Phycobiliproteins — a family of valuable,widely used fluorophores

Phycobiliproteins are brilliantly colored, highly fluorescent components of the photosynthetic light-harvesting antenna complexes of cyanobacteria (blue-green algae), red algae and cryptomonads. These proteins carry covalently attached linear tetrapyrrole pigments related structurally to biliverdin. Phycobiliproteins, purified from certain organisms, are isolated as either trimers, ()₃, of approximatelyM _r 110–120×10³ (e.g., allophycocyanins), or hexamers, ()₆, of aboutM _r 250×10³ (certain phycoerythrins). Three phycobiliproteins R-phycoerythrin, B-phycoerythrin, and allophycocyanin serve as valuable fluorescent tags with numerous applications in flow cytometry, fluorescence activated cell sorting, histochemistry and, to a limited degree, in immunoassay and detection of reactive oxygen species. These applications exploit the unique physical and spectroscopic properties of phycobiliproteins.     

Spironolactone antagonism of aldosterone action on Na+ transport and RNA metabolism in toad bladder epithelium

Summary In earlier studies, aldosterone increased the incorporation of precursors into a class of cytoplasmic RNA with the characteristics of messenger RNA (mRNA), in toad bladder epithelium. In the present studies, this effect was analyzed further with a competitive antagonist, spironolactone (SC-9420). Paired hemibladders were labeled with³H-uridine (30 min pulse–140 min chase), with or without aldosterone (3.5×10^–8 m, 7×10^–8 m) in the presence or absence of SC-9420 (7×10^–6 m, 2.5×10^–5 m) at molar ratios of 2001 to 2801. Cytoplasmic RNA, either the total phenol-SDS extract or polyadenylated-RNA (poly(A)(+)-RNA) obtained by oligo-deoxythymidylate-cellulose (oligo(dT)-cellulose) chromatography was analyzed in linear 5–20% sucrose gradients. Eight sets of experiments were completed in which the short-circuit current (scc) was monitored for 180 min and the incorporation of³H-uridine (30 min pulse–150 min chase) was simultaneously determined on pools of epithelia from 5 to 10 hemibladders. The fractional change inscc correlated linearly with the fractional change in³H-uridine of 12S cytoplasmic RNA (r=0.95,p<0.001). The poly(A)(+)-RNA fraction had no detectable rRNA or tRNA and gave a heterogeneous pattern, typical of mRNA, in the sucrose gradients. In the presence of exogenous aldosterone, SC-9420 inhibited the incorporation of³H-uridine into poly(A)(+)-RNA (particularly 12S). These results support the inference that induction of mRNA mediates the action of aldosterone on Na⁺ transport.     

Purification and properties of laccase from Ganoderma lucidum

Summary The enzyme laccase has been partially purified from the culture fluid of Ganoderma lucidum by acetone precipitation, ammonium sulphate fractionation and adsorption on alumina C gel. The enzyme has been shown to be specific for ortho and para hydroxyphenolic compounds, having Km values of 5.5×10^-5 M and 2.86×10^-5 M for catechol and hydroquinone respectively. The optimum pH for the oxidation of catechol and hydroquinone are 5.4 and 5.0 respectively. The enzyme is inactivated above 60°C and is inhibited by enzyme inhibitors and metal chelating agents like azide, cyanide etc.     

Vorläufige Versuche über die Elastizität des Atraktoplasmas

Summary Spindles of nuclei mostly from metaphases of expiants in vitro, Set free according to Dan's method by means of solutions of Duponol (a sodium lauryl sulfonate), have been studied according to their elasticity. The isolated atractoplasm has shown a static modul of elasticityE0.8 to 1.6×10⁶ dynes×cm^–2, and, sometimes, a Poisson number of0.42, that is to say the atractoplasmic gel of the spindles of nuclei is certain elastically. The values of the modul of elongation agree satisfactory with those values recently ascertained by Crick and Hughes with cytoplasm oi explanted cells.     

