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.     

Cl- -HCO3- dependent ATPase in gills of freshwater and seawater adapted eels (Anguilla anguilla L.)

The gills of both seawater and freshwater adapted eels have an ATPase activity which is stimulated by anions in the presence of Mg2+. Plasma membranes were distinguished from mitochondrial membranes with specific enzyme markers, the membrane fractions separated on a discontinuous sucrose gradient, and the ATPase activity of the plasma membranes studied. Activation by the anions of Cl- or HCO3- followed Michaelis-Menten kinetics and was competitively inhibited by SCN-. The Cl- and HCO3- activation characteristics were determined: no differences between the plasma membrane ATPase activities of freshwater and seawater-adapted fishes were observed. Maximal activity measurements after solubilization of the enzymes by Triton X 100 confirmed these findings. The function of a membrane anion-dependent ATPase in the brachial epithelium of euryhaline fish is discussed.     

Extremely low-frequency electromagnetic fields affect lipid-linked carbonic anhydrase

In the last years, the effect of extremely low-frequency electromagnetic fields (ELF-EMF) on the activity of different enzymes were investigated. Only the membrane-anchored enzymes did decrease their activity, up to 50%. In this work, the effect of ELF-EMF on bovine lung membrane carbonic anhydrase (CA) were studied. Carbonic anhydrases are a family of 14 zinc-containing isozymes catalyzing the reversible reaction: CO(2)+H(2)O = HCO(3)(- )+H(+). CA differ in catalytic activity and subcellular localization. CA IV, IX, XII, XIV, and XV are membrane bound. In particular, CA IV, which is expressed in the lung, is glycosyl phosphatidyl inositol-linked to the membrane, therefore it was a candidate to inhibition by ELF-EMF. Exposure to the membranes to a field of 75 Hz frequency and different amplitudes caused CA activity to a reproducible decrease in enzymatic activity by 17% with a threshold of about 0.74 mT. The decrease in enzymatic activity was independent of the time of permanence in the field and was completely reversible. When the source of enzyme was solubilized with Triton, the field lost its effect on CA enzymatic activity, suggesting a crucial role of the membrane, as well as of the particular linkage of the enzyme to it, in determining the conditions for CA inactivation. Results are discussed in terms of the possible physiologic effects of CA inhibition in target organs.     

Paraquat toxicity in Pisum sativum: Effects on soluble and membrane-bound proteins

The effects of paraquat (PQ) on Pisum sativum L. proteins were investigated in vivo in a new experimental system utilizing 10-day-old plant cuts.A marked decrease in the specific activity of membrane-bound Ca²⁺-dependent ATPase was recorded, while that of Mg²⁺-dependent ATPase remained unchanged. Concurrently with a drop in the total plant protein, the specific activities of the three cytoplasmic enzymes, malate dehydrogenase, hydroxypyruvate reductase and triose-phosphate isomerase, were also found to decrease. The effect on various enzymes involved in cellular defense mechanisms was also studied: glutathione reductase and superoxide dismutase activities increased, while ascorbate peroxidase was not affected.
These findings shed light on the selectivity of PQ-induced injurious processes, focusing on protein homeostasis mechanisms in the membrane and cytoplasmic compartments at the cellular level, as well as on the prominent role played by enzymatic defense systems against PQ poisoning.     

The interaction of NS3 protein of hepatitis C virus with polymethylen derivatives of nucleic bases

NS3 protein of hepatitis C virus plays the key role in the virus functioning. It possesses three enzymatic activities, namely protease activity, associated with N-terminal domain of the protein, and helicase/NTPase activities specific for C-terminal domain. Here, the effect of some polimethylenic derivatives of the nucleic bases on helicase and ATPase enzyme activities has been studied. Several of compounds tested displayed inhibitory activity towards NS3 helicase. However, most compounds demonstrated strong activating effect on ATPase activity of the enzyme as well as several other ATPases. The ATPase activating mechanism was not described earlier. The activation potency of the compounds depended on substrate/activator concentration ratio, and was maximal at the 1000:1. The activation mechanism scheme that allows us to explain phenomena observed is proposed.     

