Analytical isolation of plasma membranes of intestinal epithelial cells: Identification of Na,K-ATPase rich membranes and the distribution of enzyme activities

Summary A procedure was developed for the analytical isolation of brush border and basal lateral plasma membranes of intestinal epithelial cells. Brush border fragments were collected by low speed centrifugation, disrupted in hypertonic sorbitol, and subjected to density gradient centrifugation for separation of plasma membranes from nuclei and cole material. Sucrase specific activity in the purified brush border plasma membrane was increased fortyfold with respect to the initial homogenate. Basal lateral membrane were harvested from the low speed supernatant and resolved from other subcellular components by equilibrium density gradient centrifugation. Recovery of Na, K-ATPase activity was 94%, and 61% of the recovered activity was present in a single symmetrical peak. The specific activity of Na, K-ATPase was increased twelvefold, and it was purified with respect to sucrase, succinic dehydrogenase, NADPH-cytochromec reductase, nonspecific esterase, -glucoronidase, DNA, and RNA. The observed purification factors are comparable to results reported for other purification procedures, and the yield of Na, K-ATPase is greater by a factor of two than those reported for other procedures which produce no net increase in the Na, K-ATPase activity.Na, K-ATPase rich membranes are shown to originate from the basal lateral plasma membranes by the patterns of labeling that were produced when either isolated cells or everted gut sacs were incubated with the slowly permeating reagent³⁵S-p-(diazonium)-benzenesulfonic acid. In the former case subsequently purified Na, K-ATPase rich and sucrase rich membranes are labeled to the same extent, while in the latter there is a tenfold excess of label in the sucrase rich membranes. The plasma membrane fractions were in both cases more heavily labeled than intracellular protein.Alkaline phosphatase and calcium-stimulated ATPase were present at comparable levels on the two aspects of the epithelial cell plasma membrane, and 25% of the acid phosphatase activity was present on the basal lateral membrane, while it was absent from the brush border membrane. Less than 6% of the total Na, K-ATPase was present in brush border membranes.     

Detergent inactivation of sodium- and potassium-activated adenosinetriphosphatase of the electric eel.

The stability of the sodium- and potassium-activated adenosinetriphosphatase (Na,K-ATPase) of the electric eel, Electrophorus electricus, was studied in five detergents in an effort to establish conditions for reconstitution of this membrane protein into defined phospholipids. The Na,K-ATPase activity of purified electric organ membranes as well as the ATPase is stable for at least 1 month of storage at 0 degrees C in the absence of detergents. At low concentrations of detergents, the enzyme is also stable for several days, but irreversible inactivation occurs rapidly as the detergent concentration is further increased. This inactivation begins at well-defined threshold concentrations for each detergent, and these concentrations generally occur in the order of the detergent critical micelle concentrations. Increasing the concentration of the electric organ membranes causes a linear increase in the inactivation threshold concentrations of Lubrol WX, deoxycholate, and cholate. The onset of inactivation evidently occurs when the mole fraction of detergent associated with the membrane lipids reaches a critical value in the narrow range of 0.2-0.4, in contrast to the large differences in the bulk concentrations of these detergents. The eel Na,K-ATPase is more sensitive to detergents than the sheep kidney enzyme.     

Isolation of plasma membranes from bovine corpus luteum possessing adenylate cyclase, 125I-hCG binding and NaK-ATPase activities

Plasma membranes from bovine corpora lutea have been purified by sucrose density gradient centrifugation. The purified membranes, in addition to binding ¹²⁵I-hCG, also possess hCG-stimulated adenylate cyclase and NaK-ATPase. The relative purification of ¹²⁵I-hCG binding, adenylate cyclase and NaK-ATPase on the basis of the specific activities in the whole homogenate were 7.8, 6.4 and 2.6, respectively. The presence of both the hormone sensitive adenylate cyclase and ¹²⁵I-hCG binding activities suggest that these plasma membranes might possess the ‘receptor’ for gonadotropin.     

Analytical isolation of plasma membranes of intestinal epithelial cells: identification of Na, K-ATPase rich membranes and the distribution of enzyme activities.

