The regulation of urea amidolyase of Saccharomyces cerevisiae: mating type influence on a constitutivity mutation acting in cis.

Summary Constitutivity for the synthesis of the urea amidolyase bienzymatic complex is obtained by dur0^hmutations located in the regulatory genetic region adjacent to the dur1, dur2 gene cluster. The dur0^hmutations act only in cis and are a new case of cis effect strongly cancelled in /a diploid, similar to cargA ⁺0^hmutation shown previously to lead to arginase constitutivity. Illegitimate diploids do not show such a cancellation of constitutivity.The constitutivity produced by dur0^hmutation comprises the process of induction and the release of the glutamine effect. It does not impair the NH ₄ ⁺ effect.     

Specific induction of catabolism and its relation to repression of biosynthesis in arginine metabolism of Saccharomyces cerevisiae.

Experimental results are presented in support of the model previously proposed for specific induction of the synthesis of enzymes for arginine catabolism in Saccharomyces cerevisiae (Wiame, 1971a,b), and its connection with end-product repression of arginine biosynthetic enzymes. The data support the occurrence of negative regulation of metabolism in a eukaryote.Operator regions, one for arginase and another for ornithine transaminase, are identified. The operator mutations are fully constitutive. A mutation compatible with the occurrence of a catabolic represser, CARGR, leads to partial pleiotropic constitutivity.The connection between the induction process and the repression of biosynthetic enzymes is due to a common receptor of metabolic signals, an ambivalent repressor ARGR endowed with the property of a usual repressor for anabolic enzymes and playing the role of inducer at the level of CARGR; this cascade process simulates a positive control. argR^? mutations, by producing defective ARGR, “turn on” anabolic enzyme synthesis and “turn off” the synthesis of catabolic enzymes (Fig. 2). The dual role of ARGR is confirmed by the isolation of a mutation argRII^d which, in contrast to the defective properties caused by usual argR^? mutations, causes a dominant hyperactivity toward induction of a catabolic enzyme, but retains recessive hypoactivity toward repression of an anabolic enzyme. Such an ambivalent repressor is a function necessary for mutual, balanced exclusion between opposite metabolisms.Many operator constitutive mutations for arginase, cargA⁺O^?, change the level of enzyme to a similar value, thus defining a genetic function. One of these mutations, cargA⁺O^h, in addition to having unusual genetic behaviour, leads to production of twice as much arginase as cargA⁺O^?. This suggests the existence of another genetic region near the structural gene for this enzyme and an additional regulatory function to be analyzed in a separate paper (Dubois &; Wiame, 1978).     

Induction of trehalase activity on a nitrogen-free medium: a sporulation-specific event in the fission yeast, Schizosaccharomyces pombe

Summary Kinetic experiments with synchronously sporulating cultures of a homothallic h⁹⁰ strain of Schizosaccharomyces pombe showed that trehalase activity abruptly increased in the late sporulation process, coinciding with the appearance of visible spores. Trehalase activity was absent in vegetative cells. A set of strains different in genetic constitution at the mating type loci was tested for induction of trehalase on nitrogen-free sporulation medium. The appearance of trehalase activity on the sporulation medium was observed only in sporulating cultures; cultures of homothallic strains (h⁹⁰) and diploid strains heterozygous for mating type (h⁺/h^–), and mixed cultures of heterothallic h⁺ and h^– strains. Trehalase activity was not induced in nonsporogenic strains: heterothallic haploid strains (h⁺ and h^–), diploid strains homozygous for mating type (h⁺/h⁺ and h^–/h^–) and the homothallic strain harboring the mutation in the mat2 gene, which was unable to undergo the first meiotic division. Trehalose accumulation on the sporulation medium was observed solely in the sporulating cultures. These results led us to conclude that the induction of trehalase activity as well as the accumulation of trehalose in the medium lacking nitrogen sources was a sporulation-specific event under the control of the mating type genes.     

