached Bean and Spinach Leaves

d-[6-¹⁴C]Glucosone that had been prepared enzymically from d-[6-¹⁴C]glucose was used to compare relative efficiencies of these two sugars for l-ascorbic acid (AA) biosynthesis in detached bean (Phaseolus vulgaris L., cv California small white) apices and 4-week-old spinach (Spinacia oleracea L., cv Giant Noble) leaves. At tracer concentration, ¹⁴C from glucosone was utilized by spinach leaves for AA biosynthesis much more effectively than glucose. Carbon-14 from [6-¹⁴C]glucose underwent considerable redistribution during AA formation, whereas ¹⁴C from [6-¹⁴C]glucosone remained almost totally in carbon 6 of AA. In other experiments with spinach leaves, l-[U-¹⁴C]sorbosone was found to be equivalent to [6-¹⁴C]glucose as a source of ¹⁴C for AA. In the presence of 0.1% d-glucosone, conversion of [6-¹⁴C] glucose into labeled AA was greatly repressed. In a comparable experiment with l-sorbosone replacing d-glucosone, the effect was much less. The experiments described here give substance to the proposal that d-glucosone and l-sorbosone are putative intermediates in the conversion of d-glucose to AA in higher plants.     

Changes in Membrane Potential as a Demonstration of Selective Pore Formation in the Plasmalemma by Poly-l-Lysine Treatment

A technique which allows determination of solute pool concentrations in the cytosol was developed exploiting the interaction between a polycation and the anionic sites of the plasmalemma. It was shown that treatment of Nicotiana tabacum, cv Xanthi, cells in suspension culture with an appropriate concentration of poly-l-lysine induced pore formation selectively in the plasmalemma. The data presented in this paper shows that the plasmalemma of all the cells was affected while the tonoplast remained undamaged. This conclusion is based on the facts that treatment of the cells with the minimum amount of poly-l-lysine which just abolishes the electrogenic potential (similarly to carbonyl cyanide-p-trifluormethoxyphenylhydrazone and NaN₃) induces the leakage of only a small fraction of the K⁺ present in the cells. These effects of poly-l-lysine differ from the effects of polymyxin B which induces total leakage of low molecular weight solutes (R. Weimberg, H. R. Lerner, A. Poljakoff-Mayber 1983 J Exp Bot 34: 1333-1346) and therefore affects also the tonoplast.     

Interaction of wheat germ ca-dependent protein kinases with calmodulin antagonists and polyamines

The two soluble Ca²⁺-dependent protein kinases resolved from wheat (Triticum aestivum) embryo (protein kinases I and II) are inhibited by the phenothiazine-derived calmodulin antagonists trifluoperazine fluphenazine, and chlorpromazine. Protein kinases I and II are also inhibited by a variety of other calmodulin antagonists (including calmidazolium, amitriptyline, and iprindole), phosphodiesterase inhibitors (including flufenamic acid and papavarine) and by lanthanides. A number of compounds that inhibit mammalian Ca²⁺ - and phospholipid-activated protein kinase (protein kinase C) including quercetin, polymixin B sulfate, and polyamines (as well as phenothiazine derivatives) also inhibit protein kinases I and II. Poly-l-lysine and poly-l-ornithine activate both plant Ca²⁺-dependent protein kinases.     

Regulation of sulfate uptake by amino acids in cultured tobacco cells

The sulfur requirements of tobacco (Nicotiana tabacum L. var. Xanthi) XD cells grown in chemically defined liquid media can be satisfied by sulfate, thiosulfate, l-cyst(e)ine, l-methionine or glutathione, and somewhat less effectively by d-cyst (e) ine, d-methionine or dl-homocyst (e)ine. Sulfate uptake is inhibited after a 2 hr lag by l-cyst (e)ine, l-methionine, l-homocyst(e)ine or l-isoleucine, but not by any of the other protein amino acids, nor by d-cyst(e)ine. l-cyst(e)ine is neither a competitive nor a non-competitive inhibitor of sulfate uptake. Its action most closely resembles apparent uncompetitive inhibition. Inhibition of sulfate uptake by l-cyst(e)ine can be partially prevented by equimolar l-arginine, l-lysine, l-leucine, l-phenylalanine, l-tyrosine or l-tryptophan, but is little affected by any of the other protein amino acids. The effective amino acids are apparent competitive inhibitors of l-cyst(e)ine uptake after a 2 hr lag. Inhibition of sulfate uptake by l-methionine cannot be prevented, nor can uptake of l-methionine be inhibited by any single protein amino acid. The results suggest the occurrence of negative feedback control of sulfate assimilation by the end products, the sulfur amino acids, in cultured tobacco cells.     

