Identification of a vanadate-sensitive, membrane-bound ATPase in the archaebacterium Methanococcus voltae.

Membrane-bound ATPase activity was detected in the methanogen Methanococcus voltae. The ATPase was inhibited by vanadate, a characteristic inhibitor of E1E2 ATPases. The enzyme activity was also inhibited by diethylstilbestrol. However, it was insensitive to N,N'-dicyclohexylcarbodiimide, ouabain, and oligomycin. The enzyme displayed a high preference for ATP as substrate, was dependent on Mg2+, and had a pH optimum of approximately 7.5. The enzyme was completely solubilized with 2% Triton X-100. The enzyme was insensitive to oxygen and was stabilized by ATP. There was no homology with the Escherichia coli F0F1 ATPase at the level of DNA and protein. The membrane-bound M. voltae ATPase showed properties similar to those of E1E2 ATPases.     

Hydrolysis and synthesis of ATP by membrane-bound ATPase from a motile Streptococcus

ATPase was detected in the membranes of a motile Streptococcus. Maximal enzymic activity was observed at pH 8 and ATP/Mg²⁺ ratio of 2. Mn²⁺ and Ca²⁺ could replace Mg²⁺ to some extent. Besides ATP, GTP and ITP were substrates. The enzyme was inhibited by N,N-dicyclohexylcarbodiimide but not by sodium azide, uncouplers or bathophenanthroline.An electrochemical gradient of protons, which was artificially imposed across the membranes of Streptococcus cells by manipulation of either the K⁺ diffusion potential or the transmembrane pH gradient, led to ATP synthesis. ATP synthesis was abolished by proton conductors, an inhibitor of the ATPase or an increase in the extracellular K⁺ concentration. A comparison between the phosphate potential and the electrochemical proton gradient showed that the data found are in agreement with a stoichiometry of 2 protons translocated per molecule ATP synthesized.Abbreviations electrochemical gradient of protons - DMO 5,5-dimethyl-2,4-oxazolidinedione - CCCP carbonylcyanide m-chlorophenylhydrazone - FCCP carbonylcyanide p-trifluoromethoxyphenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DNP 2,4-dimitrophenol     

Measurement of ATP generation and decay in Mycobacterium leprae in vitro

The intracellular ATP content of Mycobacterium leprae isolated from armadillo tissue was approximately 1.5 X 10(-16) g per bacillus. During in vitro incubation of bacilli at 4 degrees C, 33 degrees C or 37 degrees C there was an exponential decrease in ATP content, the rate depending on the medium and the temperature. M. leprae incorporated phosphate into ATP and into other nucleotide materials during in vitro incubation.     

Interaction of vanadate with membrane-bound ATPase from Mycobacterium phlei

Vanadate inhibited the formation of proton gradient and membrane potential as well as Ca2+ transport by everted membrane vesicles from Mycobacterium phlei, with half-maximal inhibition occurring at 5 to 14 microM. That this is due to the inhibition of the proton-translocating ATPase was suggested by the observation that the inhibition described above occurred only when the processes were driven by the hydrolysis of ATP but not when energized by the oxidation of succinate and NADH. Furthermore, vanadate did indeed inhibit ATP hydrolysis by these membrane vesicles. Although the inhibition of ATP hydrolysis could be demonstrated only in the presence of high concentrations (e.g. 11 mM) of Mg2+, this was presumably due to the fact that we were measuring the sum of ATP hydrolysis by both coupled and partially uncoupled enzymes. This is the first reported effect of vanadate on bacterial proton-translocating ATPase.     

Kinetic analysis of proton transport by the vanadate-sensitive ATPase from maize root microsomes

Proton transport by the nitrate-insensitive, vanadate-sensitive ATPase in Kl-washed microsomes and reconstituted vesicles from maize (Zea mays L.) roots was followed by changes in acridine orange absorbance in the presence of either KNO₃ or KCl. Data from such studies obeyed a kinetic model in which net proton transport by the pump is the difference between the rate of proton transport by the action of the ATPase and the leak of protons from the vesicles in the direction opposite from the pump. After establishing the steady state proton gradient, the rate of return of transported protons was found to obey first-order kinetics when the activity of the ATPase was completely and rapidly stopped. The rate of return of these protons varied with the quencher used. When the substrate Mg:ATP was depleted by the addition of either EDTA or hexokinase, the rate at which the proton gradient collapsed was faster than when vanadate was used as the quencher. These trends were independent of the anion accompanying the K and the transport assay used.     