Gliadin activates arginase pathway in RAW264.7 cells and in human monocytes

Celiac disease (CD) is an autoimmune enteropathy triggered in susceptible individuals by the ingestion of gliadin-containing grains. Recent studies have demonstrated that macrophages play a key role in the pathogenesis of CD through the release of inflammatory mediators such as cytokines and nitric oxide (NO). Since arginine is the obliged substrate of iNOS (inducible nitric oxide synthase), the enzyme that produces large amount of NO, the aim of this work is to investigate arginine metabolic pathways in RAW264.7 murine macrophages after treatment with PT-gliadin (PTG) in the absence and in the presence of IFNγ. Our results demonstrate that, besides strengthening the IFNγ-dependent activation of iNOS, gliadin is also an inducer of arginase, the enzyme that transforms arginine into ornithine and urea. Gliadin treatment increases, indeed, the expression and the activity of arginase, leading to the production of polyamines through the subsequent induction of ornithine decarboxylase. This effect is strengthened by IFNγ. The activation of these pathways takes advantage of the increased availability of arginine due to a decreased system y⁺l-mediated efflux, likely ascribable to a reduced expression of Slc7a6 transporter. A significant induction of arginase expression is also observed in human monocytes from healthy subject upon treatment with gliadin, thus demonstrating that gluten components trigger changes in arginine metabolism in monocyte/macrophage cells.     

Regulatory mechanism of the tryptophan operon in Escherichia coli: possible interaction between trpR and trpS gene products

Summary The trpS5 mutation (a mutation in the structural gene for tryptophanyl-tRNA synthetase (TRSase) in E. coli), when present in the genetic background of strain KY913 (HfrH), results in the failure to grow at high temperature (42° C) in a complete medium. The rel (RC relaxed) marker present in this strain was found to be partly responsible for this temperature sensitivity. TRSase in such a strain was rapidly inactivated during growth at 42° C in rich media, but not in minimal media or in the presence of chloramphenicol. A partial derepression of anthranilate synthetase formation took place in the presence of excess tryptophan at growth-restricting temperatures. When some of the trpR mutations (including amber mutations) were combined with trpS5, the resulting double mutants (trpR trpS5) were temperature-insensitive, and TRSase was not inactivated at high temperature, in contrast to the trpR ⁺trpS5 strain. This effect of trpR mutations on temperature sensivity was shown not to be a secondary consequence of the constitutive expression of the trp operon. These findings suggest that the trpR ⁺ product interacts with the TRSase of the trpS5 mutant so as to bring about the growth-dependent inactivation of the enzyme. Furthermore, a special class of trpR mutants was obtained whose constitutivity with respect to the trp operon is manifested only in strains carrying trpS5 (but not trpS ⁺) grown at high temperatures. It is proposed that TRSase participates in repression trrough direct interaction with the product of the trpR gene.     

Release of the "ammonia effect" on three catabolic enzymes by NADP-specific glutamate dehydrogenaseless mutations in Saccharomyces cerevisiae

Mutations which inactivate the NADP-glutamate dehydrogenase (anabolic GDHase) pleiotropically release the ammonia inhibition (NH₄⁺ effect) on a number of distinct catabolic activities. In addition to releasing inhibition on several permeability functions (1), these mutations suppress the NH₄⁺ effect on the synthesis of arginase, urea amidolyase and allantoinase. They do not affect the NH₄⁺ effect on the NAD-glutamate dehydrogenase.Two mechanisms of action of these mutations have to be considered, namely a modification of the process of $\text{induction}$ (such as removal of inducer exclusion) and a suppression of $\text{nitrogen catabolite repression}$.     

A regulatory mutant affecting the synthesis of enzymes involved in the catabolism of nucleosides in Escherichia coli

Summary A regulatory mutant which leads to constitutive synthesis of enzymes involved in catabolism of nucleosides is described. It is unlinked to the structural genes whose activity is affected. The gene concerned is designated nucR. The amount of thymine required for growth (colony formation) of thy ^– strains is affected by the nucR mutation. The amount required by a thy ^– drm ^–strain is reduced about four fold if it carries the constitutivity mutation. The amount required by a thy ^– drm ⁺strain is increased at least two fold. These differences in nutritional requirement provide a method for selecting constitutives from non-constitutives and vice versa.Abbreviations Rib-1-P Ribose-1-phosphate - dRib-1-P deoxyribose-1-phosphate - Rib-5-P Ribose-5-phosphate - dRib-5-P deoxyribose-5-phosphate - Pi inorganic phosphate     

Interaction of benthic microalgae and macrofauna in the control of benthic metabolism, nutrient fluxes and denitrification in a shallow sub-tropical coastal embayment (western Moreton Bay, Australia)

