Post-operative paraplegia following spinal cord infarction.

Thoracic epidural analgesia is a frequently utilised technique. Neurological complications are uncommon, but of grave consequence with significant morbidity. Spinal cord infarction following epidural anaesthesia is rare. We present a case where a hypertensive patient underwent an elective sigmoid colectomy under combined general/epidural anaesthesia for a suspected malignant abdominal mass. An epidural infusion was used for intra-operative and post-operative analgesia. During surgery, the blood pressure was labile and she was hypotensive. Postoperatively, the patient became confused, pyrexial and tachycardic and developed systemic inflammatory response syndrome requiring intensive care management. She developed a flaccid paralysis at L3 level with areflexia, analgesia and impaired sensation. A spinal cord infarct in the region of the conus extending into the thoracic cord was diagnosed. Complications of epidural anaesthesia are easily recognised when they develop immediately; their relationship to the anaesthesia and the post-operative period may be misjudged or underestimated when they appear after a delay, if neurological signs are masked by lack of patient cooperation and drowsiness or if the epidural anaesthesia is prolonged by long-acting drugs. New neurological deficits should be evaluated promptly to document the evolving neurological status and further testing or intervention should be arranged if appropriate. The association with epidural anaesthesia as a cause of paraplegia is reviewed. The aetiological factors that may have contributed to this tragic neurological complication are discussed.

Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut.

The midgut of mosquito larvae maintains a specific lumen alkalinization profile with large longitudinal gradients (pH approximately 3 units*mm(-1)) in which an extremely alkaline (pH approximately 11) anterior midgut lies between near-neutral posterior midgut and gastric cecum (pH 7-8). A plasma membrane H(+) V-ATPase energizes this alkalinization but the ion carriers involved are unknown. Capillary zone electrophoresis of body samples with outlet conductivity detection showed a specific transepithelial distribution of chloride and bicarbonate/carbonate ions, with high concentrations of both anions in the midgut tissue: 68.3 +/- 5.64 and 50.8 +/- 4.21 mM, respectively. Chloride was higher in the hemolymph, 57.6 +/- 7.84, than in the lumen, 3.51 +/- 2.58, whereas bicarbonate was higher in the lumen, 58.1 +/- 7.34, than the hemolymph, 3.96 +/- 2.89. Time-lapse video assays of pH profiles in vivo revealed that ingestion of the carbonic anhydrase inhibitor acetazolamide and the ion exchange inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), at 10(-4) M eliminates lumen alkalinization. Basal application of these inhibitors in situ also reduced gradients recorded with self-referencing pH-sensitive microelectrodes near the basal membrane by approximately 65% and 85% respectively. Self-referencing chloride-selective microelectrodes revealed a specific spatial profile of transepithelial chloride transport with an efflux maximum in anterior midgut. Both acetazolamide and DIDS reduced chloride effluxes. These data suggest that an H(+) V-ATPase-energized anion exchange occurs across the apical membrane of the epithelial cells and implicate an electrophoretic Cl(-)/HCO(3)(-) exchanger and carbonic anhydrase as crucial components of the steady-state alkalinization in anterior midgut of mosquito larvae.

Structure-function relationships of A-, F- and V-ATPases.

Ion-translocating ATPases, such as the F(1)F(o)-, V(1)V(o)- and archaeal A(1)A(o) enzymes, are essential cellular energy converters which transduce the chemical energy of ATP hydrolysis into transmembrane ionic electrochemical potential differences. Based on subunit composition and primary structures of the subunits, these types of ATPases are related through evolution; however, they differ with respect to function. Recent work has focused on the three-dimensional structural relationships of the major, nucleotide-binding subunits A and B of the A(1)/V(1)-ATPases and the corresponding beta and alpha subunits of the F(1)-ATPase, and the location of the coupling subunits within the stalk that provide the physical linkage between the regions of ATP hydrolysis and ion transduction. This review focuses on the structural homologies and diversities of A(1)-, F(1)- and V(1)-ATPases, in particular on significant differences between the stalk regions of these families of enzymes.

In situ analysis of pH gradients in mosquito larvae using non-invasive, self-referencing, pH-sensitive microelectrodes.

