Effects of Na+ and K+ on Artemia salina (Na,K)-ATPase solubilized with a zwitterionic detergent, CHAPS

The membrane bound (Na,K)-ATPase prepared from Artemia salina nauplii was solubilized with a zwitterionic detergent, 3[3(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and then purified on a Bio-Gel A-1.5 m column in the presence of the detergent. 1) Upon solubilization, both NaCl and KCl protected the enzyme against loss of activity, KCl being more effective than NaCl. 2) Gel filtration of the solubilized enzyme on a Bio-Gel A-1.5 m column in the presence of 5 mM CHAPS resulted in loss of the enzyme activity even when one of the cations was added. Most of the phospholipids in the solubilized enzyme preparation were removed during the gel filtration (delipidation) and 10-25 phospholipids were left on a protomer (alpha beta) of the enzyme irrespective of the cation present during the gel filtration. With the addition of exogenous phospholipids, the activity was restored. The activity of the enzyme delipidated in the presence of KCl was restored to 3-4 times higher than in the case of that delipidated in the presence of NaCl. 3) Relipidation experiments with a fluorescent phospholipid, dansyl phosphatidylethanolamine (Dans-PE), suggested that the enzyme delipidated in the presence of KCl reassociated with phospholipids more firmly than the enzyme delipidated in the presence of NaCl. From these results we concluded that K+ stabilized the (Na,K)-ATPase more effectively than Na+, even when the enzyme was delipidated.     

Characterization of native and reconstituted hydrogen ion pumping adenosinetriphosphatase of chromaffin granules

The ATP-dependent H+ pump from adrenal chromaffin granules is, like the platelet-dense granule H+ pump, essentially insensitive to the mitochondrial ATPase inhibitors sodium azide, efrapeptin, and oligomycin and also insensitive to vanadate and ouabain, agents that inhibit the Na+,K+-ATPase. The chromaffin granule H+ pump is, however, sensitive to low concentrations of NEM (N-ethylmaleimide) and Nbd-Cl (7-chloro-4-nitro-2,1,3-benzoxadiazole). These transport ATPases may thus belong to a new class of ATP-dependent ion pumps distinct from F1F0-and phosphoenzyme-type ATPases. Comparisons of ATP hydrolysis with ATP-dependent serotonin transport suggest that approximately 80% of the ATPase activity in purified chromaffin granule membranes is coupled to H+ pumping. Most of the remaining ATPase activity is due to contaminating mitochondrial ATPase and Na+,K+-ATPase. When extracted with cholate and octyl glucoside, the H+ pump is solubilized in a monodisperse form that retains NEM-sensitive ATPase activity. When reconstituted into proteoliposomes with crude brain phospholipid, the extracted enzyme recovers ATP-dependent H+ pumping, which shows the same inhibitor sensitivity and nucleotide dependence as the native pump. These data demonstrate that the predominant ATP hydrolase of chromaffin granule membrane is also responsible for ATP-driven amine transport and granule acidification in both native and reconstituted membranes.     

Reactivation of lipid-depleted Ca2+-ATPase by a nonionic detergent.

The Ca2+-ATPase of sarcoplasmic reticulum can be reversibly delipidated by precipitation with polyethyleneglycol in the presence of deoxycholate and glycerol to as low as 4 mol of phospholipid/mol of enzyme polypeptide and can then be reactivated to 90% of its original ATPase activity by the addition of phosphatidylcholine. Furthermore, the preparation exhibits nearly the same activity if the nonionic detergent dodecyl octaoxyethyleneglycol monoether is substituted for the added phospholipid. The delipidated ATPase is soluble in the detergent and retains activity for several days. This is the first report of the Ca2+-ATPase retaining high activity with less than about 30 mol of phospholipid bound per mol of polypeptide.     

The effect of changes of the content of membrane cholesterol and the effect of benzyl alcohol on the activity of the intrinsic Mg2+-ATPase from the erythrocyte plasma membrane of the flounder (Platichthys flesus L.)

