The Lactococcal lmrP gene encodes a proton motive force-dependent drug transporter.

To genetically dissect the drug extrusion systems of Lactococcus lactis, a chromosomal DNA library was made in Escherichia coli and recombinant strains were selected for resistance to high concentrations of ethidium bromide. Recombinant strains were found to be resistant not only to ethidium bromide but also to daunomycin and tetraphenylphosphonium. The drug resistance is conferred by the lmrP gene, which encodes a hydrophobic polypeptide of 408 amino acid residues with 12 putative membrane-spanning segments. Some sequence elements in this novel membrane protein share similarity to regions in the transposon Tn10-encoded tetracycline resistance determinant TetA, the multidrug transporter Bmr from Bacillus subtilis, and the bicyclomycin resistance determinant Bcr from E. coli. Drug resistance associated with lmrP expression correlated with energy-dependent extrusion of the molecules. Drug extrusion was inhibited by ionophores that dissipate the proton motive force but not by the ATPase inhibitor ortho-vanadate. These observations are indicative for a drug-proton antiport system. A lmrP deletion mutant was constructed via homologous recombination using DNA fragments of the flanking region of the gene. The L. lactis (ΔlmrP) strain exhibited residual ethidium extrusion activity, which in contrast to the parent strain was inhibited by ortho-vanadate. The results indicate that in the absence of the functional drug-proton antiporter LmrP, L. lactis is able to overexpress another, ATP-dependent, drug extrusion system. These findings substantiate earlier studies on the isolation and characterization of drug-resistant mutants of L. lactis (Bolhuis, H., Molenaar, D., Poelarends, G., van Veen, H. W., Poolman, B., Driessen, A. J. M., and Konings, W. N.(1994) J. Bacteriol. 176, 6957-6964).

For many years antibiotics have been found effective in the treatment of several infectious diseases caused by various pathogens. The occurrence of antibiotic resistance, however, transformed many of the up to now readily treatable diseases to a new threat to public health (Cullinton, 1992;Nikaido, 1994). One of the mechanisms underlying antibiotic resistance involves the extrusion of the compounds by an efflux pump or carrier (Tennent et al., 1989;Levi, 1992;Bolhuis et al., 1994;Midgley, 1987Midgley, , 1989Miyauchi et al., 1992). For most (micro) organisms, it is not clear whether efflux is mediated by one multispecific system or by several, more or less specific systems (Hä chler et al., 1991). The most intriguing mechanisms of drug extrusion are those that can handle a wide variety of structurally unrelated compounds (antibiotics, drugs, etc.), and those are often referred to as multidrug resistance (MDR) 1 transporters. The bacterial MDR type transporters as well as several closely related specific drug extrusion systems (SDR) can be divided into four groups on the basis of their relatedness in the primary sequences, similarity in the global molecular structure, and/or mechanism of energy coupling (Nikaido, 1994). The first and largest group consists of secondary transporters that are characterized by the presence of either 12 or 14 putative transmembrane spanning segments (Paulsen and Skurray, 1993;Lewis, 1994;Marger and Saier, 1993;Lomovskaya and Lewis, 1992;Rouch et al., 1990). The second group, often referred to as the Staphylococcal multidrug resistance (Smr) family, comprises drug-proton antiporters that are about 100 amino acids long and are composed most likely of four transmembrane ␣-helices (Yerushalmi et al., 1995;Grinius and Goldberg, 1994). The third group of secondary drug extrusion transporters is formed by the resistance-nodulation division (RND) family, found in Gram-negative bacteria (Saier et al., 1994). The resistance-nodulation division systems require an accessory protein that spans the periplasm and most likely interacts with an outer membrane pore. The accessory proteins are required for the transport of the substrates to the external medium. The fourth group of efflux systems consists of ATP binding cassette transporters (Fath and Kolter, 1993;Higgins, 1993;Molenaar et al., 1992) and is best exemplified by the mammalian P-glycoprotein (MDR1). Recently, also a bacterial homolog of MDR1 was identified in Lactococcus lactis. 2 Although a number of MDR-and specific drug resistancetype of transporters have been identified in bacteria, their mechanism of action and energy coupling to transport have not been studied in great detail. The resistance to high concentrations of ethidium bromide (Eth R ), daunomycin (Dau R ), and rhodamine 6G (Rho R ), of three independently isolated mutants of L. lactis MG1363, suggested that at least two different transport mechanisms are involved in multidrug resistance in this organism (Bolhuis et al. 1994). One of the mechanisms is dependent on the proton motive force (⌬p), while the other is ATP-dependent. The ⌬p-dependent system, termed LmrP, is the first multidrug transporter for which both the membrane potential (⌬), and the proton gradient (⌬pH), has been shown * This work was supported by Grant BIO2CT-930145 from the Biotech program of the European Community. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EMBL Data Bank with accession number(s) X89779.
