Membrane-bound Adenosine Triphosphatase Research Articles

AMELIORATION OF ACETAMINOPHEN-INDUCED HEPATOTOXICITY USING SOLANUM TORVUM SW. FRUIT EXTRACT IN WISTAR RATS

Objective: Ayurveda and Chinese pharmacopeia have highlighted the traditional medicinal uses of Solanum torvum Sw. The fruits are ethnomedical used in the treatment of liver and spleen enlargement, cough, and also used as a hematopoietic, antimicrobial, and analgesic agent. In the present study, the amelioration of acetaminophen (APAP)-induced hepatotoxicity of the aqueous extract of S. torvum Sw. fruits is evaluated.
 Methods: The hepatoprotective activity of the fruit extract against APAP insult was evaluated by assessing it is in vivo antioxidants status, membrane-bound adenosine triphosphatases (ATPases), and tricarboxylic acid (TCA) cycle marker enzymes and also through histopathological studies of the liver.
 Results: Administration of the aqueous fruit extract of the plant caused a significant increase in the in vivo antioxidant status as evident from the reduction in lipid peroxidation caused by APAP and improvement in the mitochondrial membrane stability which is proved from the activity of membrane-bound ATPases and TCA cycle marker enzymes. Histological studies also supported the fact that the plant extract proved to revive the architecture of the toxin damaged liver tissues in par with silymarin. The chemical pathological changes were consistent with histopathological observations suggesting marked hepatoprotective effect of the aqueous extract of S. torvum.
 Conclusion: The results showed that the extract of S. torvum Sw. fruits has hepatoprotective potential which may be due to the antioxidant activity of its phytoconstituents, especially flavonoids, alkaloids, phenolics, etc.

Maslinic acid ameliorate electrolytes, membrane bound ATPases, antioxidants and histopathology in isoprenaline attenuated myocardial toxicity in rats

ObjectivesMaslinic acid (MA), a natural compound widely distributed in many fruits and vegetables exhibited vast biological properties. Myocardial infarction (MI) is the most common cause of cardiovascular diseases morbidity and mortality. In this study cardioprotection of MA against isoprenaline (ISO) induced biochemical and histopathological alterations have been evaluated. MethodsRats were pretreated with MA (15 mg/kg) for 7 days and MI was induced with administration of ISO (85 mg/kg) on 8th and 9th days. Gallic acid (15 mg/kg) was used as positive control. Blood and heart were collected on 10th day from sacrificed rats and subjected to biochemical and histopathological analysis. ResultsISO administration significantly increased the enzymes creatine kinase-MB, lactate dehydrogenase, and alkaline phosphatase in serum whereas significantly decreased the marker enzymes in heart homogenate. ISO administration significantly increased the electrolytes sodium and calcium whereas significantly decreased potassium in heart homogenate. ISO showed a significant decrease in membrane bound adenosine triphosphatase (ATPase) enzymes sodium/potassium, calcium and magnesium ATPase in heart homogenate. ISO also significantly decreased the antioxidants reduced glutathione, glutathione S-transferase and glutathione peroxidase in heart homogenate. Furthermore, pretreatment of MA and GA reduced the effect of ISO significantly on all parameters studied. Furthermore, the cardioprotection of MA was also supported by histopathology. ConclusionsThis is the first report revealed that MA attenuates ISO-induced cardiac toxicity by ameliorating biochemical parameters such as cardiac marker enzymes, electrolytes, membrane bound ATPases and increased the antioxidants. The possible cardioprotective mechanism is due to the stabilization of myocardial membrane and antioxidant activity of MA.

Arsenic exposure intensifies glycogen nephrosis in diabetic rats.

