Free amino acids in claw muscle and haemolymph from Australian freshwater crayfish at different stages of the moult cycle

Amino acids were measured in claw muscle and haemolymph in the freshwater decapod crustacean, Cherax destructor, at different stages of the moult cycle. The total pool of amino acids in muscles from animals in intermoult (97+/-13 mmol kg(-1) muscle), premoult (80+/-20 mmol kg(-1)) and postmoult (97+/-19 mmol kg(-1)) were not significantly different. Despite the relatively stable total pool of amino acids, there were changes in the concentrations of alanine, glutamine and proline over the moult cycle. Compared to intermoult, claw muscles from animals in premoult had a lower concentration of proline, and animals in postmoult had higher concentrations of alanine and glutamine, but lower concentrations of proline. Concentrations of alanine and glutamine in claw muscle of animals in postmoult were higher and proline concentrations lower than in the same animals during the premoult stage. The concentration of proline in haemolymph was lower in animals in premoult and postmoult compared to intermoult. The total amino acid pool in the claw muscle of Cherax destructor did not change significantly over the moult which is distinctly different to the changes in amino acids reported in the claw muscles of marine decapod crustaceans.     

Moult cycle and its chronology in Mysis mixta and Neomysis integer (Crustacea, Mysidacea): implications for growth assessment

The moult-staging technique was used to determine the main moult stages (MS) and their duration in laboratory-reared juveniles and subadults of Mysis mixta and Neomysis integer. The relative duration of each stage was similar for both species with premoult occupying the major part of the moult cycle (51% and 44% for M. mixta and N. integer, respectively), followed by postmoult (26% and 34%) and intermoult (23% and 22%). Effects of temperature and feeding regimes on the chronology of the moult cycle were investigated. When the duration of the moult cycle (MCD) was extended by manipulating the feeding regime, animals prolonged their late postmoult and early premoult stages. At 12 and 5 °C, no specific moult stage varied in relative duration as long as food supply was high. Field application of moult staging for growth assessment was tested using wild-caught M. mixta. The MCD estimated via moulting experiments was compared to that obtained by analyzing moult stage distribution combined with the experimentally obtained data on stage duration. Close correspondence between two methods of MCD assessment was observed with moult-staging technique being particularly useful in situations when conducting of experiments immediately after collection is not feasible.     

Moult cycle and morphogenesis inHyas araneus larvae (decapoda,majidae), reared in the laboratory

Moult cycle and morphogenesis in larval instars (zoea I, zoea II, megalopa) of the spider crabHyas araneus (L.) were studied in the laboratory. Changes in the epidermis and cuticle were documented photographically at daily intervals to characterize the stages of the moult cycle. Stage A (early postmoult) is a very short period during which the larva takes up water. During late postmoult (B) and intermoult (C) the endocuticle is secreted, and there is conspicuous epidermal tissue condensation and growth. The onset of early premoult (D₀) is characterized by epidermal apolysis, occurring first at the bases of the setae in the telson of zoeal instars or in the rostrum of the megalopa, respectively. Intermediate premoult (D₁) is the main period of morphogenesis, in particular of setogenesis: in the setae of the zoeal telson and carapace there is invagination or (in the zoea II) degeneration of epidermal tissues. Formation of new setae in the interior of epidermal tubules was observed in zoeal maxillipeds and in the antennae of the zoea II and megalopa instars. During late premoult (Stages D_2–4) part of the new cuticle is secreted, and the results of morphogenesis become clearly visible. For technical reasons (rigid exoskeleton) only a preliminary account of the moult cycle in the megalopa can be given. A time schedule is suggested for the stages of the moult cycle. It is estimated that postmoult (A–B) takes ca 9 to 15 % of total instar duration, intermoult (C) ca 22 to 37 %, and premoult (D) ca 48 to 69 %. There is an increasing trend of relative portions of time (% of total instar duration) from instar to instar in Stages A–C (mainly in the latter) and a decreasing trend in Stage D (mainly in D₀ and D_2–4).     