The alkaline environment, pH approximately 11, in the anterior midgut lumen of mosquito larvae is essential for normal nutrition and development. The mechanism of alkalization is, however, unknown. Although evidence from immunohistochemistry, electron microscopy and electrophysiology suggests that a V-ATPase is present in the basal membranes of the epithelial cells, its physiological role in the alkalization process has not been demonstrated. To investigate a possible role of the V-ATPase in lumen alkalization, pH gradients emanating from the hemolymph side of the midgut in semi-intact mosquito larvae were measured using non-invasive, self-referencing, ion-selective microelectrodes (SERIS). Large H+ concentration gradients, with highest concentrations close to the basal membrane (outward [H+] gradients), were found in the anterior midgut, whereas much smaller gradients, with concentrations lowest close to this membrane (inward [H+] gradients), were found in the gastric caeca and posterior midgut. Similar region-specific pH gradients, with consistent anterior-to-posterior profiles, were observed in individuals of two Aedes species, Aedes aegypti from semi-tropical Florida and Aedes canadensis from north-temperate Massachusetts. The gradients remained in a steady state for up to 6 h, the maximum duration of the recordings. Bafilomycin A1 (10(-5), 10(-7 )mol x l(-1)) on the hemolymph side greatly diminished the [H+] gradients in the anterior midgut but had no effect on the gradients in the gastric caecum and posterior midgut. These physiological data are consistent with the previous findings noted above. Together, they support the hypothesis that a basal, electrogenic H+ V-ATPase energizes luminal alkalization in the anterior midgut of larval mosquitoes.

Analgesia, virtue, and the principle of double effect.

The principle of double effect is widely used to permit the administration of narcotics and sedatives with the intent to palliate dying patients, even though the administration of these drugs may cause hastening of death. In recent medical literature, this principle's validity has been severely criticized, causing health care providers to fear providing good palliative care. Most of the criticisms levelled at the principle of double effect arise from misconceptions about its purpose and origins. This discussion will explore how virtue-based ethics can overcome the most important challenge to the principle of double effect's validity, that of its reliance on intention to determine whether the administration of analgesia is ethically acceptable.

The multigene family of the tobacco hornworm V-ATPase: novel subunits a, C, D, H, and putative isoforms.

The plasma membrane V-ATPase from Manduca sexta (Lepidoptera, Sphingidae) larval midgut is composed of at least 12 subunits, eight of which have already been identified molecularly [Wieczorek et al., J. Bioenerg. Biomembr. 31 (1999) 67-74]. Here we report primary sequences of subunits C, D, H and a, which previously had not been identified in insects. Expression of recombinant proteins, immunostaining and protein sequencing demonstrated that the corresponding proteins are subunits of the Manduca V-ATPase. Genomic Southern blot analysis indicated the existence of multiple genes encoding subunits G, a, c, d and e. Moreover, multiple transcripts were detected in Northern blots from midgut poly(A) RNA for subunits B, G, c and d. Thus, these polypeptides appear to exist as multiple isoforms that could be expressed either in different tissues or at distinct locations within a cell. By contrast subunits A, C, D, E, F and H appear to be encoded by single transcripts and therefore should be present in any Manduca V-ATPase, independent of its subcellular or cell specific origin.

Three-dimensional structure and subunit topology of the V(1) ATPase from Manduca sexta midgut.

The three-dimensional structure of the Manduca sexta midgut V(1) ATPase has been determined at 3.2 nm resolution from electron micrographs of negatively stained specimens. The V(1) complex has a barrel-like structure 11 nm in height and 13.5 nm in diameter. It is hexagonal in the top view, whereas in the side view, the six large subunits A and B are interdigitated for most of their length (9 nm). The topology and importance of the individual subunits of the V(1) complex have been explored by protease digestion, resistance to chaotropic agents, MALDI-TOF mass spectrometry, and CuCl(2)-induced disulfide formation. Treatment of V(1) with trypsin or chaotropic iodide resulted in a rapid cleavage or release of subunit D from the enzyme, indicating that this subunit is exposed in the complex. Trypsin cleavage of V(1) decreased the ATPase activity with a time course that was in line with the cleavage of subunits B, C, G, and F. When CuCl(2) was added to V(1) in the presence of CaADP, the cross-linked products A-E-F and B-H were generated. In experiments where CuCl(2) was added after preincubation of CaATP, the cross-linked products E-F and E-G were formed. These changes in cross-linking of subunit E to near-neighbor subunits support the hypothesis that these are nucleotide-dependent conformational changes of the E subunit.

Evidence for major structural changes in the Manduca sexta midgut V1 ATPase due to redox modulation. A small angle X-ray scattering study.