Extraction of membrane cholesterol and incorporation of cholesteryl hemisuccinate into the membrane affect the activity of the membrane-bound Mg2+-ATPase. Increasing the ratio of cholesterol to phospholipid from 0.30 mg/mg in the control membranes to 0.45-0.90 in the enriched membranes results in a slight increase of the activity of about 20%. Diminishing the ratio of cholesterol to phospholipid to about one tenth of the ratio of the control membrane results in a decrease of the activity to about 30% of the untreated control. Benzyl alcohol inactivates the membrane-bound enzyme. Digitonin-solubilized Mg2+-ATPase is also inactivated by benzyl alcohol. For concentrations below 20 mM the dependence of the solubilized and the membrane-bound enzymes are virtually identical, and linearly dependent on alcohol concentration. This linear relationship continues up to 70 mM for the solubilized enzyme, while inhibition of the membrane-bound form shows a slightly steeper dependence on inhibitor concentration. It is suggested that the activity of the native Mg2+-ATPase depends on the organization of the lipid phase of the membrane and that addition of benzyl alcohol or depletion of cholesterol results in a disorganization of the lipid phase which in turn results in diminished activity.     

Polar head-group and acyl side-chain requirements for phospholipid-dependent (Na-+ plus K-+)-ATPase.

The abilities of different phospholipids to reactivate a lipid-depleted (Na-+ plus K-+)-ATPase (EC 3.6.1.3) have been compared. The phospholipids contained either the same group of hydrocarbon chains withhh different polar groups, or different hydrocarbon groups wit the same polar group; they were prepared by enzymic modification of the polar group of phosphatidylserine isolated from bovine brain and of phosphatidylcholine extracted from egg. Only the acidic phospholipids reactivated the ATPase but the amount of reactivation depended on the nature of the hydrocarbon chains as well as the polar group. These findings are discussed in relations to the compositions of the two different groups of fatty acyl chains constituting the hydrocarbon portions of the phospholipids.     

Lipid composition of different preparations of brain Na+, K+-ATPase

The content and composition of phospholipids is determined in beef microsomal and synaptosomal fractions and also in these fractions preparations solubilized with triton X-100 (0.1%) and digitonin (0.2%). It is shown that the microsomal fraction is richer in phospholipids. The solubilized fragments of microsomes have less or the same amount of phospholipids per protein unit than the initial fraction of microsomes, and the solubilized fragments of synaptosomes contain a higher quantity of phospholipids than the initial fraction. The content of phospholipids in "the riton" fragments of synaptosomes is higher than in "those" of microsomes. Contrary to digitonin which solubilizes the active Na+, K+-ATPase complex of microsomes and synaptosomes, triton X-100 solubilizes the active enzyme of microsomes only. A higher total content of phospholipids in "the triton" extracts of synaptosomes does not probably correlate with the presence of Na+, K+-ATPase activity in them. But these extracts are found to contain less phosphatidylserine whose addition recovers Mg2+, Na+, K+-ATPase activity in them. The effect of phosphatidylserine is not strictly specific for "the triton" extracts of synaptosomes, this lipid activates to a definite extent the extracts of microsomes as well. It is shown that at the first stages of bull brain Na+, K+-ATPase purification the total content of phospholipids and cholesterol in the preparations increases but the composition of phospholipids remains unchanged.     

不同磷脂和糖脂对莱氏衣原体膜上Mg~（2＋）－ATPase活性的影响

莱氏衣原体膜上Ｍｇ~（２＋）－ＡＴＰａｓｅ用ＤＯＣ溶解后,经Ｓｅｐｈａｒｏｓｅ－６Ｂ和ＤＥＡＥ－ＣｅｌｌｕｌｏｓｅＤＥ－５２离子交换柱,得到了部分纯化的Ｍｇ~（２＋）ＡＴＰａｓｅ,并将此ＡＴＰａｓｅ与不同极性头部的磷脂和膜糖脂重组,研究了不同的极性头部的磷脂和膜糖脂对ＡＴＰａｓｅ活性的影响。此酶的活性不依赖酸性磷脂,ＰＧ、ＤＰＧ、大豆磷脂等明显抑制酶活性,中性磷脂ＤＭＰＣ、ＰＥ、ＰＣ则能增加酶活性,其中尤以非双层脂ＰＥ的作用最为明显。从莱氏衣原体膜上提取的糖脂（ＭＧＤＧ,ＤＧＤＧ）单独和ＡＴＰａｓｅ重组时,酶活性增加并不明显,当ＭＧＤＧ和ＤＧＤＧ以等比例混合时,能大大地增加酶活性。这表明Ｍｇ~（２＋）－ＡＴＰａｓｅ的活性很大程度上与磷脂的表面电荷及磷脂的组成相关。     

Effect of concanavalin A on the activity of membrane-bound and detergent-solubilized Mg2+ -ATPase