The immune response of the white shrimp Litopenaeus vannamei and its susceptibility to Vibrio infection in relation with the moult cycle

The white shrimp Litopenaeus vannamei (8.0-14.4 g) was examined for haemocyte count, phenoloxidase activity, respiratory burst (release of superoxide anion), phagocytic activity, and clearance efficiency to the pathogen Vibrio alginolyticus in relation with moult cycle (postmoult, A, B; intermoult, C; premoult, D(0)/D(1)D(2)/D(3)). Granular cells were the highest at C and D(0)/D(1)stage, and the lowest at A stage. Hyaline cells and THC (total haemocyte count) were higher at C stage, but lower at postmoult stages. Phenoloxidase activity was the highest at C stage, and the lowest at A stage. Respiratory burst was lower at A stage. Phagocytic activity of shrimps against V. alginolyticus decreased significantly at postmoult and premoult stages. Additionally, the clearance efficiency of shrimps to V. alginolyticus was significantly lower for shrimps at A stage than those at C stage. In another experiment, L. vannamei at different moult stages were injected with tryptic soy broth (TSB)-grown V. alginolyticus (1x10(5)cfu shrimp(-1)) and then held in 34% seawater. After 10 h, the mortality of V. alginolyticus-injected shrimps was significantly higher for shrimps at postmoult stage than those at intermoult stage. Over 48-120 h, the mortality of V. alginolyticus-injected shrimps was 50.0%, 33.3% and 40.0% at postmoult, intermoult and premoult stage, respectively. It is concluded that L. vannamei showed a decrease in resistance at A stage through a reduction of its haemocyte count, phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency against V. alginolyticus.     

Tissue accumulation and the moult cycle in juveniles of the Australian freshwater crayfish Cherax destructor

1. This paper investigates moult stage and size-specific changes in whole body composition during growth in juvenile crayfish in order to better describe the nature of growth and energy use. 2. Composition is described in terms of moult stage and size-specific wet, dry and ash-free dry weight, water, carbon, protein nitrogen, non-protein nitrogen, ash and energy. Dry weight and ash-free dry weight (AFDW) peaked in the middle and in the later stages of premoult. Both peaks were about 2–2.3 times postmoult weight. Losses in tissue weight during ecdysis were substantial in the smallest crayfish but declined with size. Water was taken up between late premoult and early postmoult. Tissue accumulation occurred primarily between B and D₁.₃, with further weight gain largely the result of fluid uptake. Ash increased immediately postmoult with a major peak occurring during intermoult. Mean organic carbon varied between 33 and 35.5% of the body and 49% of the exuvia. Chitin varied between 9 and 17% of body AFDW and made up about 50.5% of the exuvia. Protein content varied between 47 and 62% of body AFDW and about 25% of the exuvia. Carbon, chitin and protein were not affected by moult stage but protein declined with ocular carapace length (OCL) in larger crayfish. Mean caloric content varied between 19 and 22 J mg-¹ AFDW depending on size and moult stage. Caloric content increased with OCL during premoult and early intermoult then declined with size until part of the way through premoult. 3. Relationships between protein, chitin and remaining carbon (organic carbon minus chitin) were examined. It is suggested that protein and some carbon are catabolized during the moulting process, possibly to fuel metabolism. Models are presented showing changes in proximate composition over the moult cycle for two sizes of crayfish, and tissue and energy accumulation and loss over a series of moult cycles and sizes from 3.1 mm to 17 mm OCL.     

Effects of seasonality and moult cycle on the proliferation of nerve cells and on the labelling of ecdysone receptors in an estuarine crab

Decapod crustaceans show proliferation of the nerve cells in the olfactory lobe throughout their lives. However, the regulation of this process is still poorly understood, since it may vary with endogenous and exogenous factors. The objective of the present investigation was to quantify the proliferation of nerve cells and number of nerve cells with ecdysone receptors in the clusters of the central olfactory system in Neohelice granulata, according to moult stages and in different seasons (summer and winter). Three injections of bromodeoxyuridine (BrdU) were administered to the crabs. Brains were sectioned by microtome and fixed on slides for immunohistochemistry with anti-BrdU and anti-EcR antibodies. The proliferation of nerve cells was higher in winter than in summer, probably because in winter the crabs do not breed and the premoult and postmoult periods are longer. Crabs in postmoult exhibited more BrdU-labelled cells than crabs in premoult or intermoult in winter, because of a greater number of mitoses related to an increase in body size and addition of olfactory receptor neurons. The number of EcR-labelled cells was higher in premoult than in postmoult or intermoult in winter. The proliferation of nerve cells is regulated seasonally and according to moult stages.     

