Purification and characterization of an extracellular beta-glucosidase produced by Phoma sp. KCTC11825BP isolated from rotten mandarin peel

A beta-glucosidase from Phoma sp. KCTC11825BP isolated from rotten mandarin peel was purified 8.5-fold with a specific activity of 84.5 U/mg protein. The purified enzyme had a molecular mass of 440 kDa with a subunit of 110 kDa. The partial amino acid sequence of the purified beta-glucosidase evidenced high homology with the fungal beta- glucosidases belonging to glycosyl hydrolase family 3. Its optimal activity was detected at pH 4.5 and 60 degrees C, and the enzyme had a half-life of 53 h at 60 degrees C. The Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose were 0.3 mM and 3.2 mM, respectively. The enzyme was competitively inhibited by both glucose (Ki=1.7 mM) and glucono-delta-lactone (Ki=0.1 mM) when pNPG was used as the substrate. Its activity was inhibited by 41% by 10 mM Cu2+ and stimulated by 20% by 10 mM Mg2+.     

Purification of beta-glucosidase from olive (Olea europaea L.) fruit tissue with specifically designed hydrophobic interaction chromatography and characterization of the purified enzyme

An olive (Olea europaea L.) β-glucosidase was purified to apparent homogeneity by salting out with ammonium sulfate and using specifically designed sepharose-4B-L-tyrosine-1-napthylamine hydrophobic interaction chromatography. The purification was 155 fold with an overall enzyme yield of 54%. The molecular mass of the protein was estimated as ca. 65 kDa. The purified β-glucosidase was effectively active on p-/o-nitrophenyl-β-D-glucopyranosides (p-/o-NPG) with K(m) values of 2.22 and 14.11 mM and V(max) values of 370.4 and 48.5 U/mg, respectively. The enzyme was competitively inhibited by δ-gluconolactone and glucose against p-NPG as substrate. The K(i) and IC(50) values of δ-gluconolactone were determined as 0.016 mM and 0.23 mM while the enzyme was more tolerant to glucose inhibition with K(i) and IC(50) values of 6.4 mM and 105.5 mM, respectively, for p-NPG. The effect of various metal ions on the purified β-glucosidase was investigated. Of the ions tested, only the Fe(2+) increased the activity while Cd(2+) Pb(2+) Cu(2+), Ni(+), and Ag(+) exhibited different levels of inhibitory effects with K(i) and IC(50) values of 4.29×10(-4) and 0.38×10(-4), 1.26×10(-2) and 5.3×10(-3), 2.26×10(-4) and 6.1×10(-4), 1.04×10(-4) and 0.63×10(-4), 3.21×10(-3) and 3.34×10(-3) mM, respectively.     

Induction of maleate hydratase in Pseudomonas pseudoalcaligenes

Maleate hydratase (malease, EC 4.2.1.31) activity in P. pseudoalcaligenes is induced when grown on3-hydroxybenzoate. The specific malease activity was constant during the logarithmic phase in a batch culture containing 3-hydroxybenzoate as the carbon source, when 3-hydroxybenzoate-grown cells were used as inoculum. When yeast extract-grown cells were used as inoculum, the specific malease activity was correlated with growth. In both instances the specific malease activity dropped rapidly as soon as growth ceased. Maleate did not serve as a growth substrate for this microorganism, but a mutant able to grow on maleate was selected. The specific malease activity of maleate-grown cells of this mutant was not higher than the basal level of induction of malease activity.     

