Membrane ATPase Activity Research Articles

Change in plasma membrane ATPase activity during dormancy release of vegetative peach‐tree buds

Plant dormancy and dormancy breaking depend, at least partially, on peculiar short distance relationships between buds and tissues underlying buds (bud stands). In peach‐tree, it was previously observed that dormancy was related to a high nutrient absorption capacity in tissues underlying buds. This situation could be linked to higher plasma membrane ATPase activity (EC 3.6.1.3), inducing a higher nutrient absorption, in bud stands. This work consists of characterization of the plasma membrane ATPase activity in vegetative buds and bud stands during the rest period and dormancy release. During the dormant period (October and November), plasma membrane ATPase activity was found to be higher in bud stands than in buds. This was correlated with a lower amount of plasma membrane ATPase in buds compared to bud stands during this period. Moreover, plasma membrane ATPase activation by trypsin treatment was not the same in both tissues and different levels of ATPase activation could be noted within the same tissue during the different stages of dormancy release. According to these results, it can be postulated that dormancy release in peach‐tree, is related to modifications of plasma membrane ATPase properties in buds and bud stands during winter time.

Erythrocyte Na +, K +-ATPase activity in patients with congestive heart failure

In order to determine if the Na +, K +-ATPase activity in erythrocyte membranes is altered in congestive heart failure, and to examine its clinical significance with respect to other clinical variables, erythrocyte Na +, K +-ATPase activity was measured in 51 patients with left ventricular ejection fractions <40% (coronary artery disease, n=26; dilated cardiomyopathy, n=25) and 24 control patients. Na +, K +-ATPase activity was lower in both coronary artery disease and dilated cardiomyopathy groups than control group even in the absence of digitalis use. There was a significant inverse correlation between Na +, K +-ATPase activity and plasma norepinephrine. The presence of non-sustained ventricular tachycardia was associated with a lower Na +, K +-ATPase activity in both groups with congestive heart failure without digitalis use than those without ventricular tachycardia. Plasma norepinephrine was higher in patients with non-sustained ventricular tachycardia than those without in the coronary artery disease group, but not in the dilated cardiomyopathy group. Na +, K +-ATPase activity may be helpful in predicting electrophysiologic instability in patients with heart failure.

Effect of In Vitro Lipid Peroxidation on High Affinity Ca++-ATPase Activity in Neuronal Nuclear Membranes of the Newborn Piglet

Effect of In Vitro Lipid Peroxidation on High Affinity Ca ++ -ATPase Activity in Neuronal Nuclear Membranes of the Newborn Piglet

Studies of pH Regulation by Btn1p, the Yeast Homolog of Human Cln3p

Although the gene responsible for Batten disease,CLN3,was positionally cloned in 1995, the function of Cln3p and the molecular basis of the disease still remain elusive. We previously reported that the yeastSaccharomyces cerevisiaecontains a homolog to Cln3p, designated Btn1p, and that the human Cln3p complemented the pH-dependent resistance tod-(−)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol inbtn1-Δ yeast mutants. We have determined that yeast lacking Btn1p have an elevated ability to acidify media during growth that correlates with an elevated plasma membrane ATPase activity. Btn1p may be involved in maintaining pH homeostasis of yeast cells.

Ectonucleotidases of Avian Gizzard Smooth Muscle and Liver Plasma Membranes: A Comparative Study

Extracellular nucleotides, e.g., ATP, ADP, and UTP, are important signaling molecules which elicit various physiological responses in different tissues. Their degradation is catalyzed by ectonucleotidases which are located on cell surfaces. Most tissues have a mixed population of ectonucleotidases. In this report, the ATP and ADP hydrolyzing ectonucleotidases of chicken gizzard smooth muscle and liver plasma membranes were studied. The two membranes exhibited marked differences in the ratio of ATPase/ADPase activities, activation by divalent cations, thermal stability, responses to detergents and cross-linking agents, and sensitivity to several enzyme inhibitors. The ATPase activity of chicken gizzard membranes is (i) labile to heat and detergents; (ii) activated by concanavalin A and disuccinimidyl suberate, both cross-linking agents; (iii) inhibited by mercurials; and (iv) insensitive to high concentrations of azide, a known inhibitor of ecto-ATP diphosphohydrolases (ecto-ATP/Dase). In contrast, the liver membrane ATPase and ADPase activities are more stable to treatment by heat and detergents and insensitive to cross-linking agents and mercurials, but are inhibited by azide. A low ADP hydrolase activity in the gizzard membranes could be distinguished from both the gizzard ATPase and the liver ATPase/ADPase. This ADP hydrolase, which is markedly stimulated by NBD-Cl, accounts for most of the ADP hydrolysis activity in gizzard membranes. It is concluded that the major ectonucleotidase in the gizzard membranes is an ecto-ATPase whereas that in the liver membranes is an ecto-ATP/Dase. That both membranes contain a mixed population of the ecto-ATPase and ecto-ATP/Dase, but in different proportions, is further demonstrated by immunochemical characterization. The different composition of ectonucleotidases in the two membranes is expected to have an important effect on the regulation of hydrolysis of extracellular ATP as well as the concentration of extracellular adenine nucleotides in the gizzard and liver tissues.

