Μmol Pi Research Articles

Hepatic Na, K‐ATPase Development in the Rat

SummaryThe development of the enzyme Na, K‐ATPase was studied in hepatic tissue from Sprague‐Dawley rats at 18–20 days of gestation. 1, 3, 5, 7, 14, 21, and 30 days, and young adulthood (45 days). A developmental pattern was demonstrated for total and membrane‐associated Na, K‐ATPase activity. The activity in tissue homogenate, expressed per gram tissue, increased from late fetal life, 43.4 ± 1.3 μmol Pig liver−1 h−1.until 21 days of age, when an adult level of 187.3 ± 19.6 μmol Pi g liver −1 h−1 was attained. A less pronounced ontogenic pattern was observed when enzyme levels were expressed as specific activity per milligram protein. The activity profile in a crude membrane preparation was similar. The potential for stimulation of enzyme activity by glucocorticoids was studied in 11‐day‐old animals injected with cortisone acetate (10 mg 100 g body weight−1) for 3 days. Enzyme specific activity was inducible: Specific activity was greater in cortisone‐treated animals, 1.794 ± .043 μmol Pi mg protein −1 h −1, versus controls, 1.258 ± 0.043 μmol Pi mg protein−1 h−1 (p < 0.01). We postulate that this developmental pattern for hepatic Na, K‐ATPase activity may be a reflection of, or a contributing factor in, the ontogeny of sodium‐dependent hepatic transport, such as that for bile salts.

Glomerular Na+-K+-ATPase activity in acute and chronic diabetes and with aldose reductase inhibition.

Sorbitol accumulation, myo-inositol depletion, and reduced Na+-K+-ATPase activity have been identified in experimental diabetes in several tissues in which diabetic complications occur. However, a reduction in renal Na+-K+-ATPase activity has not been universally reported, prompting us to examine the influence of diabetes duration on the activity of this enzyme complex in isolated glomeruli. Additionally, we examined the ability of the aldose reductase inhibitor sorbinil to directly stimulate glomerular Na+-K+-ATPase activity in vitro, an area of interest in view of the central position that the ability of sorbinil to restore Na+-K+-ATPase activity in vivo occupies in the interpretive scheme that links associated changes in sorbitol, myo-inositol, and Na+-K+-ATPase to enhanced polyol-pathway activity. Glomerular Na+-K+-ATPase activity was significantly decreased in rats with acute (less than 18 days) streptozocin-induced diabetes but was significantly greater than control values in rats with more chronic (greater than 32 days) diabetes. In vitro addition of sorbinil (1 x 10(-7) M) directly stimulated Na+-K+-ATPase in control (0.627 +/- 0.090 vs. 0.843 +/- 0.098 mumol P1.mg-1.min-1) but not diabetic glomeruli. These results indicate that the time of examination after induction of diabetes critically influences glomerular Na+-K+-ATPase activity and suggest that sorbinil, at least in normal glomerular tissue, has a membrane-associated effect that may be independent of its aldose reductase-inhibiting property.

Erythrocyte Na-K-ATPase membrane activity in obese patients fed over a long-term period with a very-low-calorie diet

In a search for the role of long-term hypocaloric feeding on the expression of the erythrocyte Na pump in obesity, we examined three groups of subjects. Group 1 consisted of 10 obese subjects who had been under treatment for a long period of time with a very-low-calorie diet (500 kcal/d) while group 2 consisted of 10 age-, sex-, and body mass index-matched obese subjects on their usual diet; in the third group, 12 normal-weight subjects on a free diet served as controls. There was no difference between the groups in the number of erythrocyte binding sites per cell. On the contrary, the Na-K-ATPase activity was significantly lower in the obese group 1 (0.35 ± 0.09 μmol Pi × mg protein −1 × h −1) compared to that observed in the obese group 2 (0.42 ± 0.07, P < .05) and in control subjects (0.45 ± 0.06, P < .05). Sex, duration of hypocaloric feeding, and the amount of weight loss before the study in the obese group 1 seemed not to be related to the Na pump parameters. We conclude that long-term severe hypocaloric feeding may be a factor in altered erythrocyte Na-K-ATPase in obese individuals.

Why do hagfish have gill "chloride cells" when they need not regulate plasma NaCl concentration?