Effect of trifluoperazine on in vitro ATP synthesis by Mycobacterium leprae

The effect of trifluoperazine (TFP), a calmodulin antagonist, was investigated on in vitro ATP levels of human derived Mycobacterium leprae . M. leprae were obtained from biopsies from multi-bacillary forms of leprosy and were incubated in a modified Dubos medium system which supports limited in vitro synthesis of M. leprae . This incubation was carried out in the absence and presence of different concentrations of trifluoperazine. Samples for estimation of bacillary ATP levels were taken at day 0 and at 14 days of incubation. TFP inhibited ATP levels in M. leprae and this inhibitory effect was marginal at 2.5 μg ml⁻¹ (35% inhibition), highly significant at 5 μg ml⁻¹ (87% inhibition) and almost total at 10 μg ml⁻¹ (98.5% inhibition). This compound appears to have potential as an anti-leprotic drug and also as a broad spectrum anti-mycobacterial agent in view of its anti-tubercular activity reported earlier.     

Mycobacterium leprae and leprosy: a compendium.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which was discovered by G.H.A. Hansen in 1873. M. leprae is an exceptional bacterium because of its long generation time and no growth in artificial media. Entire sequencing of the bacterial genome revealed numerous pseudogenes (inactive reading frames with functional counterparts in M. tuberculosis) which might be responsible for the very limited metabolic activity of M. leprae. The clinical demonstration of the disease is determined by the quality of host immune response. Th1-type immune response helps to kill the bacteria, but hosts are encroached upon when Th2-type response is predominant. The bacteria have affinity to the peripheral nerves and are likely to cause neuropathy. M. leprae/laminin-alpha2 complexes bind to alpha/beta dystroglycan complexes expressed on the Schwann cell surface. WHO recommends a chemotherapy protocol [multidrug therapy (MDT)] which effectively controls the disease and contributes to the global elimination program. Leprosy has been stigmatized throughout history, and recent topics regarding the disease in Japan are also discussed.     

Genomic arrangement of a putative operon involved in maltose transport in the Mycobacterium tuberculosis complex and Mycobacterium leprae

A Mycobacterium bovis gene coding for a putative MalE maltose binding protein was cloned and its full-length sequence determined. Database searches revealed 99.9% identity with IpqY, encoding a putative sugar uptake protein from Mycobacterium tuberculosis strain H37Rv. The deduced protein product showed high sequence similarity to MalE-like proteins from a variety of bacterial species, including Mycobacterium leprae. Analysis of flanking database sequences from M. tuberculosis and M. leprae revealed the presence of malF-, malG- and malK-like genes. Comparison of these mycobacterial sequences with other maltose operons has allowed us to deduce a unique genomic arrangement of the genes involved in the uptake of maltose in members of the Mycobacterium tuberculosis complex and M. leprae.     

Purification of a vanadate-sensitive ATPase from clathrin-coated vesicles of bovine brain

Clathrin-coated vesicle acidification is mediated by an N-ethylmaleimide-sensitive, vanadate-resistant proton-translocating ATPase. This enzyme is a 530-kDa hetero-oligomer which catalyzes ATP-dependent proton pumping when reconstituted (Xie, X. S., and Stone, D. K. (1986) J. Biol. Chem. 261, 2492-2495). We now report the purification of a second ATPase from bovine brain clathrin-coated vesicles which is inhibited by both N-ethylmaleimide (1 mM) and vanadate (10 microM). Localization of the ATPase to clathrin-coated vesicles was demonstrated by the precipitation of ouabain-resistant, vanadate-sensitive ATPase activity with anti-clathrin antibodies. The enzyme was solubilized with 0.1% polyoxyethylene 9-lauryl ether and has been purified 700-fold to a specific activity of 42 mumol of Pi.mg of protein-1.min-1. A molecular mass of 116 kDa was determined by centrifugation in sucrose gradients prepared in H2O and D2O, by high performance liquid chromatography using gel filtration, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under reducing conditions. The ATPase is unlike any known mammalian E1E2-type ATPase in that it is not inhibited by ouabain or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and it is not activated by Na+, K+, or Ca2+.     