Erythrocyte membrane ATPase activities in patients with chronic active hepatitis B and C virus infections

The aim of the present study was to obtain erythrocyte membrane fractions from patients with chronic active hepatitis caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Na +,K + ATPase activities were significantly lower ( P<0.005) in HBV related chronic active hepatitis ( n=20, 0.134±0.073 μmol P/mg protein per h) and in HCV hepatitis ( n=11, 0.144±0.049 μmol P/mg protein per h) when compared to control group ( n=10, 0.219±0.055 μmol P/mg protein per h). Both types of chronic active hepatitis B and C have markedly altered erythrocyte membrane Na +,K + ATPase activities that can be indicative of hepatic cell damage in humans. Although HBV and HCV infections are based on diverse mechanisms of action on their effect in liver injury, they cause similar effects on membrane ATPase activities.

Characterization of the Na +-ATPase gene ( ZENA1) from the salt-tolerant yeast Zygosaccharomyces rouxii

In order to clarify the relationship between the salt tolerance of Zygosaccharomyces rouxii and the function of Na +-ATPase, a gene which exhibited homology to the Na +-ATPase gene ( ZENA1) of Saccharomyces cerevisiae was isolated from Z. rouxii. This newly isolated gene ( ZENA1) encoded a product of 1048 amino acids. The predicted amino-acid sequence of Zena1p was highly homologous to that of S. cerevisiae Ena1p and Ena2p, and Schwanniomyces occidentalis Ena1p and Ena2p, but showed low homology to that of Zpma1p, which is the product of the Z. rouxii plasma membrane H +.ATPase gene ( ZENA1). Zena1p shares the peptide motifs which have been suggested to participate in the function of ATPase. Expression of ZENA1 was observed, but was independent of NaCl shock. When ZENA1 was expressed in salt-sensitive S. cerevisiae under the regulation of a GAL1 promoter by using the expression vector pYES2, salt tolerance of the transformants was observed. The growth characteristics of Zena1Δ-disruptants of Z. rouxii and the pH profiles of their plasma membrane ATPase activity were almost the same as those of the wild-type strain, indicating that the function of Zena1p is of little relevance to the salt tolerance property of Z. rouxii. By considering the close relationship between the salt tolerance of Z. rouxii and the function of its Na +/H +-antiporter, we concluded that the extrusion of Na + across the plasma membrane in Z. rouxii cells might be carried out mainly via the function of the Na +/H +-antiporter in a high salinity environment.

Na+,K+-ATPase interaction with a brain endogenous inhibitor (endobain E)

Na +,K +-ATPase activity of rat brain synaptosomal membranes was evaluated in the presence of an inhibitory fraction II-E (termed endobain E), isolated by gel filtration and anionic exchange HPLC of a rat brain soluble fraction. We studied endobain E aging, analyzed its inhibitory potency in the absence or presence of ouabain as well as its ability to block high affinity [ 3H]ouabain binding to cerebral cortex membranes. Similar loss of endobain E activity was observed when samples were stored either dried or in solution. Endobain E fraction inhibited synaptosomal membrane Na +,K +-ATPase activity in a concentration-dependent manner and the slope of the corresponding curve strongly resembled that of ouabain. Assays performed in the presence of endobain E and ouabain indicated that the inhibitory effect was additive or less than additive, depending on their respective concentrations during preincubation and⧹or incubation. High affinity [ 3H]ouabain binding to cerebral cortex membranes proved concentration-dependent from 0.10 to 0.50 mg protein per ml; binding inhibition by endobain E was independent of protein concentration within the above range. [ 3H]ouabain binding inhibition by endobain E was concentration-dependent over a 10-fold range, an effect similar to that found for Na +,K +-ATPase inhibition. The extent of endobain E effect on Na +,K +-ATPase inhibition was much higher (90–100%) than that on [ 3H]ouabain binding blockade (50%). Findings suggest some type of interaction between endobain E and ouabain inhibitory mechanisms and favour the view that the former behaves as an endogenous ouabain.