Substitution of D-Trp32 in NPY Destabilizes the Binding Transition State to the Y1 Receptor Site in SK-N-MC Cell Membranes

The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porcine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp³²-NPY peptide at Lys⁴ was investigated using SK-N-MC neuroblastoma cell membranes containing the Y1 receptor. The release rate of the spin labeled peptides was monitored by electron spin resonance and the K_D was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr¹N⁴-proxyl]-NPY, the K_D was 8 × 10^–10 M and k_off was 2.7 × 10^–4 sec^–1 yielding a value for k_on of 3.3 × 10⁵ sec^–1 M^–1. The [Ac-Tyr¹, N⁴-proxyl,-D-Trp³²]-NPY antagonist ligand had a value of K_D equal to 1.35 × 10^–7 M and k_off was 1.7 × 10^–4 sec^–1 leading to a value for k_on of 1.2 × 10³ sec^–1 M^–1. The difference in the k_on rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-N⁴ proxyl-D-Trp³²-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.     

Restriction and modification in B. subtilis. Purification and general properties of a restriction endonuclease from strain R.

Summary All Bacillus subtilis R-type strains showing the phenomena of restriction and modification contain an endonuclease that inactivates in vitro the biological activity of a variety of DNAs lacking R-specific modification, such as transfecting SPP1, SPO2 and 105 DNA, and transforming B. subtilis 168-type DNA. The corresponding DNAs carrying R-specific modification are resistant to the enzyme. The enzyme has been purified approximately 400-fold and is essentially free from contaminating double strand-directed unspecific exo-or endonuclease activity. Only Mg²⁺ is required as cofactor. The substrate DNAs are cleaved at specific sites. The double-stranded fragments produced from SPP1 DNA (molecular weight 2.5×10⁷) have an average molecular weight of about 3×10⁵.     

Characterization of the reciprocal binding sites on human α-thrombin and factor XIII A-chain

Solution- and solid-phase techniques were used to probe Factor XIII A-chain-a-thrombin interactions. -Thrombin activated Factor XIII more efficiently (Km = 0.83 ± 0.08 × 10^-7 M; V/K = 14.90 ± 3.20 × 10^-3 min^-1) than -thrombin (Km = 6.14 ± 1.26 × 10^-7 M; V/K = 3.30 ± 1.00 × 10^-3 min^-1) or -thrombin (Km = 6.25 ± 1.15 × 10^-7 M; V/K = 3.00 ± 0.80 × 10^-3 min^-1). Immobilized FPR--thrombin bound plasma Factor XIII (Kd = 0.17 ± 0.04 × 10^-7 M) > Factor XIIIa (Kd = 0.69 ± 0.18 × 10^-7 M) > liver transglutaminase (Kd = 4.73 ± 1.01 × 10^-7 M) > Factor XIII A-chain (Kd = 49.00 ± 9.40 × 10^-7 M). FPR--thrombin and -thrombin also bound immobilized Factor XIII A-chain with affinities inversely related to protease activity: maximal binding at 1.36 × 10^-7 M and 13.6 × 10^-7 M, respectively. Plasma Factor XIII, transglutaminase, and dithiothreitol competitively inhibited Factor XIII A-chain binding to FPR--thrombin: IC₅₀ = 1.0 × 10^-7 M, 3.0 × 10^-6 M and 1.52 × 10^-4 M, respectively. Transglutaminase also inhibited Factor XIII binding to ×-thrombin (IC₅₀ = 2.0 × 10^-6 M). Thrombin-binding site was localized to G^-38-M^-731 fragment of Factor XIII A-chain, probably within homologous regions (N^-72-A^-493) of transglutaminase. R^-320-E^-579 of -thrombin was Factor XIII A-chain binding site. Intra-B-chain disulfides in -thrombin were essential for binding but not catalytic H^-363 or residues R^-382-N^-394 and R^-443-G^-475. These studies propose a structural basis for Factor XIII activation, provide a regulatory mechanism for Factor XIIIa generation, and could eventually help in the development of new structure-based inhibitors of thrombin and Factor XIIIa.     