Purification and some properties of rose (Fructus cynosbati) hips invertase

Invertase was purified from rose (Fructus cynosbati) hips by ammonium sulfate fractionation and hydroxyapatite column chromatography. The enzyme was obtained with a yield of 4.25% and about 10.48-fold purification and had a specific activity of 8.59 U/mg protein. The molecular mass of invertase was estimated to be 66.51 kDa by PAGE and 34 kDa by SDS-PAGE, indicating that the native enzyme was a homodimer. The enzyme was a glycoprotein and contained 5.86% carbohydrate. The K(m) for sucrose was 14.55 mM and the optimum pH and temperature of the enzyme were 4.5 and 40 degrees C, respectively. Sucrose was the most preferred substrate of the enzyme. The enzyme also hydrolyzed D(+) raffinose, D(+) trehalose and inulin (activity 39.88, 8.12 and 4.94%, respectively of that of sucrose), while D(+) lactose, cellobiose and D(+) maltose showed no effect on the enzyme. The substrate specificity was consistent with that for a beta-fructofuranoside, which is the most popular type in the higher plants. The enzyme was completely inhibited by HgCl2, MnCl2, MnSO4, FeCl3, Pb(NO3)2, ammonium heptamolybdate, iodoacetamide and pyridoxine hydrochloride. It was also inhibited by Ba(NO3)2 (86.32%), NH4Cl (84.91%), MgCl2 (74.45%), urea (71.63%), I2 (69.64%), LiCl (64.99%), BaCl2 (50.30%), Mg(NO3)2 (49.90%), CrCl3 (31.90%) and CuSO4 (21.45%) and but was activated by Tris (73.99%) and methionine (12.47%).     

p-Hydroxyphenylacetate decarboxylase from Clostridium difficile. A novel glycyl radical enzyme catalysing the formation of p-cresol.

The human pathogenic bacterium Clostridium difficile is a versatile organism concerning its ability to ferment amino acids. The formation of p-cresol as the main fermentation product of tyrosine by C. difficile is unique among clostridial species. The enzyme responsible for p-cresol formation is p-hydroxyphenylacetate decarboxylase. The enzyme was purified from C. difficile strain DMSZ 1296(T) and initially characterized. The N-terminal amino-acid sequence was 100% identical to an open reading frame in the unfinished genome of C. difficile strain 630. The ORF encoded a protein of the same size as the purified decarboxylase and was very similar to pyruvate formate-lyase-like proteins from Escherichia coli and Archaeoglobus fulgidus. The enzyme decarboxylated p-hydroxyphenylacetate (K(m) = 2.8 mM) and 3,4-dihydroxyphenylacetate (K(m) = 0.5 mM). It was competitively inhibited by the substrate analogues p-hydroxyphenylacetylamide and p-hydroxymandelate with K(i) values of 0.7 mM and 0.48 mM, respectively. The protein was readily and irreversibly inactivated by molecular oxygen. Although the purified enzyme was active in the presence of sodium sulfide, there are some indications for an as yet unidentified low molecular mass cofactor that is required for catalytic activity in vivo. Based on the identification of p-hydroxyphenylacetate decarboxylase as a novel glycyl radical enzyme and the substrate specificity of the enzyme, a catalytic mechanism involving ketyl radicals as intermediates is proposed.     

A highly N-glycosylated chitin deacetylase derived from a novel strain of Mortierella sp. DY-52

Chitin deacetylase (CDA), the enzyme that catalyzes the hydrolysis of acetamido groups of GlcNAc in chitin, was purified from culture filtrate of the fungus Mortierella sp. DY-52 and characterized. The extracellular enzyme is likely to be a highly N-glycosylated protein with a pI of 4.2-4.8. Its apparent molecular weight was determined to be about 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 67 kDa by size-exclusion chromatography. The enzyme had an optimum pH of 6.0 and an optimum temperature of 60 °C. Enzyme activity was slightly inhibited by 1-10 mM Co(2+) and strongly inhibited by 10 mM Cu(2+). It required at least two GlcNAc residues for catalysis. When (GlcNAc)(6) was used as substrate, K(m) and V(max) were determined to be 1.1 mM and 54.6 μmol min(-1) respectively.     