Amino Acid and vitamin requirements of several bacteroides strains

Nutritional studies were performed on nine Bacteroides strains, by use of the methodology and media of anaerobic rumen microbiology. Ristella perfoetens CCI required l-arginine hydrochloride, l-tryptophan, l-leucine, l-histidine hydrochloride, l-cysteine hydrochloride, dl-valine, dl-tyrosine, and the vitamin calcium-d-pantothenate, since scant turbidity developed in media without these nutrients. R. perfoetens was stimulated by glycine, dl-lysine hydrochloride, dl-isoleucine, l-proline, l-glutamic acid, dl-alanine, dl-phenylalanine, dl-methionine, and the vitamins nicotinamide and p-aminobenzoic acid, since maximal turbidity developed more slowly in media without these nutrients than in complete medium. Medium A-23, which was devised for R. perfoetens, contained salts, 0.0002% nicotinamide and calcium d-pantothenate, 0.00001% p-aminobenzoic acid, 0.044% l-tryptophan, 0.09% l-glutamic acid, and 0.1% of the other 13 amino acids listed above. Zuberella clostridiformis and seven strains of R. pseudoinsolita did not require vitamins, and showed no absolute requirement for any one amino acid. Various strains produced maximal turbidity more slowly in media deficient in l-proline, glycine, l-glutamic acid, dl-serine, l-histidine hydrochloride, dl-alanine, or l-cysteine hydrochloride, than in complete medium. These eight strains grew optimally in medium A-23 plus 0.1% dl-serine but without vitamins.     

Effect of Chitosan on Membrane Permeability of Suspension-Cultured Glycine max and Phaseolus vulgaris Cells

Treatment of suspension-cultured Glycine max cv Harosoy 63 cells with soluble chitosan (20-500 micrograms per milliliter) increased membrane permeability as shown by leakage of electrolytes, protein, and UV absorbing material. Severe damage to the cell membrane by chitosan (100 and 500 μg/ml) was also indicated by reduced staining with fluorescein diacetate and the leakage of fluorescein from preloaded cells. Other basic polymers (poly-l-lysine, histone, DEAE-dextran, protamine sulfate, and glycol chitosan) also increased permeability, whereas the basic monomers l-lysine and d-glucosamine, and acidic or neutral polymers were not active. Chitosan-induced leakage was inhibited by divalent cations, the order of effectiveness being Ba²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺. Na polygalacturonate and Na poly-l-aspartate also reduced polycation-induced leakage, probably by formation of polycation-polyanion complexes. A chitosan-polygalacturonate complex precipitated on mixing solutions of the two polymers containing approximately equal numbers of galacturonate and glucosamine residues, but not with either polymer in excess. A similar concentration-dependent precipitation of chitosan by Na poly-l-aspartate was found. Leakage from Phaseolus vulgaris cv Grandessa cells was also induced by chitosan, and was inhibited by Ca²⁺ and Na polygalacturonate.     

Biosynthesis of (+)-Tartaric Acid from l-[4-C]Ascorbic Acid in Grape and Geranium

The metabolic fate of l-[4-¹⁴C]ascorbic acid has been examined in the grape (Vitis labrusca L.) and lemon geranium (Pelargonium crispum L. L'Hér. cv. Prince Rupert) under conditions comparable to data from l-[1-¹⁴C]ascorbic acid and l-[6-¹⁴C]ascorbic acid experiments. In detached grape leaves and immature berries, l-[4-¹⁴C]ascorbic acid and l-[1-¹⁴C]ascorbic acid were equivalent precursors to carboxyl labeled (+)-tartaric acid. In geranium apices, l-[4-¹⁴C]ascorbic acid yielded internal labeled (+)-tartaric acid while l-[6-¹⁴C]ascorbic acid gave an equivalent conversion to carboxyl labeled (+)-tartaric acid. These findings clearly show that two distinct processes for the synthesis of (+)-tartaric acid from l-ascorbic acid exist in plants identified as (+)-tartaric acid accumulators. In grape leaves and immature berries, (+)-tartaric acid synthesis proceeds via preservation of a four-carbon fragment derived from carbons 1 through 4 of l-ascorbic acid while carbons 3 through 6 yield (+)-tartaric acid in geranium apices.     