Two enzymes implicated in branchial ion transport, Na+-K+-ATPase and carbonic anhydrase, were localized in gill ionocytes ("chloride cells") of the Pacific hagfish, Eptatretus stouti, by light microscopic histochemical techniques. In hagfish, ouabain-sensitive Na+-K+-ATPase activity was confined to apical halves of ionocytes, where most of the cytoplasmic tubular system is located. In marine teleosts, Na+-K+-ATPase was noted in chloride cells and erythrocytes. Acetazolamide and potassium cyanate sensitive carbonic anhydrase activity occurred throughout the cytoplasm and nucleus of hagfish ionocytes. Biochemical assay of hagfish gill homogenates for Na+-K+-ATPase yielded a specific activity of 3.1 μmol Pi∙mg protein−1∙h−1 at 37 °C. This resembles values we obtained for freshwater fish (Carassius auratus: 3.3 μmol Pi∙mg protein−1∙h−1; Tilapia shirana: 3.7 μmol Pi∙mg protein−1∙h−1), and is less than values we obtained for marine teleosts (Pomacentrus spp.: 13 μmol Pi∙mg protein−1∙h−1; Gillichthys mirabilis: 6.7 μmol Pi∙mg protein−1∙h−1). Hagfish resemble freshwater teleosts in many other gill features related to ion transport. The presence of carbonic anhydrase in gill ionocytes of hagfish supports the proposal that these cells function in acid–base regulation, i.e., that they exchange H+ for Na+ and [Formula: see text] for Cl−.

Preparation of a highly active ATPase of the mesophilic cyanobacterium Spirulina maxima

In this work, we report new studies on the ATPase attached to the photosynthetic membranes of the mesophilic cyanobacterium Spirulina maxima. This enzyme does not display persistent latency as had been previously reported for the ATPase of Spirulina platensis. The enzyme is readily activated by the careful application of methods currently used to activate chloroplast CF1. Photosynthetic membranes of Spirulina maxima show a Mg(2+)-dependent ATPase activity of 195±25 μmol Pi (mg chl)(-1) h(-1) after a light plus dithiothreitol (DDT) treatment. Methanol treatment of these membranes elicits Mg(2+)-dependent ATPase activity of 222±18 μmol Pi (mg chl)(-1) h(-1).Here, we also describe the purification of the soluble coupling factor AF1 of Spirulina maxima. This enzyme is unique among mesophilic cyanobacterial F1 preparations in regard to its high specific Ca(2+)-dependent ATPase activity after heat treatment (14.75±1.91 μmol Pi (mg prot)(-1) min(-1)) and its room temperature stability.

Effects of anaerobic growth conditions on phosphorus tissue concentrations and absorption rates of southern pine seedlings

A non-circulating, continuously flowing solution culture was used to examine the long- and short-term effects of anaerobic growth conditions on phosphorus uptake in 12-week-old seedlings of three pine species. Sand pine (Pinus clausa (Engelm.) Sarg.) and a drought-hardy loblolly pine (P. taeda L.) had the largest reductions in biomass after 8 weeks in anaerobic solution, whereas the more flood-tolerant pond pine (P. serotina Michx.) and wet-site loblolly pine seedlings were least affected. Anaerobic growth conditions reduced P concentrations in the shoot and increased P concentrations in the root. Short-term (32)P-uptake experiments were conducted with intact seedlings to determine absorption rates (influx) of inorganic phosphate (Pi) by aerobically and anaerobically grown pine seedlings. Influx of Pi was weakly correlated with shoot fresh weight, root fresh weight, root P and shoot P per gram fresh weight root, suggesting that internal P levels and biomass influenced Pi absorption. The highest Pi absorption rates were found in anaerobically grown seedlings under anaerobic (32)P-uptake conditions. Of these, sand pine had the highest absorption rate in 50 microM KH(2)PO(4) (0.96 micromol Pi g(-1) FW root h(-1)), and the wet-site loblolly pine exhibited the lowest rate (0.24 micromol Pi g(-1) FW root h(-1)). Aerobically grown seedlings had similar Pi absorption rates that were not significantly affected by O(2) concentration in the (32)P-uptake solution.