Purification and biochemical characterization of an extracellular beta-glucosidase from the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm

An extracellular beta-glucosidase was purified from culture filtrates of the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm grown on 1.0% (w/v) carboxymethyl-cellulose using ammonium sulfate precipitation, ion-exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is monomeric with a molecular weight of 64.2 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and has a pI of 8.55. The enzyme catalyzes the hydrolysis of p-nitrophenyl-beta-D-glucopyranoside (PNPG) as the substrate, with a K(m) of 1.52 mM, and V(max) of 3.21 U min mg(-1) protein. Glucose competitively inhibited beta-glucosidase with a K(i) value of 0.79 mM. Optimal activity with PNPG as the substrate was at pH 5.0 and 50 degrees C. The enzyme was stable at pH 5.0 at temperatures up to 50 degrees C. The purified beta-glucosidase was active against PNPG, cellobiose, sophorose, laminaribiose and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel or o-nitrophenyl-beta-d-galactopyranoside. The activity of beta-glucosidase was stimulated by Ca(2+), Co(2+), Mg(2+), Mn(2+), glycerol, dimethyl sulfoxide (DMSO), dithiothreitol and EDTA, and strongly inhibited by Hg(2+). The internal amino acid sequences of D. eschscholziibeta-glucosidase have similarity to the sequences of the family 3 beta-glucosyl hydrolase.     

Purification and characterization of an intracellular beta-glucosidase from the protoplast fusant of Aspergillus oryzae and Aspergillus niger

Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed using 35% polyethylene glycol 4.000 with 0.01 mM CaCl2. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular beta-glucosidase (EC 3.2.1.21) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme had an optimum pH of 5.4 and temperature of 65 degrees C, respectively. This enzyme showed relatively high stability against pH and temperature and was stable in the pH range of 3.0-6.6. Na+, K+, Ca2+, Mg2+ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe3+. The enzyme activity was strongly inhibited by glucose, the end product ofglucoside hydrolysis. The K(m) and V(max) values against salicin as substrate were 0.035 mM and 1.7215 micromol min(-1), respectively.     

Characterization of sialidase from bloodstream forms of Trypanosoma vivax

Sialidase (EC: 3.2.1.18) from Trypanosoma vivax (Agari Strain) was isolated from bloodstream forms of the parasite and purified to apparent electrophoretic homogeneity. The enzyme was purified 77-fold with a yield of 32% and co-eluted as a 66-kDa protein from a Sephadex G 110 column. The T. vivax sialidase was optimally active at 37 degrees C with an activation energy (E(a)) of 26.2 kJ mole(-1). The pH activity profile was broad with optimal activity at 6.5. The enzyme was activated by dithiothreitol and strongly inhibited by para-hydroxy mercuricbenzoate thus implicating a sulfhydryl group as a possible active site residue of the enzyme. Theenzyme hydrolysed Neu5Ac2,3lac and fetuin. It was inactive towards Neu5Ac2,6lac, colomic acid and the gangliosides GM1, and GDI. Initial velocity studies, for the determination of kinetic constants with fetuin as substrate gave a V(max) of 142.86 micromol h(-1) mg(-1) and a K(M) of 0.45 mM. The K(M) and V(max) with Neu5Ac-2,3lac were 0.17 mM and 840 micromole h(-1) mg(-1) respectively. The T. vivax sialidase was inhibited competitively by both 2,3 dideoxy neuraminic acid (Neu5Ac2,3en) and para-hydroxy oxamic acid. When ghost RBCs were used as substrates, the enzyme desialylated the RBCs from camel, goat, and zebu bull. The RBCs from dog, mouse and ndama bull were resistant to hydrolysis.     

MALDI mass sequencing and characterization of filarialglutathione-S-transferase

Glutathione-S-transferase has been detected in the somatic extract and excretory-secretory products of different life stages of Setaria cervi, a bovine filarial parasite. The enzyme was subjected to MALDI-TOF followed by mass spectrometry and the nearest match found was Pleuronectes platessa GST. Molecular mass of the purified enzyme was approximately 26 kDa as determined by SDS-PAGE and MALDI-TOF. Setaria cervi GST exhibited high activity towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene and glutathione as substrate revealed a K(m) of 2.22 mM and 0.61 mM, respectively. The activity was inhibited significantly by Cibacron blue and alpha-tocopherol.     