Extremely low-frequency electromagnetic fields affect lipid-linked Carbonic anhydrase

In the last years, the effect of extremely low-frequency electromagnetic fields (ELF-EMF) on the activity of different enzymes were investigated. Only the membrane-anchored enzymes did decrease their activity, up to 50%. In this work, the effect of ELF-EMF on bovine lung membrane carbonic anhydrase (CA) were studied. Carbonic anhydrases are a family of 14 zinc-containing isozymes catalyzing the reversible reaction: CO₂+H₂O = HCO₃^? +H⁺. CA differ in catalytic activity and subcellular localization. CA IV, IX, XII, XIV, and XV are membrane bound. In particular, CA IV, which is expressed in the lung, is glycosyl phosphatidyl inositol-linked to the membrane, therefore it was a candidate to inhibition by ELF-EMF. Exposure to the membranes to a field of 75 Hz frequency and different amplitudes caused CA activity to a reproducible decrease in enzymatic activity by 17% with a threshold of about 0.74 mT. The decrease in enzymatic activity was independent of the time of permanence in the field and was completely reversible. When the source of enzyme was solubilized with Triton, the field lost its effect on CA enzymatic activity, suggesting a crucial role of the membrane, as well as of the particular linkage of the enzyme to it, in determining the conditions for CA inactivation. Results are discussed in terms of the possible physiologic effects of CA inhibition in target organs.     

Analysis of Staphylococcus aureus cytoplasmic membrane proteins by isoelectric focusing

Cytoplasmic membranes were isolated from late-exponential phase Staphylococcus aureus 6538 P and the membrane proteins examined under non-denaturing conditions by thin-layer isoelectric focusing (TLIEF) in a pH 3.5–9.5 gradient. Isolated membrane preparations retained protein integrity as judged by the demostration of membrane bound adenosine triphosphatase (ATPase) activity in addition to four solubilzed membrane enzyme markers. Membranes were effectively solubilized with 2.5% Triton X-100 (final concentration). Examination of Triton X-100 solubilized membrane preparations established the presence of 22 membrane proteins with isoelectric points between 3.7 and 6.0. The focused proteins displayed the following enzymatic activities and isoelectric points by zymogram methods: ATPase (EC 3.6.1.3), 4.20; malate dehydrogenase (EC 1.1.1.37), 3.90; lactate dehydrogenase (EC 1.1.1.27), 3.85; two membrane proteins exhibited multiple bands upon enzymatic staining: NADH dehydrogenase (EC 1.6.99.3), 4.25, 4.35; succinate dehydrogenase (EC 1.3.99.1), 4.85, 5.10, 5.35.     

Adenosinetriphosphatase and 5-Nucleotidase Activities in the Plasma Membrane of Liver Cells as Revealed by Electron Microscopy

The sites of reaction product resulting from ATPase and 5-nucleotidase activities remaining in parenchymatous cells of osmium-fixed rat liver were studied by electron microscopy of thin sections. These indicate that both ATPase and 5-nucleotidase activities are localized in the plasma membrane where it folds to form the microvilli of the bile canaliculus, and that 5-nucleotidase activity is also present in the microvilli at the sinusoidal aspects of the cells. It is suggested that these enzymes, particularly ATPase, may play a role in molecular transport or in some kind of membrane activity at the cell surface. Of special interest is the apparent differential localization of these enzymes at the absorptive and secretory regions of the plasma membrane of the cell. It may be of interest to study changes in these enzyme localizations in pathologic states, as a sign of changed cell function. Some of the difficulties in the interpretation of enzyme reaction products seen in electron micrographs are discussed.     

Alterations in the activities of subcellular fractions marker enzymes in rat liver and brain by hydrocortisone and corticosterone treatment