The shape and overall dimensions of the oxidized and reduced form of the V(1) ATPase from Manduca sexta were investigated by synchrotron radiation x-ray solution scattering. The radius of gyration of the oxidized and reduced complex differ noticeably, with dimensions of 6. 20 +/- 0.06 and 5.84 +/- 0.06 nm, respectively, whereas the maximum dimensions remain constant at 22.0 +/- 0.1 nm. Comparison of the low resolution shapes of both forms, determined ab initio, indicates that the main structural alteration occurs in the head piece, where the major subunits A and B are located, and at the bottom of the stalk. In conjunction with the solution scattering data, decreased susceptibility to tryptic digestion and tryptophan fluorescence of the reduced V(1) molecule provide the first strong evidence for major structural changes in the V(1) ATPase because of redox modulation.

A novel electrogenic amino acid transporter is activated by K+ or Na+, is alkaline pH-dependent, and is Cl--independent.

A new eukaryotic nutrient amino acid transporter has been cloned from an epithelium that is exposed to high voltages and alkaline pH. The full-length cDNA encoding this novel CAATCH1 (cation-anion-activated Amino acid transporter/channel) was isolated using a polymerase chain reaction-based strategy, and its expression product in Xenopus oocytes displayed a combination of several unique, unanticipated functional properties. CAATCH1 electrophysiological properties resembled those of Na(+),Cl(-)-coupled neurotransmitter amine transporters, although CAATCH1 was cloned from a gut absorptive epithelium rather than from an excitable tissue. Amino acids such as l-proline, l-threonine, and l-methionine elicited complex current-voltage relationships in alkaline pH-dependent CAATCH1 that were reminiscent of the behavior of the dopamine, serotonin, and norepinephrine transporters (DAT, SERT, NET) in the presence of their substrates and pharmacological inhibitors such as cocaine or antidepressants. These I-V relationships indicated a combination of substrate-associated carrier current plus an independent CAATCH1-associated leakage current that could be blocked by certain amino acids. However, unlike all structurally related proteins, CAATCH1 activity is absolutely independent of Cl(-). Unlike related KAAT1, CAATCH1 possesses a methionine-inhibitable constitutive leakage current and is able to switch its narrow substrate selectivity, preferring threonine in the presence of K(+) but preferring proline in the presence of Na(+).

Structure and regulation of insect plasma membrane H(+)V-ATPase.

H(+) V-ATPases (V-ATPases) are found in two principal locations, in endomembranes and in plasma membranes. The plasma membrane V-ATPase from the midgut of larval Manduca sexta is the sole energizer of all transepithelial secondary transport processes. At least two properties make the lepidopteran midgut a model tissue for studies of general aspects of V-ATPases. First, it is a rich source for purification of the enzyme and therefore for structural studies: 20 larvae provide up to 0.5 mg of holoenzyme, and soluble, cytosolic V(1) complexes can be obtained in even greater amounts of up to 2 mg. Second, midgut ion-tranport processes are strictly controlled by the regulation of the V-ATPase, which is the sole energizer of all ion transport in this epithelium. Recent advances in our understanding the structure of the V(1) and V(o) complexes and of the regulation of the enzyme's biosynthesis and ion-transport activity will be discussed.

Animal plasma membrane energization by proton-motive V-ATPases.

Proton-translocating, vacuolar-type ATPases, well known energizers of eukaryotic, vacuolar membranes, now emerge as energizers of many plasma membranes. Just as Na(+) gradients, imposed by Na(+)/K(+) ATPases, energize basolateral plasma membranes of epithelia, so voltage gradients, imposed by H(+) V-ATPases, energize apical plasma membranes. The energized membranes acidify or alkalinize compartments, absorb or secrete ions and fluids, and underwrite cellular homeostasis. V-ATPases acidify extracellular spaces of single cells such as phagocytes and osteoclasts and of polarized epithelia, such as vertebrate kidney and epididymis. They alkalinize extracellular spaces of lepidopteran midgut. V-ATPases energize fluid secretion by insect Malpighian tubules and fluid absorption by insect oocytes. They hyperpolarize external plasma membranes for Na(+) uptake by amphibian skin and fish gills. Indeed, it is likely that ion uptake by osmotically active membranes of all fresh water organisms is energized by V-ATPases. Awareness of plasma membrane energization by V-ATPases provides new perspectives for basic science and presents new opportunities for medicine and agriculture.

Antibody to H(+) V-ATPase subunit E colocalizes with portasomes in alkaline larval midgut of a freshwater mosquito (Aedes aegypti).