Plasma membranes were isolated after binding liver and hepatoma cells to polylysine-coated polyacrylamide beads, and the effect of concanavalin A on the membrane-bound Mg2+ -ATPase and the Mg2+ -ATPase solubilized by octaethylene glycol monododecyl ether (C12E8) was studied. In the experiment of membrane-bound Mg2+ -ATPase, plasma membranes were pretreated with Concanavalin A and the activity was assayed. Concanavalin A stimulated the activity of both liver and hepatoma enzymes assayed above 20 degrees C. Concanavalin A abolished the negative temperature dependency characteristic of liver plasma membrane Mg2+ -ATPase. On the other hand, Concanavalin A prevented the rapid inactivation due to storage at -20 degrees C, which was characteristic of hepatoma plasma membrane Mg2+ -ATPase. With solubilized Mg2+ -ATPase from liver plasma membranes, the negative temperature dependency was not observed. Concanavalin A, which was added to the assay medium, stimulated the activity of the enzyme solubilized in C12E8 at a high ionic strength. However, Concanavalin A failed to show any effect on the enzyme solubilized in C12E8 at a low ionic strength. With solubilized Mg2+ -ATPase from hepatoma plasma membranes, Concanavalin A could not prevent the inactivation of the enzyme during incubation at -20 degrees C.     

p-NITROPHENYL PHOSPHATASES OF A MEMBRANE FRACTION FROM BOVINE CEREBRAL CORTEX

Abstract— Three different types of p -nitrophenyl phosphatases (NPPases) were solubilized by deoxycholate treatment from a membrane fraction of bovine cerebral cortex, and their characteristics were determined. Of these three NPPases (acid, Mg²⁺-activated, and K⁺, Mg²⁺-activated), only K-Mg NPPase was stimulated about two-fold by phospholipid and was inhibited by unsaturated neutral lipids and fatty acids. Unlike Na⁺-K⁺-Mg^a+-activated ATPase, the enzyme did not absolutely require phospholipid for its activity, but was similarly thermolabile and was protected by phospholipid from thermal inactivation. Acid NPPase was separable from the other two NPPases by ammonium sulphate fractionation, and partly solubilized by dialysis against ATP-mercaptoethanol solution. Hg²⁺ inhibited equally all three NPPases, but Ca²⁺ inhibited only Mg and K-Mg NPPases. Ouabain was effective on K-Mg NPPase alone.     

Inhibition of the H+-ATPase in bovine adrenal chromaffin granule ghosts by diethylstilbestrol. Evidence for a tight coupling between ATP hydrolysis and proton translocation

Diethylstilbestrol (DES) was found to inhibit reversibly the hydrolysis of MgATP (80% at 100 microM) and proton pump activity (I50 approximately equal to 15 microM, complete at 100 microM) in chromaffin granule ghosts. The parallel inhibition suggests a tight kinetic coupling between the two activities. The Mg2+-ATPase activity, but not proton pumping, was partially restored by N,N'-dicyclohexylcarbodiimide, indicating that the two inhibitors in combination cause a partial uncoupling. The non-competitive type of inhibition shows that the action of DES is distal to the site of ATP binding and hydrolysis. Although unspecific, the interaction of DES with the chromaffin granule membrane seems primarily to affect the H+-ATPase.     

Purification and characterization of the membrane (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B

The membrane (Na⁺ + Mg²⁺)-ATPase of Acholeplasma laidlawii B has been solubilized with a Brij-58/sodium deoxycholate mixture and purified by a combination of gel filtration and ion-exchange chromatography. The purified, partially delipidated ATPase has a specific activity of 195 μmol P_i/mg protein per h, which could be enhanced by 25% upon the addition of exogenous phospholipids. The kinetic properties of the purified enzyme are similar to those of the native membrane-bound enzyme, suggesting that it has not been substantially altered during the purification procedure. The enzyme is an assembly of five polypeptide species and its kinetic properties suggest that it is dissimilar to other known ATPases.     