Kinetics behaviors of Na,K-ATPase: comparison of solubilized and DPPC:DPPE-liposome reconstituted enzyme

We describe and compare the main kinetic characteristics of rabbit kidney Na,K-ATPase incorporated inside-out in DPPC:DPPE-liposomes with the C(12)E(8) solubilized and purified form. In proteoliposomes, we observed that the ATP hydrolysis of the enzyme is favored and also its affinity for Na(+)-binding sites increases, keeping the negative cooperativity with two classes of hydrolysis sites: one of high affinity (K(0.5)=6 microM and 4 microM for reconstituted enzyme and purified form, respectively) and another of low affinity (K(0.5)=0.4 mM and 1.4 mM for reconstituted enzyme and purified form, respectively). Our data showed a biphasic curve for ATP hydrolysis, suggesting the presence of (alphabeta)(2) oligomer in reconstituted Na,K-ATPase similar to the solubilized enzyme. The Mg(2+) concentration dependence in the proteoliposomes stimulated the Na,K-ATPase activity up to 476 U/mg with a K(0.5) value of 0.4 mM. The Na(+) ions also presented a single saturation curve with V(M)=551 U/mg and K(0.5)=0.2 mM with cooperative effects. The activity was also stimulated by K(+) ions through a single curve of saturation sites (K(0.5)=2.8 mM), with cooperative effects and V(M)=641 U/mg. The lipid microenvironment close to the proteic structure and the K(+) internal to the liposome has a key role in enzyme regulation, affecting its kinetic parameters while it can also modulate the enzyme's affinity for substrate and ions.     

Purification and characterization of isocitrate lyase from the wood-destroying basidiomycete Fomitopsis palustris grown on glucose

Isocitrate lyase (EC 4.1.3.1), a key enzyme in the glyoxylate cycle, was purified 76-fold with 23% yield as an electrophoretically homogeneous protein from the wood-destroying basidiomycete Fomitopsis palustris grown on glucose. The native enzyme has a molecular mass of 186 kDa, consisting of three identical subunits of 60 kDa. The K(m) for DL-isocitrate was found to be 1.6 mM at the optimum pH (7.0). The enzyme required Mg(2+) (K(m) 92 microM) and sulfhydryl compounds for optimal activity. The enzyme activity was strongly inhibited by oxalate and itaconate with a K(i) of 37 and 68 microM, respectively. The inhibition by the glycolysis and tricarboxylic acid cycle intermediates and related compounds suggested that the isocitrate lyase was a regulatory enzyme playing a crucial role in the fungal growth.     

Allantoate amidinohydrolase (Allantoicase) from Chlamydomonas reinhardtii: its purification and catalytic and molecular characterization

An allantoate-degrading enzyme has been purified to electrophoretic homogeneity for the first time from a photosynthetic organism, the unicellular green algae Chlamydomonas reinhardtii. The purification procedure included a differential protein extraction followed by conventional steps such as ammonium sulfate fractionation, gel filtration, anion exchange chromatography, and preparative electrophoresis. Under the routine assay conditions (7 mM allantoate), specific activity for the purified enzyme was 185 U/mg, which rose to 225 U/mg under kinetic considerations (saturating substrate). Therefore, a turnover number of 4.5 x 10(4) min(-1) can be deduced for the 200-kDa protein. The enzyme is a true allantoicase (EC 3.5.3.4) that catalyzes the degradation of allantoate to (-)ureidoglycolate and (+)ureidoglycolate to glyoxylate. The enzyme exhibited hyperbolic kinetic for allantoate and ureidoglycolate with K(m) values of 2 and 0.7 mM, respectively. V(max) of the reaction with allantoate as substrate was nine times higher than that with ureidoglycolate. The native enzyme has a molecular weight of 200 kDa and consists of six identical or similar-sized subunits of 34 kDa each, organized in two trimers of 100 kDa. Each subunit has five cysteine residues, four of which are involved in disulfide bonds, with a total of 12 disulfide bonds in the 200-kDa protein. Allantoate inhibits competitively the reaction with ureidoglycolate as substrate. In addition, buffers and group-specific reagents affect the activity in the same manner irrespective of the substrate used. Those results suggest that both substrates use the same active site. The effect of group-specific reagents suggest that the amino acids histidine, tyrosine, and cysteine are essentials for the allantoicase activity with both substrates.     