Progressive inactivation of plasma membrane (Ca2++Mg2+)ATPase by Cd2+ in the absence of ATP and reversible inhibition during catalysis.

The influence of Cd2+ ions on plasma membrane (Ca2++Mg2+)ATPase activity from red cells was investigated. When the membranes were preincubated with Cd2+ in the absence of ATP, basal (Ca2++Mg2+)ATPase activity (no calmodulin) was slowly and irreversibly inhibited (inactivated) following first-order kinetics (k/Ki = 0.0057 microM-1 min-1 at [Cd2+] = 0.25-1 microM). However, preincubation with Cd2+ did not affect the degree of stimulation by calmodulin added to the assay medium together with ATP. Inactivation was not released by prolonged exposure of membranes to EGTA prior to catalysis, but it was strongly attenuated when the pH in the preincubation medium was lowered from 7.2 to 6.4. When the reaction was started by supplying membranes simultaneously with Cd2+ and ATP (no preincubation), (Ca2++Mg2+)ATPase was inhibited by increasing concentrations of the CdATP complex ([CdATP]50 = 7.2 microM). In this condition, however, even total inhibition of the pump was almost completely released after addition of enough EGTA to decrease CdATP concentrations to the nanomolar range. These results, taken together, indicate that inactivation of the unphosphorylated enzyme by Cd2+ is influenced by dissociation of amino acid residues exhibiting pK between 6.0 and 7.0, and that recognition by the pump of the physiological modulator calmodulin is preserved in the preincubated pump molecules which did not undergo inactivation. In addition, they show that the catalytic site is a target for reversible inhibition of the pump by CdATP and that occupancy of the nucleotide binding site prevents inactivation.

Stability of theEscherichia coliATP Synthase F0F1Complex Is Dependent on Interactions between γGln-269 and the β Subunit Loop βAsp-301–βAsp-305

The role of the conserved sequence motif301DDLTDP306in the F0F1ATP synthase β subunit was assessed by mutagenic analysis in theEscherichia colienzyme. Mutations gave variable effects on F1sector activity, stability, and membrane binding to the F0sector. Upon solubilization, F1sectors of the βD302E and βD305E mutants (βAsp-302 and βAsp-305 replaced by glutamate) dissociated into subunits, while mutants with other β305 substitutions failed to assemble. Membrane ATPase activities of β301 and 302 mutants were 20–70% of wild type. Replacements of the γ subunit Gln-269 had similar effects. The membrane ATPase activities of the γQ269E or γQ269D mutants were significantly lower and their F1sectors dissociated into subunits upon solubilization. These results suggest that the β301–305 loop and the γ subunit region around Gln-269 form a key region for the assembly of α3β3γ complex. These results are consistent with the X-ray crystallographic structure of bovine F1(J. P. Abrahams, A. G. W. Leslie, R. Lutter, and J. E. Walker (1994)Nature370, 621–628) where the β301DDLTD305loop directly interacts with γGln-269.

PH homeostasis and ATP synthesis: studies of two processes that necessitate inward proton translocation in extremely alkaliphilic Bacillus species.