Phospholipid and calmodulin activation of solubilized calcium-transport ATPase from human erythrocytes: regulation by magnesium

The effect of phospholipids on Triton X-100 solubilized (Ca2+ + Mg2+)-ATPase from human erythrocyte membranes has been examined. The enzyme activity was increased by phosphatidylinositol, phosphatidylserine, and phosphatidic acid at both low (2 micrometer) and high (65 micrometer) free Ca2+ concentrations, while phosphatidylcholine had little effect and phosphatidylethanolamine and cardiolipin inhibited the (Ca2+ + Mg2+)-ATPase activity at all Ca2+ concentrations studied. The diacylglycerol, diolein, inhibited the enzyme at high, but not low, Ca2+ concentrations. Low concentrations of phospholipase A2 (1-2 international units) also activated the solubilized enzyme, at least in part by releasing free fatty acids, as the activation was mimicked by oleic acid (1-2 mumol/mg protein) and was abolished by fatty acid depleted bovine serum albumin. The combined activation by saturating levels of phosphatidylserine and calmodulin was additive at 6.5 mM MgCl2, and probably occurred at distinct sites on a regulatory component of the enzyme. The activation by both effectors was antagonized by MgCl2 at similar concentrations. Analysis of various models suggested that phosphatidylserine had two effects on (Ca2+ + Mg2+)-ATPase activity. First, a low Ca2+ affinity form of the enzyme was converted to a high Ca2+ affinity form, which was more sensitive to Ca2+ inhibition. Second, it increased the turnover of the enzyme, probably by enhancing its dephosphorylation, which was mimicked in this study by the Ca2+-dependent p-nitrophenylphosphatase partial reaction.     

Purification and properties of a vanadate- and N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes

A vanadate- and N-ethylmaleimide-sensitive ATPase was purified about 500-fold from chromaffin granule membranes. The purified preparation contained a single major polypeptide with an apparent molecular mass of about 115 kDa, which was copurified with the ATPase activity. Immunological studies revealed that this polypeptide has no relation to subunit I (115 kDa) of the H+-ATPase from chromaffin granules. The ATPase activity of the enzyme is inhibited about 50% by 100 microM N-ethylmaleimide or 5 microM vanadate. The enzyme is not sensitive to dicyclohexylcarbodiimide, ouabain, SCH28080, and omeprazole, which distinguishes it from Na+/K+-ATPase and the gastric K+/H+-ATPase. ATP and 2-deoxy ATP are equally effective substrates for the enzyme. However, the enzyme exhibited only 10% activity with GTP as a substrate. UV illumination of the purified enzyme in the presence of [alpha-32P]ATP exclusively labeled the 115 kDa protein. This labeling was increased by Mg2+ and strongly inhibited by Ca2+ ions. Similarly, the ATPase activity was dependent on Mg2+ and inhibited by the presence of Ca2+ ions. The ATPase activity of the enzyme was largely insensitive to monovalent anions and cations, except for F-, which inhibited the vanadate-sensitive ATPase. Incubation of the enzyme in the presence of [14C]N-ethylmaleimide labeled the 115-kDa polypeptide, and this labeling could be prevented by the addition of ATP during the incubation. A reciprocal experiment showed that preincubation with N-ethylmaleimide inhibited the labeling of the 115-kDa polypeptide by [alpha-32P]ATP by UV illumination. This suggests a close proximity between the ATP-binding site and an essential sulfhydryl group. A possible connection between the isolated ATPase and organelle movement is discussed.     

Transient kinetics of a Ca2+-induced fluorescence change from membrane-associated and solubilized sarcoplasmic reticulum Ca2+-ATPase

The kinetics and extent of the fluorescence change induced by Ca2+ interaction with the Ca2+-ATPase from sarcoplasmic reticulum have been compared by stopped flow fluorimetry for three preparations: sarcoplasmic reticulum; purified ATPase in membrane vesicles; and solubilized, delipidated ATPase. The kinetics of Ca2+ release and binding for both purified preparations could be described by a single exponential as has been observed for sarcoplasmic reticulum. The rate and extent of the fluorescence change for the solubilized and membrane-associated preparations are shown to be quite similar to those of the sarcoplasmic reticulum. From these results, it is concluded that all of the Ca2+-induced fluoescence change in sarcoplasmic reticulum originates from the Ca2+-ATPase. In addition, since the change in fluorescence is probably result of a conformational change in the ATPase during the Ca2+ pumping cycle, the results provide additional evidence that monomeric Ca2+-ATPase may be capable of Ca2+ transport since the delipidated preparation is monomeric under the conditions used for these experiments. Finally, it is concluded that phospholipid bilayer is not essential for this conformational change.     