Rapid purification and biochemical characterization of glucose kinase from Streptomyces peucetius var. caesius

Glucose kinase catalyzes the ATP-dependent phosphorylation of glucose. Streptomyces peucetius var. caesius glucose kinase was purified 292-fold to homogeneity. The enzyme has cytosolic localization and is composed of four identical subunits, each of 31 kDa. The purified enzyme easily dissociates into dimers. However, in the presence of 100 mM glucose the enzyme maintains its tetrameric form. Maximum activity was found at 42 degrees C and pH 7.5. Isoelectric focusing of the enzyme showed a pl of 8.4. The N- and C-terminal amino acid sequences were MGLTIGVD and VYFAREPDPIM, respectively. The kinetic mechanism of S. peucetius var. caesius glucose kinase appears to be a rapid equilibrium ordered type, i.e., ordered addition of substrates to the enzyme, where the first substrate is d-glucose. The K(m) values for d-glucose and MgATP(2-) were 1.6 +/- 0.2 and 0.8 +/- 0.1 mM, respectively. Mg(2+) in excess of 10 mM inhibits enzyme activity.     

Purification and characterization of a thermostable α-galactosidase from Thielavia terrestris NRRL 8126 in solid state fermentation

Several seeds and husks of some plants belonging to leguminosae, Graminae, Compositae and Palmae were evaluated as carbon substrates to produce α-galactosidase (α-Gal) by the thermophilic fungus, Thielavia terrestris NRRL 8126 in solid substrate fermentation. The results showed that Cicer arietinum (chick pea seed) was the best substrate for α-Gal production. The crude enzyme was precipitated by ammonium sulphate (60%) and purified by gel filtration on sephadex G-100 followed by ion exchange chromatography on DEAE-Cellulose. The final purification fold of the enzyme was 30.42. The temperature and pH optima of purified α-Gal from Thielavia terrestris were 70 °C and 6.5, respectively. The enzyme showed high thermal stability at 70 °C and 75 °C and the half-life of the α-Gal at 90 °C was 45 min. Km of the purified enzyme was 1.31 mM. The purified enzyme was inhibited by Ag2+, Hg2+, Zn2+, Ba2+, Mg2+, Mn2+ and Fe2+ at 5 mM and 10 mM. Also, EDTA, sodium arsenate, L-cysteine and iodoacetate inhibited the enzyme activity. On the other hand, Ca2+, Cu2+, K+ and Na+ slightly enhanced the enzyme activity at 5 mM while at 10 mM they caused inhibition. The molecular weight of the α-Gal was estimated to be 82 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for biotechnological and medicinal applications.     

Purification and partial characterization of polygalacturonase from Streptomyces lydicus

Polygalacturonase produced by Streptomyces lydicus was purified to homogeneity by ultrafiltration and a combination of ion exchange and gel filtration chromatographic procedures. The purified enzyme was an exo-polygalacturonase with a molecular weight of 43 kDa. It was optimally active at 50 degrees C and pH 6.0. The enzyme was stable from pH 4.0 to 7.0 and at or below 45 degrees C for 90 min. K(m) value for polygalacturonic acid was 1.63 mg/mL and the corresponding V(max) was 677.8 microM min(-1) mg(-1). The inhibition constant (K(i)) for gluconic acid d-lactone was 20.75 mM. Purified enzyme had been inhibited by N-bromosuccinimide, while l-tryptophan could induce enzyme activity, indicating the involvement of tryptophan at the active site.     