Purification and Properties of Alkaline Proteinase from Aspergillus oryzae

Alkaline proteinase was purified from culture extract of a strain of Aspergillus oryzae. The process consists of the Amberlite IRC-50 adsorption, column chromatography on DEAE-cellulose and CM-cellulose and Sephadex G-100 gel filtration. The molecular weight of the enzyme was estimated to be about 23,000 by a gel filtration method. Alkaline proteinase showed neither carboxypeptidase activity nor aminopeptidase activity, but degraded 10101010 poly-l,α-glutamic acid, poly-l-lysine, 10101010 and 10101010. The enzyme was completely inhibited by diisopropylphos-phorofluoridate (10^?2 m) or potato inhibitor (250 μg/ml).     

Synthesis of l-(+)-Tartaric Acid from l-Ascorbic Acid via 5-Keto-d-Gluconic Acid in Grapes

5-Keto-l-idionic acid (5-keto-d-gluconic acid, d-xylo-5-hexulosonic acid) was found as a metabolic product of l-ascorbic acid in slices of immature grapes, Vitis labrusca L. cv `Delaware'. Specifically labeled compounds, recognized as metabolic products of l-ascorbic acid in grapes, were fed to young grape tissues to investigate the metabolic pathway from l-ascorbic acid to l-(+)-tartaric acid.     

A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily: PRODUCTION,FUNCTIONAL CHARACTERIZATION,AND STRUCTURE MODELING*

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.     

Purification and Properties of Neutral Proteinase I from Aspergillus oryzaePurification and Properties of Neutral Proteinase II from Aspergillus oryzae

Neutral proteinase I (the first peak in DEAE-cellulose chromatogrraphy) was purified from the Amberlite IRC-50 adsorbed fraction by chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. It shows an optimum pH of 7.0 for milk casein. The enzyme was found to be stable in the pH range of 5.5 to 12.0. The molecular weight of the enzyme was estimated to be about 41,000 by gel filtration. The enzyme had neither aminopeptidase nor carboxypeptidase activity, but degraded carbobenzoxy-glycyl-phenyl-alanine amide, poly-l-lysine and poly-l,α-glutamic acid. The enzyme was inhibited by ethylenediaminetetraacetate, but not inhibited by diisopropylphosphorofluoridate and potato inhibitor.     

l-Lysine Catabolism Is Controlled by l-Arginine and ArgR in Pseudomonas aeruginosa PAO1