Conformational basis of the phospholipid requirement for the activity of SR Ca(2+)-ATPase

The delipidated sarcoplasmic reticulum (SR) Ca(2+)-ATPase was reconstituted into proteoliposomes containing different phospholipids. The result demonstrated the necessity of phosphatidylcholine (PC) for optimal ATPase activity and phosphatidylethanolamine (PE) for the optimal calcium transport activity. Fluorescence intensity of Fluorescein 5-isothiocyanate (FITC)-labeled enzyme at Lys515 as well as the measurement of the distance between 5-((2-[(iodoacetyl) amino] ethyl) amino)naphthalene-1-sulphonic acid (IAEDANS) label sites (Cys674/670) and Pr3+ demonstrated a conformational change of cytoplasmic domain, consequently, leading to the variation of the enzyme function with the proteoliposomes composition. Both the intrinsic fluorescence of Trp and its dynamic quenching by HB decreased with increasing PE content, revealing the conformational change of transmembrane domain. Time-resolved fluorescence study characterized three classes of Trp residues, which showed distinctive variation with the change in phospholipid composition. The phospholipid headgroup size caused the conformational change of SR Ca(2+)-ATPase, subsequent the ATPase activity and Ca2+ uptake.     

Accessibility of phospholipids in the chromaffin granule membrane.

1. The accessibility of phospholipids in the membrane of the adrenomedullary storage vesicles (chromaffin granules) has been studied. 2. The reaction of 2,4,6-trinitrobenzenesulphonic acid with both intact granules and their ghosts, results in the labelling of 70% of the phosphatidylethanolamine. 3. The action of phospholipase A2 (from bee venom), phospholipase C (from Bacillus cereus) and sphingomyelinase C (from Staphylococcus aureus) on granules and their ghosts was followed as a function of time. No significant difference was observed between the intact granules and their ghosts. 4. In the intact granules the various treatments led to varying amounts of lysis although again no evidence was obtained that such lysis in any way increased the amount of accessible phospholipid. 5. Highly purified granule preparations were also compared with the so-called "large granule" fraction and no significant differences were detected. 6. Approx. 67% of phosphatidylethanolamine + phosphatidic acid, 50% of phosphatidylserine + phosphatidylinositol, 65% of phosphatidylcholine and 20% of sphingomyelin is accessible to enzymatic degradation. In total, approx. 50% of all the phospholipids reacted. 7. It is also shown that, unlike in enzymatic treatment, all the phosphatidylcholine can be exchanged in the presence of a phospholipid exchange protein (prepared from beef liver). 8. It is concluded that transmembrane movement of phosphatidylcholine is slow in isolated membranes of chromaffin granules. The presence of the exchange protein, however, in conjunction with membrane proteins and specific phospholipid arrangements may catalyse this transmembrane movement.     

sn-1,2-Diacylglycerol kinase of Escherichia coli. Mixed micellar analysis of the phospholipid cofactor requirement and divalent cation dependence

Efficient delivery of hydrophobic water-insoluble substrates and cofactors to membrane-bound enzymes is a recurring problem which has impeded kinetic analyses. Kinetic analysis of the Escherichia coli sn-1,2-diacylglycerol kinase, an extremely hydrophobic integral membrane protein of 122 residues, was facilitated by the development of a mixed micellar assay. beta-Octyl glucoside micelles quantitatively solubilized diacylglycerol kinase from membranes of strains which overproduced the enzyme up to 250-fold and provided an effective method to disperse and deliver the hydrophobic water-insoluble substrate, sn-1,2-dioleoyglycerol. Diacylglycerol kinase was active in mixed micelles containing octyl glucoside and dioleoyglycerol. Several phospholipids stimulated activity up to 6-fold, suggesting a cofactor function. Activation by phospholipids was not stereospecific and was mimicked partially by fatty acids. Half-maximal activation was observed at 1 mol % cardiolipin, suggesting that a small number of phospholipids are sufficient to activate the enzyme. Activity was dependent on the mole fractions of dioleoylglycerol and phospholipid in the mixed micelles, but independent of micelle number. Several lines of evidence indicated that the transfer of diacylglycerol between micelles was much more rapid than its utilization by the enzyme. Diacylglycerol kinase exhibited Michaelis-Menten kinetics with respect to diacylglycerol and MgATP. A second Mg2+ ion (in addition to MgATP) was required for activity. When Mg2+ was excluded from the assay, Mn2+, Zn2+, Cd2+, and Co2+ supported activity to lesser extents. These data establish a suitable system for in-depth kinetic analysis of the E. coli diacylglycerol kinase and its phospholipid cofactor requirements.     