The pH profile, gross structure, ultrastructure and immunolabeling of the mosquito (Aedes aegypti) larval midgut are described as a first step in analyzing the role of plasma membrane H(+ )V-ATPase in the alkalization of the gut, nutrient uptake and ionic regulation. Binding of an antibody to H(+ )V-ATPase subunit E colocalizes with 'portasomes' (approximately 10 nm in diameter), which are thought to correspond to the V(1) part of the H(+) V-ATPase. In gastric caeca (pH 8), both antibody-binding sites and portasomes are located apically; in the anterior midgut (pH 10-11), they are located basally; and in the posterior midgut (pH approximately equal to 8) they are again located apically. The hypothesis that the energization of alkalization is mediated by an H(+) V-ATPase is supported by the inability of larvae to maintain the high pH after 72 h in 10 (micro)M bafilomycin B1. Confirming earlier reports, the two principal epithelial cell types are designated as 'columnar' and 'cuboidal' cells. The apical plasma membranes (microvilli) of epithelial cells in the gastric caeca and basal infoldings of anterior midgut are invaded by mitochondria that lie within approximately 20 nm of the portasome-studded plasma membranes. The colocalization of V-ATPase-immunolabeling sites and portasomes to specific plasma membranes within so-called 'mitochondria-rich' cells of gastric caeca and anterior midgut suggests that midgut alkalization in mosquitoes is achieved by molecular mechanisms similar to those that have been described in caterpillars, even though the gross structure of the midgut and the localization of the V-ATPase are dissimilar in the two species. In caterpillars, the high alkalinity is thought to break down dietary tannins, which block nutrient absorption; it may play a similar role in plant-detritus-feeding mosquito larvae. The colocalization of immunolabeling sites and portasomes, together with the presence of long, 'absorptive-type' microvilli in the posterior midgut, suggest that the V-ATPase energizes nutrient uptake there.

Molecular architecture of Manduca sexta midgut V1 ATPase visualized by electron microscopy.

The structure of the V1 ATPase from the tobacco hornworm Manduca sexta has been determined from electron micrographs of isolated, negatively stained specimens. The resulting images clearly show a pseudohexagonal arrangement of six equal-sized protein densities, presumably representing the three copies each of subunits A and B, which comprise the headpiece of the enzyme. A seventh density could be observed either centrally or asymmetrically to the hexamer. The maximum diameter of the V1 complex in the hexagonal projection is 13 nm with each of the six peripheral densities being 3-4 nm in diameter.

A novel insect V-ATPase subunit M9.7 is glycosylated extensively.

Plasma membrane V-ATPase isolated from midgut and Malpighian tubules of the tobacco hornworm, Manduca sexta, contains a novel prominent 20-kDa polypeptide. Based on N-terminal protein sequencing, we cloned a corresponding cDNA. The deduced hydrophobic protein consisted of 88 amino acids with a molecular mass of only 9.7 kDa. Immunoblots of the recombinant 9.7-kDa polypeptide, using a monoclonal anti- body to the 20-kDa polypeptide, confirmed that the correct cDNA had been cloned. The 20-kDa polypeptide is glycosylated, as deduced from lectin staining. Treatment with N-glycosidase A resulted in the appearance of two additional protein bands of 16 and 10 kDa which both were immunoreactive to the 20-kDa polypeptide-specific monoclonal antibody. Thus, extensive N-glycosylation of the novel Vo subunit M9.7 accounts for half of its molecular mass observed in SDS-polyacrylamide gel electrophoresis. M9.7 exhibits some similarities to the yeast protein Vma21p which resides in the endoplasmic reticulum and is required for the assembly of the Vo complex. However, as deduced from immunoblots as well as from activities of the V-ATPase and endoplasmic reticulum marker enzymes in different membrane preparations, M9.7 is, in contrast to the yeast polypeptide, a constitutive subunit of the mature plasma membrane V-ATPase of M. sexta.

The plasma membrane H+-V-ATPase from tobacco hornworm midgut.

The midgut plasma membrane V-ATPase from larval Manduca sexta, the tobacco hornworm, is the sole energizer of any epithelial ion transport in this tissue and is responsible for the alkalinization of the gut lumen up to a pH of more than 11. This mini-review deals with those topics of research on this enzyme which may have contributed or are expected to contribute novel and general aspects to the field of V-ATPases. Topics dealt with include novel subunits or the quaternary structure of the V1 complex, as well as the regulation of the enzyme's function by reversible dissociation of the V1 from the V0 complexes and by genetic control on the transcriptional and posttranscriptional level.

Vacuolar and plasma membrane proton-adenosinetriphosphatases.