It is known that either arsenic exposure or diabetes can impact renal function. However, it is unclear how these combined factors may influence kidney functions. Therefore, we evaluated morphological, functional, and oxidative parameters in the kidney of diabetic rats exposed to arsenic. Healthy male Wistar rats and streptozotocin-induced diabetic rats were exposed to 0 and 10mg/L arsenate through drinking water for 40days. Renal tissue was assessed using morphometry, mitosis and apoptosis markers, mineral proportion, oxidative stress markers, as well as the activity of antioxidant enzymes and membrane-bound adenosine triphosphatases. Arsenate intake altered glucose levels in healthy animals, but it did not reach hyperglycemic conditions. In diabetic animals, arsenate led to a remarkable increase of glycogen nephrosis in distal tubules. In these animals, additionally, the activity of catalase and glutathione S-transferase, besides the proportion of Fe, Cu, and K in renal tissue, was altered. Nevertheless, arsenate did not accumulate in the kidney and did not impact on other parameters previously altered by diabetes, including levels of malondialdehyde, Na, urea, creatinine, and apoptosis and mitosis markers. In conclusion, besides the intensification of glycogen nephrosis, the kidney was able to handle arsenate toxicity at this point, preventing arsenic deposition in the exposed groups and the impairment of renal function.

Membrane stabilization effect and histological changes in the heart in experimental myocardial rats with Zanthoxulym armatum fruit


 
 
 Membrane bound adenosine triphosphatases (ATPases) shed a massive function into the contraction and relaxation of the heart muscle via keeping the normal ion levels within the myocyte. The current study aims to assess the potency of Zanthoxylum armatum (Z. armatum) fruit on membrane bound ATPases and ions in Isoproterenol (ISO) induced myocardial infracted rats. The hydroethanolic extract of Z. armatum fruit was administered at a dose of 200 and 400mg/kg body weight for 30 days to male Wistar albino rats. On 28th and 29th day, ISO (8.5mg/100g body weight) used to be administered to induce myocardial infarction (MI). ISO treated rats confirmed a significant increase in the levels of tissue sodium (Na+) and calcium (Ca2+) ions and membrane bound Ca2+ ATPase then Mg2+ ATPase activity. A significant decrease in tissue potassium (K+), Na +/K+ ATPase was observed which indicates membrane destabilization. Pretreatment with hydroethanolic extract of Z. armatum fruit to ISO induced rats significantly (p<0.05) prevented the altered membrane bound enzymes to near normal status. The findings of the present study indicate the protective effect of Z. armatum fruit on altered ion pumps and destabilization on the cardiac membrane into ISO induced MI rats which might also be due to the presence of phytoconstituents.
 
 

Galangin ameliorates changes of membrane–bound enzymes in rats with streptozotocin–induced hyperglycemia

Objective: To assess the protective effect of galangin on membrane bound enzymes in rats with streptozotocin-induced diabetes. Methods: A single low dose of streptozotocin was injected to adult male albino rats to induce hyperglycemia. Galangin (8 mg/kg) or glibenclamide 600 μg/kg as a standard drug was given orally once daily for 45 days by gavage. Membrane-bound adenosine triphosphatases were determined including total ATPase, sodium-potassium-ATPase, calcium-ATPase and magnesium-ATPase in erythrocytes and tissues (kidney, liver, and heart). Results: The levels of total ATPases, sodium-potassium-ATPase, calcium-ATPase and magnesium-ATPase in erythrocytes and tissues were significantly altered in diabetic rats as compared to that in normal rats. After 45 days of treatment with galangin or glibenclamide, the levels of these enzymes were similar to that of normal control rats. Conclusions: Oral administration of galangin or glibenclamide can improve activities of these membrane-bound ATPases towards normal levels. Mechanism of galangin needs to be further explored in future.