Effects of digitoxin and hypokalaemia on pancreatic NaHCO3secretion and pancreatic Na, K-ATPase activity

To study the role played by Na,K-ATPase in the pancreatic secretion of NaHCO3, experiments were performed in 20 anaesthetized, secretin-infused pigs (3.0 clinical units X kg b. wt. X h-I). The relationship between pancreatic NaHCO3 secretion and arterial pH was obtained before and during Na,K-ATPase inhibition by digitoxin and hypokalaemia. Na,K-ATPase activity in pancreatic tissue homogenate averaged 5.45 (5.02-6.68) mumol Pi X mg X protein X h-I. Retrograde injection of 0.5 ml 1.4 X 10(-4) mol X l-I digitoxin into pancreatic ducts reduced pancreatic Na,K-ATPase activity by 3I(I8-47)%, while intra-arterial injection of 0.2 mg X kg b. wt-I digitoxin reduced pancreatic Na,K-ATPase activity by 50(45-56)%. Digitoxin and hypokalaemia reduced the rate of pancreatic NaHCO3 and shifted the normal, proportional relationship between NaHCO3 secretion and arterial pH towards higher pH. Hypokalaemia reduced Na,K-ATPase activity and NaHCO3 secretion in proportion. These effects indicate that Na,K-ATPase helps to sustain the requisite electrochemical potential gradients for driving H+ ions, and hence HCO-3 ions, out of secretory cells.

Bicarbonate-stimulated ATPase activity of bovine liver alkaline phosphatase

A commercial preparation of bovine hepatic alkaline phosphatase was found to have a Mg2+-stimulated ATPase activity. The pH optimum was 8.5, the Km for ATP was 4.2 X 10(-5) M and the Vmax was 88.3 mumol Pi . h-1 . mg-1. HCO3- had a stimulatory effect on Mg2+ -ATPase activity. Other anions had no effect or an inhibitory effect while Na+, K+ and ouabain had no effect. Purification of the commercial preparation by gel filtration and affinity chromatography yielded a fraction with alkaline phosphatase and (Mg2+ + HCO3-)ATPase activities that had been enriched respectively 27-fold and 23-fold; both activities were inhibited by levamisole (93.1% and 93.8%, respectively) and the purified fraction was found to be a single protein on sodium dodecyl sulfate polyacrylamide gel electrophoresis. These results suggest that alkaline phosphatase and (Mg2+ + HCO3-)ATPase may be properties of the same liver protein that might be involved in biliary HCO3- transport and bile secretion.

INCREASED CALCIUM PUMP TURNOVER RATE IN CARDIAC SARCOPLASMIC RETICULUM IN FETAL SHEEP

We have previously reported decreased Ca uptake and Ca-dependent ATP hydrolysis in cardiac sarcoplasmic reticulum (SR) vesicles isolated from fetal sheep as compared to SR vesicles from maternal sheep. These indices of SR Ca-handling are dependent upon the relative amounts of ATPase protein present but are expressed as μmoles of Pi or Ca per mg SR protein. To determine if the apparently decreased Ca tranport capacity in the fetal vesicles is due to the presence of varying amounts of other SR proteins, we measured acylphosphoprotein (EP) concentrations and then calculated enzyme turnover rates in SR vesicles isolated from fetal (0.7 gestation) and maternal sheep. EP concentrations were measured using specially developed gel electrophoresis techniques. The enzyme turnover rate is the ratio between Vmax of CaATPase and EP concentration and represents actual turnover of Ca pump sites.These data suggest a significantly higher apparent turnover rate for the Ca pumps on the fetal SR vesicles. We conclude that there is an intrinsic difference in Ca transport function between these SR preparations that is independent of possible differences in protein composition between the preparations.

The effect of IAA on the activity of Ca+2-ATPase from maize chloroplasts

Summary From maize chloroplasts we isolated and purified an electrophoretically homogeneous Ca 2+ -ATPase preparation, with specific activity of 5.14 μmoles Pi · mg −1 protein · mg −1 , and with complete subunit composition. IAA at 10 −3 -10 −10 M concentrations failed to activate Ca +2 -ATPase in vitro , whereas the enzymic activity was pronouncedly stimulated when leaf fragments were — prior to enzyme isolation — incubated with 10 −5 M IAA for 60 min. A highly purified plasmatic membrane fraction was obtained by differential centrifugation in discontinuous sucrose gradient. These plasmatic membranes were found to contain a closely bound factor, which was released in the presence of IAA (10 −5 M) and activated Ca +2 -ATPase.

