Peptidoglycan synthesis in L-phase variants of Bacillus licheniformis and Bacillus subtilis.

Stable L-phase variants isolated from Bacillus licheniformis and Bacillus subtilis, when grown in osmotically stabilized media, do not synthesize peptidoglycan but have been found to accumulate the nucleotide precursors of this polymer. The enzymes involved in the synthesis of these precursors and the later membrane-bound stages of peptidoglycan synthesis have been investigated, and the L-phase variants have been shown to contain lesions, which provide a rational explanation for the absence of peptidoglycan and for the nature of the precursor accumulated. The majority of the L-phase variants contained a single enzymic defect, but two strains were isolated with double lesions. Five out of seven strains examined accumulated uridine 5'-diphosphate (UDP)-MurAc-L-ala-D-glu and were unable to synthesize diaminopimelic acid as a consequence of a defect in aspartate-beta-semialdehyde dehydrogenase activity. Two strains were deficient in UDP-MurAc: L-alanine ligase and accumulated UDP-MurAc. One strain accumulated the complete nucleotide precursor UDP-MurAc-L-ala-D-glu-mA2pm-D-ala-D-ala and was deficient in phospho-N-acetylmuramyl pentapeptide translocase. A second strain also had this lesion, together with defective aspartate-beta-semialdehyde dehydrogenase activity. The other enzymes of peptidoglycan synthesis were present in the L-phase variants, with activities similar to those found in the parent bacilli grown under identical conditions. Membrane preparations from certain of the L-phase variants were also capable of synthesizing the secondary polymers poly(glycerol phosphate) teichoic acid and teichuronic acid and also a polymer of N-acetylglucosamine.     

The Lantibiotic NAI-107 Binds to Bactoprenol-bound Cell Wall Precursors and Impairs Membrane Functions

The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell.     

Growth of Streptococcal Protoplasts and L-Colonies on Membrane Filters

Membrane filters (Millipore Corp.; pore sizes 1.2 to 0.22 mum) were placed on the surface of L-phase growth medium solidified with agar. The filter and the surrounding medium were inoculated with either protoplasts or stable broth-grown L-phase variants obtained from Streptococcus faecium strain F24. The L-phase inoculum gave rise to viable L-colonies on the filters and on the medium, whereas protoplasts gave colony formation only on the medium. However, when the Millipore filters were covered by a layer of solid L-phase medium, 75 mum or greater in depth, before inoculation with protoplasts, colony formation resulted but with atypical morphology. In contrast, inoculation of protoplasts on Nuclepore and Sartorius membrane filters did give rise to L-colonies on the surface and underneath the filters after 2 days of incubation at 37 C. Submicroscopic, viable L-phase elements produced during colony formation were capable of passing through membrane filters with pore channels as small as 0.22 mum; these elements required transfer from underneath the filters to fresh agar medium in order to develop into L-phase colonies. Membrane filters were also placed on the surface of L-phase growth medium solidified with gelatin. Inoculation of the filters and surrounding medium with a lysozyme-prepared protoplast suspension gave rise to streptococci on the surface of the filters and on the medium. However, inoculation with the stable broth-grown L-phase variants gave rise to atypical colonies on the medium and only small patches of abortive growth on the filters.     

Evidence for an S-layer protein pool in the peptidoglycan of Bacillus stearothermophilus.

Intact cells of Bacillus stearothermophilus PV72 revealed, after conventional thin-sectioning procedures, the typical cell wall profile of S-layer-carrying gram-positive eubacteria consisting of a ca. 10-nm-thick peptidoglycan-containing layer and a ca. 10-nm-thick S layer. Cell wall preparations obtained by breaking the cells and removing the cytoplasmic membrane by treatment with Triton X-100 revealed a triple-layer structure, with an additional S layer on the inner surface of the peptidoglycan. This profile is characteristic for cell wall preparations of many S-layer-carrying gram-positive eubacteria. Among several variants of strain PV72 obtained upon single colony isolation, we investigated the variant PV72 86-I, which does not exhibit an inner S layer on isolated cell walls but instead possesses a profile identical to that observed for intact cells. In the course of a controlled mild autolysis of isolated cell walls, S-layer subunits were released from the peptidoglycan of the variant and assembled into an additional S layer on the inner surface of the walls, leading to a three-layer cell wall profile as observed for cell wall preparations of the parent strain. In comparison to conventionally processed bacteria, freeze-substituted cells of strain PV72 and the variant strain revealed in thin sections a ca. 18-nm-wide electron-dense peptidoglycan-containing layer closely associated with the S layer. The demonstration of a pool of S-layer subunits in such a thin peptidoglycan layer in an amount at least sufficient for generating one coherent lattice on the cell surface indicated that the subunits must have occupied much of the free space in the wall fabric of both the parent strain and the variant. It can even be speculated that the rate of synthesis and translation of the S-layer protein is influenced by the packing density of the S-layer subunits in the periplasm of the cell wall delineated by the outer S layer and the cytoplasmic membrane. Our data indicate that the matrix of the rigid wall layer inhibits the assembly of the S-layer subunits which are in transit to the outside.     