Lysosomal H+-translocating ATPase has a similar subunit structure to chromaffin granule H+-ATPase complex

Subunit structure of the lysosomal H+-ATPase was investigated using cold inactivation, immunological cross-reactivity with antibodies against individual subunits of the H+-ATPase from chromaffin granules and chemical modification with N,N'-dicyclohexyl[14C]carbodiimide. The lysosomal H+-ATPase was irreversibly inhibited when incubated at 0 degrees C in the presence of chloride or nitrate and MgATP. Inactivation in the cold resulted in the release of several polypeptides (72, 57, 41, 34 and 33 kDa) from the membrane, which had the same electrophoretic mobility as the corresponding subunits of chromaffin granule H+-ATPase. Cross-reactivity of antibodies revealed that the 72, 57 and 34 kDa polypeptides were immunologically identical to the corresponding subunits of chromaffin granule H+-ATPase. Dicyclohexylcarbodiimide, which inhibits proton translocation in the vacuolar ATPase, predominantly labeled two polypeptides of 18 and 15 kDa, which compose the membrane sector of the enzyme. These results suggest that the lysosomal H+-ATPase is a multimeric enzyme, whose subunit structure is similar to the chromaffin granule H+-ATPase. The subunit structure of other vacuolar H+-ATPases, revealed by cold inactivation and immunological cross-reactivity, is also presented.     

The influence of changes in the phospholipid pattern of intact fibroblasts on the activities of four membrane-bound enzymes.

Human skin fibroblasts, grown to confluency in the presence of 32P for random labelling of the phospholipids, showed upon 24 h incubation in the presence of either 8 mM L-serine or 4 mM ethanolamine an increased content of phosphatidylserine (150% of control cells) or phosphatidylethanolamine (116% of control cells), respectively. Concomitantly the phosphatidylcholine correspondingly decreased. Upon cell harvesting and gentle enzyme preparation the base-treated cells demonstrated a significantly higher unstimulated, fluoride- and thyrotropin-stimulated activity of adenylate cyclase. The activities of total ATPase, ouabain-sensitive ATPase, 5'-nucleotidase and gamma-glutamyltransferase remained unaltered. When subjecting enzyme preparations from fibroblasts to ultrasonication the activity of adenylate cyclase decreased progressively with energy applied, whereas the activities of the other enzymes were unaltered ((K+ + Na+)-ATPase, 5'-nucleotidase) or even increased (Mg2+-ATPase, gamma-glutamyltransferase). The results have a bearing upon the regulatory function of the phospholipid microenvironment of membrane-bound enzymes.     

The lipid requirement of the (Ca2+ + Mg2+)-ATPase in the human erythrocyte membrane, as studied by various highly purified phospholipases.

1. When complete hydrolysis of glycerophosphlipids and sphingomyelin in the outer membrane leaflet is brought about by treatment of intact red blood cells with phospholipase A2 and sphingomyelinase C, the (Ca2+ + Mg2+)-ATPase activity is not affected. 2. Complete hydrolysis of sphingomyelin, by treatment of leaky ghosts with spingomyelinase C, does not lead to an inactivation of the (Ca2+ + Mg2+)-ATPase. 3. Treatment of ghosts with phospholipase A2 (from either procine pancreas of Naja naja venom), under conditions causing an essentially complete hydrolysis of the total glycerophospholipid fraction of the membrane, results in inactivation of the (Ca2+ + Mg2+)-ATPase by some 80--85%. The residual activity is lost when the produced lyso-compounds (and fatty acids) are removed by subsequent treatment of the ghosts with bovine serum albumin. 4. The degree of inactivation of the (Ca2+ + Mg2+)-ATPase, caused by treatment of ghosts with phospholipase C, is directly proportional to the percentage by which the glycerophospholipid fraction in the inner membrane layer is degraded. 5. After essentially complete inactivation of the (Ca2+ + Mg2+)-ATPase by treatment of ghosts with phospholipase C from Bacillus cereus, the enzyme is reactivated by the addition of any of the glycerophospholipids, phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine or lysophosphatidylcholine, but not by addition of sphingomyeline, free fatty acids or the detergent Triton X-100. 6. It is concluded that only the glycerophospholipids in the human erythrocyte membrane are involved in the maintenance of the (Ca2+ + Mg2+)-ATPase activity, and in particular that fraction of these phospholipids located in the inner half of the membrane.