Alkaliphilic Bacillus species that are isolated from nonmarine, moderate salt, and moderate temperature environments offer the opportunity to explore strategies that have developed for solving the energetic challenges of aerobic growth at pH values between 10 and 11. Such bacteria share many structural, metabolic, genomic, and regulatory features with nonextremophilic species such as Bacillus subtilis. Comparative studies can therefore illuminate the specific features of gene organization and special features of gene products that are homologs of those found in non-extremophiles, and potentially identify novel gene products of importance in alkaliphily. We have focused our studies on the facultative alkaliphile Bacillus firmus OF4, which is routinely grown on malate-containing medium at either pH 7.5 or 10.5. Current work is directed toward clarification of the characteristics and energetics of membrane-associated proteins that must catalyze inward proton movements. One group of such proteins are the Na+/H+ antiporters that enable cells to adapt to a sudden upward shift in pH and to maintain a cytoplasmic pH that is 2-2.3 units below the external pH in the most alkaline range of pH for growth. Another is the proton-translocating ATP synthase that catalyzes robust production of ATP under conditions in which the external proton concentration and the bulk chemiosmotic driving force are low. Three gene loci that are candidates for Na+/H+ antiporter encoding genes with roles in Na(+)-dependent pH homeostasis have been identified. All of them have homologs in B. subtilis, in which pH homeostasis can be carried out with either K+ or Na+. The physiological importance of one of the B. firmus OF4 loci, nhaC, has been studied by targeted gene disruption, and the same approach is being extended to the others. The atp genes that encode the alkaliphile's F1F0-ATP synthase are found to have interesting motifs in areas of putative importance for proton translocation. As an initial step in studies that will probe the importance and possible roles of these motifs, the entire atp operon from B. firmus OF4 has been cloned and functionally expressed in an Escherichia coli mutant that has a full deletion of its atp genes. The transformant does not exhibit growth on succinate, but shows reproducible, modest increases in the aerobic growth yields on glucose as well as membrane ATPase activity that exhibits characteristics of the alkaliphile enzyme.

Influence of probes for calcium–calmodulin and protein kinase C signalling on the plasma membrane Ca2+–ATPase activity of rat synaptosomes and leukocyte membranes

The influence of selected inhibitors of calcium signalling on the plasma membrane Ca2+-ATPase activity of rat synaptosomes and peritoneal leukocyte membranes was studied. The calmodulin inhibitor calmidazolium was an efficient inhibitor (50%) of the synaptosomal Ca2+-ATPase activity in a manner competitive with phosphatidylserine. The inhibition by CGS 9343B (30%) was not counteracted by phosphatidylserine. The intracellular calcium antagonist TMB-8 and the protein kinase inhibitor staurosporine and the derivatives CGP 41251 and CGP 42700 hardly affected the synaptosomal Ca2+-ATPase activity. The flavonoid quercetin was a more effective inhibitor of the ATPase activity of synaptosomal than of leukocyte membranes. Phloretin, at relatively high concentrations, caused only a modest inhibition of synaptosomes. The protein kinase C inhibitor sphingosine was a weak inhibitor of the synaptosomal but an effective inhibitor of the leukocyte membrane Ca2+-ATPase activity. The antineoplastic ether phospholipids BM 41.440 (ilmofosine) and ET-18-OCH3 (edelfosine) effectively inhibited the leukocyte membranes whereas the ATPase activity of synaptosomes was significantly increased by 20 μM and slightly inhibited by higher concentrations of these agents. The analogue hexadecylphosphocholine (miltefosine) did not affect the ATPase activity of the synaptosomes and only inhibited that of the leukocyte membranes at concentrations above 20 μM. These results show that several test substances of current interest affect the activity of the plasma membrane Ca2+-ATPase. The effects depend on the origin of the membranes. The investigation does not permit a distinction between direct effects on the enzyme and an interference with its membrane environment although the latter is indicated for the ether phospholipids.

A Missense Mutation of the Gene for Na+,K+-ATPase α-Subunit Causes Abnormal Feeding Behavior inCaenorhabditis elegans

The Na+,K+-ATPase activity of membranes from a behavioral mutant ofC. eleganswas found to be about one-third that of the wild-type. The levels of mRNA and polypeptide of Na+,K+-ATPase α-subunit in the mutant were as high as those in the wild-type, but the level of the phosphorylated intermediate of the Na+,K+ATPase in the mutant worm was 80% lower than that in the wild-type. A single predicted amino acid replacement (Leu to Phe) was found in a highly conserved region in the α-subunit that is involved in the formation of phosphorylated intermediate. The abnormal feeding behavior of the mutant worm may be attributed to this missense mutation.