A procedure was developed for the analytical isolation of brush border and basal lateral plasma membranes of intestinal epithelial cells. Brush border fragments were collected by low speed centrifugation, disrupted in hypertonic sorbitol, and subjected to density gradient centrifugation for separation of plasma membranes from nuclei and core material. Sucrase specific activity in the purified brush border plasma membranes was increased fortyfold with respect to the initial homogenate. Basal lateral membrane were harvested from the low speed supernatant and resolved from other subcellular components by equilibrium density gradient centrifugation. Recovery of Na, K-ATPase activity was 94%, and 61% of the recovered activity was present in a single symmetrical peak. The specific activity of Na, K-ATPase was increased twelvefold, and it was purified with respect to sucrase, succinic dehydrogenase, NADPH-cytochrome c reductase, nonspecific esterase, beta-glucuronidase, DNA, and RNA. The observed purification factors are comparable to results reported for other purification procedures, and the yield of Na, K-ATPase is greater by a factor of two than those reported for other procedures which produce no net increase in the Na, K-ATPase activity. Na, K-ATPase rich membranes are shown to originate from the basal lateral plasma membranes by the patterns of labeling that were produced when either isolated cells or everted gut sacs were incubated with the slowly permeating reagent 35S-p-(diazonium)-benzenesulfonic acid. In the former case subsequently purified Na, K-ATPase rich and sucrase rich membranes are labeled to the same extent, while in the latter there is a tenfold excess of label in the sucrase rich membranes. The plasma membrane fractions were in both cases more heavily labeled than intracellular protein. Alkaline phosphatase and calcium-stimulated ATPase were present at comparable levels on the two aspects of the epithelial cell plasma membrane, and 25% of the acid phosphatase activity was present on the basal lateral membrane, while it was absent from the brush border membrane. Less than 6% of the total Na, K-ATPase was present in brush border membranes.     

A simplified purification and some properties of ribulose 1,5-diphosphate carboxylase from barley

A rapid procedure was developed for purifying ribulose 1,5-diphosphate carboxylase from barley leaves. After (NH₄)₂SO₄ fractionation, the unique sedimentation properties of the enzyme were exploited to effect a single step purification to 90% homogeneity. High speed centrifugation pelleted the enzyme with complete recovery of activity. Residual impurities were then removed by diethylaminoethyl cellulose chromatography and density gradient centrifugation. The purified protein exhibited size heterogeneity due to polymerization. The polymerization products were enzymatically active aggregates of ribulose 1,5-diphosphate carboxylase and were precipitated by an antibody specific for the enzyme.     

SDS purification of porcine H,K-ATPase from gastric mucosa

A highly purified membrane fraction of H,K-ATPase was isolated from hog gastric mucosa by using differential centrifugation, sodium dodecyl sulfate (SDS:0.125%) treatment and density-gradient centrifugation. The final fraction showed a major band at 97 kD by SDS-gel electrophoresis. This purified H,K-ATPase sedimented at the interface of a 28-35% sucrose step gradient and displayed a specific activity of 140-170 mumol Pi/h/mg protein and a ratio of K-stimulated ATPase activity to Mg-stimulated ATPase activity of 6.5-8.7. The apparent Km for ATP was 0.154 mM and the Km for K+ was o.6 mM. The enzymatic activity recovered from this purification procedure was K(+)-ionophore-independent. SDS treatment in the presence of 2.5 mM ATP did not change the kinetic properties of the isolated enzyme. Exclusion of ATP during SDS solubilization diminished the enzymatic activity by 90%, indicating that ATP protection is essential for the full recovery of enzymatic activity. In summary, mild SDS solubilization can be used to purify relatively large quantities of active H,K-ATPase to near homogeneity without altering the enzyme's kinetic properties.     

Molecular and kinetic properties of purified γ-glutamyl transpeptidase from yeast (Saccharomyces cerevisiae)

The enzyme γ-glutamyl transpeptidase was purified from the yeast Saccharomyces cerevisiae by a procedure involving protamine sulfate treatment, chromatography on DEAE-Sephadex A 50, salt fractionation, successive chromatography on Sephadex G 150 and lentil lectin sepharose 6B. The procedure achieves 25 % yield and 4200-fold purification. The final preparation is a glycoprotein (M, 90 000) containing 31.4 % carbohydrates and composed of two non-identical subunits (M, 64 000 and 23 000). The specificity patterns of the yeast enzyme are rather similar to those of mammalian and higher plant transpeptidases. The enzyme mechanism might be of the double displacement (ping-pong) type.     

Purification of rat chondrosarcoma xylosyltransferase

The chondroitin sulfate chain-initiating enzyme, UDP-d-xylose:core protein β-d-xylosyltransferase has been purified over 600-fold from the high-speed supernatant fraction of a rat chondrosarcoma. The purification procedure involved differential centrifugation, gel chromatography on Sephadex G-200, and affinity chromatography on a matrix consisting of core protein bound to Sepharose. The purified enzyme was homogenous by electrophoretic and immunological criteria, had a molecular weight between 95,000 and 100,000 and contained approximately 10% carbohydrate. The K_m value for UDP-xylose was 1 × 10^?5, m and for the core-protein acceptor was 330 mg/liter.     

Comparison of detergent-solubilized and membrane-bound forms of kidney (Na + K)-ATPase

The detergent solubilization of dog kidney (Na + K)-ATPase has been investigated. The nonionic detergents, Brij 58, C₁₂E₈, and Lubrol WX were tested for their ability to produce active, soluble enzyme. Lubrol WX gave the best results. Enzyme so treated is found in the supernatant fraction after centrifugation at 100,000g for 1 h. It has the same or slightly greater specific activity, the same subunit composition as judged by SDS-gel electrophoresis, and very similar kinetic parameters with respect to sodium, potassium, ATP, pNPP, and ouabain as the membrane-bound enzyme. The Lubrol-treated enzyme is stable for at least 5 days at 4 °C. The phospholipid content of the Lubrol-treated enzyme is decreased, as might be expected, by about 50%. Limited tryptic proteolysis and fluorescence changes seen after modification with FITC indicate that the solubilized (Na + K)-ATPase undergoes the same conformational transitions as the membrane enzyme. Our results indicate that kidney enzyme solubilized as described here is nondenatured and fully active, and therefore a valuable preparation for spectroscopic and other approaches for study of this enzyme.     

Purification and Characterization of RNA Polymerase from Fremyella diplosiphon

We have purified and characterized a DNA-dependent RNA polymerase from the blue-green alga Fremyella diplosiphon. This enzyme, purified by gel filtration, DEAE-cellulose chromatography, and glycerol gradient centrifugation, is comprised of five polypeptide subunits. Their masses are 161,000, 134,000, 91,000, 72,000, and 41,000 daltons. Preparative electrophoresis of the purified enzyme on nondenaturing gels separates the 41,000-dalton polypeptide from the rest of the enzyme. The enzyme is extremely labile in the presence of a variety of salts of strong acids and bases; the purification procedure was devised to avoid exposure to such compounds.     

Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase : VII. Comparison of the properties of the membrane-bound and partially purified soluble and insoluble forms of the enzyme

Several kinetic parameters of the beef brain NaK ATPase in different physical states at four stages of purification have been compared. These stages were: (a) NaI-treated microsomes (2.5% pure), (b) Lubrol extracts of NaI-treated microsomes (5% pure), (c) a fraction isolated by zonal centrifugation of the Lubrol extracts (10% pure), and (d) an ammonium sulfate fraction (50% pure). The latter two fractions were insoluble and contained 30% and 16% bound Lubrol, respectively.     

Phosphorylation of the alpha-subunit of Na,K-ATPase from duck salt glands by cAMP-dependent protein kinase inhibits the enzyme activity

Although it was shown earlier that phosphorylation of Na,K-ATPase by cAMP-dependent protein kinase (PKA) occurs in intact cells, the purified enzyme in vitro is phosphorylated by PKA only after treatment by detergent. This is accompanied by an unfortunate side effect of the detergent that results in complete loss of Na,K-ATPase activity. To reveal the effect of Na,K-ATPase phosphorylation by PKA on the enzyme activity in vitro, the effects of different detergents and ligands on the stoichiometry of the phosphorylation and activity of Na,K-ATPase from duck salt glands (11-isoenzyme) were comparatively studied. Chaps was shown to cause the least inhibition of the enzyme. In the presence of 0.4% Chaps at 1 : 10 protein/detergent ratio in medium containing 100 mM KCl and 0.3 mM ATP, PKA phosphorylates serine residue(s) of the Na,K-ATPase with stoichiometry 0.6 mol P_i/mol of -subunit. Phosphorylation of Na,K-ATPase by PKA in the presence of the detergent inhibits the Na,K-ATPase. A correlation was found between the inclusion of P_i into the -subunit and the loss of activity of the Na,K-ATPase.     

Studies concerning the possible reconstitution of an active cation pump across an artificial membrane

Summary The cortical tissue of rat brain was fractionated through zonal centrifugation in a continuous sucrose density gradient, yielding a variety of morphologically distinct membrane fragments derived from nerve-end particles possessing variable levels of activity of Na, K-dependent Mg-sensitive ATPase (Na, K-ATPase) and other enzymes. Upon addition of certain of the zonal fractions, particularly those rich in the ATPase and acetylcholinesterase activities, to one side of planar artificial membranes, formed from mixtures of oxidized cholesterol and alkanes and bathed in a solution containing sodium, potassium, and magnesium ions, direct current membrane resistance fell from one to three orders of magnitude. Subsequent addition of ATP to the same side of the membrane to which the ATPase was added (thecis side) led to the development of net short-circuit current flow and open-circuit potential across the membrane (thecis side being negative with respect to thetrans side). Development of the short-circuit current and open-circuit potential is dependent upon the presence of all the substrates of Na, K-ATPase as well as that of the enzyme itself. The net current flow is inhibited and the open-circuit potential discharged by the addition of ouabain to thetrans side of the membrane, of phospholipase A to thecis side, or of trypsin to either side of the membrane. These observations provide circumstantial evidence for the reconstitution of the active cation pump across the artificial bilayer. Efforts to effect a similar reconstitution across membranes of this and other compositions employing Na, K-ATPase preparations from beef heart, beef brain, cat brain, human red cells, rabbit kidney, and rat brain microsomes failed.Career Development Awardee of the National Institutes of Health, Grant No. GM 10248.     

Na,K-ATPase structure/function relationships probed by the denaturant urea

Urea interacts with the Na,K-ATPase, leading to reversible as well as irreversible inhibition of the hydrolytic activity. The enzyme purified from shark rectal glands is more sensitive to urea than Na,K-ATPase purified from pig kidney. An immediate and reversible inhibition under steady-state conditions of hydrolytic activity at 37 °C is demonstrated for the three reactions studied: the overall Na,K-ATPase activity, the Na-ATPase activity observed in the absence of K⁺ as well as the K⁺-dependent phosphatase reaction (K-pNPPase) seen in the absence of Na⁺. Half-maximal inhibition is seen with about 1 M urea for shark enzyme and about 2 M urea for pig enzyme. In the presence of substrates there is also an irreversible inhibition in addition to the reversible process, and we show that ATP protects against the irreversible inhibition for both the Na,K-ATPase and Na-ATPase reaction, whereas the substrate paranitrophenylphosphate leads to a slight increase in the rate of irreversible inhibition of the K-pNPPase. The rate of the irreversible inactivation in the absence of substrates is much more rapid for shark enzyme than for pig enzyme. The larger number of potentially urea-sensitive hydrogen bonds in shark enzyme compared to pig enzyme suggests that interference with the extensive hydrogen bonding network might account for the higher urea sensitivity of shark enzyme. The reversible inactivation is interpreted in terms of domain interactions and domain accessibilities using as templates the available crystal structures of Na,K-ATPase. It is suggested that a few interdomain hydrogen bonds are those mainly affected by urea during reversible inactivation.     

Purification and Properties of Amine Oxidase from Epicotyls of Pisum sativum

A procedure has been developed for the purification of amine oxidase (E.C. 1.4.3.4) from etiolated pea epicotyls (Pisum sativum cv. Little Marvel). The enzyme is sensitive to copper chelating reagents and carbonyl reagents, but is not inhibited by sulfhydryl reagents. The purified enzyme has a molecular weight of 1.85 × 10⁵, as determined by sedimentation equilibrium centrifugation, and has been shown to be specifically stimulated by phosphate.     

Method to Obtain a Chlorophyll-free Preparation of Intact Mitochondria from Spinach Leaves

Mitochondria from green leaves of spinach have been prepared using a three-step procedure involving differential centrifugation, partition in an aqueous dextran polyethylene glycol two-phase system and Percoll gradient centrifugation. The mitochondrial fractions after the different steps of purification were compared. The final mitochondrial preparation was totally free from chloroplast material measured as chlorophyll content. The enrichment of mitochondria in relation to peroxisomes and microsomes was approximately 12 and 33 times, respectively, based on NAD:isocitrate dehydrogenase activity, glycolate oxidase activity, and NADPH:cytochrome c oxidoreductase activity. The apparent intactness of the inner and the outer mitochondrial membranes was higher than 90% as measured by latency of enzyme activities. The mitochondria showed high respiratory rates with respiratory control and the ADP/O ratios approached the theoretical limits.     

Melittin induces both time-dependent aggregation and inhibition of Na,K-ATPase from duck salt glands however these two processes appear to occur independently

Using cupric phenanthroline as a cross-linking agent, we have shown that melittin induced time-dependent aggregations of Na,K-ATPase in microsomal fractions and in preparations of purified Na,K-ATPase from duck salt glands. Incubation of melittin with these preparations also led to the progressive loss of Na,K-ATPase activity. At melittin/protein molar ratio of 5:1, we did not observe inhibition of Na,K-ATPase in the microsomal fraction but the process of enzyme aggregation occurred. At higher melittin/protein molar ratios (10:1 and 30:1), the inhibition of the enzyme and its aggregation proceeded simultaneously but the rates of these processes and maximal values achieved were different. At a melittin/protein ratio of 30:1, Na,K-ATPase inhibition may be described as a biexponential curve with the values for pseudo-first order rate constants being 2.7 and 0.15 min⁻¹. However, the aggregation may be presented by a monoexponential curve with a pseudo-first order rate constant of 0.15 min⁻¹. In purified preparations of Na,K-ATPase, the maximal aggregation (about 90%) was achieved at a melittin/protein molar ratio of 2:1, and a further increase in the melittin/protein ratio increased the rate of aggregation but did not affect the value of maximal aggregation. The results show that melittin induced both aggregation and inhibition of Na,K-ATPase but these two processes proceeded independently.     

Glutathionylation of the alpha-subunit of Na,K-ATPase from rat heart by oxidized glutathione inhibits the enzyme

A partially purified Na,K-ATPase preparation from rat heart containing α1- and α2-isoforms of the enzyme was shown to include both subunits in S-glutathionylated state. Glutathionylation of the α1-subunit (but not of the α2-subunit) was partially removed when the preparation was isolated in the presence of dithiothreitol. The addition of oxidized glutathione irreversibly inhibited both isoforms. Inhibition of the enzyme containing the α1-subunit was biphasic, and the rate constants of the inhibition were 3745 ± 360 and 246 ± 18 M^?1·min^?1. ATP, ADP, and AMP protected the Na,K-ATPase against inactivation by oxidized glutathione.     

Purification and Characterization of a Chloroplast Outer-Envelope-Bound, ATP-Dependent Protein Kinase

An ATP-dependent protein kinase was partially purified from isolated outer envelope membranes of pea (Pisum sativum L., Progress No. 9) chloroplasts. The purified kinase had a molecular weight of 70 kilodaltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was of the cyclic nucleotide and Ca²⁺, calmodulin-independent type. The purification involved the detergent solubilization of purified outer envelopes by 0.5% cholate and 1% octylglycoside, followed by centrifugation on a linear 6 to 25% sucrose gradient. Active enzyme fractions were further purified by affinity chromatography on histone III-S Sepharose 4B and ion exchange chromatography on diethylaminoethyl cellulose. The protein kinase eluted at 100 millimolar and 50 millimolar NaCl, respectively. The protein kinase was essentially pure as judged by Western blot analysis. The enzyme has a K_M of 450 micromolar for ATP and a V_max of 25 picomoles of ³²P incorporated into histone III-S per minute per microgram. Inhibition by ADP is competitive (K_i 150 micromolar).     

Purification and some properties of gamma-glutamyltransferase from human liver.

The purification of gamma-glutamyltransferase ((gamma-glutamyl)-peptide: amino acid gamma-glutamyltransferase, EC 2.3.2.2) from normal human liver is described. The procedure includes solubilization of enzyme from membranes using deoxycholate and Lubrol W, treatment with acetone and butanol, and affinity chromatography on immobilized concanavalin A. Treatment with papain was used to release the enzyme from aggregates of lipid and protein, prior to further purification. On overall purification of 9400 was achieved and analytical polyacrylamide gel electrophoresis indicated that the final product was homogeneous, and had a molecular weight of 110 000. Two subunits were identified on dodecyl sulfate gel electrophoresis with estimated molecular weights of 47 000 and 22 000. The kinetic properties studied for the purified enzyme were similar to those found for partially purified (not papain-treated) enzyme, and resembled those of serum gamma-glutamyltransferase. The true KM values for the liver enzyme were estimated to 0.81 mM for gamma-glutamyl-p-nitroanilide and to 12.4 mM for glycyl-glycine.