Ethanol acetylation by mycelium-bound carboxylesterase of Aspergillus oryzae: estimation of thermodynamic parameters and integral productivity

The results collected at different temperatures for ethanol acetylation by cell-bound carboxylesterase from lyophilized cells of Aspergillus oryzae have been used to investigate the kinetics and thermodynamics of this esterification in n-heptane. The occurrence of reversible unfolding followed by irreversible denaturation of the enzyme has been proposed to explain the increase in the starting rate of ethyl acetate formation with temperature observed up to 55 °C and the consequent fall beyond this threshold. The Arrhenius model has been used to estimate the apparent activation enthalpies of both the acetylation reaction (H = 29–33 kJ mol^–1) and reversible enzyme unfolding (H _u = 56–63 kJ mol^–1). The results of residual activity tests performed with cells previously exposed at different temperatures for variable times enabled us also to estimate the first-order rate constant of irreversible denaturation (2.40 × 10^–3 h^–1 < k _d < 8.11 × 10^–3 h^–1) as well as the related thermodynamic parameters (H _d = 22 kJ mol^–1; S _d = –0.29 kJ mol^–1 K^–1). This last phenomenon proved particularly slow for the system under consideration, probably because the biocatalyst link to the mycelium was able to improve its thermostability. In view of future continuous application, the effects of operating time, starting substrate concentration and temperature on the theoretical integral productivity of a fixed-bed column filled with this biocatalyst have been investigated.     

Interactions of growth inhibitory factor with hydroxyl and superoxide radicals

To show the effects of growth inhibitory factor (Cu₄Zn₃MT-III) involved in the scavenging of reactive oxygen species (ROS), a pulse radiolytic study was employed using N₂O-saturated Cu₄Zn₃MT-III aqueous solutions. It was demonstrated that the oxidizing OH radical efficiently reacted with Cu₄Zn₃MT-III by forming a thiyl radical RS with a second-order constant of 1.46×10¹¹ mol l^–1s^–1, which was determined by competition kinetics against KSCN. The thiyl radical RS reacted rapidly and reversibly with a thiolate in Cu₄Zn₃MT-III to form radical anion RSSR ^– with a constant of 1.65×10⁹ mol lL^–1s^–1 per thiolate, while the constant of the decay of this radical anion was 2.72×10⁵ s^–1, and the equilibrium constant of the formation for RSSR ^– was 6.08×10³ mol^–1 l. These values were close to those of Cd₅Zn₂MT-II. The SOD activity of Cu₄Zn₃MT-III to quench O₂ ^–was assayed by the riboflavine-methionine-nitrobluetetrazolium (NBT) method which catalyzed the dismutation of superoxide (O₂ ^–) at pH 7.8 with an IC₅₀ value of 1.50×10^–6 M for Cu₄Zn₃MT-III and 1.62×10^–6 M for Cd₅Zn₂MT-II. Additionally, the down-regulation of GIF may be a main factor in the decrease of the scavenging ability for the free OH and O₂ ^–radicals, which is possibly associated with the pathogenesis of neurodegenerative disease.     

In vitro propagation of the bamboo (Bambusa tulda Roxb.) through shoot proliferation

An efficient protocol has been developed for the in vitro propagation of Bambusa tulda through shoot proliferation. Shoots from 3-week-old aseptically grown seedlings were used to initiate cultures. Multiple shoots were obtained on liquid Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (8×10^–6M) and kinetin (4×10^–6M). Continuous shoot proliferation at a rate of 4–5 fold every three weeks was achieved through forced axillary branching. More than 90% of the shoots could be rooted on a modified MS medium containing indoleacetic acid (1×10^–5M) and coumarin (6.8×10^–5M). Following simple hardening procedures, the in vitro raised plants were transferred to the soil with more than 80% success.Abbreviations BAP 6-benzylaminopurine - 2-ip 6-,-dimethylallylaminopurine - Kn kinetin - IAA 3-indoleacetic acid - IBA 3-indolebutyric acid - NAA 1-naphthaleneacetic acid     

Magnetic susceptibilities of lake sediment and soils on the shoreline of Lake Naivasha,Kenya

On steep, unvegetated slopes, sands (particle sizes 0.063 to 2 mm) and gravels (2–64 mm) erode from the shoreline of Lake Naivasha (Kenya) and enter the lake basin. This occurred freely where fringing papyrus (Cyperus papyrus) swamp had been cleared in favour of landing jetties or agriculture. Gravel-sized particles have been recovered up to 60 m offshore and sands to 80 m. In an area where papyrus was undisturbed and the swamp margin was 52 m wide, gravel did not enter the lake and sands penetrated to around 35 m. Large particles are much easier to trace to source and to manage than the finer silts and clays (<0.63 m) that form the bulk of Lake Naivasha's sediment. The pattern of mass specific magnetic susceptibilities for the <63 m fraction of lake sediment around the southern shoreline of the lake suggests that fine particles enter open water directly from the shoreline and are not transported from the lake's only perennial inflow, the River Malewa in the north. Such particles originate from a hinterland that supports high intensity horticulture and are therefore a potential source of contamination. Mean ± SD susceptibility (_If) immediately offshore papyrus fringe was 0.49 ± 0.08 × 10^-6 m³ kg^-1 compared with higher values of 1.33 ± 0.14 × 10^-6 m³ kg^-1 where there was no papyrus barrier (P<0.0001). The value for five sites in the middle parts of the lake was 0.45 ± 0.02 × 10^-6 m³ kg^-1 with 1.38 ± 0.10 × 10^-6 m³ kg^-1 near the mouth of the River Malewa. The results of this study are evidence, therefore, that conservation of a continuous papyrus margin of about 50 m width is a priority for intercepting particulate material.     