Cultivation and inorganic carbon uptake of the rare desmid Oocardium stratum (Conjugatophyceae)

:The desmid Oocardium stratum is sporadically and exclusively found in active meteogene travertine habitats. The restriction to these peculiar biotopes is probably linked to specific carbon requirements. We studied inorganic carbon (Ci) uptake under controlled laboratory conditions. First, we developed a method for isolating and cultivating O. stratum. Colonies visible to the naked eye (green pinhead-shaped formations) were collected from a tufa spring in Lower Austria. We then grew unialgal cultures on solidified agar prepared with Wood's Hole culture medium, buffered with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) to pH 7 and enriched with CaCO3. Scanning and transmission electron microscopy were applied to study the condition of cells shortly after they started in vitro growth. For Ci uptake, pH-drift experiments were performed including the external carbonic anhydrase inhibitor acetazolamide and the inhibitor of membrane-bound adenosine triphosphatase (ATPase) sodium orthovanadate. We compared the Ci affinity of Oocardium to Acutodesmus acuminatus and Chlorella sp. grown under identical conditions. Whereas A. acuminatus was common in slightly alkaline, eutrophic surface waters, Chlorella sp. was isolated from a soil sample. Owing to its peculiar habitat with abundant free CO2, we expected a clear preference for CO2, no enzymatic catalysis of Ci uptake and absence of active HCO3− uptake for Oocardium. Our experiments confirmed these hypotheses. In contrast to A. acuminatus, we found no HCO3− uptake in Chlorella sp. and O. stratum. Moreover, enzyme inhibition assays did not suggest activity of those enzymes that are commonly responsible for active Ci uptake.

Modulating effects of hesperidin on key carbohydrate-metabolizing enzymes, lipid profile, and membrane-bound adenosine triphosphatases against 7,12-dimethylbenz(a)anthracene-induced breast carcinogenesis

The aim of this study was to document the effect of hesperidin on the key enzyme activities of carbohydrate metabolism, lipid profile, and membrane-bound adenosine triphosphatases (ATPases) during 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast carcinogenesis. Hesperidin has been reported to have multiple biological properties. Breast cancer was induced by single dose of DMBA (20mg/kg body weight (bw)). The results revealed that there was a significant increase in the activities of hexokinase, phosphoglucoisomerase, and aldolase and a concomitant decrease in the activities of glucose-6-phosphatase and fructose-1,6-diphosphatase in cancer-induced animals. The activities of ATPases were found to be decreased both in erythrocyte membrane and in the liver of mammary cancer-bearing animals. The lipid profiles such as total cholesterol, free cholesterol, phospholipids, triglycerides, and free fatty acids significantly increased and in contrast the ester cholesterol in plasma was found to be decreased, whereas it was found to be elevated in the liver of cancer-bearing groups. The altered levels of the above-mentioned biochemical parameters in cancer-bearing animals were significantly ameliorated by the administration of hesperidin at the dosage of 30mg/kg bw for 45days. The histopathological analysis of breast and liver tissues were well supported the modulatory property of hesperidin, and this might be associated with normalizing the gluconeogenesis process, stabilization of cell membranes, and modulation of lipid biosynthesis.

(−) Epicatechin prevents alterations in lysosomal glycohydrolases, cathepsins and reduces myocardial infarct size in isoproterenol-induced myocardial infarcted rats

The preventive effects of (−) epicatechin on oxidative stress, cardiac mitochondrial damage, altered membrane bound adenosine triphosphatases and minerals were reported previously in isoproterenol-induced myocardial infarction model. Leakage of lysosomal glycohydrolases and cathepsins play an important role in the pathology of myocardial infarction. This study was aimed to evaluate the preventive effects of (−) epicatechin on alterations in lysosomal glycohydrolases, cathepsins and myocardial infarct size in isoproterenol-induced myocardial infarcted rats. Male albino Wistar rats were pretreated with (−) epicatechin (20mg/kg body weight) daily for a period of 21 days. After the pretreatment period, isoproterenol (100mg/kg body weight) was injected subcutaneously into the rats at an interval of 24h for two days to induce myocardial infarction. The levels of serum cardiac troponin-I and the activities of serum and heart lysosomal enzymes (β-glucuronidase, β-N-acetyl glucosaminidase, β-galactosidase, cathepsin-B and cathepsin-D) were increased significantly (P<0.05) and the activities of β-glucuronidase and cathepsin-D in the heart lysosomal fractions were significantly (P<0.05) decreased in isoproterenol-induced myocardial infarcted rats. The in vitro study revealed the potent antioxidant action of (−) epicatechin. Pretreatment with (−) epicatechin daily for a period of 21 days prevented the leakage of cardiac marker, lysosomal glycohydrolases, cathepsins, and reduced infarct size, thereby protecting the lysosomal membranes in isoproterenol-induced myocardial infarcted rats, by virtue of its membrane stabilizing property.