‡ To whom correspondence should be addressed. to function as driving force for drug extrusion. In this paper, we describe the gene cloning, characterization, mutant construction, and functional analysis of LmrP.

MATERIALS AND METHODS
Growth of the Organisms-Bacterial strains and plasmids used in this study are listed in Table I. L. lactis strains were grown at 30°C on M17 medium (Difco) supplemented with glucose (25 mM) and erythromycin (5 g/ml) when appropriate. Escherichia coli was grown aerobically at 37°C on Luria Broth (Sambrook et al., 1989) without further additions or with carbenicillin (50 g/ml) or erythromycin (100 g/ml). For the determination of growth rates in the presence of various toxic compounds, the E. coli strains were grown semianaerobically in microplates; growth was monitored at 30°C on Luria Broth containing 25 mM glucose. Growth rates were estimated as described previously (Bolhuis et al., 1994).
DNA Manipulation and Cloning of the lmrP Gene-General procedures for cloning and DNA manipulations were performed essentially as described by Sambrook et al. (1989). For the cloning of lmrP, chromosomal DNA from L. lactis was isolated as described previously (Leenhouts et al., 1990) and partially digested with HindIII. DNA fragments ranging from 2 to 5 kb were isolated from a 1% agarose gel using the Qiaex DNA extraction kit (Qiagen) and, subsequently, ligated into the HindIII site of the expression/cloning vector pKK223-3. The ligation mixture was used to transform electro-competent E. coli HB101 via electroporation. Transformants were plated out on Luria Broth plates containing 50 g/ml carbenicillin plus 100 g/ml ethidium bromide. Resistant colonies were used for further analysis.
Sequencing Analysis-Double-stranded DNA was sequenced in both directions using the dideoxy chain-termination procedure (Sanger et al., 1977) and the T7 DNA sequencing kit (Pharmacia Biotech Inc.). Standard oligonucleotides for pBluescript SK IIϪ and 17-19-bp-long synthetic oligonucleotides, complementary to already determined sequences, were used as primers. Computer-assisted sequence analysis was performed using PCGENE (release 6.8, Genofit). Protein secondary structure was predicted from hydropathy profiling according to Kyte and Doolittle (1992) and the positive-inside rule of von Heijne (1986).
Construction of the lmrP Deletion Mutant-The 771-bp NdeI-ClaI fragment (Fig. 1A), comprising an internal lmrP fragment, was deleted from pSKLMR3.2. The vector ends were made blunt using S1 exonuclease and religated. The resulting plasmid pSKLMRNC was digested with BamHI plus KpnI, yielding a 2.2-kb fragment containing the lmrP deletion and its flanking regions. This fragment was ligated into pORI280 and transformed to E. coli EC1000, which carries the repA gene of L. lactis on the chromosome, thereby allowing the pORI280 derivative to replicate in E. coli (Leenhouts and Venema, 1993;Hagting et al., 1994). The resulting construct pORILMRNC was isolated from E. coli EC1000 and transformed to L. lactis MG1363. As this strain does not contain the essential replication factor (RepA), selection for growth in the presence of erythromycin forces the plasmid to integrate into the chromosome by homologous recombination. Positive colonies were selected on the basis of erythromycin resistance and a ␤-galactosidase positive phenotype using M17 X-gal agar plates. A number of the blue colonies were subsequently grown for about 30 generations under nonselective conditions in M17 medium lacking erythromycin. Nonselective growth allows a second recombination event to occur, which results in the deletion of either the wild-type gene lmrP or pORILMRNC. In both cases, the strains are erythromycin-sensitive and ␤-galactosidase-negative (white colonies on M17 X-gal agar plates). A number of clones were selected, and the ⌬lmrP mutation was confirmed by the polymerase chain reaction (PCR) as well as Southern hybridization experiments.