The effect of subcutaneous injection of hydrocortisone and corticosterone on the activity values of some subcellular fractions marker enzymes from rat liver and brain was investigated and compared with controls (without treatment with hormones). The following enzymes were studied (subcellular fraction are shown between parentheses): N-acetyl-beta-D-glucosaminidase and beta-glucuronidase (lysosomes); succinate dehydrogenase = SDH (mitochondria); glucose-6-phosphatase (endoplasmic reticulum); 5'-nucleotidase and Na+-K+-Mg2+ ATPase (plasma membrane). The specific activity of lysosomal enzymes from liver showed no change when rats were injected either with hydrocortisone or corticosterone. The same enzymes from brain showed significant increases in their activities with both hydrocortisone or corticosterone except beta-glucuronidase; this enzyme gave activity values remaining between the control levels, after treatment with corticosterone. The activity of mitochondrial SDH was increased after corticosterone injection either in liver or brain. After hydrocortisone injection, its activity rises significantly in brain (72%), but it falls in liver compared to the control values. Glucose-6-phosphatase behaves similarly in brain or liver fractions; its activity increases always after corticosterone treatment and decreases by hydrocortisone. The plasma membrane marker enzymes did not change practically in brain fractions, excepted Na+-K+-Mg2+ ATPase which tends to rise its activity after hydrocortisone injection. In liver fractions, both 5'-nucleotidase and Na+-K+-Mg2+ ATPase activities increase either by corticosterone or hydrocortisone treatment, except 5'-nucleotidase which specific activity decreases in liver after hydrocortisone treatment.     

Roles of homooligomerization and membrane association in ATPase and proteolytic activities of FtsH in vitro.

Escherichia coli FtsH is a membrane-bound and ATP-dependent protease which degrades some soluble and integral membrane proteins. The N-terminal region of FtsH mediates membrane association as well as homooligomeric interaction of this enzyme. Previously, we studied in vivo functionality of FtsH derivatives, in which the N-terminal membrane region was either deleted (FtsH(DeltaTM)), replaced by a leucine zipper (Zip-FtsH(DeltaTM)), or replaced by a lactose permease transmembrane segment (LacY-FtsH). It was indicated that homooligomerization is required for the minimum proteolytic activity, whereas a transmembrane sequence is required for membrane protein degradation. Here we characterized these proteins in vitro. Although these mutant enzymes were very low in their activities, they were significantly stimulated by dimethyl sulfoxide, which enabled us to characterize their activities. LacY-FtsH degraded both soluble and membrane proteins, but Zip-FtsH(DeltaTM) only degraded soluble proteins. These proteins also exhibited significant ATPase activities. However, FtsH(DeltaTM) remained inactive both in ATPase and in protease activities even in the presence of dimethyl sulfoxide. The monomeric FtsH(DeltaTM) was able to bind ATP and a denatured protein. These results indicate that subunit association is important for the enzymatic catalysis by FtsH and that the additional presence of the transmembrane sequence is required for this enzyme to degrade a membrane protein even under detergent-solubilized conditions.     

Characterization of the plasma membrane ATPase of Saccharomyces cerevisiae

1. The distribution of ATPase and several marker enzymes was examined after differential and sucrose gradient centrifugation of yeast homogenates. 2. An ATPase activity not sensitive to oligomycin is found exclusively associated with a particulate fraction equilibrating at densities of 1.23-1.25. This particulate material shows the chemical and enzymatic characteristics of the yeast plasma membrane. 3. The pH optimum of the plasma membrane ATPase is 5.6, as compared with 8.5 for the mitochondrial ATPase. In addition to oligomycin, the enzyme is not sensitive to other inhibitors of the mitochondrial ATPase as azide, dicyclohexylcarbodiimide and the mitochondrial ATPase inhibitor protein. It is inhibited by p-chloromercuryphenyl sulfonate, fluoride, quercetin and by the antibiotic Dio-9 but is not affected by ouabain. 4. The plasma membrane ATPase shows a high affinity for ATP (Km = 0.1 mM) and is very specific for this compound, hydrolyzing other nucleotide triphosphates less than 25% as rapidly. No activity was detected with ADP. 5. The enzyme requires a divalent cation for activity and Mg2+ is the most effective. It is not significantly stimulated by K+ or bicarbonate and Ca2+ is inhibitory. 6. The activity cannot be assayed in intact cells unless they are permeabilized with toluene. This suggest that the active site is on the cytoplasmic side of the plasma membrane.     