In vitro synthesis of peptidoglycan precursors modified with N-acetylputrescine by Cyanophora paradoxa cyanelle envelope membranes

The photosynthetic organelles (cyanelles) of the protist Cyanophora paradoxa are surrounded by a peptidoglycan wall, modified through amidation with N-acetylputrescine. Cyanelle envelope membrane preparations were shown to catalyze the lipid-linked steps of peptidoglycan biosynthesis as well as the putrescinylation and subsequent acetylation, occurring at the stage of lipid I and/or lipid II.     

Peptidoglycan synthesis by partly autolyzed cells of Bacillus subtilis W23.

Partly autolyzed, osmotically stabilized cells of Bacillus subtilis W23 synthesized peptidoglycan from the exogenously supplied nucleotide precursors UDP-N-acetylglucosamine and UDP-N-acetylmuramyl pentapeptide. Freshly harvested cells did not synthesize peptidoglycan. The peptidoglycan formed was entirely hydrolyzed by N-acetylmuramoylhydrolase, and its synthesis was inhibited by the antibiotics bacitracin, vancomycin, and tunicamycin. Peptidoglycan formation was optimal at 37 degrees C and pH 8.5, and the specific activity of 7.0 nmol of N-acetylglucosamine incorporated per mg of membrane protein per h at pH 7.5 was probably decreased by the action of endogenous wall autolysins. No cross-linked peptidoglycan was formed. In addition, a lysozyme-resistant polymer was also formed from UDP-N-acetylglucosamine alone. Peptidoglycan synthesis was inhibited by trypsin and p-chloromercuribenzenesulfonic acid, and we conclude that it occurred at the outer surface of the membrane. Although phospho-N-acetylmuramyl pentapeptide translocase activity was detected on the outside surface of the membrane, no transphosphorylation mechanism was observed for the translocation of UDP-N-acetylglucosamine. Peptidoglycan was similarly formed with partly autolyzed preparations of B. subtilis NCIB 3610, B. subtilis 168, B. megaterium KM, and B. licheniformis ATCC 9945. Intact protoplasts of B. subtilis W23 did not synthesize peptidoglycan from externally supplied nucleotides although the lipid intermediate was formed which was inhibited by tunicamycin and bacitracin. It was therefore considered that the lipid cycle had been completed, and the absence of peptidoglycan synthesis was believed to be due to the presence of lysozyme adhering to the protoplast membrane. The significance of these results and similar observations for teichoic acid synthesis (Bertram et al., J. Bacteriol. 148:406-412, 1981) is discussed in relation to the translocation of bacterial cell wall polymers.     

Role of the Eikenella corrodens pilA locus in pilus function and phase variation

The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolate E. corrodens VA1. Earlier work defined the pilA locus which includes pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin, whereas pilB predicts a putative pilus assembly protein. The role of hagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilA mutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated for pilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation of hagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function.     

Lipodepsipeptide empedopeptin inhibits cell wall biosynthesis through Ca2+-dependent complex formation with peptidoglycan precursors

Empedopeptin is a natural lipodepsipeptide antibiotic with potent antibacterial activity against multiresistant Gram-positive bacteria including methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae in vitro and in animal models of bacterial infection. Here, we describe its so far elusive mechanism of antibacterial action. Empedopeptin selectively interferes with late stages of cell wall biosynthesis in intact bacterial cells as demonstrated by inhibition of N-acetylglucosamine incorporation into polymeric cell wall and the accumulation of the ultimate soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide in the cytoplasm. Using membrane preparations and the complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes and their respective purified substrates, we show that empedopeptin forms complexes with undecaprenyl pyrophosphate containing peptidoglycan precursors. The primary physiological target of empedopeptin is undecaprenyl pyrophosphate-N-acetylmuramic acid(pentapeptide)-N-acetylglucosamine (lipid II), which is readily accessible at the outside of the cell and which forms a complex with the antibiotic in a 1:2 molar stoichiometry. Lipid II is bound in a region that involves at least the pyrophosphate group, the first sugar, and the proximal parts of stem peptide and undecaprenyl chain. Undecaprenyl pyrophosphate and also teichoic acid precursors are bound with lower affinity and constitute additional targets. Calcium ions are crucial for the antibacterial activity of empedopeptin as they promote stronger interaction with its targets and with negatively charged phospholipids in the membrane. Based on the high structural similarity of empedopeptin to the tripropeptins and plusbacins, we propose this mechanism of action for the whole compound class.     