In comparison to other pseudomonads, Pseudomonas aeruginosa grows poorly in l-lysine as a sole source of nutrient. In this study, the ldcA gene (lysine decarboxylase A; PA1818), previously identified as a member of the ArgR regulon of l-arginine metabolism, was found essential for l-lysine catabolism in this organism. LdcA was purified to homogeneity from a recombinant strain of Escherichia coli, and the results of enzyme characterization revealed that this pyridoxal-5-phosphate-dependent decarboxylase takes l-lysine, but not l-arginine, as a substrate. At an optimal pH of 8.5, cooperative substrate activation by l-lysine was depicted from kinetics studies, with calculated K_m and V_max values of 0.73 mM and 2.2 μmole/mg/min, respectively. Contrarily, the ldcA promoter was induced by exogenous l-arginine but not by l-lysine in the wild-type strain PAO1, and the binding of ArgR to this promoter region was demonstrated by electromobility shift assays. This peculiar arginine control on lysine utilization was also noted from uptake experiments in which incorporation of radioactively labeled l-lysine was enhanced in cells grown in the presence of l-arginine but not l-lysine. Rapid growth on l-lysine was detected in a mutant devoid of the main arginine catabolic pathway and with a higher basal level of the intracellular l-arginine pool and hence elevated ArgR-responsive regulons, including ldcA. Growth on l-lysine as a nitrogen source can also be enhanced when the aruH gene encoding an arginine/lysine:pyruvate transaminase was expressed constitutively from plasmids; however, no growth of the ldcA mutant on l-lysine suggests a minor role of this transaminase in l-lysine catabolism. In summary, this study reveals a tight connection of lysine catabolism to the arginine regulatory network, and the lack of lysine-responsive control on lysine uptake and decarboxylation provides an explanation of l-lysine as a poor nutrient for P. aeruginosa.Decarboxylation of amino acids, including lysine, arginine, and glutamate, is important for bacterial survival under low pH (2, 7, 19). Lysine is abundant in the rhizosphere where fluorescent Pseudomonas preferentially resides, and serves as a nitrogen and carbon source to these organisms (28). In microbes, lysine catabolism can be initiated either through monooxygenase, decarboxylase, or transaminase activities. The monooxygenase pathway has been considered the major route for l-lysine utilization in Pseudomonas putida, and davBATD encoding enzymes for the first four steps of the pathway have been characterized (25, 26). In contrast, Pseudomonas aeruginosa cannot use exogenous l-lysine efficiently for growth (5, 24). It has been reported that enzymatic activities for the first two steps of the monooxygenase pathway are not detectable in P. aeruginosa, and no davBA orthologs can be identified from this organism (24, 25).Mutants of P. aeruginosa with improved growth on l-lysine and a high level of lysine decarboxylase activity can be isolated by repeated subcultures in l-lysine (5). This suggests that in P. aeruginosa, l-lysine utilization might be mediated by the lysine decarboxylase pathway with cadaverine and 5-aminovalerate as intermediates (Fig. ​(Fig.1).1). Alternatively, conversion of l-lysine into 5-aminovalerate may also be accomplished by a coupled reaction catalyzed by AruH and AruI. The AruH and AruI enzymes were reported as arginine:pyruvate transaminase and 2-ketoarginine decarboxylase, respectively (36). Interestingly, transamination by AruH using l-lysine as an amino group donor can also be detected in vitro (35). The reaction product α-keto-ɛ-aminohexanonate can potentially be decarboxylated into 5-aminovalerate by AruI, providing an alternative route for lysine degradation.Open in a separate windowFIG. 1.Lysine catabolic pathways. l-lysine decarboxylase pathway is shown at center. Broken arrows represent lysine monooxygenase pathway from P. putida which is not present in P. aeruginosa.In this study, we showed that the lysine decarboxylase pathway is the main route for lysine utilization under arginine control. Expression of the ldcAB operon encoding l-lysine decarboxylase and a putative lysine/cadaverine antiporter was analyzed regarding its response to l-lysine, l-arginine, and the arginine-responsive regulator ArgR. Enzyme characterization was performed to verify the function of LdcA as l-lysine decarboxylase. Arginine control on lysine incorporation was also investigated by genetic studies and uptake experiments. The peculiar role of ArgR controlling arginine and lysine uptake and catabolism provides the explanation for poor growth in lysine, and it implies a higher level of complexity in metabolic networks of pseudomonads.     

Circadian Rhythm in Amino Acid Uptake by Synechococcus RF-1

In the prokaryote Synechococcus RF-1, circadian changes in the uptake of l-leucine and 2-amino isobutyric acid were observed. Uptake rates in the light period were higher than in the dark period for cultures entrained by 12/12 hour light/dark cycles. The periodic changes in l-leucine uptake persisted for at least 72 hours into continuous light (L/L). The rhythm had a free-running period of about 24 hours in L/L at 29°C. A single dark treatment of 12 hours could initiate rhythmic leucine uptake in an L/L culture. The phase of rhythm could be shifted by a pulse of low temperature (0°C). The free-running periodicity was “temperature-compensated” from 21 to 37°C. A 24 hour depletion of extracellular Ca²⁺ before the free-running L/L condition reduced the variation in uptake rate but had little effect on the periodicity of the rhythm. The periodicity was also not affected by the introduction of 25 mm NaNO₃. The uptake rates for 20 natural amino acids were studied at 12 hour intervals in cultures exposed to 12/12 hour light/dark cycles. For eight of these amino acids (l-Val, l-Leu, l-Ile, l-Pro, l-Phe, l-Trp, l-Met, and l-Tyr), the light/dark uptake rate ratios had values greater than 3 and the rhythm persisted in L/L.     

Amino Acid Isomerization in the Production of l-Phenylalanine from d-Phenylalanine by Bacteria

To establish an advantageous method for the production of l-amino acids, microbial isomerization of d- and dl-amino acids to l-amino acids was studied. Screening experiments on a number of microorganisms showed that cell suspensions of Pseudomonas fluorescens and P. miyamizu were capable of isomerizing d- and dl-phenylalanines to l-phenylalanine. Various conditions suitable for isomerization by these organisms were investigated. Cells grown in a medium containing d-phenylalanine showed highest isomerization activity, and almost completely converted d- or dl-phenylalanine into l-phenylalanine within 24 to 48 hr of incubation. Enzymatic studies on this isomerizing system suggested that the isomerization of d- or dl-phenylalanine is not catalyzed by a single enzyme, “amino acid isomerase,” but the conversion proceeds by a two step system as follows: d-pheylalanine is oxidized to phenylpyruvic acid by d-amino acid oxidase, and the acid is converted to l-phenylalanine by transamination or reductive amination.     