An investigation of herbicide interaction with the H+-ATPase activity of plant plasma membranes

This study has investigated the activity of several herbicide classes at the plant plasma membrane. Two-phase partitioning was used to prepare highly purified plasma membrane vesicles from the monocotyledon weed black-grass (Alopecurus myosuroides Huds.) and the dicotyledon crop sugar beet (Beta vulgaris L. cv. Celt). The purity of the plasma membrane H + -ATPase activity was characterised with respect to inhibitors, pH and substrate specificity. In both species, contamination of the plasma membrane by tonoplast fragments was largely eliminated and chlorophyll was absent. In addition, the plasma membrane H + -ATPase from black-grass and sugar beet exhibited high vanadate sensitivity and a sharp pH profile around 6.5. Subsequently, H + -ATPase activity was assayed in the presence (100 μM) and absence of four graminicide classes and auxin-type herbicides. Graminicides, including the aryloxyphenoxypropionate diclofop-methyl and the thiocarbamate triallate, inhibited H + -ATPase activity by 50-80% in both species. However, other graminicides, including the cyclohexanediones and the chloroacetamide alachlor, had no affect. Similarly, auxin-type herbicides such as 2,4-D and MCPA did not inhibit H + -ATPase activity. Results are discussed in relation to the proposed mode of action of these herbicides.

Effect of Seed Freezing and Storage Conditions on Plasma Membrane Properties of Norway Spruce (Piceo abies Karst.) Seedlings

Abstract:Norway spruce (Picea abies Karst.) seeds were frozen and stored for 15 months at + 3, − 25, − 75 or − 196°C. After storage, seeds were germinated for 9−14 days to determine viability and plasma membrane protein composition, H+‐ATPase activity and fluidity. The results indicate no significant differences in viability of seed 14 days after germination. Biochemical analyses revealed increased plasma membrane fluidity in 9‐day‐old Norway spruce seedlings raised from seeds pretreated at − 75 °C. and changes in the temperature profile of membrane fluidity in seedlings after pre‐treatment of seeds at − 25 °C. On the other hand, the same treatments did not result in changes in plasma membrane protein content, protein composition or ATPase activity. There was also no difference in plasma membrane H+‐ATPase activity assayed in the presence of different ATP hydrolysis inhibitors. Based on the presented results, and other experimental data, we suggest that during early seedling growth, adaptation of seeds to − 25 and − 75°C freezing and/or storage temperature results in stability of the plasma membrane protein function and composition and increased fluidity or changes in the temperature‐dependent fluidity profile of these membranes.

Comparison of Mg 2+-dependent ATP hydrolase activities of pea nodule symbiosomes and of pea root plasmalemma, obtained by an aqueous polymer two-phase system

The characteristics of the Mg 2+-dependent ATPase activity from the peribacteroid membrane of pea symbiosomes was compared with that from pea root plasma membranes. Enzyme inhibitors, optimum reaction pH, substrate specificity and antibody recognition were the main parameters examined. Both the symbiosomes and the root plasma membrane were purified with an aqueous polymer two-phase system (APS). The final concentration of the APS for the purification of symbiosomes were: 6.3% w/w dextran T500, 6.3% w/w poly(ethylene glycol) 3350, 5 m M KH 2PO 4–K 2HPO 4, 5 m M KCl, 0.33 M sucrose, (pH 7.85); for the root plasma membrane was: 6.2% (w/w) dextran T500, 6.2% poly(ethylene glycol) 3350, 330 m M sucrose, 5 m M K 2HPO 4 and 4 m M KCl (pH 7.8). The lack of contamination of pea symbiosomes with endoplasmic reticulum, mitochondria, broken bacteroids and/or tonoplast vesicles was established. Similarly, the aqueous two-phase system used here provided a fairly enriched root plasma membrane with low cross-contamination from other sources. Both symbiosomal and root plasma membrane ATPase activities were highly specific to ATP. The symbiosome ATPase apparently corresponds to an E 1E 2–ATPase mechanism, similar to that found at the plasma membrane. The similarity between these two ATPases was further supported by immuno-analysis. Mg 2+-ATPase of pea symbiosome and root plasma membranes were very similar, by all parameters tested.

Ca(2+)-ATPases of Saccharomyces cerevisiae: diversity and possible role in protein sorting.