Regulation of sodium in the shore crabCarcinus maenas, adapted to environments of constant and changing salinities

The activity of Na-K-ATPase was determined in the posterior gills of the shore crabCarcinus maenas during a period following transfer from 35 to 10 ‰ salinity and vice versa at 15 °C. After transfer from high to low salinity, Na-K-ATPase activity increased from 3.2 to 7.0 μmoles Pi mg protein−1 h−1 within a period of 2 to 3 weeks. Transfer of crabs from low to high salinity resulted in reduction of activity from 7.4 to 4.5 μmoles Pi mg protein−1 h−1 within about the same period. The relatively slow response following salinity change indicates that the amounts of Na-K-ATPase in the gills may play a role in hyperionic Na regulation in relatively constant brackish-water environments. Instant responses to salinity result from activation and inhibition of Na-K-ATPase activity by Na. Gill Na-K-ATPase is activated by the Na concentration of the incubation medium to attain a steep maximum at about 75 mM Na, which corresponds to the lowest environmental Na levels tolerated byC. maenas equivalent to a salinity of ca 6 ‰. Activity greatly decreased towards higher Na levels, equivalent to the salinity of normal sea water, at which hyperregulation no longer occurs. Selective addition of either Na or Cl to brackish water of 9 ‰ S resulted in effective hyperregulation of the non-increased ion, and passive distribution between medium and blood of the increased ion. These data indicate that under appropriate conditions the normally coupled transport of Na and Cl may be uncoupled and take place independently of each other.

Seasonal Changes in Gill (Na+K)-ATPase Activity in Atlantic Salmon Retained in Fresh Water after Smolting

Gill (Na+K)-adenosine triphosphatase (ATPase) activity of Atlantic salmon Salmo salar was monitored to determine how the activity of this enzyme varied seasonally when smolts were retained in fresh water. In early June, after the fish had smolted, ATPase activity varied from 27 to 30 μmoles phosphate (Pi)˙mg protein (Pr)−1˙hour−1. It steadily declined to just above 6 μmoles Pi˙mg Pr−1˙hour−1 by October, remained low during winter, and began to rise again in March. A rise in enzyme activity during autumn, documented by others for Pacific salmon Oncorhynchus spp., was not evident among Atlantic salmon. Received July 20, 1982 Accepted May 31, 1983

Characterization of phosphate absorption in maize root cortex segments

Uptake of phosphate ions by 1 mm segments of isolated maize root cortex layers was studied. Cortex segments (from roots of 8 days old maize plants) absorb phosphate ions from 1 mM KH2PO4 in 0.2 mM CaSCO4 at the average rate of 34.3 ±3.2 μg Pi g−1 (fr. m.) h−1,i.e. 0.35± 0.02 μmol Pi g−1 (fr. m.) h−1. Phosphate uptake considerably increases after a certain period of “augmentation”,i.e. washing in aerated 0.2 mM CaSO4. This increase is completely blocked by the presence of 10 μg ml−1 cycloheximide. The relation of uptake rate to phosphate concentration in the medium was shown to have 3 phases in the concentration range of 0.02 - 40 mM. Transition points were found between 0.8–1 mM and 10–20 mM. Following Km and Vmax values were found: Km[mM] : 0.37 - 3.82 - 27.67 Vmax[μg Pi g−1 (fr. m.) h−1] : 3.33 - 39.40 - 66.67 We have found no sharp pH optimum for phosphate uptake. It proceeds at almost constant rate till pH 6.0 and then the uptake rate drops with increasing pH. At low phosphate concentrations (1 mM) the lowest uptake rate was found at 5 and 13 °C, while the uptake is higher at 5 °C than at 13 °C at phosphate concentrations higher than 1 mM. At these concentrations uptake rate at 35 °C is lower than at 25 °C. Phosphate uptake considerably decreased in anaerobic conditions. DNP and iodoacetate (0.1 mM) completely blocked phosphate uptake from 1 mM KH2PO4, while uptake from 5 and 10 mM KH2PO4 was left unaffected by these substances. The inhibitors of active - SH groups NEM and PCMB inhibited phosphate uptake: 10−3 M NEM by 81.6%, 104 M NEM by 42% and 10−4 M PCMB by 42%.