Effect of quercetin on daunorubicin-induced heart mitochondria changes in rats

Cancer therapy with daunorubicin is limited by its cardiotoxicity. It has been suggested that daunorubicin-induced free radical generation can be involved. The precise molecular mechanism of daunorubicin-induced cardiotoxicity is still not well understood but it is believed that mitochondria play an important role in this process. It has been reported that flavonoids with antioxidant properties may prevent anthracycline-induced cardiotoxicity. In this work, we investigated the effects of daunorubicin and quercetin on mitochondrial enzyme activities such as ATPase, glutathione peroxidase (GPx) and glutathione reductase (GR). Moreover, we also studied the changes of outer mitochondrial membrane using synchronous fluorescence spectra. The activity of ATPase and GR were significantly increased after daunorubicin application. Pretreatment with quercetin significantly alleviated this increase. On the other hand, GPx activity was significantly decreased and quercetin prevented this decrease. Treatment with quercetin alone had no significant effect on the enzyme activity studied. Quercetin also completely prevented daunorubicin-induced changes in fluorescence of the outer mitochondrial membrane. In conclusion, our data indicate that quercetin may be useful in mitigating daunorubicin-induced cardiotoxicity.     

Phospholipase C axis is the preferential pathway leading to PKC activation following PTH or PTHrP stimulation in human term placenta

In the present report we describe an NTPDase 1 (ATP diphosphohydrolase; ecto-apyrase; EC 3.6.1.5) in rat hippocampal slices. The effect of glutamate on the ATPase and ADPase activities in rat hippocampal slices of different ages was also studied since adenosine, the final product of an enzymatic chain that includes NTPDase 1 and 5'-nucleotidase, can act upon A1 receptors in turn decreasing the release of glutamate. Hippocampal slices from 7, 14, 20-23 and 60 day-old rats were prepared and ATPase and ADPase activities were measured. The parallelism of ATPase and ADPase activities in all parameters tested indicated the presence of an ATP diphosphohydrolase. In addition, a Chevillard plot indicated that ATP and ADP are hydrolyzed at the same active site on the enzyme. ATPase activity was significantly activated by glutamate in 20-23 and 60 day-old rats, but ADPase activity was not activated. These results could indicate distinct behavior of the ATPase and ADPase activities of NTPDase 1 in relation to glutamate or the simultaneous action of the ecto-ATPase. Activation of ATPase activity by glutamate may constitute an important role in this developmental period, possibly protecting against the neurotoxicity induced by ATP, as well as producing high levels of ADP, by increasing adenosine production, a neuroprotective compound.     

Phosphatidylcholine/sphingomyelin metabolism crosstalk inside the nucleus

It is known that phospholipids represent a minor component of chromatin. It has been highlighted recently that these lipids are metabolized directly inside the nucleus, thanks to the presence of enzymes related to their metabolism, such as neutral sphingomyelinase, sphingomyelin synthase, reverse sphingomyelin synthase and phosphatidylcholine-specific phospholipase C. The chromatin enzymatic activities change during cell proliferation, differentiation and/or apoptosis, independently from the enzyme activities present in nuclear membrane, microsomes or cell membranes. This present study aimed to investigate crosstalk in lipid metabolism in nuclear membrane and chromatin isolated from rat liver in vitro and in vivo. The effect of neutral sphingomyelinase activity on phosphatidylcholine-specific phospholipase C and sphingomyelin synthase, which enrich the intranuclear diacylglycerol pool, and the effect of phosphatidylcholine-specific phospholipase C activity on neutral sphingomyelinase and reverse sphingomyelin synthase, which enrich the intranuclear ceramide pool, was investigated. The results show that in chromatin, there exists a phosphatidylcholine/sphingomyelin metabolism crosstalk which regulates the intranuclear ceramide/diacylglycerol pool. The enzyme activities were inhibited by D609, which demonstrated the specificity of this crosstalk. Chromatin lipid metabolism is activated in vivo during cell proliferation, indicating that it could play a role in cell function. The possible mechanism of crosstalk is discussed here, with consideration to recent advances in the field.     