Polymerase Chain Reaction-Chromosomal DNA and synthetic oligonucleotide primers were used at a concentration of 200 ng/100 l of total PCR reaction mixture. The reactions were performed with Vent DNA-polymerase (New England Biolabs) using denaturation, annealing, and proliferation temperatures of 94, 45, and 73°C, respectively. PCR products were analyzed by ethidium-stained agarose gel-electrophoresis.
Ethidium and Daunomycin Transport in Whole Cells-The ethidium and daunomycin transport assays are based on the fluorescence properties of the compounds upon interaction with DNA/RNA as described before (Bolhuis et al., 1994). A washed cell-suspension (various buffers) with an A 660 of 0.5 was incubated with 10 M of ethidium bromide or daunomycin, and the fluorescence was followed using excitation and emission wavelengths of 500 and 580 nm, respectively, for ethidium bromide, and 480 and 590 nm, respectively, for daunomycin. The fluorescence was measured with a Perkin Elmer LS 50B fluorometer with computer-controlled data acquisition and storage.

Cloning of the Lactococcal Drug Resistance
Determinant lmrP-It has been shown that L. lactis MG1363 contains at least two different transport systems that are involved in efflux-mediated multidrug resistance (Bolhuis et al., 1994). To genetically dissect the different efflux activities, strategies were developed to clone the transporter genes by complemen-   sequence with a calculated free energy ⌬G of Ϫ7.6 kcal was identified 28 bp downstream of the TAA stop codon. A second putative ORF (ORF2) was found downstream of lmrP. However, deletion of up to 200 bp 5Ј of the NruI site (Fig. 1A) did not affect the ethidium resistance in E. coli HB101, whereas removal of the 800-bp HindIII-EcoRI fragment (promoter region and 5Ј end of lmrP) totally abolished the ethidium resistance (data not shown). These experiments indicate that lmrP is essential and sufficient in conferring ethidium resistance to E. coli.
Properties of the lmrP Gene Product-The amino acid composition of the LmrP protein corresponds with that of a membrane protein being rich in hydrophobic residues (Val, Leu, Ile, Phe, Met, and Ala), constituting 53% of the total number of residues. The hydropathy profile of LmrP, according to the method of Kyte and Doolittle (1982), classifies the polypeptide as an integral membrane protein with 12 hydrophobic regions large enough to span the plasma membrane (Fig. 1B, underlined and boldface). A secondary structure model of LmrP based on the hydropathy profile and the positive-inside rule of von Heijne (1986) is shown in Fig. 2. Both the carboxyl-and the amino-terminal ends as well as a large central hydrophilic loop between helix 6 and 7 are predicted to be on the cytoplasmic side of the membrane.
Multidrug Resistance Linked to lmrP Expression in E. coli-The lmrP gene was isolated for its ability to confer ethidium bromide resistance to E. coli HB101. To check whether the lmrP gene product is involved in the efflux of multiple drugs, growth of E. coli was monitored in the presence of various concentrations of drugs. For these experiments pSKLMR3.2 was transferred to E. coli CS1562, containing a TolC integration mutation (tolC6::Tn10), which makes the strain hypersusceptible to various (hydrophobic) toxic compounds due to an impaired barrier function of the outer membrane. Overnight cultures of E. coli CS1562/pSKLMR3.2 and the control strain CS1562/pBluescript SKII Ϫ , grown on Luria Broth, were transferred to 96-well microplates containing 250 l of LB medium supplemented with glucose (25 mM), ampicillin (50 g/ml), tetracycline (20 g/ml; Tn10 selection marker), and various concentrations of ethidium bromide, daunomycin, or tetraphenylphosphonium (TPP ϩ ). The cells were grown semianaerobically for 15 h at 30°C, during which the maximal growth rate was determined. The relative growth rate of E. coli CS1562/pSKLMR3.2 in the presence of ethidium bromide, daunomycin, and TPP ϩ was increased relative to that of the control strain, indicating that the lmrP gene product affects resistance to these compounds (Fig. 3).