Involvement of free radicals in the pathophysiology of ischemic heart disease

Acute myocardial ischemia results in a decrease in developed tension and an increase in resting tension. A breakdown of the excitation–contraction coupling system can explain the behavior of ischemic muscle at a subcellular level. We have identified a specific defect in the sarcoplasmic reticulum (SR) from the ischemic myocardium; i.e., the uncoupling of calcium transport from ATP hydrolysis. The mediators of this excitation–contraction uncoupling process have not been identified. It is now established that the intracellular pH of the ischemic myocardium is in the range of 6.4 but the role of protons and potential role of free radicals have not been identified. We have hypothesized that protons and free radicals may interact to produce the excitation–contraction uncoupling of the ischemic myocardium. Cardiac SR was isolated from the wall of canine left ventricle and calcium uptake velocity and Ca2+-stimulated Mg2+-dependent ATPase activity determined. Increasing proton concentration between pH 7.0 and 6.4 significantly reduced calcium uptake rates (pH 7.0 = 0.95 ± 0.02; 6.4 = 0.50 ± 0.02 μmol Ca2+∙mg protein−1∙min−1; p &lt; 0.01) with no effect on ATPase activity. Calculated coupling ratios (micromoles Ca2+/micromoles Pi) decreased from 0.87 ± 0.06 at pH 7.0 to 0.51 ± 0.05 at pH 6.4. At pH 7.0, the generation of exogenous free radicals from the xanthine (X) – xanthine oxidase (XO) system significantly depressed both calcium uptake rates (control = 0.95 ± 0.02; X + XO = 0.15 ± 0.02) and ATPase activity (control = 1.05 ± 0.02; X + XO = 0.30 ± 0.01 μmol Pi∙mg protein−1∙min−1; p &lt; 0.01). The decreases in calcium uptake and in ATPase activity were completely reversible with superoxide dismutase (SOD). At pH 6.4 in the presence of xanthine and xanthine oxidase, there is a further depression of calcium uptake rates (control = 0.50 ± 0.02; X + XO = 0.11 ± 0.01; p &lt; 0.05) but there is no SOD-reversible component. The addition of SOD + 20 mM mannitol normalized calcium transport at pH 6.4. The calculated coupling ratio at pH 6.4 in the presence of free radicals was 0.13. This value is identical with that reported for the ischemic myocardium. In the absence of an exogenous free-radical generating system, preincubation of whole heart homogenate at pH 6.4 resulted in a significant depression of calcium transport. At 1 min preincubation at pH 6.4, the return of the pH to 7.0 reversed the inhibiting effects of pH 6.4. At 5 min preincubation at pH 6.4, the system required both normalization to pH 7.0 and SOD to reverse the inhibiting effects of acidosis. At 15-min preincubation, the system required both SOD and 20 mM mannitol to reverse the depressant effects of acidosis. It is concluded that at pH 7.0, the xanthine – xanthine oxidase system generates superoxide anion [Formula: see text] which depresses both calcium uptake and ATPase activity. At pH 6.4, this system generates the more lethal hydroxylradical (∙OH) which uncouples calcium transport from ATP hydrolysis in a manner analogous to that observed in the ischemic myocardium. In the whole heart homogenate, acidosis results in a three phase reaction; an initial reversible effect of protons, followed by the production of [Formula: see text] and finally with continued acidosis, the generation of the ∙OH. Combining our observations with the mechanical function of the ischemic myocardium, it is hypothesized that free-radical generation, most probably the ∙OH, may form the link between the reversible and irreversible phase of myocardial ischemia.