Ammonia excretion of the sand-shrimpCrangon crangon (L.) during the moult cycle

Summary Ammonia excretion in the shrimpCrangon crangon (L.) shows a cyclical pattern during the moult cycle. At the early premoult (stage D₀) there is a 7 to 17% increase over that characteristic at intermoult; at late premoult (stage D₂) excretion decreases to a minimum; immediately after ecdysis, the excretion rate is 1.5 to 2.6 times higher than at the intermoult stage (Table 1).These variations appear to be correlated with protein breakdown and protein synthesis, and possibly with periods of high metabolic activity (growth resulting from cell multiplication).Ammonia excretion of animals in standing water is higher than of those in running sea-water. Excretion might possibly be influenced by a semi-lunar rhythm.     

The moult cycle of the terrestrial isopod Armadillo officinalis Duméril, 1816 (Crustacea: Isopoda: Oniscidea)

The present work focuses on the moult cycle of Armadillo officinalis. For a 100‐day period, 134 animals were observed and routinely examined with the aim of detecting distinctive morphological characters in the several stages and substages of the moult cycle and of disclosing their duration. Statistical tests and Poisson regression models with robust standard errors were used to investigate differences and relationships between moult and the size and gender of the animals. The appearance of the calcium carbonate deposits on the pereon sternites during the premoult stage was documented in detail, and three main substages were identified. The average duration of the premoult and of the biphasic ecdysis was about 12 and 1.5 days, respectively. This observation period, however, did not allow to establish a determined average duration of the intermoult stage, which was extremely variable. This stage lasted for 2 months or more in most of the cases observed, but about 1‐month‐long intermoult stages were also recorded. No statistically significant association was found between the number of moults and gender and size of the animals.     

Haemolymph constituents and osmolality as functions of moult stage,body weight,and feeding status in marron,Cherax cainii () and yabbies,Cherax destructor (Clark, 1936)

The study investigates the change in osmolality and haemolymph constituents in marron Cherax cainii and yabbies Cherax destructor associated with moult stages, body weights and their feeding status. A total of 582 haemolymph samples from 5 moult stages (postmoult-AB, intermoult-C, and premoult stages – D₀, D₁, D₂), two body weight classes (2–15 g and 61–75 g) and nutritional status were used for analysis of osmolality, protein, glucose, and ionic concentrations of potassium and chloride following the standard biochemical procedures. The haemolymph protein, glucose, potassium and chloride levels were highest at intermoult and early premoult stages, and lowest at postmoult in both crayfish species. Except protein, no significant differences were seen in analyzed parameters between various weight classes and two species. Haemolymph osmolality, protein and glucose were significantly higher in fed crayfish, whereas no variations in haemolymph potassium and chloride concentrations were observed between the fed and unfed crayfish. Maximum osmolality was recorded at 7–8 h after feeding in both crayfish species. The results showed that the biochemical changes in the haemolymph of marron and yabbies are related to moult stages, body weight and feeding and thus can be used as tools for determining suitable diets.     

Chronic environmental pollution alters adenylate levels in needles and fine roots of Scots pine

Contents of ATP, ADP, AMP, inorganic phosphate, and values of ATP/ADP ratio, adenylate energy charge (AEC), phosphorylation potential (PP) and adenylate kinase activity were analysed in needles and fine roots of Scots pine trees grown at the polluted and control (free of acute air pollution) site. Also chemical properties of the soil and mineral elements in needles from both sites were analysed. In comparison with the control, developing needles from the polluted site contained less ATP, the same amount of ADP and more AMP, and had lower values of ATP/ADP, AEC and PP. In one-year-old needles from the polluted site no change or a decrease in ATP was recorded, while ADP decreased, AMP increased, AEC did not change, and ATP/ADP ratio and PP were higher. In fine roots from the polluted site AMP level was higher, while ATP, ADP, ATP/ADP ratio, PP and AEC were lower than in the control.     

Effect of bilateral eyestalk ablation on ovarian development and moulting in early and late intermoult stages of female spiny lobster Panulirus homarus (Linnaeus, 1758)

Abstract

The present study describes the effect of bilateral eyestalk ablation (BESA) on reproduction and moulting of spiny lobster Panulirus homarus females in their early and late intermoult stages. The lobsters obtained from the wild were conditioned for the experiment. The experiments were conducted at the Calicut research centre of Central Marine Fisheries Research Institute, India. Eyestalk ablation of females was done by ligation. The responses of the bilaterally eyestalk-ablated lobsters were statistically analysed. BESA conducted on the early and late intermoult stages resulted in the simultaneous acceleration of the somatic growth and reproductive processes with higher emphasis for oogenesis in lobsters ablated in the early intermoult phase and comparatively lower activity in those ablated in the late intermoult phase. Ablation in late intermoult phase resulted in faster entry into the premoult stage compared to the control.     