LmrP-mediated Ethidium and Daunomycin Efflux-To demonstrate that lmrP expression catalyzes energy-dependent drug extrusion, ethidium,and daunomycin transport was fol-lowed by monitoring the intracellular concentration of the compounds in L. lactis. The 3.2-kb HindIII fragment of pSKLMR3.2 was ligated into the HindIII site of the E. coli-L. lactis shuttle vector pGK13. This construct, termed pGKLMR3.2, was transformed to the L. lactis MG1363 wildtype strain. Assays of ethidium uptake in energized cells as well as efflux upon energization of cells preloaded with ethidium bromide demonstrated that lmrP expression resulted in a lower intracellular ethidium concentration. The ethidium efflux was inhibited by reserpine, a known inhibitor of the mammalian P-glycoprotein (data not shown). In a typical ethidium efflux experiment, a decrease of fluorescence in time was found upon energization with glucose from cells preincubated with ethidium (Fig. 4). The L. lactis strain containing pGKLMR3.2 showed a higher fluorescence decrease than the wild-type strain containing the control vector (Fig. 4). As shown previously (Bolhuis et al., 1994), ethidium efflux by the secondary drug transport activity was affected by ionophores that dissipate the components of the ⌬p. The addition of valinomycin, which selectively dissipates the ⌬, resulted in an increased ethidium efflux ( Fig. 4; Val). Dissipation of the ⌬ not only results in an increase in the ⌬pH with a concomitant increase in ethidium efflux but also inhibits the passive uptake of the cationic ethidium into the cell (data not shown). Dissipation of the ⌬pH upon addition of the ionophore nigericin, inhibited ethidium efflux in both the wild-type and the strain expressing LmrP (Fig. 4; Nig). Active ethidium extrusion from FIG. 2. Secondary structure model of LmrP. The model is based on the hydropathy-profile of the amino acid sequence and the distribution of the arginine and lysine residues (ϩ) according to the "positive inside rule" by Von Heijne (1986). The residues constituting the conserved sequence motif A in the first cytoplasmic loop (CL1) and motif B in transmembrane helix V are shaded.

FIG. 3.
Resistance of E. coli CS1562 to toxic compounds with and without expression of the lmrP gene. Cells carrying pSKLMR3.2 (E) or pBluescript SKII Ϫ (q), were grown semianaerobically at 30°C on Luria Broth, supplemented with glucose (25 mM), carbenicillin (50 g/ml), and ethidium (A), daunomycin (B), or TPP ϩ (C) at various concentrations. The relative growth rate is plotted as a function of the drug concentration. The growth rates in the absence of added drug corresponded to 0.54 and 0.63 h Ϫ1 for E. coli CS1562(pSKLMR3.2) and E. coli CS1562(pBluescript SKII Ϫ ), respectively.
both strains was not affected by the ATPase inhibitor orthovanadate (see below, Fig. 6). Comparable results were obtained with the fluorescent chemotherapeutic agent daunomycin as transport substrate (data not shown), confirming that lmrP encodes a transport protein catalyzing ⌬p-dependent extrusion of multiple drugs.
Construction and Analysis of the lmrP Deletion Mutant-To establish the in vivo role of LmrP in L. lactis, a deletion mutant was constructed via homologous recombination as described under "Materials and Methods." The integration event was confirmed via PCR analysis, using two different sets of primers to distinguish between the wild-type and deleted gene. Chromosomal DNA isolated from wild-type and putative deletion mutants was used as template DNA for the PCR reaction. One set of oligonucleotide primers (LMR28/LMR29) is complementary to sequences outside of the deleted fragment, and these yielded a 1340-bp PCR product with template DNA from the wild-type strain DNA and a 560-bp product with DNA from ⌬lmrP (Fig. 5, A and B, lanes 4 and 2). The second set of oligonucleotide primers (LMR28/LMR2), complementary to sequences outside (LMR28) and inside (LMR2) the deleted fragment, gave a 730-bp PCR product with wild-type DNA while, as expected, no product was obtained with DNA from the ⌬lmrP strain (Fig. 5, A and B, lanes 3 and 1).