Sodium regulation in the shore crab Carcinus maenas as related to ambient salinity

The activity of Na−K-ATPase was measured in crude homogenates prepared from various organs (leg muscle, pincer muscle, heart, testes, digestive gland, hypodermis, gills 1–9) of shore crabs, Carcinus maenas L., acclimated to salinities ranging between 10 and 50‰ S (in steps of 10‰ S). In all salinities tested, Na−K-ATPase activity was highest in posterior gills 7–9 (10–12 μmol Pi mg protein-1 h-1), followed by anterior gills 1–6 (ca. 2.5 μmol Pi mg protein-1 h-1) and the other organs (in most cases far below 2μmol Pi mg protein-1 h-1). In gills only, Na−K-ATPase activity was salinity-dependent, with the highest values in the lowest salinities and vice versa. In gills 7–9, Na−K-ATPase activity was increased more than threefold following a reduction in salinity from 50 to 10‰ S. Na−K-ATPase activity, expressed as percentage of total ATPase activity, amounted to 60–80% in gills, about 60% in hypodermis and 20–40% in the other organs. Ouabain, a specific inhibitor of Na−K-ATPase activity, reduced serum osmolalities in crabs kept at 9–10‰ S only when injected into the hemolymph (1 and 5 · 10-5 M), but had no effect when dissolved in ambient water (10-4 M). The results obtained underline that crustacean gills are the main organs for ionic regulation, and confirm the hypothesis of the central role of the Na−K-ATPase in active Na uptake as the basic mechanism of hyperregulation in dilute media. Reduction of serum osmolalities following injection of ouabain into the hemolymph confirms previous reports on localization of the sodium pump in the basolateral parts of epithelial cells.

PHYTASE OF MOLDS USED IN ORIENTAL FOOD FERMENTATION

ABSTRACTMolds commonly used in Oriental food fermentation have been examined for their ability to produce phytase. Except for Mucor dispersus NRRL 3103 and Actinomucor elegans NRRL 3104, all the other molds tested produced both extra‐ and intracellular phytase. The enzyme produced by Aspergillus oryzae NRRL 1988 was purified by acetone fractionation, gel filtration, and diethylaminoethyl‐cellulose chromatography. The phytase was successfully separated from an acid phosphatase, and a 44‐fold increase in specific activity was observed. The pH optimum of 5.3 characterizes the enzyme as an acid phosphohydrolase, and its maximum activity at 50°C suggests a relatively high thermostability. The enzyme is also fairly stable over a pH range 3.5‐7.8 at 25°C. The A. oryzae phytase is active with either phytic acid or glycerophosphate. as substrate, but it hydrolyzes phytic acid twice as fast as it does glycerophosphate. However, the enzyme is partially inhibited by high concentration of phytic acid. The Km value of A. oryzae phytase was estimated as 0.47 mM and Vmax was 11.9 μmoles Pi per mg per mg protein.

Effects of chromium compounds on plasma membrane Mg 2+-ATPase activity of BHK cells

An ATPase activity stimulated by divalent ions (Mg 2+, Ca 2+, Mn 2+, Zn 2+) has been observed in intact hamster fibroblasts cultured in vitro (BHK line). Such activity has been determined by the incubation (30 min at 37°C) of washed cell suspensions (about 1 mg of proteins) in a medium containing 100 mM NaCl, 20 mM KCl, 15 mM Tris—HCl (pH 7.4), 10 mM NaHCO 3, 5 mM glucose and equimolar concentrations of ATP and divalent cation. Mg 2+-ATPase activity is insensitive to ouabain and lacks specificity towards nucleoside triphosphate substrates. AMP and ADP are not hydrolyzed under these conditions. Apparent K m of 0.76 mM and V max of 1.46 μmol Pi · mg proteins −1 · h −1 have been calculated for Mg-ATP complex. This ATPase is an ectoenzyme, therefore its activity could be used as a suitable index of the action of chemicals like chromium compounds known for their cytotoxic effects on membrane functions. Salts of trivalent (CrCl 3) and hexavalent (K 2Cr 2O 7) chromium at concentrations ranging from 1 mM to 5 mM inhibit Mg 2+-ATPase. The inhibition by K 2Cr 2O 7 is observed after pretreatment of the cells with this compound followed by its absence from the assay medium “per se” for Mg 2+-ATPase, and it is referred to the alterations of membrane bound enzyme structures by the oxidizing hexavalent chromium. The inhibition by CrCl 3 is mainly evident when this compound is present in the incubation medium, and is referred to the interaction of trivalent chromium with Mg 2+-ATP as it is partially reversed by increasing Mg 2+-ATP concentration.