Effects of reduced salinities on development and bioenergetics of early larval shore crab, Carcinus maenas

The shore crab, Carcinus maenas L. (Portunidae), is a coastal and estuarine species, which can live and reproduce under brackish water conditions; freshly hatched larvae have been observed in the field at salinities below 15‰. In the present laboratory study, the tolerance of hypo-osmotic stress was experimentally investigated in early larvae of a marine (North Sea) population of C. maenas reared at four different salinities (15, 20, 25, 32‰). Two and 4 days after hatching, the Zoea I larvae were moult-staged microscopically, and their rates of respiration and growth (changes in dry weight, W, carbon, C, nitrogen, N, and hydrogen, H) were measured. Survival and development were monitored until the megalopa was reached: 15‰ did not allow for development beyond the first zoeal stage, while metamorphosis to the megalopa was reached at salinities ≥20‰. At 20‰, development was significantly delayed and mortality enhanced as compared with 25 and 32‰. Rates of growth and respiration decreased during exposure to reduced salinities ≤25‰. Hence, the suppression of growth could not be explained as a consequence of enhanced metabolic losses per larva. Instead, a partial C budget indicates that the Zoea I larvae suffered from decreased capabilities of assimilating ingested and subsequently converting assimilated matter to tissue growth. Net growth efficiency (K₂, C-based) was at 25 and 32‰ initially high (>60% during the postmoult and intermoult stages of the Zoea I moult cycle), but decreased during the later stages (down to ≤30% in premoult). An inverse pattern of C partitioning was observed at ≤20‰, with initially low K₂ values (≤21% during the first 2 days of the moult cycle), and a later increase (up to ≥46% in premoult). Thus, larval growth was initially suppressed under conditions of reduced salinity, but this was later (during premoult) partially compensated for by an increase in C assimilation and K₂. Our observations indicate that Zoea I shore crab larvae react during the late stages of their moulting cycle less sensitively against reduced salinities than during postmoult and intermoult. This suggests that the transition between moult cycle stages C and D₀ may be a critical point for effects of hypo-osmotic stress, similarly as already known in relation to effects of nutritional stress. Negative effects were found also when freshly hatched Zoea I shore crab larvae were exposed only transitorily (for 24–72 h) to 20‰, with significantly lower rates of survival, development, growth, respiration, and K₂. These effects increased with increasing duration of initial exposure to reduced salinity.     

Adenylate energy charge, arginine phosphate and ATPase activity in juvenile Homarus americanus during the molt cycle

Neither gill nor hepatopancreas exhibited significant differences in Na+, K+-ATPase activity with molt stage. Hepatopancreatic residual ATPase activity was significantly higher (F = 6.273) in post-molt animals; while gill residual ATPase activity exhibited no significant differences. Muscle AEC did not change with molt stage, but levels of ATP (F = 8.050) and ADP (F = 4.130) were significantly higher in premolt (D3 pleopod stage 5.0-5.5) animals; while levels of arginine phosphate (F = 6.981) were significantly higher in post-molt animals. Arginine phosphate/ATP and ATP/ADP ratios were highest in post-molt animals, but were not statistically significant. Although not significant, changes in Na+, K+-ATPase activity and AEC did suggest alterations in: enzyme activity that correlate with known osmotic compensations occurring during the water uptake and hardening/mineralization processes; and energy metabolism which occur during the molt cycle, respectively.     

Adenylic nucleotides and energy charge during the embryonic development of Bufo arenarum

The contents of ATP, ADP and AMP were determined by HPLC and adenylic energy charge (AEC) was estimated during different stages of the embryonic development of Bufo arenarum up to the tailbud stage. All the developmental stages studied showed a high ATP content (about 1.04-1.48 nmol/emb.). The concentration of ADP was low (0.025-0.041 nmol/emb.) but rose slightly at the neural tube stage. AMP was undetectable before the tailbud stage. AEC values were almost constant (about 0.987-0.992) throughout the period studied. Only a fall at the tailbud stage could be detected which can be related to this more advanced cellular differentiation stage.     