Benthic biogeochemistry and macrofauna were investigated six times over 1 year in a shallow sub-tropical embayment. Benthic fluxes of oxygen (annual mean ?918 μmol O₂ m^?2 h^?1), ammonium (NH₄ ⁺), nitrate (NO₃ ^?), dissolved organic nitrogen, dinitrogen gas (N₂), and dissolved inorganic phosphorus were positively related to OM supply (N mineralisation) and inversely related to benthic light (N assimilation). Ammonium (NH₄ ⁺), NO₃ ^? and N₂ fluxes (annual means +14.6, +15.9 and 44.6 μmol N m^?2 h^?1) accounted for 14, 16 and 53 % of the annual benthic N remineralisation respectively. Denitrification was dominated by coupled nitrification–denitrification throughout the study. Potential assimilation of nitrogen by benthic microalgae (BMA) accounted for between 1 and 30 % of remineralised N, and was greatest during winter when bottom light was higher. Macrofauna biomass tended to be highest at intermediate benthic respiration rates (?1,000 μmol O₂ m^?2 h^?1), and appeared to become limited as respiration increased above this point. While bioturbation did not significantly affect net fluxes, macrofauna biomass was correlated with increased light rates of NH₄ ⁺ flux which may have masked reductions in NH₄ ⁺ flux associated with BMA assimilation during the light. Peaks in net N₂ fluxes at intermediate respiration rates are suggested to be associated with the stimulation of potential denitrification sites due to bioturbation by burrowing macrofauna. NO₃ ^? fluxes suggest that nitrification was not significantly limited within respiration range measured during this study, however comparisons with other parts of Moreton Bay suggest that limitation of coupled nitrification–denitrification may occur in sub-tropical systems at respiration rates exceeding ?1,500 μmol O₂ m^?2 h^?1.     

Ammonium influence on nitrogen assimilating enzymes and protein accumulation in suspension cultures of Paul's Scarlet rose

The influence of NH₄⁺ on protein accumulation was examined by growing suspension cultures of Rosa cv. Paul's Scarlet on two defined media. Both contained 1920 μmol of NO₃^? but only one contained 72.8 μmol of NH₄⁺. At the conclusion of a 14-day growth period, cultures grown with NH₄⁺ possessed twice as much protein as cultures grown without NH₄⁺. The influence of NH₄⁺ did not appear to be a substrate effect, since the amount of NH₄⁺ provided accounted for only 10% of the nitrogen recovered in protein. The provision of NH₄⁺ in the starting medium increased the activity (μmol substrate. h^?1· g^?1 fr wt) of glutamate dehydrogenase and glutamate synthase, and reduced the activity of glutamine synthetase. A comparison of the total activity per culture for each of these enzymes with the rate of nitrogen incorporation into protein showed that the enzymatic potential of glutamine synthetase and glutamate dehydrogenase greatly exceeded the actual in vivo rate of nitrogen assimilation through the respective pathways. Thus it was concluded that the availability of either of these enzymes does not limit nitrogen assimilation in rose cells and the fluctuations in their level brought about by NH₄⁺ was of no physiological importance. The activity of glutamate synthase per culture approximated the rate of nitrogen incorporation into protein during early stages of growth, and for that reason may have limited nitrogen assimilation or caused a diversion of nitrogen through the alternative pathway to glutamate catalyzed by glutamate dehydrogenase.     

lac Repressor-operator interaction. IX. The binding of lac repressor to operators containing Oc mutations

Representative members of the six classes of operator constitutive (O^c) point mutations, which have been mapped and well characterized in vivo, were crossed into λφ80 lac phages. The phage DNAs containing the O^c mutations were used to measure the affinity of the lac repressor (R) for each O^c operator by determining the half-lives of the different RO^c complexes in vitro. The results provide evidence that: (a) the higher the constitutive level of β-galactosidase in vivo, as the result of an O^c mutation, the lower the affinity of the lac repressor for that O^c operator, with a maximum difference of two orders of magnitude in affinity of the repressor for the highest O^c tested as compared to the wild type O⁺ operator; (b) the six classes of O^c operators appear to be twofold degenerate, in that two members of each class, which were previously distinguished by mapping, have the same affinity for the lac repressor; (c) an inducer and an anti-inducer have the same effect on the RO^c complexes as on the RO⁺ complexes; (d) the relationship between induction ratios in vivo and the binding constant of the repressor for each O^c mutation in vitro does not follow the mass action equation but rather a more complex dependency, which is discussed.These results suggest a functional symmetry in the lac operator.     