Identical Nutritional Requirements of Streptococcus faecium F24 and a Derived Stable L-Phase Variant

The nutritional requirements of Streptococcus faecium F24 and a lysozyme-produced stable L-phase variant of this strain appear to be identical. Thus, conversion of bacteria to the stable L-phase can occur without changes in nutritional requirements.     

Biosynthesis of cell wall peptidoglycan and polysaccharide antigens by protoplasts of type III group B Streptococcus.

The formation of a nascent peptidoglycan-group-specific antigen of type III group B Streptococcus at the cell membrane level was demonstrated with an M-1 mutanolysin-prepared protoplast system. Protoplasts of group B streptococci in suitably stabilized medium (20% sucrose) readily incorporated [3H]acetate into cell surface macromolecules. Four major polysaccharides were isolated from the protoplast cultural supernatant fluid: the peptidoglycan group-specific antigen polymer, the group B-specific antigen, and the low-molecular-weight and high-molecular-weight forms of the type III polysaccharide antigen. Biosynthesis of all four polymers was not affected by the action of chloramphenicol, indicating protein synthesis was not required for the production of polysaccharide in this system. However, all but the low-molecular-weight type III antigen were inhibited by the action of bacitracin, suggesting that three of the polymers share a common synthesis-assembly site in the membrane. Attachment of the high-molecular-weight antigen to the nascent peptidoglycan-group B antigen complex did not occur in the protoplast system, suggesting that a more complex cell wall matrix may be necessary before linkage of the high-molecular-weight antigen takes place.     

The synthesis of peptidoglycan in an autolysin-deficient mutant of Bacillus licheniformis N.C.T.C. 6346 and the effect of β-lactam antibiotics, bacitracin and vancomycin

The synthesis of peptidoglycan by cell-free membrane and membrane+wall preparations from an autolysin-deficient, beta-lactamase-negative mutant of Bacillus licheniformis N.C.T.C. 6346 was studied. The membrane preparation synthesized un-cross-linked polymer, the formation of which was not inhibited by beta-lactam antibiotics. Release of d-alanine by the action of d-alanine carboxypeptidase was inhibited variably according to the antibiotic. This inhibition was reversed by neutral hydroxylamine but not by the action of beta-lactamases or by washing. Bacitracin inhibited peptidoglycan synthesis, but not the d-alanine carboxypeptidase. Examination of peptidoglycan synthesized in the presence of excess of bacitracin showed that synthesis was not restricted to the addition of one disaccharide-pentapeptide unit at each synthetic site, an average of 2-3 disaccharide-pentapeptide units being added. Peptidoglycan synthesis was three- to four-fold more sensitive to vancomycin than was the release of d-alanine by the action of the carboxypeptidase. Incorporation of newly synthesized peptidoglycan into pre-existing cell wall was studied in membrane+wall preparations. This incorporation was catalysed by a benzylpenicillin- and cephaloridine-sensitive transpeptidase. The concentrations of these antibiotics giving 50% inhibition of incorporation were almost identical with those required to inhibit growth of the bacillus. Inhibition of the transpeptidase was reversed by treatment with beta-lactamase or by washing.     

Peptidoglycan biosynthesis in Micrococcus luteus (sodonensis): transglycosidase and phosphodiesterase activities in membrane preparations.

Two enzyme activities involved in the biosynthesis of peptidoglycan in Micrococcus luteus (sodonensis), a transglycosidase and a phosphodiesterase, have been demonstrated in isolated membrane preparations. The transglycosidase activity promotes the in vitro synthesis of an uncross-bridged peptidoglycan that is completely susceptible to lysozyme. This in vitro-synthesized peptidoglycan consists of 76% "soluble" and 24% "insoluble" material. The soluble peptidoglycan is primarily a single low-molecular-weight species of approximately 20 disaccharide peptide units. "Insoluble" peptidoglycan, which likely represents newly synthesized material incorporated into an existing cell wall, was solubilized by butanol extraction, and the two were compared. The phosphodiesterase activity demonstrated in this system cleaves uridine diphosphate-N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-lysyl-D-alanyl-D-alanine to yield N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-lysyl-D-alanyl-D-alanine plus uridine 5'-monophosphate plus inorganic phosphate. This phosphodiesterase activity, not detected under normal transglycosidase assay conditions, is a recycling mechanism and acts indirectly through formation and subsequent cleavage of a lipid-linked intermediate.     