Cell-specific differences in the susceptibility of potential cellular targets of human origin derived from blood and lung following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The induction of cytochrome P4501A (CYP1A1) enzyme activity is one of the best-studied direct effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds and has been shown to be a sensitive biomarker of exposure to polycyclic aromatic hydrocarbons (PAH) in different experimental animal species as well as in humans. TCDD has also been shown to modulate cytokine gene expression in human keratinocytes, including IL-1, TGF- and TFG-₂. In the present studies, the aim was to determine whether different cellular targets of human origin differed in susceptibility to TCDD as measured by CYP1A1 activity and mRNA expression, and whether cytokine gene induction/suppression correlated with TCDD susceptibility. Human airway epithelial cells, alveolar macrophages (AM), peripheral blood monocytes and lymphocytes (PBL) were exposed to 10^-10–10^-7 mol/L TCDD. CYP1A1 enzyme activity was determined by ethoxyresorufin-O-deethylase (EROD) activity, mRNA expression of CYP1A1 was measured by semiquantitative PCR assay. The secretion and/or gene expression of specific cytokines, including IL-6, IL-8, and IL-1 were also examined. Overall, there was a clear correlation between TCDD-induced enzyme activity and CYP1A1 mRNA levels, which were dose-dependently increased in the bronchoepithelial cells and PBL. The human airway epithelial cells (BEAS-S6 cell line and primary cells) appeared to be the most inducible cellular target, with up to 50-fold increases at 10^-8 mol/L TCDD with an EC₅₀ of 3×10^-11 mol/L TCDD. The pokeweed mitogen-activated peripheral blood lymphocytes revealed approximately 5-fold less capacity in CYP1A1 activity, with high interindividual variabilities (EC₅₀ 3×10^-9 mol/L TCDD). In contrast, CYP1A1 enzyme activity in both AM and purified peripheral blood monocytes, which were costimulated with LPS and/or GM-CSF, could not be detected. CYP1A1 mRNA levels, however, were detectable and only marginally enhanced in response to TCDD. The ability of all these cells to express and produce the proinflammatory cytokines IL-6 and IL-8 was neither enhanced nor impaired by TCDD. These results indicate that cell types found in human lung and peripheral blood vary in susceptibility to TCDD, with the lung epithelium being highly susceptible and the alveolar macrophage being nonsusceptible. However, expression and production of specific cytokines such as IL-6 and IL-8, which may potentiate inflammatory processes and/or work as mitogens, does not appear to be influenced by TCDD.     

Cytospectrophotometric study of the kinetics of histochemical reaction of GABA-transaminase in cryostat sections of rat cerebellar cortex

Summary A modification of the histochemical method for demonstration of GABA-transaminase is proposed. Substrate and cofactor concentrations are chosen on the basis of a kinetic investigation in cryostat sections of the rat cerebellar cortex. Enzymatic reactions were measured by quantitative microspectrophotometry. Michaelis constants for -oxoglutarate in the Purkinje cell layer and granular layer (K_m 1.7×10^–3 M) and white matter (K_m 3,8×10^–3 M) are found. It is shown that -oxoglutarate in concentrations higher than 5.2×10^–3 M (1 mg/ml) suppresses the reaction in sections by competitive inhibition. The advisability of addition of malonate, PMS and cyanide to the incubation medium is confirmed. It is suggested that there are some isoenzymes of GABA-transaminase with predominant localization either in neurons or glia.     