Positive Regulation in a Eukaryote, a Study of the uaY Gene of ASPERGILLUS NIDULANS: I. Characterization of Alleles, Dominance and Complementation Studies, and a Fine Structure Map of the uaY -oxpA Cluster

In this paper we characterize genetically a positive eukaryotic regulatory gene: the uaY gene of the ascomycete Aspergillus nidulans. Several steps in the uptake and degradation of purines are under the control of the uaY gene (summarized in Scazzocchio and Gorton 1977). In the present paper 12 uaY^- mutations are characterized with respect to their inducibility for adenine deaminase, xanthine dehydrogenase (purine hydroxylase I) and urate oxidase and by the absence of the uric acid-xanthine permease scored in vivo by resistance to 2-thiouric acid. While 10 mutations are uniformly unleaky, two others are almost wild type for the induction of urate oxidase. A fine structure map of the uaY gene shows that the two "leaky" mutations are not clustered. The fine structure mapping unambiguously positions six uaY alleles and provides preliminary but interesting trends regarding the pattern of gene conversion in the uaY gene. The enzyme levels in all uaY^-/uaY⁺ heterozygous diploids are intermediate between the corresponding uaY^-/uaY^- and uaY⁺/uaY⁺ homozygous diploids, suggesting that one functional copy of the uaY gene is able to mediate the complete induction of only one set of structural genes. No complementation was found between any two uaY^- alleles. This establishes that the mutations showing either of the phenotypes are alleles in the same gene; it fails to provide evidence for intracistronic complementation. A mutation, oxpA5, causes resistance to the xanthine analogue oxypurinol (4, 6-dihydroxypyrazolo-(3, 4-d)-pyrimidine) and partial constitutivity of adenine deaminase, xanthine dehydrogenase (purine hydroxylase I) and urate oxidase. The constitutive phenotype is suppressed by mutations blocking the synthesis of intracellular inducers. The mutation is recessive and complements fully with the 11 uaY^- mutations tested. It maps to the left of all 12 uaY mutations to which it has been crossed. The data indicate that both the resistance and constitutivity arise from one mutational event in a gene, oxpA, different from uaY and possibly adjacent to it. We propose that the oxpA gene codes for a protein involved in limiting the flow of inducers into the cell nucleus. Thus oxpA and uaY constitute a regulatory gene cluster, indicating that uaY is the regulatory gene.     

Absence of involvement of glutamine synthetase and of NAD-linked glutamate dehydrogenase in the nitrogen catabolite repression of arginase and other enzymes in Saccharomyces cerevisiae

The escape of several enzymes from “ammonia catabolite repression” in gdhA^? (NADP-linked glutamate-dehydrogenase-less) mutants, as well as in gdhCR mutants of Saccharomyces cerevisiae, does not involve glutamine synthetase, either as a positive or as a negative control element. A glutamine-synthetase-less mutant (gln^?) was used in this demonstration.In addition to its derepressing effect on the NAD-linked glutamate dehydrogenase, the gdhCR mutation releases “nitrogen catabolite repression” on arginase and allatoinase, as well as glutamine repression on glutamine synthetase. A gdhCS mutation was used to demonstrate that these effects are not mediated through the NAD-linked glutamate dehydrogenase.     

Nitrogen deprivation induces cross-tolerance of Poa annua callus to salt stress

Alternative respiration pathway (AP) is an important pathway which can be induced by environment stresses in plants. In the present study, we show a new mechanism involving the AP in nitrogen deprivation-induced tolerance of Poa annua callus to salt stress. The AP capacity markedly increased under a 600 mM NaCl treatment or nitrogen deprivation pretreatment and reached a maximum under the nitrogen deprivation pretreatment combined with the NaCl treatment (–N+NaCl). Malondialdehyde (MDA) and H₂O₂ content and Na⁺/K⁺ ratio significantly increased under the 600 mM NaCl treatment but less under the–N+NaCl treatment. Moreover, both the nitrogen deprivation and the NaCl stress stimulated the plasma membrane (PM) H⁺-ATPase activity and increased pyruvate content. The maximal stimulating effect was found under the–N+NaCl treatment. When the AP capacity was reduced by salicylhydroxamic acid (SHAM, an inhibitor of AP), content of MDA and H₂O₂ and Na⁺/K⁺ ratio dramatically increased, whereas PM H⁺-ATPase activity decreased. Moreover, exogenous application of pyruvate produced a similar effect as the nitrogen deprivation pretreatment. The effects of SHAM on the Poa annua callus were counteracted by catalase (a H₂O₂ scavenger) and diphenylene iodonium (a plasma membrane NADPH oxidase inhibitor). Taken together, our results suggest that the nitrogen deprivation enhanced the capacity of AP by increasing pyruvate content, which in turn prevented the Poa annua callus from salt-induced oxidative damages and Na⁺ over-uptake.     