Purification and regulation of glucose-6-phosphate dehydrogenase from Bacillus licheniformis

d-Glucose-6-phosphate nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase (EC 1.1.1.49) from Bacillus licheniformis has been purified approximately 600-fold. The enzyme appears to be constitutive and exhibits activity with either oxidized NAD (NAD(+)) or oxidized NADP (NADP(+)) as electron acceptor. The enzyme has a pH optimum of 9.0 and has an absolute requirement for cations, either monovalent or divalent. The enzyme exhibits a K(m) of approximately 5 muM for NADP(+), 3 mM for NAD(+), and 0.2 mM for glucose-6-phosphate. Reduced NADP (NADPH) is a competitive inhibitor with respect to NADP(+) (K(m) = 10 muM). Phosphoenolpyruvate (K(m) = 1.6 mM), adenosine 5'-triphosphate (K(m) = 0.5 mM), adenosine diphosphate (K(m) = 1.5 mM), and adenosine 5'-monophosphate (K(m) = 3.0 mM) are competitive inhibitors with respect to NAD(+). The molecular weight as estimated from sucrose density centrifugation and molecular sieve chromatography is 1.1 x 10(5). Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is composed of two similar subunits of approximately 6 x 10(4) molecular weight. The intracellular levels of glucose-6-phosphate, NAD(+), and NADP(+) were measured and found to be approximately 1 mM, 0.9 mM, and 0.2 mM, respectively, during logarithmic growth. From a consideration of the substrate pool sizes and types of inhibitors, we conclude that this single constitutive enzyme may function in two roles in the cell-NADH production for energetics and NADPH production for reductive biosynthesis.     

N-acetyl alpha-D-glucosaminidase from the venom of African puff adder (Bitis arietans).

The activity of N-acetyl-alpha-D-glucosaminidase from venom of the African puff adder (Bitis arietans) has been detected. The enzyme from the venom was purified by chromatography on Q-sepharose, CM-cellulose, and N-acetyl-alpha-D-glucosamine-agarose affinity column. The enzyme has a molecular weight of 102 kDa determined by size exclusion chromatography on Sephacryl 200. It migrated as a 51-kDa band on SDS polyacrylamide gels. The enzyme is maximally active at pH 5.5 and 40 degrees C. The B. arietans NAGase hydrolyzed exclusively terminally linked alpha-(1-4) GlcNAc residues from nonreducing ends of oligosaccharides. It hydrolysed chito-oligosaccharide, MU-GlcNAc and chitobiose with K(M) values of 0.15 mM and 1.22 mM, respectively. Swollen chitin and oligosaccharide above (GlcNAc)(4) were not hydrolysed by the enzyme. B. arietans NAGase was strongly inhibited noncompetitively by Hg(2+), competitively by 1-thio-beta-D-GlcNAc and N-acetyl glucosamine (NAG) with K(i) of 0.55, 0.25 and 8 mM, respectively. Colombin the active component of antivenom preparation from Aristolodia albida inhibited the enzyme competitively with K(i) of 0.6 mM. Delineation of the active site by chemical modification revealed the involvement of His and Trp in the catalysis of the enzyme.     

Characterization and partial isolation of ouabain-insensitive Na(+) -ATPase in MDCK I cells

We show that MDCK I cells express, besides the classical (Na(+)+K(+))ATPase, a Na(+)-stimulated ATPase activity with the following characteristics: (1) K(0.5) for Na(+) 7.5+/-1.5 mM and V(max) 23.12+/-1.1 nmol Pi/mg per min; (2) insensitive to 1 mM ouabain and 30 mM KCl; and (3) inhibited by furosemide and vanadate (IC(50) 42.1+/-8.0 and 4.3+/-0.3 microM, respectively). This enzyme forms a Na(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate with molecular weight of 100 kDa. Immunoprecipitation of the (Na(+)+K(+))ATPase with monoclonal anti-alpha(1) antibody reduced its activity in the supernatant by 90%; the Na(+)-ATPase activity was completely maintained. In addition, the formation of the Na(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate intermediate occurred at the same magnitude as that observed before immunoprecipitation. These data suggest that Na(+)-ATPase and (Na(+)+K(+))ATPase activities are independent, with Na(+)-ATPase belonging to a different enzyme entity.