Effect of leptin administration on membrane-bound adenosine triphosphatase activity in ethanol-induced experimental liver toxicity

Hepatic injury elicits intracellular stress that leads to peroxidation of membrane lipids accompanied by alteration of structural and functional characteristics of the membrane, which affects the activity of membrane-bound ATPases. We have explored the effect of leptin on hepatic marker enzyme and membrane-bound adenosine triphosphatases in ethanol-induced liver toxicity in mice. The experimental groups were control, leptin (230 microg kg(-1), i.p. every alternate day for last 15 days), alcohol (6.32 g kg(-1), by intragastric intubation for 45 days), and alcohol plus leptin. Ethanol feeding to mice significantly (P < 0.05) elevated the plasma leptin, alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT) and hepatic lipid hydroperoxides (LOOH), and plasma and hepatic total ATPases, Na(+), K(+)-ATPase and Mg(2+)-ATPase. There was a significant decrease in Ca(2+)-ATPase and reduced glutathione (GSH). Leptin injections to ethanol-fed animals further elevated the levels of hepatic LOOH, plasma and hepatic total ATPases, Na(+), K(+)-ATPase and Mg(2+)-ATPase, while the Ca(2)-ATPase and GSH were decreased significantly. In addition, leptin administration was found to increase the plasma levels of leptin, ALT, ALP, GGT, Na(+) and inorganic phosphorous, and decrease the levels of K(+) and Ca(2+) in ethanol-fed mice. These findings were consistent with our histological observations, confirming that leptin enhanced liver ailments in ethanol-supplemented mice.

Pretreatment with a combination of quercetin and α-tocopherol ameliorates adenosine triphosphatases and lysosomal enzymes in myocardial infarcted rats

Aims Membrane bound adenosine triphosphatases (ATPases) and lysosomal enzymes play an important role in the pathology of myocardial infarction. This study was aimed to evaluate the combined preventive effects of quercetin and α-tocopherol on membrane bound ATPases and lysosomal enzymes in isoproterenol induced myocardial infarcted rats. Main methods Male Wistar rats were pretreated with a combination of quercetin (10 mg/kg) and α-tocopherol (10 mg/kg) daily for 14 days. After the pretreatment period, isoproterenol (100 mg/kg) was injected to rats at an interval of 24 h for two days to induce myocardial infarction. The activities of ATPases and lysosomal enzymes were assayed. Key findings Isoproterenol treated rats showed decreased levels of heart creatine kinase and lactate dehydrogenase. The activity of sodium potassium adenosine triphosphatase was decreased and the activities of magnesium adenosine triphosphatase and calcium adenosine triphosphatase were increased in isoproterenol treated rats. Also, the activities of β-glucuronidase, β-N-acetylglucosaminidase, β-galactosidase, cathepsin-B and D were increased (serum and heart), but the activities of β-glucuronidase and cathepsin-D were decreased in lysosomal fraction and increased in cytosolic fraction of the heart in isoproterenol treated rats. Furthermore, the heart lipid peroxidation products were increased in isoproterenol treated rats. Combined pretreatment with quercetin and α-tocopherol to isoproterenol treated rats normalized all the biochemical parameters studied. The observed effects are due to their membrane stabilizing property and this property might be due to decreased lipid peroxidation. Significance Our study demonstrated that combined pretreatment was better than single pretreatment. This study may have significant impact on myocardial infarcted patients.