Mycobacterium leprae and phenoloxidase activity.

Our earlier studies indicated that the enzyme o-diphenoloxidase was absent in Mycobacterium leprae separated from depromatous human tissues. At that time the bacilli were not available from any other source. The existence or absence of this enzyme in M. leprae recovered from infected armadillo tissues were reinvestigated. The intact cells which were metabolically active, failed to oxidize DOPA. Likewise, DOPA and its derivatives were not oxidized by the enzymatically active cell-free preparations from M. leprae. Upon incubation of DOPA for more than 2 h with whole cell suspensions or particulate fractions, there was no development of colour with an absorption maximum of 540 nm as has been reported for an intermediate of DOPA oxidation. However, DOPA and several phenolic compounds were very actively oxidized by mushroom tyrosinase. The results suggested that M. leprae is deficient in o-diphenoloxidase, and this enzyme is not an intrinsic characteristic of this mycobacterium.     

The involvement of a vanadate-sensitive ATPase in plasma membranes of a salt tolerant cyanobacterium

Plasma membranes of the marine cyanobacterium Spirulina subsalsa were tested for ATPase activity, and for involvement in salt stress. Transition of cells from saline to hypersaline medium enhances the respiratory activity associated with extrusion of Na⁺ and Cl⁻, and persisting salt stress induces synthesis of respiratory enzymes in the plasma membranes. The membranes possess an ATPase, specific for ATP and Mg²⁺ and sensitive to orthovanadate and dicyclohexylcarbodiimide. Immunoblot analysis of plasma membrane polypeptides from Spirulina subsalsa with anti- Arabidopsis H⁺-ATPase serum identified a single polypeptide of 100 kDa, which cross-reacted with the antibodies. An unusual feature of this ATPase is a specific stimulation by Na⁺ ions. Prolonged adaptation of S. subsals cells to hypersaline conditions induced an increase in ATPase activity in subsequent plasma membrane preparations, as well as a higher content of the 100 kDa polypeptide. It is suggested that the ATPase investigated is an H⁺-pump, which is involved in extrusion of Na⁺ and in conferring resistance to salt stress.     

The vanadate-sensitive ATPase of Streptococcus faecalis pumps potassium in a reconstituted system

The vanadate-sensitive ATPase of Streptococcus faecalis, purified to homogeneity, was reconstituted into soybean phospholipid vesicles in a functional state. Freeze-fracture electron micrographs revealed a relatively uniform population of unilamellar liposomes of 50-100 nm in diameter, with particles protruding from both fracture faces. Transport studies with 42K+ and with a K+-selective electrode showed that the ATP-ase catalyzes electrogenic potassium extrusion in proteoliposomes. The following parameters for potassium transport in the reconstituted system were determined: K+/ATP stoichiometry = 1, Km for potassium = 1.4 mM, Vmax = 0.1 mumol/min/mg. The ATPase could be activated by an electrical membrane potential, vesicle interior positive. This ATPase thus appears to function as a potential regulated, ATP-driven pump that serves in electrogenic potassium accumulation by the bacterial cell.     

Comparative analyses of transport proteins encoded within the genomes of Mycobacterium tuberculosis and Mycobacterium leprae

The co-emergence of multidrug resistant pathogenic bacterial strains and the Human Immunodeficiency Virus pandemic has made tuberculosis a leading public health threat. The causative agent is Mycobacterium tuberculosis (Mtu), a facultative intracellular parasite. Mycobacterium leprae (Mle), a related organism that causes leprosy, is an obligate intracellular parasite. Given that different transporters are required for bacterial growth and persistence under a variety of growth conditions, we conducted comparative analyses of transport proteins encoded within the genomes of these two organisms. A minimal set of genes required for intracellular and extracellular life was identified. Drug efflux systems utilizing primary active transport mechanisms have been preferentially retained in Mle and still others preferentially lost. Transporters associated with environmental adaptation found in Mtu were mostly lost in Mle. These findings provide starting points for experimental studies that may elucidate the dependencies of pathogenesis on transport for these two pathogenic mycobacteria. They also lead to suggestions regarding transporters that function in intra- versus extra-cellular growth.     