The effect of season and location on phosphoadenylate concentrations and adenylate energy charge in two species of freshwater clams

Summary Concentrations of phosphoadenylate nucleotides and the adenylate energy charge ((ATP+1/2ADP)/(ATP+ADP+AMP)) have been suggested as sensitive integrating measures of the energy state of organisms. This synoptic study investigated the seasonal and spatial variation of phosphoadenylate concentrations and AEC in two freshwater bivalve molluscs, the paper-shell clam, Anodonta imbecillis and the asian clam, Corbicula fluminea. Concentrations of all three adenylates, as well as the total adenylate concentration and adenylate energy charge of both species varied seasonally. These fluctuations were closely related to reproductive periods in both species. Total adenylate concentrations and ATP concentrations were slightly negatively correlated with shell length in A. imbecillis but the ADP and AMP concentrations and AEC were not significantly correlated with shell length. In C. fluminea the AEC was negatively correlated were positively correlated with shell length. Neither species exhibited significant differences in AEC between two collection locations. When C. fluminea collected from the Savannah River were acclimated and fed in the laboratory their AEC increased significantly.     

Adenylate and nicotinamide nucleotides in developing soybean seeds during seed-fill

Profiles of adenylate and nicotinamide nucleotides in soybean seeds were determined during seed-fill. The ATP content per seed increased during the early seed-filling stages to a level of 10 to 12 micrograms per seed. Seed ATP decreased after 40 days of development and reached its lowest level of less than 1 microgram at maturity. The ATP:ADP ratios were relatively constant at all seed development stages. Sharp increases in AMP levels during the late seed-fill stages were paralleled with a disappearance of ATP and ADP pools resulting in a reduced seed energy charge. Energy charge varied from the highest value of 0.78 at mid-seed-fill to less than 0.10 at maturity.     

Growth patterns,chemical composition and oxygen consumption in early juvenileHyas araneus (Decapoda: Majidae) reared in the laboratory

Early (instar I and II) juveniles of the spider crabHyas araneus were reared under constant conditions (12 °C, 32‰S) in the laboratory, and their growth, biochemical composition, and respiration were studied. Every second day, dry weight (W), ash-free dry weight (AFW), and contents of ash, organic and inorganic carbon (C), nitrogen (N), hydrogen (H), protein, chitin, lipid, and carbohydrates were measured, as well as oxygen consumption. Changes in the absolute amounts of W. AFW, and C, N, and H during the moulting cycle are described with various regression equations as functions of age within a given instar. These patterns of growth differ in part from those that have been observed during previous studies in larval stages of the same and some other decapod species, possibly indicating different growth strategies in larvae and juveniles. There were clear periodic changes in ash (% of W) and inorganic C (as % of total C), with initially very low and then steeply increasing values in postmoult, a maximum in intermoult, and decreasing figures during the premoult phase of each moulting cycle. Similar patterns were observed in the chitin fraction, reaching a maximum of 16% of W (31% of AFW). Ash, inorganic C, and chitin represent the major components of the exoskeleton and hence, changes in their amounts are associated with the formation and loss of cuticle material. Consequently, a high percentage of mineral matter was lost with the exuvia (76% of the late premoult [LPM] ash content, 74% of inorganic C), but relatively small fractions of LPM organic matter (15% of AFW, 11% of organic C, 5–6% of N and H). These cyclic changes in the cuticle caused an inverse pattern of variation in the percentage values (% of W) of AFW, organic C, N, H, and biochemical constituents other than chitin. When these measures of living biomass were related to, exclusively, the organic body fraction (AFM), much less variation was found during individual moulting cycles, with values of about 43–52% in organic C, 9–10% in N, 6–9% H, 31–49% of AFW in protein, 3–10% in lipid, and <1% in carbohydrates. All these constituents showed, on the average, a decreasing tendency during the first two crab instars, whereas N remained fairly constant. It cannot be explained at present, what other elements and biochemical compounds, respectively, might replace these decreasing components of AFW. Decreasing tendencies during juvenile growth were observed also in the organic C/N and in the lipid/protein weight ratios, both indicating that the proportion of lipid decreased at a higher rate than that of protein. Changes were observed also in the composition of inorganic matter, with significantly lower inorganic C in early postmoult (2–4% of ash) than in later stages of the moult cycle (about 9%). This reflected probably an increase in the degree of calcification, i.e. in the calcium carbonate content of the exoskeleton. As a fraction of total C, inorganic C reached maximum values of 17 and 20% in the crab I and II instars, respectively. The energy content of juvenile spider crabs was estimated independently from organic C and biochemical constituents, with a significant correlation between these values. However, the former estimates of energy were, on the average, significantly lower than the latter (slope of the regression ≠1). Since organic C should be a reliable integrator of organic substances, but the sum of protein, lipid, chitin, and carbohydrates amounted to only 60–91% of AFW, it is concluded that the observed discrepancy between these two estimates of energy was caused by energy from biochemical constituents that had not been determined in our analyses. Thus, energy values obtained from these biochemical fractions alone may underestimate the actual amount of organic matter and energy. Respiration per individual in juvenile spider crabs was higher than that in larval stages of the same species (previous studies), but their W-specific values of oxygen consumption (QO₂) were lower than in conspecific larvae (0.6–2μg O₂·[mg W]⁻¹). QO₂ showed a consistent periodic pattern in relation to the moult cycle: maximum values in early postmoult, followed by a rapid decrease, and constant values in the intermoult and premoult phases. This variation is interpreted as an effect mainly of cyclic changes in the amounts of cuticle materials which are metabolically inactive. From growth and respiration values (both expressed in units of organic C), net growth efficiency, K₂, values may be calculated. In contrast to previous findings in larval stages, K₂ showed an increasing trend during growth of the first two juvenile instars ofH. araneus.     