Lactobacilli facilitate maintenance of intestinal membrane integrity during Shigella dysenteriae 1 infection in rats

Objective Lactobacilli are used in various dairy products and fermented foods for their potential health beneficial effects. Recently we reported the protective role of Lactobacillus rhamnosus and Lactobacillus acidophilus during Shigella dysenteriae 1 infection. Nevertheless, investigations on the membrane-stabilizing effect of L. rhamnosus and L. acidophilus have not been done. Hence, the present study evaluated the effect of L. rhamnosus and L. acidophilus on the maintenance of intestinal membrane integrity during S. dysenteriae 1–induced diarrhea in rats. Methods Rats were divided into eight groups ( n = 6 in each group). Induced rats received single oral dose of S. dysenteriae (12 × 10 8 colony-forming units [cfu]/mL). Treated rats received L. rhamnosus (1 × 10 7cfu/mL) or L. acidophilus (1 × 10 7cfu/mL) orally for 4 d, alone or in combination, followed by Shigella administration. At the end of the experimental period, animals were sacrificed and the assay of membrane-bound adenosine triphosphatases (Na +/K +-ATPase, Ca 2+-ATPase, and total ATPase), immunoblot analysis of tight junctional proteins (claudin-1 and occludin), and transmission electron microscopic studies were performed. Results Induced rats showed a significant ( P < 0.05) reduction in the membrane-bound ATPases and reduced expression of tight junction proteins in the membrane, coupled with their increased expression in the cytosol, indicating membrane damage. Transmission electron microscopic studies correlated with biochemical parameters. Pretreatment with combination of L. rhamnosus and L. acidophilus significantly prevented these changes. Conclusion Lactobacillus rhamnosus and L. acidophilus synergistically offered better protection to the intestinal membrane when compared with individual treatments with these strains during S. dysenteriae 1 infection.

Bacterial target sites for biocide action

Although biocides have been used for a century, the number of products containing biocides has recently increased dramatically with public awareness of hygiene issues. The antimicrobial efficacy of biocides is now well documented; however, there is still a lack of understanding of their antimicrobial mechanisms of action. There is a wide range of biocides showing different levels of antimicrobial activity. It is generally accepted that, in contrast to chemotherapeutic agents, biocides have multiple target sites within the microbial cell and the overall damage to these target sites results in the bactericidal effect. Information about the antimicrobial efficacy of a biocide (i.e. the eta-value) might give some useful indications about the overall mode of action of a biocide. Bacteriostatic effects, usually achieved by a lower concentration of a biocide, might correspond to a reversible activity on the cytoplasmic membrane and/or the impairment of enzymatic activity. The bacteriostatic mechanism(s) of action of a biocide is less documented and a primary (unique?) target site within the cell might be involved. Understanding the mechanism(s) of action of a biocide has become an important issue with the emergence of bacterial resistance to biocides and the suggestion that biocide and antibiotic resistance in bacteria might be linked. There is still a lack of understanding of the mode of action of biocides, especially when used at low concentrations (i.e. minimal inhibitory concentration (MIC) or sublethal). Although this information might not be required for highly reactive biocides (e.g. alkylating and oxidizing agents) and biocides used at high concentrations, the use of biocides as preservatives or in products at sublethal concentrations, in which a bacteriostatic rather than a bactericidal activity is achieved, is driving the need to better understand microbial target sites. Understanding the mechanisms of action of biocides serves several purposes: (i) it will help to design antimicrobial formulations with an improved antimicrobial efficacy and (ii) it will ensure the prevention of the emergence of microbial resistance.

Functional characterization of an extremely thermophilic ATPase in membranes of the crenarchaeon Acidianus ambivalens.