Differential effect of ammonium on the induction of nitrate and nitrite reductase activities in roots of barley (Hordeum vulgare) seedlings

The effect of exogenous NH₄⁺ on the induction of nitrate reductase activity (NRA; EC 1.6.6.1) and nitrite reductase activity (NiRA; EC 1.7.7.1) in roots of 8-day-old intact barley (Hordeum vulgare L.) seedlings was studied. Enzyme activities were induced with 0.1, 1 or 10 mM NO₃⁺ in the presence of 0, 1 or 10 mM NH₄⁺, Exogenous NH₄⁺ partially inhibited the induction of NRA when roots were exposed to 0.1 mM, but not to 1 or 10 mM NO₃⁺, In contrast, the induction of NiRA was inhibited by NH₄⁺ at all NO₃⁺ levels. Maximum inhibition of the enzyme activities occurred at 1.0 mM NH₄⁺ Pre-treatment with NH₄⁺ had no effect on the subsequent induction of NRA in the absence of additional NH₄⁺ whereas the induction of NiRA in NH₄⁺-pretreated roots was inhibited in the absence of NH₄⁺ At 10 mM NO₃⁺ L-methionine sulfoximine stimulated the induction of NRA whether or not exogenous NH₄⁺ was present. In contrast, the induction of NiRA was inhibited by L-methionine sulfoximine irrespective of NH₄⁺ supply. During the postinduction phase, exogenous NH₄⁺ decreased NRA in roots supplied with 0.1 mM but not with 1mM NH₃⁺ whereas, NiRA was unaffected by NH₄⁺ at either substrate concentration. The results indicate that exogenous NH₄⁺ regulates the induction of NRA in roots by limiting the availability of NO₃⁺. Conversely, it has a direct effect, independent of the availability of NO₃⁺, on the induction of NiRA. The lack of an NH₄⁺ effect on NiRA during the postinduction phase is apparently due to a slower turnover rate of that enzyme.     

Nitrogen Metabolism of the Marine Microalga Chlorella autotrophica

The levels of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in Chlorella autotrophica (clone 580) are strongly regulated by the nitrogen source and salt concentration of the medium. GS is present at high levels in NO₃⁻-grown cells, and at maximum levels in nitrogen-starved cells. However, the levels of GS in these cells are somewhat decreased by increasing salinity. Cells growing on NH₄⁺ have high NADPH-GDH activity, the levels of which increase with increasing NH₄⁺ supply, while GS decreases to a very low level under these conditions. Salinity intensifies the induction of NADPH-GDH activity in NH₄⁺-grown cells. The levels of NADH-GDH are low in this alga, but present under all growth conditions. Methionine sulfoximine (MSX) has little effect on growth and nitrogen assimilation of the alga in the presence of NH₄⁺.     

Increased arginase activity during lymphocyte mitogenesis

A sensitive assay for arginase activity was developed using [guanidino-¹⁴C]arginine as substrate and measuring the production of ¹⁴CO₂ from [¹⁴C]urea in the presence of urease. Arginase activity was measured in bovine lymphocytes after activation by Concanavalin A. The specific enzymatic activity of arginase doubled in 6 hours and increased nearly 4-fold by 24 hours after stimulation. It is suggested that the role of arginase in these cells is to provide ornithine as substrate for the synthesis of putrescine, precursor of the polyamines spermidine and spermine.     