To analyze the drug resistance phenotype of L. lactis MG1363(⌬lmrP), the mutant strain was studied further in growth and transport assays. Within the ethidium bromide concentration range tested (0 -25 M), the L. lactis deletion mutant MG1363(⌬lmrP) was only slightly more sensitive than L. lactis MG1363(wild-type), whereas L. lactis MG1363/ pGKLMR3.2 was more resistant to ethidium bromide than the wild-type (data not shown). Since L. lactis possesses an ATPdependent efflux activity in addition to the ⌬p-driven efflux activity, it is possible that alterations in the expression of the ATP-dependent system could (partially) have masked the drug resistance phenotype of L. lactis MG1363(⌬lmrP) and MG1363/ pGKLMR3.2. Indeed, in the presence of ortho-vanadate, the inhibitor of the ATP-dependent efflux activity, the ⌬lmrP strain accumulated more ethidium than the wild-type strain (Fig. 6, ϩ). Overexpression of LmrP from pGKLMR3.2 reduced the intracellular ethidium levels to the same extent irrespective of whether or not the cells were preincubated with ortho-vanadate. (Fig. 6; Ϯ).
Comparison of the lmrP Nucleotide Sequence from Wild-type, Eth R , Dau R , and Rho R Strains of L. lactis-In order to establish whether the MDR phenotypes of Eth R , Dau R , and Rho R were due to mutation(s) in the promoter region and/or the structural gene for lmrP, the corresponding DNA sequences were compared with that of the wild-type. The genes from L. lactis MG1363 (wild-type), Dau R , and Rho R were amplified by PCR using Vent DNA polymerase. The 1400-bp product was digested with ClaI, and the two products (850 and 550 bp) were ligated into pSKN, a derivative of pBluescript SKII Ϫ containing a unique NcoI site in the multiple cloning site. The nucleotide sequence of lmrP from L. lactis MG1363 (wild-type) and the MDR strains Eth R , Dau R and Rho R appeared to be identical. In addition, the amplified and sequenced 490-bp PCR product from the chromosome of these strains, containing the promoter region and ribosomal binding site, did not reveal any differences in the DNA region that could explain the differences in drug resistance of the various strains.

DISCUSSION
In this paper, we report the isolation of a chromosomal DNA fragment from L. lactis, which effects an increased resistance to ethidium bromide, daunomycin, and TPP ϩ when expressed in E. coli. This DNA fragment contains an ORF that specifies an integral membrane protein (LmrP) with 12 putative ␣-helical membrane spanning segments. When the amino acid se-  1 and 3) and LMR28/LMR29 (lanes 2 and 4) were used to distinguish between the wild-type and the deletion mutant of lmrP. The PCR products were separated by agarose (1%) gel-electrophoresis (B). quence of the predicted LmrP polypeptide was compared with proteins in various data bases, the search did not reveal significant similarity to any known protein. However, when, the primary sequence of LmrP was directly compared with known drug transporters and other transport proteins, using the COMPARE program of PCGENE, which is based on the Dayhoff MDM-78 comparison matrix (Schwartz and Dayhoff, 1987), some homologous proteins were identified. The sequence comparisons revealed similarity (19 -24% identical residues) between LmrP and the multidrug transporters Bmr of Bacillus subtilis (Neyfakh et al., 1991) and NorA of Staphylococcus aureus (Yoshida et al., 1990) as well as the transporters involved in tetracycline resistance and bicyclomycin resistance in E. coli (Bentley et al., 1993). Multiple alignment of these proteins revealed two conserved sequence motifs (Fig. 2, A and B) that are located at similar positions within the putative secondary structure of the proteins. Motif A is identical to the consensus sequence GXXXDRXGR(K/R) (Henderson, 1991), which is found in various (unrelated) secondary transport proteins and is present in the cytoplasmic loop of LmrP between transmembrane segments 2 and 3. It has been suggested that this loop is of structural importance to the polypeptide, but it might also be involved in the conformational change required for the opening and closing of the transport channel (Paulsen and Skurray, 1993). Functional studies of site-directed mutants of the transposon Tn10-encoded metal-tetracycline/proton antiporter (Tn10-Tet) showed that the negatively charged aspartate (Asp 66 ) and the positively charged arginine (Arg 70 ) are essential for transport (Yamaguchi et al., 1993). Motif B has residues in common with the conserved sequence GpilGPvlGG found at the end of transmembrane segment 5, which, so far, has only been observed in drug extrusion systems (Lewis, 1994;Paulsen and Skurray, 1993). Alignment of the carboxyl-terminal half of LmrP with its amino-terminal half displays about 18% identical residues. Among the identical residues are an aspartate (Asp 286 ) and a lysine (Lys 291 ) in the cytoplasmic loop between transmembrane segment 8 and 9, which align with Asp 68 and Lys 73 of the GXXXDRXGR(K/R) consensus sequence.