A plasma membrane-bound adenosine triphosphatase with specific activities up to 0.2 micromol min(-1) (mg protein)(-1) at 80 degrees C was detected in the thermoacidophilic crenarchaeon Acidianus ambivalens (DSM 3772). The enzymatic activity exhibited a broad pH-optimum in the neutral range with two suboptima at pH 5.5 and 7.0, respectively. Sulfite activation resulted in only one pH optimum at 6.25. In the presence of the divalent cations Mg2+ and Mn2+ the ATPase activity was maximal. Remarkably, the hydrolytic rates of GTP and ITP were substantially higher than for ATP. ADP and pyrophosphate were only hydrolyzed with small rates, whereas AMP was not hydrolyzed at all. Both activities could be weakly inhibited by the classical F-type ATPase inhibitor N,N'-dicyclohexylcarbodiimide, whereas azide had no influence at all. The classical inhibitor of V-type ATPases, nitrate, also exerted a small inhibitory effect. The strongly specific V-type ATPase inhibitor concanamycin A, however, showed no effect at all. The P-type ATPase inhibitor vanadate had no inhibitory effect on the ATPase activity at pH 7.0, whereas a remarkable inhibition at high concentrations could be observed for the activity at pH 5.5. Arrhenius plots for both membrane bound ATPase activities were linear up to 95 degrees C, reflecting the enormous thermostability of the enzyme.

Reduction of membrane-bound ATPase activity in aLactobacillus helveticusstrain with slower growth at low pH

One acid-sensitive variant of Lactobacillus helveticus, designated L. helveticus CPN4, was isolated from L. helveticus CP790 using neomycin resistance as a selective marker. In a milk medium, the variant strain showed similar growth to the parent strain until the pH reached 4.5, afterwards growth was slower than the parent strain. The variant strain had lower activity of membrane bound adenosine triphosphatase (ATPase, EC 3.6.1.3) over several batch growth ranges from pH 3.5 to 7.5. Membrane vesicles of the variant strain showed lower uptake of quinacrine than vesicles of the parent strain. The variant strain had lower uptake ability for tyrosine, leucine and alanine.

Reduction of membrane-bound ATPase activity in a Lactobacillus helveticus strain with slower growth at low pH

One acid-sensitive variant of Lactobacillus helveticus, designated L. helveticus CPN4, was isolated from L. helveticus CP790 using neomycin resistance as a selective marker. In a milk medium, the variant strain showed similar growth to the parent strain until the pH reached 4.5, afterwards growth was slower than the parent strain. The variant strain had lower activity of membrane bound adenosine triphosphatase (ATPase, EC 3.6.1.3) over several batch growth ranges from pH 3.5 to 7.5. Membrane vesicles of the variant strain showed lower uptake of quinacrine than vesicles of the parent strain. The variant strain had lower uptake ability for tyrosine, leucine and alanine.

Effect of selected phenolic compounds on the membrane-bound adenosine triphosphatase of Staphylococcus aureus

The effect of selected phenolic compounds on the membrane-bound ATPase activity of two strains of Staphylococcus aureus was studied. These phenolic compounds were: butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ), propyl gallate (PG), p-coumaric, ferulic, caffeic acids, methyl paraben and propyl paraben. Cytoplasmic membrane-bound ATPase activity was measured in the presence of 0, 150, 300, 600 and 1200 μg ml −1 of each phenolic compound. Among all the compounds studied, only BHA was found to significantly stimulate the activity of the enzyme. In contrast, some of the compounds were found to significantly inhibit the activity of the enzyme as was the case of TBHQ, PG, p-coumaric acid, ferulic acid and caffeic acid. The remaining compounds did not influence the activity of the ATPase at any of the concentrations tested. Strain LP ATPase as stimulated less by BHA and inhibited to a greater extent by TBHQ and PG than was the enzyme from strain A100. In contrast, LP ATPase was less inhibited by p-coumaric acid and not affected by either ferulic or caffeic acid.