The vacuolar H+-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes. V-ATPases have similar structure and mechanism of action with F-ATPase and several of their subunits evolved from common ancestors. In eukaryotic cells, F-ATPases are confined to the semi-autonomous organelles, chloroplasts, and mitochondria, which contain their own genes that encode some of the F-ATPase subunits. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as ATP-dependent proton pumps. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. The mechanistic and structural relations between the two enzymes prompted us to suggest similar functional units in V-ATPase as was proposed to F-ATPase and to assign some of the V-ATPase subunit to one of four parts of a mechanochemical machine: a catalytic unit, a shaft, a hook, and a proton turbine. It was the yeast genetics that allowed the identification of special properties of individual subunits and the discovery of factors that are involved in the enzyme biogenesis and assembly. The V-ATPases play a major role as energizers of animal plasma membranes, especially apical plasma membranes of epithelial cells. This role was first recognized in plasma membranes of lepidopteran midgut and vertebrate kidney. The list of animals with plasma membranes that are energized by V-ATPases now includes members of most, if not all, animal phyla. This includes the classical Na+ absorption by frog skin, male fertility through acidification of the sperm acrosome and the male reproductive tract, bone resorption by mammalian osteoclasts, and regulation of eye pressure. V-ATPase may function in Na+ uptake by trout gills and energizes water secretion by contractile vacuoles in Dictyostelium. V-ATPase was first detected in organelles connected with the vacuolar system. It is the main if not the only primary energy source for numerous transport systems in these organelles. The driving force for the accumulation of neurotransmitters into synaptic vesicles is pmf generated by V-ATPase. The acidification of lysosomes, which are required for the proper function of most of their enzymes, is provided by V-ATPase. The enzyme is also vital for the proper function of endosomes and the Golgi apparatus. In contrast to yeast vacuoles that maintain an internal pH of approximately 5.5, it is believed that the vacuoles of lemon fruit may have a pH as low as 2. Similarly, some brown and red alga maintain internal pH as low as 0.1 in their vacuoles. One of the outstanding questions in the field is how such a conserved enzyme as the V-ATPase can fulfill such diverse functions.

Cloning and characterization of a potassium-coupled amino acid transporter.

Active solute uptake in bacteria, fungi, plants, and animals is known to be mediated by cotransporters that are driven by Na+ or H+ gradients. The present work extends the Na+ and H+ dogma by including the H+ and K+ paradigm. Lepidopteran insect larvae have a high K+ and a low Na+ content, and their midgut cells lack Na+/K+ ATPase. Instead, an H+ translocating, vacuolar-type ATPase generates a voltage of approximately -240 mV across the apical plasma membrane of so-called goblet cells, which drives H+ back into the cells in exchange for K+, resulting in net K+ secretion into the lumen. The resulting inwardly directed K+ electrochemical gradient serves as a driving force for active amino acid uptake into adjacent columnar cells. By using expression cloning with Xenopus laevis oocytes, we have isolated a cDNA that encodes a K+-coupled amino acid transporter (KAAT1). We have cloned this protein from a larval lepidopteran midgut (Manduca sexta) cDNA library. KAAT1 is expressed in absorptive columnar cells of the midgut and in labial glands. When expressed in Xenopus oocytes, KAAT1 induced electrogenic transport of neutral amino acids but excludes alpha-(methylamino)isobutyric acid and charged amino acids resembling the mammalian system B. K+, Na+, and to a lesser extent Li+ were accepted as cotransported ions, but K+ is the principal cation, by far, in living caterpillars. Moreover, uptake was Cl(-)-dependent, and the K+/Na+ selectivity increased with hyperpolarization of oocytes, reflecting the increased K+/Na+ selectivity with hyperpolarization observed in midgut tissue. KAAT1 has 634 amino acid residues with 12 putative membrane spanning domains and shows a low level of identity with members of the Na+ and Cl(-)-coupled neurotransmitter transporter family.

Sense and antisense RNA for the membrane associated 40 kDa subunit M40 of the insect V-ATPase.

For the first time a cDNA encoding the membrane associated subunit M40 of an invertebrate V-ATPase has been isolated and sequenced, based on a cDNA library from larval midgut of the tobacco hornworm, Manduca sexta. Immunoblotting with monospecific antibodies raised against the recombinant M40 polypeptide demonstrated that it is a subunit of the insect plasma membrane V-ATPase. Since M40 subunits had been identified only in endosomal V-ATPases till now, this result indicates that they are constitutive members of all, endomembrane and plasma membrane V-ATPases. A phagemid clone representing a polyadenylated antisense transcript was also isolated and sequenced. Using RT-PCR, endogenous antisense RNA was detected in poly(A) RNA isolated from the larval midgut. Since Southern blots indicated a single gene locus, both the antisense RNA as well as the sense mRNA encoding subunit M40 seem to originate from the same gene.