The PMR1 gene of Saccharomyces cerevisiae is thought to encode a putative Ca(2+)-ATPase [1]. Membranes isolated from wild-type cells and from pmr1 null mutant of S. cerevisiae were fractionated on sucrose density gradients. In the pmr1 mutant we found a decrease in activity of the P-type ATPase and of ATP-dependent, protonophore-insensitive Ca2+ transport in light membranes, that comigrate with the Golgi marker GDPase. We conclude that the product of the PMR1 gene (Pmr1p) is indeed a Ca(2+)-ATPase of the Golgi and Golgi-like membranes. Surprisingly, the pmr1 null mutation abolished Ca(2+)-ATPase activity in Golgi and/or Golgi-like membranes only to 50% under conditions where they are separated from vacuolar membranes. This indicates that an additional Ca(2+)-ATPase is localized in Golgi and/or Golgi-like membranes. Moreover, a third Ca(2+)-ATPase is found in the ER and ER-like membranes. The data are consistent with the assumption that these Ca(2+)-ATPases are encoded by gene(s) different from PMR1. Disruption of PMR1 Ca(2+)-ATPase causes significant redistribution of enzyme activities and of total protein in compartments of the secretory pathway. A decrease in activity is observed for three integral membrane proteins: NADPH cytochrome c reductase, dolichyl phosphate mannose synthase, and Ca(2+)-ATPase, and also for total protein in Golgi, Golgi-like compartments and in vacuoles, whereas a corresponding increase of these activities is observed in endoplasmic reticulum and endoplasmic reticulum-like membranes. We assume that Ca(2+)-ATPases and sufficient Ca2+ gradients across the organellar membranes are important for the correct sorting of proteins to the various compartments of the secretory apparatus.

Ca 2+-ATPases of Saccharomyces cerevisiae: diversity and possible role in protein sorting

The PMR1 gene of Saccharomyces cerevisiae is thought to encode a putative Ca 2+-ATPase [1]. Membranes isolated from wild-type cells and from pmr1 null mutant of S. cerevisiae were fractionated on sucrose density gradients. In the pmr1 mutant we found a decrease in activity of the P-type ATPase and of ATP-dependent, protonophore-insensitive Ca 2+ transport in light membranes, that comigrate with the Golgi marker GDPase. We conclude that the product of the PMR1 gene (Pmr1p) is indeed a Ca 2+-ATPase of the Golgi and Golgi-like membranes. Surprisingly, the pmr1 null mutation abolished Ca 2+-ATPase activity in Golgi and/or Golgi-like membranes only to 50% under conditions where they are separated from vacuolar membranes. This indicates that an additional Ca 2+-ATPase is localized in Golgi and/or Golgi-like membranes. Moreover, a third Ca 2+-ATPase is found in the ER and ER-like membranes. The data are consistent with the assumption that these Ca 2+-ATPases are encoded by gene(s) different from PMR1. Disruption of PMR1 Ca 2+-ATPase causes significant redistribution of enzyme activities and of total protein in compartments of the secretory pathway. A decrease in activity is observed for three integral membrane proteins: NADPH cytochrome c reductase, dolichyl phosphate mannose synthase, and Ca 2+-ATPase, and also for total protein in Golgi, Golgi-like compartments and in vacuoles, whereas a corresponding increase of these activities is observed in endoplasmic reticulum and endoplasmic reticulum-like membranes. We assume that Ca 2+-ATPases and sufficient Ca 2+ gradients across the organellar membranes are important for the correct sorting of proteins to the various compartments of the secretory apparatus.

Mode of action of torilin in multidrug-resistant cancer cell lines.

The mechanism of action of multidrug-resistance reversing activity of torilin was studied. In vitro experiments for the accumulation and efflux of vinblastine clearly indicated that MDR reversing effects of torilin would directly be associated with the increase of the intracellular accumulation of anticancer drugs by blocking the drug efflux. Furthermore, torilin increased the membrane ATPase activity from KB-V1 cells, suggesting that torilin might function by inhibiting drug transport mediated by P-glycoprotein.

A novel phosphorylated lipid counteracts activation of the renal plasma membrane (Ca2+ + Mg2+)ATPase by endogenous phosphatidylinositol-4-phosphate.

The plasma membrane (Ca2+ + Mg2+)ATPase is activated by acidic phospholipids in reconstituted systems. In this report it is shown that reversible phosphorylation of endogenous phosphatidylinositol regulates the renal plasma membrane (Ca2+ + Mg2+)ATPase, and that a novel phosphorylated lipid that can be isolated from the same membrane strongly counteracts the stimulatory effect of phosphatidylinositol-4-phosphate.