Membrane adenosine triphosphatase activities in rat pancreas

The membrane ATPase activities present in rat pancreas were studied to investigate the possible role of ATPase enzymes in HCO3(-) secretion in the pancreas. It was found that all the HCO3(-)-sensitive (anion-sensitive) ATPase activity was accountable as pancreatic mitochondrial ATPase, thus supporting the view that a distinct plasma membrane 'bicarbonate-ATPase' is not involved in HCO3(-) secretion in pancreas. A remarkably high Mg+- and CA2+-requiring ATPase activity (30 mumol ATP hydrolysed/min per mg) was found in the plasma membrane fraction (rho = 1.10-1.13). This activity has been characterized in some detail. It is inhibited by p-fluorosulfonylbenzoyladenosine, an affinity label analogue of ATP and the analogue appears to label covalently a protein of Mr approximately 35 000. The (Ca2+ + Mg2+)-ATPase activity did not form a 'phosphorylated-intermediate' and was vanadate-insensitive. These and other tests have served to demonstrate that the (Ca2+ + Mg2+)-ATPase activity is different in properties from (Na+ + K+)-ATPase, Ca2+-ATPase, (H+ + K+)-ATPase or mitochondrial H+-ATPase. Apart from the (Ca2+ + Mg2+)-ATPase of plasma membrane and mitochondrial ATPase, the only other membrane ATPase activities noted were (Na+ + K+)-ATPase, which occurred in the same fractions as the (Ca2+ + Mg2+)-AtPase at rho = 1.10-1.13 and was of surprisingly low activity, and an ATPase activity in light membrane fractions (rho - 1.08-1.09) derived from zymogen granule membranes. At this time, therefore, there is no obvious candidate for an ATPase activity at the luminal surface of pancreatic cells which is directly involved in ion transport, but the results presented here direct attention to the high activity (Ca2+ + Mg2+)-ATPase in the plasma membrane fraction.     

Spectrophotometric and cytochemical analyses of phosphatase activity in Beta vulgaris L

Spectrophotometric and cytochemical methods were used to investigate the localization and/or the sensitivity of phosphatase activities in aldehyde-fixed beet leaves and membrane fractions. The nonspecific acid phosphatase substrates, p-nitrophenyl phosphate and beta-glycerol phosphate, each exhibited unique spectrophotometric patterns of hydrolysis as a function of pH. Additionally, beta-glycerol phosphatase activity was primarily present on the tonoplast, whereas p-nitrophenyl phosphatase was present on the plasma membrane. Because of the unique pH response of each enzyme and their different localization, we conclude that they cannot be entirely "nonspecific." The spectrophotometric pattern of ATP hydrolysis differed from that of p-nitrophenol phosphate in that it decreased at pH 5.0-5.5 and was greatly inhibited by 10 mM sodium fluoride; however, both activities were on the plasma membrane. Therefore, we conclude that these activities represent either two enzymes or only one enzyme that differs in its ability to hydrolyze these two substrates. Generally, enzymatically produced lead deposits on the plasma membrane of non-vascular cells were as frequent and large as those on phloem cells; frequently, deposits on sieve element plasma membranes were relatively small. We therefore conclude that there is no evidence for the presence of relatively intense ATPase activity on the plasma membrane of phloem cells in beet leaf, in contrast to other species. Studies with membrane fractions indicated that formaldehyde could completely inhibit the inhibitor-sensitive phosphatase activities in mitochondrial and vacuolar fractions while preserving significant activity in the plasma membrane fraction.     

Myofibrillar and membrane-bound enzymes in skeletal muscle from myodystrophic mice.

1. Experiments were carried out to examine the biochemical changes, such as contractile protein biochemistry and membrane bound enzyme alterations associated with skeletal muscles of myd/myd. 2. Our studies demonstrate that there was a progressive decline in myofibrillar ATPase activity, and this decrease is greatest in 30 weeks old animals of myd/myd as compared to controls. 3. The proteolytic activity of myofibrils isolated from myd/myd was significantly higher than controls. 4. There was no significant difference in Ca2+ ATPase activity of myosin and actin-activated myosin ATPase activity of myd/myd and their controls. 5. Mg2+ ATPase and Na(+)+K(+)-ATPase of myodystrophic SL showed significant increase compared to controls. 6. Isoproterenol stimulated adenylate cyclase activity was significantly lower in the SL of dystrophic mice compared to controls. 7. GTP+isoproterenol stimulate adenylate cyclase was significantly higher in control SL and SR when compared to SL and SR isolated from myd/myd. 8. Guanylate cyclase activity was greater in myodystrophic mice both in the absence and presence of Triton X-100. cGMP and cAMP phosphodiesterase activities were greater in dystrophic mice as compared to controls. 9. These observations suggest that there are significant changes in myofibrillar ATPase, myofibrillar protease and membrane bound enzymes of myd/myd compared to control.     