Repression of nitrogen fixation in Klebsiella pneumoniae at high temperature

Clostridium perfringens 11268 CDR (Rifr Tcs), the strain transformed in our experiments, was generated by curing a spontaneous, rifampicin-resistant mutant of C. perfringens 11268 (Rifr Tcr). High-temperature growth yielded tetracycline-sensitive, rifampicin-resistant cells which no longer contained pCW3, a 42.8-kilobase plasmid. The tetracycline-sensitive, rod-shaped cell was then converted to an L-phase variant by growth in the presence of penicillin G (10 micrograms/ml) and 0.4 M sucrose. After several passages, the antibiotic was removed from the medium, and cells continued to grow as L-phase variants. Another large plasmid, pJU124 (38.8 kilobases), which confers tetracycline resistance, was used for transformation. Transformation of L-phase variants of C. perfringens 11268 CDR (Rifr Tcs) was mediated by polyethylene glycol. Transformation frequency is a nonlinear function of DNA concentration. Restriction analysis showed that the plasmid isolated from the transformants was identical to that supplied. Stable L-phase variants do not revert to rod-shaped cells, but autoplasts can be both transformed and reverted.     

Biosynthesis of spin-labeled peptidoglycan: spin-spin interactions.

Membrane preparations from Gaffkya homari catalyzed the in vitro biosynthesis of soluble uncross-linked spin-labeled peptidoglycan, a uniformly labeled polynitroxide, from the spin-labeled nucleotide UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-2,2,5,5-tetramethyl-1-pyrrolin-1-oxyl-3-carbonyl)-DAla-DAla (I) and UDP-GlcNAc. Soluble spin-labeled peptidoglycan was separated from membrane fragments and its spin-labeled precursor by centrifugation and gel filtration. The molecular weight distribution of the polymer was examined by agarose gel filtration. Spin-labeled [14C]peptidoglycan was polydisperse with a peak of radioactivity corresponding to a molecular weight of 5.0 X 10(5). The electron spin resonance spectrum of spin-labeled peptidoglycan was extensively broadened by spin-spin exchange interactions. These interactions were modified by changes in temperature, reduction by ascorbate, hydrolysis by lysozyme, and complexation with the antibiotic, vancomycin. Spin-spin exchange was reduced or eliminated in spin-labeled peptidoglycan by the random reduction of free radicals by ascorbate. A rotational correlation time of 0.37 ns was calculated for the probe in partially reduced spin-labeled peptidoglycan. This compares to a correlation time of 0.13 ns for the substrate (I). Raising the temperature increases spin-spin exchange line broadening. No transition points were observed for spin-labeled peptidoglycan as measured by this method. Degradati on of spin-labeled peptidoglycan by lysozyme eliminated the observed spin-spin exchange and yielded products with a mobility similar to I. Complexation of spin-labeled peptidoglycan with vancomycin resulted in both pronounced free-radical immobilization and a decrease in spin-spin exchange. The exchange effects are consistent with distance measurements in molecular models for peptidoglycan.     

Cytoplasmic steps of peptidoglycan biosynthesis

The biosynthesis of bacterial cell wall peptidoglycan is a complex process that involves enzyme reactions that take place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner side (synthesis of lipid-linked intermediates) and outer side (polymerization reactions) of the cytoplasmic membrane. This review deals with the cytoplasmic steps of peptidoglycan biosynthesis, which can be divided into four sets of reactions that lead to the syntheses of (1) UDP-N-acetylglucosamine from fructose 6-phosphate, (2) UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine, (3) UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid and (4) D-glutamic acid and dipeptide D-alanyl-D-alanine. Recent data concerning the different enzymes involved are presented. Moreover, special attention is given to (1) the chemical and enzymatic synthesis of the nucleotide precursor substrates that are not commercially available and (2) the search for specific inhibitors that could act as antibacterial compounds.     

Fatty acid remodeling: a novel reaction sequence in the biosynthesis of trypanosome glycosyl phosphatidylinositol membrane anchors.