Effects of starvation on moult cycle and hepatopancreas of Stage I lobster(Homarus americanus) larvae

Effects of feeding and starvation on the moult cycle and on the ultrastructure of hepatopancreas cells were studied in Stage I lobster larvae (Homarus americanus Milne-Edwards). The relative significance of yolk and first food was quite different in larvae originating from two females. This difference was evident also in the amounts of stored lipid in the R-cells of the larval hepatopancreas. Most larvae from one hatch were, in principle, able to develop exclusively with yolk reserves (without food) to the second instar. The larvae from the second hatch showed lecithotrophic development only to the transition between late intermoult and early premoult (Stages C/D₀ of Drachs's moult cycle) of the first larval instar. When initial starvation in this group lasted for 3 days or more, the point of no return (PNR) was exceeded. After the PNR, consumption of food was still possible, but development ceased in the transition C/D₀ or in late premoult (D_3–4). It is suggested that these stages of the moult cycle are critical points were cessation of development and increased mortality are particularly likely in early larval lobsters under nutritional stress. Examination of hepatopancreas R-cells suggested that the PNR is caused by an irreversible loss of the ability to restore lipid reserves depleted during initial starvation. Initial periods of starvation ending before the PNR prolonged mainly Stage D₀ of the same instar (I). During this delay, structural changes in the R-cells caused by the preceding period of starvation were reversed: reduced lipid inclusions, swollen mitochondria, an increased number of residual bodies indicating autolysis, and a reduction of the microvillous processes. Continually starved larvae which showed lecithotrophic development throughout the first instar and were then re-fed after moulting successfully, had later a prolonged intermoult (Stage C) period in the second instar. This shows that, despite occasional lecithotrophy, food is an important factor in early larval development of the lobster.     

Pre-steady state kinetic studies on H(+)-ATPase from Candida albicans.

The mechanism of ATP hydrolysis by plasma membrane H(+)-ATPase from Candida albicans has been investigated by following the kinetics of H(+) liberation/absorption and the UV difference spectrum in a stopped flow spectrophotometer. A distinct pre-steady state phase of ATP hydrolysis could be defined. While the rapid mixing of P(i) and ATPase produced no transient pH changes, the mixing of ADP leads to the release of 1 H(+) per molecule of ATPase. Rapid mixing of ATP with ATPase releases about 2 H(+) per molecule of ATPase, of which around 1.3 H(+) are reabsorbed. The magnitudes of both H(+) release and absorption were found to be independent of ATP concentration. The rate of H(+) release (k(f)) shows ATP dependence while the rate of H(+) absorption is independent of ATP concentration. The rate of H(+) liberation with ADP, on a concentration basis, was far less as compared with ATP, indicating a low affinity of the ATPase for ADP. No change in the difference spectrum was observed with ADP. The stoichiometry of ATP binding to PM-ATPase was found to be unity from UV-difference spectrum studies. The k(f) values for H(+) release and for the appearance of a difference spectrum following the addition of ATP were found to be similar beyond a 1:1 ratio of ATP:ATPase. The results obtained lead us to propose a 4-step kinetic scheme for the mechanism of ATP hydrolysis.