Isolation and characterization of the ecto-5′-nucleotidase from a rat glioblastoma cell line

Summary 5-Nucleotidase has been purified from rat glioblastoma cells (Rugli cells). The enzyme has been solubilized from plasma membranes by using Triton X-100 and CHAPS. Two affinity chromatographies on concanavalin A and 5-AMP-Sepharose render the purified enzyme with a high specific activity (76.36 mol AMP-min^–1-mg^–1). The purified enzyme gives a single polypeptide band on SDS-PAGE with an apparent molecular mass of 74 kDa. Active forms with an apparent molecular mass of 135 kDa and 268 kDa are observed when the purified enzyme is analyzed by gel filtration in the presence of either 0.6% sodium deoxycholate or 0.1% Triton X-100, respectively. The purified 5-nucleotidase presents optimum activity at pH 7.8–8.1 either in the presence or in the absence of Me²⁺. A linear Arrhenius plot is observed in the 25–46° C temperature range and an activation energy of 33.7 KJ/mol is calculated. The enzyme is inhibited by EDTA; the activity is partially restored by different divalent cations as Zn²⁺, Mn²⁺, and Co²⁺. The hydrolysis of nucleosides 5-monophosphate shows Michaelis kinetic. The enzyme is inhibited by nucleosides di- and triphosphate. 5-Nucleotidase is a glycoprotein, being its activity inhibited at different extent by various lectins.     

Effect of antagonists of ethylene action on binding of ethylene in cut carnations

Five hours after cut carnations had been treated with a pulse of 1 or 4 mM silver thiosulfate (STS), in vivo ethylene binding in petals was inhibited by 22 and 29%, respectively. When binding was measured 4 days after the 4-mM STS treatment, binding was inhibited by 81%. 2,5-Norbornadiene, which substantially delays carnation senescence, inhibited ethylene binding by 41% at a concentration of 1000 l/l. The Kd for ethylene binding in carnations was estimated to be 0.1 l/l in petals and 0.09 l/l in leaves. The concentration of binding sites was estimated to be 6.0×10^–9 mol/kg of petals and 2.0×10^–9 mol/kg of leaves     

An improved selection method for λlacZ − phages based on galactose sensitivity

The determination of thelacZ mutant frequency in gt10lacZ phage vectors isolated from the transgenic mouse strain 40.6 (MutaMouse), requires the screening of large numbers of phages on -galactosidase activity. Existing methods rely on distinguishing a few white plaques on X-gal containing plates amongst a multide of blue ones which is both time-consuming and expensive. The new screening method described here employs the galactose sensitiveEscherichia coli C lacZ recA galE strain into which a multicopy plasmid has been introduced, which results in over-expression of thegalK andgalT genes. In the presence of phenyl--d-galactopyranoside, a substrate for -galactosidase, this leads to the suppression of lacZ ⁺ phage propagation without affecting the ability of lacZ ^– phages to form plaques. With this method it is possible to screen 1.5×10⁶ phages on a single 9-cm Petri dish. Furthermore, the need for blue/white screening has been eliminated.     

A novel method for quantifying cell migration through tissue engineering scaffolds: evaluation of modified collagen matrices

A novel method to quantify cell migration through potential tissue engineering 3-d scaffolds is described. The migration assay uses a dot-blotting apparatus into which the tissue engineering matrix is placed on top of a nitrocellulose membrane. This assay was used to evaluate human dermal fibroblast migration through four porcine collagen matrices with varying pore diameters and pitch lengths. Fibroblasts were placed on the matrix surface, at between 1 ×10³–3 × 10³ cells mm^–2, and left for 18 h to allow migration. The nitrocellulose membrane was stained with haematoxylin, the membrane digitised and the pixel intensity of the stained cells quantified. We showed that for all matrix variants, migration was more effective with a higher initial seeding density. The application of varying initial cell densities resulted in the greatest extent of cell migration through the matrix variant with pores of 30 m diameter and 400 m pitch length (i.e. 10.3% migration at 1 ×10³ cells mm^–2). This method was coupled with confocal microscopy to evaluate the depth of cell migration within the matrix. At a depth of 20 m cell numbers were similar to those on the matrix surface: at a depth of 100 m only a few cells were observed.     