Inhibition of neuronal Cl-stimulated Mg-ATPase by alpha-human atrial natriuretic polypeptide.

Effects of alpha-human atrial natriuretic polypeptide (alpha-hANP) on neuronal membrane-bound adenosine triphosphatases (ATPases) (Na, K-ATPase, Cl-stimulated Mg-ATPase and anion-insensitive Mg-ATPase) were examined using rat brain microsomes. This peptide inhibited Cl-stimulated Mg-ATPase in a dose-dependent manner. The inhibition was non-competitive with respect to ATP, and was not affected by Cl-, the most potent stimulator of this enzyme. The activities of Na,K-ATPase and anion-insensitive Mg-ATPase were not changed by alpha-hANP at all.

Insulin stimulates renal glomerular sodium-potassium adenosine triphosphatase activity

The effect of insulin on total and ouabain-inhibited membrane-bound adenosine triphosphatase (ATPase) activity in renal glomeruli isolated from adult white rats was examined. In concentrations of 1–10 μg/ml, insulin significantly stimulated the ouabain-inhibited (Na + + K +)-ATPase activity, without affecting total (composite) ATPase activity. These results, coupled with previous findings demonstrating that glomerular (Na + + K +)-ATPase activity is reduced in acute streptozotocin diabetes, suggest that the renal glomerulus is a target tissue with respect to this biologic effect of insulin.

New methodology for assessment of the Langerhans cell network

A new procedure is described for staining Langerhans cells (LCs) based on the ability of anti-S-100 antibody to stain both epidermal LCs and melanocytes, while L-Dopa stains only melanocytes. This procedure can be used on paraffin-embedded skin sections and is therefore advantageous for examination of pathological skin specimens. In order to determine how best to quantitate LCs in skin sections the distribution of LCs has been investigated using an improved method for preparation of epidermal sheets from mouse skin. Epidermal LCs stained for their surface membrane-bound enzyme adenosine triphosphatase were observed to link with each other via their dendrites, forming a single cell layer which undulates throughout the epidermis. It is therefore proposed that LCs in skin sections should be enumerated per unit length, after identification in paraffin-embedded sections double stained with anti-S-100 antibody and L-Dopa.

Structural relatedness of three ion-transport adenosine triphosphatases around their active sites of phosphorylation.

Three membrane-bound adenosine triphosphatases were investigated for homology in the sequence of four amino acids about the active site of phosphorylation. The ATPases were as follows: sodium-potassium-dependent ATPase from dog kidney, Na,K-ATPase; hydrogen-potassium-dependent ATPase from hog gastric mucosa, H,K-ATPase, an ATPase similar to Na,K-ATPase; and an ATPase activity in the plasma membrane of corn, Zea mays, roots (CR-ATPase), a higher plant ATPase. A membrane preparation containing an ATPase of Acholeplasma laidlawii, a prokaryote, (AL) was also investigated. For most of the experiments, the preparations were phosphorylated from [gamma-32P]ATP, denatured in acid, and subjected to proteolytic digestion. Radioactive phosphopeptides were separated by high voltage paper electrophoresis and characterized by sensitivity to chemical reagents. In gastric H,K-ATPase, the aspartate residue at the active site was determined directly by labeling with [3H]borohydride. A common sequence around the active site was found for Na,K-ATPase, H,K-ATPase, and CR-ATPase. This sequence, -Cys-(Ser/Thr)-Asp(P)-Lys-, is similar to that in the calcium ion-transport ATPase of sarcoplasmic reticulum. The AL membrane preparation showed an acylphosphate that turned over rapidly after a chase of labeled membranes with unlabeled ATP. The corresponding sequence was different from that of the three ATPases. An acylphosphate was on two polypeptides with molecular weights of about 80,000 and 60,000; these appear not to correspond to subunits of a Na+-stimulated ATPase in this organism (Lewis, R. N. A. H., and McElhaney, R. N. (1983) Biochim. Biophys. Acta 735, 113-122).