Entamoeba histolytica: ultrastructural localization of Ca2+-dependent nucleotidases

Localization of nucleotidases dependent on Ca²⁺ was investigated cytochemically in axenically cultivated trophozoites of Entamoeba histolytica, strain HM-1:IMSS, with an electron microscope. Ca²⁺-dependent ATPase (EC 3.6.1.3) activity was found on the plasma membrane and on the inner surface of the limiting membrane of a few cytoplasmic vacuoles. Ca²⁺-dependent ADPase, Ca²⁺-dependent thiamine pyrophosphatase, and acid phosphatase (EC 3.1.3.2) activities were detected on the inner surface of the limiting membrane of most of the cytoplasmic vacuoles but not on the plasma membrane. Cytoplasmic vacuoles with these enzymatic activities seemed similar in morphological characteristics. Moreover, the reaction product formed by Ca²⁺-dependent ADPase, Ca²⁺-dependent thiamine pyrophosphatase and acid phosphatase was demonstrable on the inner surface of the limiting membrane of vacuoles containing ingested red blood cells. The reaction product formed by these enzymes was also observed on the periphery of ingested red blood cells. The findings suggest that cytoplasmic vacuoles with these enzymatic activities are lysosomal in nature, probably phagolysosomes; therefore, the enzymes appear to be at least partially associated with primary lysosomes of E. histolytica.     

Rabbit platelet calcium ATPase differs from the human erythrocyte (Ca2+ + Mg2+)-ATPase in its response to three purified phospholipases A2, exogenous phospholipids and calmodulin

Human erythrocyte (Ca2+ + Mg2+)-ATPase and calcium ATPase of rabbit platelets were compared by their responses to a variety of treatments. These included three purified phospholipases A2 (acidic, neutral and basic) from Agkistrodon halys blomhoffii, as well as several phospholipids and lysophospholipids. The erythrocyte enzyme was stimulated 2-3-fold by all three phospholipases with maximal stimulation occurring at different concentrations of the three enzymes. The basic phospholipase was the most potent, followed by the neutral and acidic enzymes in that order. The calcium ATPase activity of the platelet was also stimulated by phospholipase treatment, but only by 10-20%. The stimulatory activity was attributable to hydrolysis of a very small portion of the total membrane phospholipid. Inactivation of the phospholipases by heating or chemical modification with p-bromophenacyl bromide abolished their ability to stimulate. Addition of polyphosphoinositides stimulated both ATPases. However, another acidic phospholipid, lysophosphatidic acid, stimulated only the erythrocyte enzyme and failed to affect the platelet calcium ATPase. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) had no effect on either enzyme, while the platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), its lyso compound and lysoPC inhibited both ATPases. Calmodulin stimulated the erythrocyte enzyme, but did not affect the platelet calcium ATPase. These results demonstrate that the protein-lipid interactions operative in the erythrocyte and platelet calcium ATPases are quite different.     

HELA CELL PLASMA MEMBRANES : I. 5'-Nucleotidase and Ouabain-Sensitive ATPase as Markers for Plasma Membranes

A method for the preparation of HeLa cell plasma membrane ghosts is described. The purity of the plasma membrane fraction was examined by phase contrast and electron microscopy, by chemical analysis, and by assay of marker enzymes. Data on the composition of the plasma membrane fraction are given. It was observed that the distribution pattern of 5'-nucleotidase activity among the subcellular fractions differed from that of ouabain-sensitive ATPase. In addition, the specific activity of 5'-nucleotidase did not follow the distribution of the membrane ghosts. Thus, this enzyme would seem unsuitable as a plasma membrane marker. A complete balance sheet for marker enzyme activities during the fractionation is necessary for the calculation of increase in specific activity because the activities of both 5'-nucleotidase and ouabain-sensitive ATPase might change during the fractionation procedures.