On the involvement of singlet oxygen in mutation induction by 8-methoxypsoralen and UVA irradiation in Escherichia coli K-12

The possible mutagenic effects induced by singlet oxygen, which is formed during UVA irradiation of bacterial cells pretreated with 8-methoxypsoralen (8-MOP), were investigated. As genetic endpoint, back mutation from arg₅₆^? to arg⁺ was assayed in strain Escherichia coli K-12/343/113/uvrB; this system, in preliminary experiments, was rather sensitive to 8-MOP-induced photodynamic effects. To assess the involvement of singlet oxygen (¹O₂) in the mutation induction process, 2 tests were applied, namely, comparative mutation induction in D₂O and in H₂O media (pH 7.0) and quenching of ¹O₂ with 1,4-diazabicyclo[2.2.2]octane (DABCO).When photodynamy was performed with the indicator cells suspended in D₂O buffer, the mutagenic effect was substantially higher than that obtained with cells suspended in H₂O buffer; this increase was even more pronounced when the incubation mixtures were thoroughly oxygenated before irradiation. D₂O itself was not mutagenic under the present experimental conditions. Addition of DABCO in concentrations of 0.1–10 mM to the irradiation mixtures effectively reduced the number of 8-MOP-induced mutant yields by about 40%. DABCO itself had no effect on cell viability or on spontaneous mutation frequency under our experimental conditions.From these 2 sets of results, and from the preliminary findings that the photomutagenic effect of 8-MOP is higher in the uvrB derivative than in the corresponding excision-repair-proficient parent strain, which is in concordance with previous observations in other E coli strains, it can be concluded that ¹O₂ generated upon UVA irradiation of 8-MOP solutions is probably responsible for part of the observed genetic effects.     

Experimental demonstration of the roles of bacteria and bacterivorous protozoa in plankton nutrient cycles

We have used a model food chain composed of a natural bacterial assemblage, a pennate diatom and a bacterivorous microflagellate to investigate the factors controlling the relative importance of bacteria and protozoa as sources for regenerated nitrogen in plankton communities. In bacterized diatom cultures in which diatom growth was nitrogen-limited, the carbon:nitrogen (C:N) ratio of the bacterial substrate greatly affected which population was responsible for the uptake of nitrogen. When nitrogen was added as NH ₄ ⁺ and the cultures were supplemented with glucose, the bacteria competed successfully with the algae for NH ₄ ⁺ and prevented the growth of algae by rapidly assimilating all NH ₄ ⁺ in the cultures. Bacterivorous protozoa inoculated into these cultures grazed the bacterial population and remineralized NH ₄ ⁺ , thus relieving the nitrogen limitation of algal growth and allowing an increase in algal biomass. In contrast, bacteria in cultures supplemented with the amino acid glycine (C:N = 2) were major remineralizers of nitrogen, and the influence of protozoan grazing was minimal. We conclude that the relative importance of bacteria and protozoa as nutrient regenerators in the detrital food loop is dependent largely on the overall carbon:nutrient ratio of the bacterial substrate. The role of bacterivorous protozoa as remineralizers of a growth-limiting nutrient is maximal in situations where the carbon:nutrient ratio of the bacterial substrate is high.     

Essential amino acid residues in the central transmembrane domains and loops for energy coupling of Streptomyces coelicolor A3(2) H-pyrophosphatase

The H⁺-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14−17 transmembrane domains, and is found in a range of organisms. We focused on the second quarter region of Streptomyces coelicolor A3(2) H⁺-pyrophosphatase, which contains long conserved cytoplasmic loops. We prepared a library of 1536 mutants that were assayed for pyrophosphate hydrolysis and proton translocation. Mutant enzymes with low substrate hydrolysis and proton-pump activities were selected and their DNAs sequenced. Of these, 34 were single-residue substitution mutants. We generated 29 site-directed mutant enzymes and assayed their activity. The mutation of 10 residues in the fifth transmembrane domain resulted in low coupling efficiencies, and a mutation of Gly¹⁹⁸ showed neither hydrolysis nor pumping activity. Four residues in cytoplasmic loop e were essential for substrate hydrolysis and efficient H⁺ translocation. Pro¹⁸⁹, Asp²⁸¹, and Val³⁵¹ in the periplasmic loops were critical for enzyme function. Mutation of Ala³⁵⁷ in periplasmic loop h caused a selective reduction of proton-pump activity. These low-efficiency mutants reflect dysfunction of the energy-conversion and/or proton-translocation activities of H⁺-pyrophosphatase. Four critical residues were also found in transmembrane domain 6, three in transmembrane domain 7, and five in transmembrane domains 8 and 9. These results suggest that transmembrane domain 5 is involved in enzyme function, and that energy coupling is affected by several residues in the transmembrane domains, as well as in the cytoplasmic and periplasmic loops. H⁺-pyrophosphatase activity might involve dynamic linkage between the hydrophilic and transmembrane domains.