Although lmrP was originally cloned from an ethidium-resistant strain of L. lactis ML3, the nucleotide sequence was identical to that of the wild-type and the other multidrugresistant mutants of L. lactis MG1363. Also, the promoter regions upstream of the structural gene were identical, and DNA hybridization experiments indicated that the observed increase in drug resistance and drug efflux is not due to amplification of the lmrP gene on the chromosome. As shown previously (Bolhuis et al., 1994), the MDR phenotype of the drug-resistant mutants Eth R , Dau R , and Rho R resulted from increased drug extrusion from the cell. These mutant strains have a comparable drug resistance spectrum, but differ with respect to their mode of energy coupling to active drug efflux. Whereas ethidium extrusion from the Eth R strain is mainly driven by an ATP-dependent transport activity, a significant contribution of both the ATP-dependent and the ⌬p-driven transport systems was observed in the ethidium extrusion from Dau R and Rho R (Bolhuis et al., 1994). Since the nucleotide sequence of lmrP in the MDR strains is identical, the increased resistance in these strains must be due either to an increased expression of lmrP, possible affected by mutation of a transcription factor, or to an alternative drug extrusion activity. It has been shown that expression of the homologous tetracyclinemetal/drug antiporter TetA is repressed by the repressor TetR in the absence of tetracycline (Hillen and Berens, 1994). Regulation of transcription of the operon for the EmrAB multidrug resistance determinant from E. coli is mediated by the repressor EmrR (Lomovskaya et al., 1995). Regulation of gene expression by transcription activation is found for the B. subtilis MDR transporter Bmr (Ahmed et al. 1994). The possible involvement of similar regulatory proteins in the enhanced drug resistance in L. lactis will be the subject of future research.
The lmrP gene was cloned in E. coli HB101 by selection for resistance to high concentrations of ethidium bromide. In the hypersensitive E. coli CS1562, expression of lmrP not only increased resistance to ethidium but also to unrelated hydrophobic compounds like TPP ϩ and daunomycin. Increased ethidium resistance was also observed when lmrP was present on a plasmid in L. lactis MG1363, but the effect was less pronounced. The lower LmrP-dependent ethidium resistance in L. lactis is most likely the result of the relatively low copy number of the pGK13 vector in L. lactis as compared with the vector used in E. coli (Kok et al., 1984). Transport studies in L. lactis showed that LmrP catalyzes the energy-dependent efflux of ethidium and daunomycin. The energy-dependent efflux of ethidium from the wild-type strain and L. lactis MG1363(pGKLMR3.2) was completely inhibited upon dissipation of the ⌬p but unaffected by the ATPase inhibitor orthovanadate. These observations are consistent with a drug-proton antiport mechanism for LmrP. Deletion of part of the lmrP gene from the chromosome of L. lactis MG1363 (wild-type strain) affected drug extrusion when the cells were pretreated with ortho-vanadate. However, the ⌬lmrP mutation did not result in significant changes in the drug resistance phenotype. These results suggest an increased expression (or activity) of the ATP-dependent drug transporter (Bolhuis et al., 1994). It is possible that this increased expression is essential to survive the otherwise lethal effect of the deletion of lmrP. The occurrence of efflux-mediated resistance to various hydrophobic toxic compounds and antibiotics in both pathogens and nonpathogens implicates a general mechanism and an important physiological function. This function could involve a general detoxification mechanism against naturally occurring hydro- FIG. 6. Ethidium uptake in L. lactis MG1363 (wild-type), L. lactis MG1363/pGKLMR3.2, and L. lactis MG1363/⌬lmrP. Cells were preincubated for 10 min in a phosphate-free buffer (50 mM HEPES, 25 mM K 2 SO 4 , 5 mM MgSO 4 ; pH 7.4) in the presence (ϩ) or absence (Ϫ) of 0.5 mM ortho-vanadate. L-Arginine (10 mM) was added as source of metabolic energy to allow ortho-vanadate to be taken up. The assay was started upon addition of 10 M of ethidium bromide to the cell suspension.