Sodium Transport, Ouabain Binding, and (Na+/K+)-ATPase Activity in Down's Syndrome Platelets

Summary: The mechanism of low serotonin content in Down's syndrome (DS) platelets was studied by comparing the activity of (Na+/K+)-ATPase, ouabain binding, and rate of outward movement of sodium in platelets from DS and control subjects. Ouabain inhibited (Na+/K+)-ATPase activity in sodium iodide-extracted platelet membranes from 10 normal subjects had an average value of 2.87 ± 0.20 μmol Pi./hr/109 platelets whereas the activity from DS platelet membranes was 2.13 ± 0.19 μmol Pi/hr/109 platelets (P < 0.02). Total ATPase activity in normal platelet membranes was 3.58 ± 0.33 μmol Pi/hr/109 platelets whereas that from DS membranes was 2.49 ± 0.35 μmol Pi/hr/109 platelets (P < 0.02). (Na+/K+)-ATPase activity is decreased in DS platelets. Ouabain binds specifically to (Na+/K+)-ATPase sites on membranes. The amount of ouabain bound by DS platelets is less than that by control platelets. The average amount of ouabain bound was 1.08 ± 0.04 × 10-15 mol/109 platelets for controls and 0.86 ± 0.02 × 10-15 mol/109 platelets for DS (P < 0.001). There appear to be fewer (Na+/K+)-ATPase sites on DS platelets compared to normal. The outward movement of Na+ in platelets is catalyzed by the enzyme (Na+K+)-ATPase. The average ouabain-sensitive outward movement of sodium in normal platelets was 9.03 ± 0.46 μmol/hr/109 platelets. For DS subjects the average outward movement was 50% of normal, 4.41 ± 0.14 /μmol/hr/109 platelets. The rate of outward Na+ transport is significantly less (P < 0.001) in DS compared to normal platelets. Decreased serotonin content of DS platelets may be secondary to decreased (Na+/K+)-ATPase activity, which results in decreased rate of outward Na+ transport and increased Na+ content. As serotonin is cotransported with Na+, the decreased rate of serotonin uptake may be secondary to increased Na+ content of DS platelets. Speculation: If decreased (Na+/K+)-ATPase activity, decreased rate of Na+ outward transport, increased Na+ content, and decreased rate of serotonin uptake were also present in synaptosomes of DS patients, decrease in amine content might occur and result in altered ncurotransmission.

Role of gill Na+ + K+ ‐dependent ATPase in acclimation of blue crabs (Callinectes sapidus) to low salinity

AbstractMale blue crabs (Callinectes sapidus Rathbun) were acclimated to salinities of 5 and 34 o/oo. Microsomal preparations of gills from these animals possessed Na+ + K+‐dependent ATPase specific activities of 2.33 ± 0.11 and 1.29 ± 0.11 μmoles Pi min per mg protein respectively, implicating the Na+ + K+‐dependent ATPase in the previously recognized ability of the blue crab to maintain high hemolymph sodium levels in low salinity environments. The specific ATPase activity of gill microsomes increased significantly within two and one‐half hours after transfer of crabs from high to low salinity, prior to stabilization of hemolymph sodium levels. The ATPase isolated from crabs acclimated to the two different salinities were both highly sensitive to ouabain, and required either K+ or NH4+ as well as Na+. Sodium‐dependent phosphorylation of both enzymes was blocked by K+ or NH4+. Polyacrylamide gel electrophoresis of the sodium dodecyl sulfate‐solubilized ATPases revealed one band of radioactivity in each case, with an apparent molecular weight of 104,000. The ability of NH4+ to serve as an effective counterion supports the hypothesis that, in blue crabs acclimated to low salinity, Na+ uptake may be coupled to NH4+ excretion.

Phosphoprotein phosphatase activity of the progesterone-induced purple glycoprotein of the procine uterus

The iron-containing, progesterone-induced, purple glycoprotein from the pig uterus catalyzes the hydrolysis of phosphate groups from phosphvitin. This enzyme which can be purified in large amounts, has a pH optimum at 4.6 and an apparent K m of 0.1 mM for bound phosphate. Its V max with phosphvitin as substrate is about 200, μmoles Pi released/min/μmole enzyme and is at least twenty-fold lower than its activity towards p-nitrophenylphosphate. The enzyme is activated by 2-mercaptoethanol and inhibited by molybdate, fluoride and arsenate. The resemblance of this protein to a number of other acid phosphatases of similar substrate specificity in other organisms, suggests that a broad class of such enzymes might exist in nature.