A small sensor for detecting animal burrows and monitoring burrow water conductivity

An electrical conductivity probe for measuring ground conductivity is described. The probe measures bulk ground conductivity in situ and can assist in locating animal burrows on a centimeter scale and in monitoring conductivity of burrow waters over long period of times. It is shown how burrow caves are located by their conductivity contrast relative to the soil. The conductivity of the water in a burrow cave 70cm under the swamp surface has been recorded over 15 days. The conductivity dropped during/after periods of significant rainfall, and rapidly increased during tidal inundation of the swamp. At times with neither freshwater nor saltwater input through the openings of the burrow on the surface, the conductivity slowly increased presumably due to diffusion of salt through the burrow walls. The diffusion constant was estimated to be 2×10^–9m²/s, being comparable to previously determined diffusion constants for diffusion of salt within the substrate.     

Thymidine and leucine incorporation in soil bacteria with different cell size

Thymidine and leucine incorporation into macromolecules of soil bacteria extracted by homogenization-centrifugation were measured after size-fractionation of the bacterial suspension through different sized filters (1.0, 0.8, 0.6, 0.4 m). The specific thymidine incorporation rate was highest for the unfiltered and 1.0 m filtered suspensions (approximately 10 × 10^–21 mol thymidine bacteria^–1 h^–1), but decreased to 1.39 × 10^–21 mol bacteria^–1 h^–1 for bacteria passing the 0.4 m filter. The proportion of culturable bacteria (percent colony forming units/acridine orange direct counts) also decreased with bacterial cell size from 5.0% for the unfiltered bacterial suspension to 0.8% in the 0.4 µm filtrate. A strong linear correlation (r ² = 0.995) was found between the specific thymidine incorporation rate and the proportion of culturable bacteria. Leucine incorporation gave similar results to the thymidine incorporation. No effects of cell size on the degree of isotope dilution or unspecific labeling of other macromolecules were found either for the thymidine or the leucine incorporation technique. These data indicate that small bacteria, although more numerous than larger ones, not only constitute a smaller proportion of the soil bacterial biomass than larger bacteria, but also contribute to a lesser degree to carbon transformations in soil.     

Structure and function of the yeast vacuolar membrane proton ATPase

Our current work on a vacuolar membrane proton ATPase in the yeastSaccharomyces cerevisiae has revealed that it is a third type of H⁺-translocating ATPase in the organism. A three-subunit ATPase, which has been purified to near homogeneity from vacuolar membrane vesicles, shares with the native, membrane-bound enzyme common enzymological properties of substrate specificities and inhibitor sensitivities and are clearly distinct from two established types of proton ATPase, the mitochondrial F₀F₁-type ATP synthase and the plasma membrane E₁E₂-type H⁺-ATPase. The vacuolar membrane H⁺-ATPase is composed of three major subunits, subunita (M _r =67 kDa),b (57kDa), andc (20 kDa). Subunita is the catalytic site and subunitc functions as a channel for proton translocation in the enzyme complex. The function of subunitb has not yet been identified. The functional molecular masses of the H⁺-ATPase under two kinetic conditions have been determined to be 0.9–1.1×10⁵ daltons for single-cycle hydrolysis of ATP and 4.1–5.3×10⁵ daltons for multicycle hydrolysis of ATP, respectively.N,N-Dicyclohexylcarbodiimide does not inhibit the former reaction but strongly inhibits the latter reaction. The kinetics of single-cycle hydrolysis of ATP indicates the formation of an enzyme-ATP complex and subsequent hydrolysis of the bound ATP to ADP and Pi at a 7-chloro-4-nitrobenzo-2-oxa-1,3-diazolesensitive catalytic site. Cloning of structural genes for the three subunits of the H⁺-ATPase (VMA1, VMA2, andVMA3) and their nucleotide sequence determination have been accomplished, which provide greater advantages for molecular biological studies on the structure-function relationship and biogenesis of the enzyme complex. Bioenergetic aspects of the vacuole as a main, acidic compartment ensuring ionic homeostasis in the cytosol have been described.Abbreviations CCCP carbonyl cyanidem-chlorophenyl hydrazone - DCCD N,N-dicyclohexylcarbondiimide - DES diethylstilbestrol - DIDS 4,4-diisothiocyano-2,2-stilbene disulfonic acid - NBD-Cl 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole - Pi inorganic phosphate - SDS sodium dodecylsulfate - SF6847 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile - SITS 4-acetamide-4-isothiocyanatostilbene-2,2-disulfonic acid - ZW3-14 N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate