Purification and characterization of laccase from Monocillium indicum Saxena

Summary An ascomycete Monocillium indicum Saxena producing extracellular laccase was isolated. The culture filtrate on native polyacrylamide gel electrophoresis (PAGE) revealed four bands of activity, one of which was a major one. The major laccase band, a glycoprotein, was purified and characterized. Gel filtration chromatography showed that the relative molecular weight (M_r) of laccase was 100 000. On sodium dodecyl sulphate (SDS)-PAGE the major laccase band further resolved into three proteins of M_r 72 000, 56 000 and 24 000. The enzyme had a pH optimum of 3.0 and was active on a number of o-phenols and aromatic acids. The 72 000 M_r protein was found to share common immunological properties with laccases of Coriolus versicolor, Agaricus bisporus and lignin peroxidase of Phanerochaete chrysosporium. Correspondence to: K. Koteswara Rao     

Simple high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin

Haptoglobin is an acute-phase protein and its plasma levels increase consistently in response to infection and inflammation. Some evidence has demonstrated that haptoglobin is involved in the immune responses. In this study, we established a novel high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin. The procedure required an ammonium sulfate fractionation and a HPLC Superose 12 gel-permeation chromatography. Purified porcine haptoglobin possessed one heavy (β) and light chain (α) on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, under reducing conditions, with a M_r (molecular mass) of about 42 000 and 14 000 for heavy (β) and light chains (α), respectively. Although the N-terminal amino acid sequence of porcine heavy chain of haptoglobin has never been reported previously, the analyses of N-terminal amino acid sequence showed a great sequence similarity to that of human and other animal species. In addition, Western blot using our specific antibody prepared against porcine M_r 42 000 chain did react with human haptoglobin and likewise, the antibody against human haptoglobin also cross-reacted with purified porcine M_r 42 000 chain. Thus, it confirmed that the identity of the porcine protein purified from our procedures was as haptoglobin.     

Various forms of mouse lactoferrins: purification and characterization

This work was conducted to study the microheterogeneity of mouse lactoferrin (LF). Two forms, LF₁ and LF₂, could be purified from uterine luminal fluid by ion-exchange HPLC on a Protein PAK SP 5PW column. Another form, LF₃, was purified from the epididymis homogenate by affinity chromatography on a column of Protein A-Sepharose coupled with the purified LF₂ antibody that was prepared to give no crossreaction with serum albumin. Both LF₁ and LF₂ showed a M_r 74 000 band while LF₃ gave a M_r 70 000 band on reducing SDS–PAGE. All of them were reduced to a M_r 68 000 band after they had been digested with N-glycosidase F. The data from automated Edman degradation confirmed the completely identical 19 amino acid sequences in the N-terminal regions of these three LFs, except the lack of N-terminal Lys–Ala of LF₂/LF₃ in LF₁. LF in tissue homogenates was immunodetected by Western blot procedure using the purified LF₂ antibody. Different amounts of LF with a molecular mass of the 70 000 or 74 000 were distributed in the non-sexual organs such as kidney, spleen, lung, heart and liver and the sexual glands including epididymis, vagina, uterus, ovary and prostate. No LF was detected in stomach, intestine, testis and seminal vesicle.     

Rapid purification and properties of human glycodelin (endometrial α2-globulin)

The method presented can easily produce milligram amounts of glycodelin from pregnancy endometrium, with a 19% yield. It involves anion-exchange chromatography, gel permeation and chromatofocusing; it results in one stainable band at M_r 28 000 after sodium dodecyl sulphate–polyacrylamide electrophoresis, as well as after immunoblot analysis, performed using an affinity-purified IgG fraction from an antiserum against glycodelin. In spite of this, the corresponding gel isoelectric focusing pattern gives four stainable bands with pI values between 4.55 and 5.2. Western immunoblot analysis of tissue extracts indicates the presence of glycodelin epitopes associated with materials heavier than the native protein. Circular dichroism spectra of the highly purified protein in water solutions indicate a large amount of β-sheet conformation, whereas those obtained with different proportions of 2-propanol in water, show an increased proportion of α-helix conformation.     

Intermediate filament proteins in echinoderm coelomocytes

The presence and organization of intermediate filament (IF) proteins in petaloid coelomocytes from two species of echinoderms, the sea urchin Strongylocentrotus droebachiensis and the sea cucumber Cucumaria frondosa, were studied. Two monoclonal antibodies (IFA and Ah6) and one polyclonal antibody (W3-1) that together recognize invertebrate as well as vertebrate IF proteins were used to probe coelomocytes by immunofluorescence and immunoblotting methods. All three antibodies cross-reacted with a single M_r 68 000 sea urchin lamin, as well as two putative lamin isoforms of approximately M_r 70 000 and 68 000 in sea cucumber coelomocytes. Both IFA and Ah6 labeled granular material in the cytoplasm of sea urchin coelomocytes; by contrast, IFA labeling revealed a striking network of reticular material irregularly arrayed within the central regions of the sea cucumber coelomocyte cytoplasm. In addition, foci of Ah6-positive material were present in coelomocyte nuclei from both species. Comparison of immunoblotting patterns among whole cell and isolated nuclear preparations suggest that the cytoplasmic IF-like material is composed of M_r 46 000 and 58 000 polypeptides, while M_r 215 000 and 185 000 proteins are candidates for the immunoreactive nuclear foci. Further study of the functions of these non-filamentous arrays of IF proteins may furnish valuable insights into the evolution of IF function within vertebrate cells, particularly with respect to certain cytoplasmic and nuclear regulatory functions with which IF proteins have been speculated to be involved.     

Purification of an endopeptidase involved with storage-protein degradation in Phaseolus vulgaris L. cotyledons

Phaseolin, the major seed storage protein of Phaseolus vulgaris L., is degraded in the cotyledons in the first 7–10 d following seed germination. We assayed cotyledon extracts for protease activity by using [³H]phaseolin as a substrate and then fractionated the digestion mixtures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in order to identify the cleavage products. The cotyledons of 4-d-old seedlings contain an endopeptidase which cleaves the polypeptides of [³H]phaseolin (apparent molecular weights=51 000, 48 000, 46 000 and 43 000) into three discrete clusters of proteolytic fragments (M _rs=27 000, 25 000 and 23 000). Endopeptidase activity is not detected in the cotyledons until the protein content of these organs starts to decline, shortly after the first day of seedling growth. Endopeptidase activity increases to a maximum level in the cotyledons of 5-d-old seedlings and then declines to a minimum value by day 10. The enzyme was purified 335-fold by ammonium-sulfate precipitation, organomercurial-agarose chromatography, gel filtration and ion-exchange chromatography. The endopeptidase constitutes 0.3% of the protein content in the cotyledons of 4-d-old seedlings. It is a cysteine protease with a single polypeptide chain (M _r=30 000). Optimum hydrolysis of [³H]phaseolin occurs at pH 5. The enzyme is irreversibly inactivated at pH values above 7 and at temperatures above 45° C. The endopeptidase attacks only a limited number of peptide bonds in [³H]phaseolin, without causing any appreciable change in the native molecular weight of the storage protein. The endopeptidase is also able to hydrolyze the bean-seed lectin, phytohemagglutinin. Thus, this enzyme may play a general role in degrading cotyledon proteins of P. vulgaris following seed germination.Abbreviations Da dalton - DTT dithiothreitol - M _r apparent molecular weight - PAGE polyacrylamide gel electrophoresis - PHA phytohemagglutinin - SDS sodium dodecyl sulfate     

Karyophobic proteins : A category of abundant soluble proteins which accumulate in the cytoplasm

The cytoplasm of oocytes of Xenopus laevis is enriched in several soluble proteins which are either absent from the nucleus or are present there at very low concentrations. These molecules, collectively referred to as karyophobic (from the Greek verbs oβιν and oβλoθαi which are meant here in the sense of “to be afraid of” or “to avoid”) proteins represent more than 20% of the total soluble cytoplasmic proteins and include some of the most abundant soluble cellular components. They may be recovered from high-speed supernatant (S-100) fractions and, following sucrose gradient centrifugation, most of them appear in the form of complexes smaller than 8.5S. On denaturation in urea and two-dimensional gel electrophoresis these proteins appear to be comprised of polypeptides of widely different sizes (ca M_r 15 000–230 000) and isoelectric points covering a broad range of pH values (4.2–8.0). Gel filtration and isoelectric focusing of native karyophobic proteins show that the majority occur in acidic complexes smaller than M_r 150 000, including one case of a small karyophobic protein (C9; M_r 30 000). In contrast to karyophilic proteins and proteins equilibrating between nucleus and cytoplasm karyophobic soluble proteins from [³⁵S]methionine-labelled ooplasms, when injected into unlabelled oocytes, remain in the cytoplasm. Human proteins with a similar karyophobic behaviour have been identified in fractions of soluble proteins from HeLa cells; there, the major karyophobic protein (HCa, M_r 36 000) is also one of the most abundant soluble proteins.We conclude that the specific nucleocytoplasmic compartmentalization of soluble proteins is governed not only by the principles of exclusion of large molecules from nuclear uptake and the existence of karyophilic signals in certain proteins but that a series of soluble, globular proteins exist in the cytoplasm, which have other molecular features which selectively exclude them from distribution over the nucleus. The possible functional role of the selective enrichment of these abundant proteins, which so far have escaped attention, in establishing a cytoplasmic milieu is discussed.     

Midgut amylase,lysozyme, aminopeptidase,and trehalase from larvae and adults of Musca domestica

Based on polyacrylamide gel electrophoresis, density-gradient ultracentrifugation and thermal inactivation, there is only one major molecular species of each of the following larval enzymes (soluble in water or solubilized in Triton X-100): membrane-bound aminopeptidase (pH optimum 8.5; K_m 0.21 mM L-leucine p-nitroanilide; M_r 322,000), amylase (pH optimum 6.5; K_m 0.14% starch; M_r 66,000), lysozyme (pH optimum 3.5; K_m 0.3 mg/ml; Mr 24,000); and membrane-bound trehalase (pH optimum 5.0; K_m 1.09 mM trehalose; M_r 94,000). Except for lysozyme, the properties of adult digestive enzymes are different from those described for larval enzymes. Larval aminopeptidase and trehalase were purified by electrophoresis and larval lysozyme (contaminated with amylase) by density-gradient ultracentrifugation, and were used to raise antibodies in a rabbit. Antibodies raised against larval aminopeptidase, trehalase, and amylase did not recognize the imaginal enzymes, whereas those against larval lysozyme recognize imaginal lysozyme. The data suggest that the genes coding for digestive enzymes (except for lysozyme) are different in larvae and imagoes.     

Purification and characterization of three major hemolymph proteins of an insect,Calpodes ethlius (lepidoptera,hesperiidae)

Like many other Lepidoptera, fifth-stage Calpodes larvae have three major hemolymph proteins. Their molecular weights were estimated by 3-15% nondenaturing polyacrylamide gel electrophoresis (N-PAGE) as 470,000 (arylphorin; Ar), 580,000 (storage protein 2; SP2) and 720,000 (storage protein 1; SP1). Carbohydrate is associated with all three, but only Ar has lipid. The three proteins have been purified by preparative N-PAGE and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. On 3-15% SDS gels, Ar dissociated into 82,000 M_r subunits, SP2 into 86,000 M_r subunits, and SP1 into both 86,000 and 90,000 M_r subunits. The 470,000 M_r protein is identified as Ar because it is rich in aromatic amino acids. The 580,000 and 720,000 M_r proteins are rich in glycine and are called storage proteins. Electron microscopy of negatively stained preparations shows that each polymer has a different geometrical arrangement of subunits. SP1 is a cube made from eight subunits. SP2 is a hexamer in the form of a pentahedral prism. Ar is probably an octahedron made from six subunits. All three geometrical arrangements could permit the presence of a central carrying space.     

Purification and characterization of the messenger ribonucleoprotein-associated casein kinase II of Artemia salina cryptobiotic gastrulae

The mRNP-associated protein kinase is purified to near homogeneity by ion-exchange chromatography on phosphocellulose and affinity chromatography on casein-Sepharose 4B and ATP-agarose. The cyclic nucleotide-independent enzyme phosphorylates casein using either ATP or GTP. The enzyme exists in two forms composed of subunits with M_r 36 500 (α) and 28 000 (β) and of subunits with M_r 36 500 (α), 33 000 (α′) and 28 000 (β). The undegraded enzyme has an M_r of 136 000 ± 7000. The enzyme is inhibited by heparin and hemin and stimulated by spermine. The mRNP-associated protein kinase may be classified as a casein kinase II. Main mRNP protein phosphate acceptors have M_r values of 112 000, 72 000, 65 000, 53 000, 38 000, 28 000, 23 500 and 21 000. Phosphorylation of the M_r 38 000 poly(A)-binding protein resulted in the generation of different acidic ionic species. From the observed inhibition of the translational activity after phosphorylation by the mRNP-associated protein kinase a function in the repression of mRNP is proposed.     

Purification and immunochemical characterization of human adrenal tyrosine hydroxylase

Tyrosine hydroxylase (TH) was purified from the soluble fraction of human adrenal glands. The enzyme in human adrenal glands that was purified to apparent homogeneity had an apparent M_r of about 280,000. Sodium dodecyl sulfate (SDS) gel electrophoresis gave a single band with a M_r of 60,000 similar to the M_r of bovine adrenal enzyme. The enzyme is considered to be composed of four identical subunits. The specific activity of the final preparation was approximately 310 nmol 3,4-dihydroxyphenylalanine (DOPA) formed/min/mg protein. The use of the “Western Blot” method showed that human adrenal TH did not aggregate as rapidly as bovine adrenal TH.     

The purification and partial amino acid sequence of a polypeptide from the glutelin fraction of rice grains; homology to pea legumin

The glutelin fraction was extracted from grain meals of rice (Oryzea sativa) with 50 mM Tris-HCl buffer (pH 8.8) containing 6 M urea and 10 mM 2-mercaptoethanol. Polypeptides of glutelin were separated and purified by ion-exchange chromatography under denaturing conditions. Analysis by two-dimensional gel electrophoresis showed that 2 major polypeptides of the rice glutelin fraction, M_r 36 000 and 22 000, were linked in disulphide bonded pairs containing one M_r 36 000 and one M_r 22 000 subunit. A partial amino acid sequence of the purified M_r 22 000 glutelin subunit showed it to be homologous to the β-subunit of pea legumin, a storage protein which also contains disulphide-linked subunit pairs (M_r 38 000 and M_r 22 000). It is therefore proposed that the major component of rice glutelin is a legumin-like protein.     

Purification of an elicitor-induced glucan synthase (callose synthase) from suspension cultures of French bean (Phaseolus vulgaris L.): purification and immunolocation of a probable Mr-65 000 subunit of the enzyme

Membrane preparations from suspension-cultured cells of French bean (Phaseolus vulgaris L.) contained callose synthase (EC 2.4.1.34) activity which was preserved upon solubilisation. Following elicitor treatment of cell cultures, increased activity could be extracted and this increase was maintained during purification. The enzyme was purified by high-pressure liquid chromatography and active fractions showed a variable association of two polypeptides of relative molecular masses (M_r) 55 000 and 65 000, the latter being in excess. The M_r-65 000 polypeptide was purified to homogeneity and an antibody raised to it. This antibody showed complex effects on callose synthase activity when incubated with membrane and soluble extracts. In comparison with other systems, the M_r-55 000 subunit is likely to represent the catalytic subunit while the M_r-65 000 polypeptide is a possible regulatory subunit. The M_r-65 000 polypeptide was immunolocated in membranes at sites of callose synthesis in the plant, in cell plates, in sieve plates, at the plasma membrane-wall interface of wounded cells and in papillae in infected cells. Received: 18 January 1997 / Accepted: 8 May 1997     

Sodium dodecyl sulphate gel electrophoretic preparation of protein standard human apolipoprotein B-48

Quantitation of plasma apo B-48 is currently performed by densitometric analysis of SDS–PAGE zones stained with Coomassie Brilliant Blue, using standard solutions of purified apo B-48. Here, preparative gel electrophoresis with a continuous elution system was used for purifying apo B-48. A chylomicron fraction was isolated by 107 000 g ultracentrifugation of a chylous ascite. The proteins were delipidated and precipitated in ethanol–diethyl ether (3:1, v/v), subjected to preparative electrophoresis in a 5% polyacrylamide gel and eluted in 0.1% SDS. The peak containing apo B-48 was eluted at a retention time of 445–480 min. The purity of apo B-48 in this fraction was assessed by the detection of a single band (M_r 260 000) after silver staining and Coomassie staining of 4–15% gradient SDS–PAGE. It was confirmed by the absence of apo B-100 contaminant in Western blot of the purified protein preparation. A linear relationship was observed between the densitometric analysis of SDS–PAGE bands and the apo B-48 in a protein range of 0–3 μg. In conclusion, preparative gel electrophoresis was used in a single step purification of apo B-48 that was adapted to the preparation of a standard solution.     

Effect of Cl− on the function of the Cl−-activated arginine aminopeptidase purified from human erythrocytes

The Cl^?-activated arginine aminopeptidase was purified from human erythrocytes using electrofocusing in granulated gel, gel permeation chromatography, and affinity chromatography. The purified enzyme showed a molecular weight of 105,000 ± 3000 and was homogenous according to several criteria. A subunit structure was revealed during sodium lauryl sulfate electrophoresis, the main form being of M_r 24,500 ± 1300. The enzyme was considered to be a tetramer consisting of four monomers of equal molecular weight. Cl^? affected the hydrolysis of peptides and synthetic substrates differently, the Cl^? activation being less marked with peptide substrates. The catalysis obeyed regular Michaelis-Menten kinetics and Cl^? affected both the K_m and V values. Arg-Phe and bradykinin showed no cooperativity in the hydrolysis of Arg-2-naphthylamide catalyzed by the Cl^?-activated arginine aminopeptidase. Cl^? affected the enzyme structure reflected by changes in the uv-absorption spectra in the presence and without added Cl^?.     

Partial purification and characterization of the mitochondrial and peroxisomal isozymes of enoyl-coenzyme a hydratase from germinating pea seedlings

Distinct organellar forms of the β-oxidation enzyme enoyl-coenzyme A (CoA) hydratase were partially purified and characterized from 2-day germinated pea (Pisum sativum L.) seedlings. The purification was accomplished by disruption of purified mitochondria or peroxisomes, (NH₄)₂SO₄ fractionation, and gel permeation chromatography using a column of Sephacryl S-300. The organellar isozymes had distinct kinetic constants for the substrates 2-butenoyl-CoA and 2-octenoyl-CoA, and could be easily distinguished by differences in thermostability and salt activation. The peroxisomal isozyme had a native M_r of 75,000 and appeared to be a typical bifunctional enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, while the mitochondrial isozyme had a native M_r of 57,000 and did not have associated dehydrogenase activity. Western blots of total pea mitochondrial proteins gave a positive signal when probed with anti-rat liver mitochondrial enoyl-CoA hydratase antibodies but there was no signal when blots of total peroxisomal proteins were probed.     

Purification and characterization of a multifunctional calmodulin-dependent protein kinase from canine myocardial cytosol

A calmodulin-dependent protein kinase from canine myocardial cytosol was purified 1150-fold to apparent homogeneity with a 1.5% yield. The purified enzyme had a M_r of 550,000 with a sedimentation coefficient of 16.6 S, and showed a single protein band with a M_r of 55,000 (55K protein), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 1.6 μmol/mg protein/min, and K_a values of 67 nM and 1.1 μM for calmodulin and Ca²⁺, respectively, using chicken gizzard myosin light chain as substrate. Calmodulin bound to the 55K protein. The purified enzyme had a broad substrate specificity. Endogenous proteins including glycogen synthase, phospholamban, and troponin I from the canine heart were phosphorylated by the enzyme. These results suggest that the purified enzyme works as a multifunctional protein kinase in the Ca²⁺, calmodulin-dependent cellular functions of the canine myocardium, and that the enzyme resembles enzymes detected in the brain, liver, and skeletal muscle.     

Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO₂ to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M _r of 30000. The chloroplastic isoenzyme (M _r 220000) is also an octamer composed of two different subunits with M _r estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the M _r-27 000 subunit was three amino acids shorter than that of the M _r-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl^–, I^–, N ₃ ^- and NO ₃ ^- ) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.Abbreviations BSA bovine serum albumin - CA carbonic anhydrase - PMSF phenylmethylsulphonyl fluoride - BAM benzamidine - DTT dithiothreitol - 2-ME 2-mercaptoethanol - PVDF polyvinylidene difluoride The authors thanks P. Carrier and Dr. B. Dimon for technical assistance with the mass-spectrometry measurements.     

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.     

Phosphorylation of the α-subunit of (Na+ + K+)-ATPase by carbachol in tissue slices and the role of phosphoproteins in stimulus-secretion coupling

This study examined the changes in protein phosphorylation in response to cholinergic (muscarinic) stimulation of salivary secretion in the rat submandibular gland. Carbachol stimulation was associated with phosphorylation in a number of protein bands as detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and autoradiography. The molecular masses (M_r) of two proteins, in which the amount of phosphorylation more than doubled in response to carbachol, were 22 000 and 96 000. The M_r 96 000 protein precipitated at 120 000 × g while most of the M_r 22 000 protein remained in the supernatant at this speed. The effect of carbachol on the phosphorylation of the M_r 22 000 and 96 000 proteins was blocked by atropine, indicating that the cholinergic receptor involved is muscarinic. The time course of phosphorylation of the M_r 22 000 protein consisted of a rapid incrase in phosphorylation within the first min of carbachol stimulation. This increased phosphorylation persisted for less than 1 min. The increased phosphoryaltion of the M_r 96 000 protein also occurred within the first min but it persisted for at least 10 min. However, removal of the muscarinic agonist, carbachol, resulted in the rapid dephosphorylation of this protein. When the plasma membranes were purified, the M_r 96 000 protein was phosphorylated by ATP in the presence of Na⁺ and Mg²⁺. It was dephosphorylated by K⁺. This proves that the M_r 96 000 dalton protein is the α-subunit of the (Na⁺ + K⁺)-ATPase.