Active transport of calcium in membrane vesicles from Mycobacterium phlei.

Active transport of calcium ions has been demonstrated in inside-out membrane vesicles from Mycobacterium phlei mediated by respiratory linked substrates as well as by ATP hydrolysis. The uptake of calcium exhibited an apparent Km of 80 microM and V of 16.6 nmol calcium uptake x min-1 x mg protein-1. A fortyfold concentration gradient for calcium ions was calculated for both the ATP-induced and the respiration-induced transport of calcium. Removal of coupling-factor-latent ATPase resulted in the complete loss of ATP-driven Ca2+ transport whereas the respiration-driven uptake was reduced by 40-50%. The uptake of calcium was inhibited by the proton conducting ionophores carbonylcyanide m-chlorophenylhydrazone and Gramicidin-D. The accumulated calcium was freely exchangeable with external calcium and was rapidly released by the addition of inhibitors of energy transduction, proton-translocating uncouplers or the ionophore A23187. The uptake of the weak base, methylamine, upon the oxidation of respiratory-linked substrates or the hydrolysis of ATP showed the generation of a protein gradient (inside acidic) which was partially collapsed on the addition of calcium ions. These results suggest that a Ca2+/H+ antiport mechanism may be responsible for the transport of calcium.     

Comparative ultrastructure of Mycobacterium leprae and Mycobacterium lepraemurium cell envelopes.

The structural properties of the cell envelopes of Mycobacterium leprae and Mycobacterium lepraemurium were investigated by freeze-fracture, freeze-etching, and negative-staining techniques. Freeze-fracture split the cell wall and exposed the internal features of the peptidoglycolipid mycosidic filamentous network. The cell membrane was also split into two asymmetric faces. The external fracture face was characterized by linear arrays of intramembranous particles, whereas the protoplasmic fracture face showed randomly distributed clusters of particulate entities. Comparative analysis of the ultrastructural features observed in M. leprae and M. lepraemurium indicated that the organization of the cell envelope in these two species differed particularly with respect to the amount and complexity of the superficial peptidoglycolipid and mycosidic integument, which is poorly developed in the mycobacterium responsible for human disease.     

High resolution shadowing of Mycobacterium leprae.

Metal shadow casting techniques for transmission electron microscopic examination was used to determine the morphological characteristics of Mycobacterium leprae in untreated and treated patients. This technique is used to visualize bacterial surface structures by thermal evaporation of platinum alloys under moderate vacuum. This method gives a high contrast image at relatively low resolution and is useful for correlating micro-morphology quantitatively to early therapeutic effects of anti-leprosy drugs. Using these techniques in untreated cases, the surface structures of M. leprae were uniformly filled with relatively homogenous protoplasm surrounded by a cell wall. Most of the bacilli had thick cell walls with prominent banded and fibrous structures on the surface of the cell body. The cell wall was not detached in any of the solid bacilli in untreated cases. The bacilli varied in size and some of them were swollen in their mid-portion. Some bacilli were very short and completely filled with cytoplasm; therefore, these short bacilli were counted as solid bacilli in electron microscopic morphological index (EM-MI) determination. During treatment, mainly the cytoplasms of the bacilli were affected, and degeneration was observed. Ultrastructurally, the cytoplasm was shrunken and detached from the cell wall indicating mild degeneration. After moderate degeneration, the cytoplasm appeared fragmented. In advanced degeneration, all structures except the cell walls collapsed completely and no fibrous or band structures were visible on the surfaces of the cell walls. Therefore, these bacilli were counted as non-solid bacilli for EM-MI determination. This study shows that transmission electron shadowing gives more accurate counts than standard light microscopy of intact M. leprae bacilli in patient specimens.