The trypanosome variant surface glycoprotein (VSG) is anchored to the plasma membrane via a glycosyl phosphatidylinositol (GPI). The GPI is synthesized as a precursor, glycolipid A, that is subsequently linked to the VSG polypeptide. The VSG anchor is unusual, compared with anchors in other cell types, in that its fatty acid moieties are exclusively myristic acid. To investigate the mechanism for myristate specificity we used a cell-free system for GPI biosynthesis. One product of this system, glycolipid A', is indistinguishable from glycolipid A except that its fatty acids are more hydrophobic than myristate. Glycolipid A' is converted to glycolipid A through highly specific fatty acid remodeling reactions involving deacylation and subsequent reacylation with myristate. Therefore, myristoylation occurs in the final phase of trypanosome GPI biosynthesis.     

Characteristics of the active and inactive variants of Actinomyces sp. 26-115, a producer of actinomycin C

Two natural variants, i.e. No. 1 and No. 2, not producing actinomycin were isolated from cultures of the actinomycin C-producing organism Actinomyces sp. 26-115. Variant No. 1 differed from the active variant by the growth dynamics and colony morphology. Variant No. 2 was close to the active variant by the growth dynamics. It was shown with electron microscopy that the cells of variant No. 1 differed from those of the active variant in the number and form of the mycelial septa, more even and compact structure of the cell walls and higher sensitivity to actinomycin. Still, they were more stable to the effect of lysozyme and ultrasound. The cell walls of the inactive variant No. 1 gradually lost teichoic acid during development, while the loss of peptidoglycan was observed only on transfer to the stationary phase. The cell walls of the active variant lost teichoic acid and peptidoglycan at the same time on transfer to the stationary phase. Peptidoglycans of both variants contained diaminopimelic acid (the configuration of which was not determined) and glycine (1:1) as differentiating amino acids. The two adjacent tetrapeptides were joined with one glycine radical. The peptidoglycan peptide chains of both variants contained muramic, glutamic and diaminopimelic acids and alanine (1:1:1:2). The peptidoglycans of the inactive variant No. 1 contained in addition valine and isoleucine. However, it is hardly probable that they are contained by the peptidoglycan peptide chains.     

An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis

MreB proteins play a major role during morphogenesis of rod‐shaped bacteria by organizing biosynthesis of the peptidoglycan cell wall. However, the mechanisms underlying this process are not well understood. In Bacillus subtilis, membrane‐associated MreB polymers have been shown to be associated to elongation‐specific complexes containing transmembrane morphogenetic factors and extracellular cell wall assembly proteins. We have now found that an early intracellular step of cell wall synthesis is also associated to MreB. We show that the previously uncharacterized protein YkuR (renamed DapI) is required for synthesis of meso‐diaminopimelate (m‐DAP), an essential constituent of the peptidoglycan precursor, and that it physically interacts with MreB. Highly inclined laminated optical sheet microscopy revealed that YkuR forms uniformly distributed foci that exhibit fast motion in the cytoplasm, and are not detected in cells lacking MreB. We propose a model in which soluble MreB organizes intracellular steps of peptidoglycan synthesis in the cytoplasm to feed the membrane‐associated cell wall synthesizing machineries.     

Characteristics of ATPase from sugarcane protoplast and vacuole membranes

Characteristics of membrane-associated ATPase from commercial Hawaiian varieties of sugarcane ( Saccharum spp. hybrids) were investigated in preparations from sugarcane cell suspension culture and from stalk tissues of the intact plant. In order to examine comparable preparations, protoplasts and vacuoles, in turn, were obtained from both sources. ATPase from preparations of crude protoplast membranes and tonoplast had a pH optimum of 6 to 6.5. The relative effectiveness of divalent cations in stimulating ATPase was Mg²⁺ > Mn²⁺≥ Co²⁺ > Ca²⁺≥ Zn²⁺. Enzyme activity was not stimulated by K⁺, nor by other monovalent cations. Protoplasts and vacuoles from both sources showed significant acid phosphatase activity. Acid phosphatase activity was inhibited by molybdate, but ATPase activity was unaffected. Membrane preparations from protoplasts contained inorganic pyrophosphatase, but enzyme activity was low or not present in tonoplast preparations. Cell suspension and stalk tissue preparations hydrolyzed a large number of nucleoside di- and triphosphates. The hydrolysis is most likely due to a series of enzymes rather than a single enzyme. ATPase from protoplast and tonoplast preparations was inhibited 30–50% by diethylstilbestrol and sodium ortho-vanadate and was unaffected by ionophores. This study illustrates the complexity of phosphohydrolase activities in membrane preparations from sugarcane. The study, however, also illustrates substantial similarity in the behavior of these enzymes, whether they are derived from the plant itself or from cell cultures originating from comparable tissues of the plant.