The deposition and stability of pectin/protein and pectin/poly-l-lysine/protein multilayers

The sequential deposition of pectin and protein – bovine serum albumin (BSA), β-lactoglobulin (BLG) and gelatin – to form multilayer structures was examined by Fourier transform infrared-attenuated total reflection spectroscopy (FTIR-ATR) and a quartz crystal microbalance with dissipation monitoring (QCMD). With each layer deposited there was a progressive increase in mass deposited, with a more substantial deposition of protein. Pectin deposition led to a relatively hydrated, open structure which permitted binding of protein within the layer when the biopolymers carried an opposite net charge. On increasing the pH, disassembly of the structures occurred within the vicinity of the isoelectric point of the globular proteins. No disassembly was observed for the pectin/gelatin multilayer. When a globular protein was substituted for a poly-l-lysine layer in a pectin/poly-l-lysine multilayer it was displaced by the subsequent deposition of a poly-l-lysine layer, the more highly charged polycation displacing the relatively low charged polyampholyte. The pectin/poly-l-lysine/protein multilayers remained intact upon titration to pH 8.0.     

Engineering Filamentous Fungi for Conversion of d-Galacturonic Acid to l-Galactonic Acid

d-Galacturonic acid, the main monomer of pectin, is an attractive substrate for bioconversions, since pectin-rich biomass is abundantly available and pectin is easily hydrolyzed. l-Galactonic acid is an intermediate in the eukaryotic pathway for d-galacturonic acid catabolism, but extracellular accumulation of l-galactonic acid has not been reported. By deleting the gene encoding l-galactonic acid dehydratase (lgd1 or gaaB) in two filamentous fungi, strains were obtained that converted d-galacturonic acid to l-galactonic acid. Both Trichoderma reesei Δlgd1 and Aspergillus niger ΔgaaB strains produced l-galactonate at yields of 0.6 to 0.9 g per g of substrate consumed. Although T. reesei Δlgd1 could produce l-galactonate at pH 5.5, a lower pH was necessary for A. niger ΔgaaB. Provision of a cosubstrate improved the production rate and titer in both strains. Intracellular accumulation of l-galactonate (40 to 70 mg g biomass⁻¹) suggested that export may be limiting. Deletion of the l-galactonate dehydratase from A. niger was found to delay induction of d-galacturonate reductase and overexpression of the reductase improved initial production rates. Deletion of the l-galactonate dehydratase from A. niger also delayed or prevented induction of the putative d-galacturonate transporter An14g04280. In addition, A. niger ΔgaaB produced l-galactonate from polygalacturonate as efficiently as from the monomer.     

Reticulocyte lipoxygenase, ingensin, and ATP-dependent proteolysis

Lipoxygenase purified from rabbit reticulocyte lysate has a molecular mass of 68 kDa on SDS gel and a pI of 5.97. Lipoxygenase is inhibited by nordihydroguaiaretic acid (NDGA), 3-amino-1-(m-(trifluoromethyl)phenyl)-2-pyrazoline (BW755C), 5,8,11,14-eicosatetraynoic acid (ETYA), salicylhydroxamate (SHAM) or hemin. Metal ions or nucleotides do not affect its activity. The addition of certain of these inhibitors to the reticulocyte extract also inhibited the ATP-dependent proteolysis of casein, one of the distinct characteristics of reticulocytes. No clear correlation between lipoxygenase activity and ATP-dependent proteolysis could be detected. Hemin and NDGA inhibited both processes, but the concentrations necessary for inhibition were quite different. SHAM completely inhibited lipoxygenase, but not proteolysis. o-Phenanthroline inhibited ATP-dependent proteolysis, but had no effect on lipoxygenase activity. We have also purified a high-molecular-mass protease, ingensin, from reticulocyte extract. This protease accounted for more than 90% of the casein-degrading activity in reticulocyte extract. NDGA inhibited ingensin at the same concentrations required for inhibition of ATP-dependent proteolysis. These results suggest that lipoxygenase is not indispensable for the ATP-dependent proteolysis and the novel high-molecular-mass protease, ingensin, may be involved in the process.