Animal plasma membrane energization by chemiosmotic H+ V-ATPases.

Proton-motive forces are thought to be less important than sodium-motive forces in energizing animal membranes. On the supply side, proton-motive forces across mitochondrial inner membranes are well-known energizers of ATP synthesis, catalyzed by F-type ATP synthases. However, on the demand side, proton-motive forces, generated from ATP by V-ATPases, are not widely accepted as energizers of animal membranes; instead, sodium-motive forces, generated by P-ATPases, are thought to predominate. During the 1980s, Anraku, Nelson, Forgac and others showed that proton-motive forces from H+ V-ATPases energize endomembranes of all eukaryotic cells; in most cases, chloride ions accompany the protons and the output compartment is acidified. Unexpectedly, numerous examples of animal plasma membrane energization by proton-motive forces are now appearing. In many insect epithelia, H+ V-ATPases generate transmembrane voltages which secondarily drive sensory signalling, fluid secretion and even alkalization, rather than acidification. Plasma membranes of phagocytes and osteoclasts as well as polarized membranes of epithelia in vertebrate kidney, bladder and epididymis, even apical membranes of frog skin epithelial cells, are now known to be energized by proton-motive forces. The list of proton-energized animal plasma membranes grows daily and includes cancer cells. The localization of H+ V-ATPases either on endomembranes or on plasma membranes may reflect a key event in their evolution. Proton-motive ATPases, like the H+ A-ATPases in present-day archaebacteria, appear to be ancestors of both H+ F-ATP synthases and H+ V-ATPases. On the basis of a greater than 25% overall sequence identity and much higher identity in the nucleotide-binding and regulatory sites, Nelson and others have argued that the A and B subunits of V-ATPases, like the corresponding beta and alpha subunits of F-ATP synthases, derive from common 'A-ATPase-like' ancestral subunits. They postulate that oxygen, introduced into the earth's atmosphere by cyanobacteria, was a selective agent as these key subunits diverged during evolution. Forgac has focused the issue more sharply by showing that the catalytic 'A' subunit of H+ V-ATPases has tow key sulfhydryl residues that are proximal to each other in the tertiary structure; these residues form a disulfide bond under oxidizing conditions, thereby inactivating the enzyme. The corresponding beta subunit of H+ F-ATPases lacks such sulfhydryl residues. Perhaps because their plasma membranes are the site of oxygen-dependent ATP synthesis, which would select against their sulfhydryl-containing regulatory sites, eubacterial cells lack H+ V-ATPases. This retention of the regulatory cysteine residue in the active sites during evolution may explain why H+ V-ATPases. are commonly found in the reducing atmosphere of the cytoplasm, where they would be active, rather than in the putatively oxidizing atmosphere of many plasma membranes, where they would be inactive. It may also explain why animal plasma membrane H+ V-ATPases are commonly found in 'mitochondria-rich' cells. We suggest that the high oxygen affinity of cytochrome oxidase leads to localized reducing conditions near mitochondria which would allow H+ V-ATPases to remain active in plasma membranes of such cells. Moreover, this 'redox modulation mechanism' may obviate the need to evoke two types of enzyme to explain selective targeting of H+ V-ATPases to plasma membranes or endomembranes: membrane that contains a single form of H+ V-ATPase may cycle between the membranes of the cytoplasmic organelles and the cell surface, the enzyme being active only when reducing conditions remove the disulfide bonding restraint.

Regulation of proton-translocating V-ATPases.

Vacuolar-type ATPases (V-ATPases) are proton-translocating enzymes that occur in the endomembranes of all eukaryotes and in the plasma membranes of many eukaryotes. They are multisubunit, heteromeric proteins composed of two structural domains, a peripheral, catalytic V1 domain and a membrane-spanning V0 domain. Both the multitude of locations and the heteromultimeric structure make it likely that the expression and the activity of V-ATPases are regulated in various ways. Regulation of gene expression encompasses control of transcription as well as control at the post-transcriptional level. Regulation of enzyme activity encompasses many diverse mechanisms such as disassembly/reassembly of V1 and V0 domains, oxidation of SH groups, control by activator and inhibitor proteins or by small signalling molecules, and sorting of the holoenzyme or its subunits to target membranes.