An improved purification method for cytoplasmic dynein

An improved method has been devised for the purification of cytoplasmic dynein from sea urchin eggs (Strongylocentrotus droebachiensis and S purpuratus). This protocol introduces three changes over a previously published procedure (Hisanaga and Sakai: J Biochem 93:87, 1983)--the substitution of diethylaminoethyl (DEAE)-cellulose for hydroxylapatite chromatography, the elimination of sucrose density gradient centrifugation, and the use of phosphocellulose chromatography. These changes reduce the time and increase the efficiency of the purification procedure. The purified egg cytoplasmic dynein has enzymatic properties in common with axonemal dynein, including ionic specificity (Ca++ATPase/Mg++ ATPase = 0.8) and inhibition by sodium vanadate and erythro-9-2,3-hydroxynonyl adenine (EHNA). As assayed by silver staining of polyacrylamide gels, the cytoplasmic dynein is composed of two high molecular weight polypeptides (greater than 300 kilodaltons) that comigrate with flagellar dynein heavy chains, and lesser amounts of three lower molecular weight bands. None of these polypeptides appears to contain bound carbohydrate. The purification procedure can be modified slightly to allow the preparation of cytoplasmic dynein in only 2 days from as little as 3-5 ml of packed eggs, a 20-fold reduction over the previous method. This more rapid and efficient method will facilitate the investigation of cytoplasmic dynein in other systems where starting material is limited, including tissue culture cells and nerve axoplasm.     

CYTOPLASMIC DYNEIN OF THE SEA URCHIN EGG I. PARTIAL PURIFICATION AND CHARACTERIZATION*

Cytoplasmic ATPase of sea urchin eggs was partially purified by ammonium sulfate fractionation, DEAE-cellulose chromatography, gel-filtration chromatography and sucrose density gradient centrifugation. The specific activity increased to 0.7 μmole/min/mg protein indicating 100 fold purification. The ATPase had a sedimentation constant of 12S and was highly specific for ATP. The enzyme fraction contained neither (Na, K)-ATPase, Ca-ATPase, oligomycin-sensitive ATPase, phosphatases, nor myosin. This cytoplasmic ATPase was inhibited by a low concentration of vanadate (V). Half-maximal inhibition was observed at a vanadate concentration of 1 μM at low ionic strength. The inhibition was almost totally reversed by addition of norepinephrine. The vanadate-sensitivity of cytoplasmic ATPase decreased with increasing KCl concentration. The activation by Mg²⁺ or Ca²⁺, and dependence of the activity on KCl concentration characteristic of dyneins from sea urchin sperm flagella and the embryonic cilia were observed with cytoplasmic ATPase. These results allowed the cytoplasmic ATPase to be classified as a dynein. In addition, this designation was reinforced by the fact that an oligomeric 23S form of cytoplasmic dynein was identified in the cytoplasm as well as in the isolated mitotic apparatus.     

Homology of egg and flagellar dynein. Comparison of ATP-binding sites and primary structure

Unfertilized sea urchin eggs contain a Mg2+-ATPase which shares physical and enzymatic characteristics with dynein, the enzyme which powers ciliary and flagellar movement. To further investigate the homology of the egg ATPase and axonemal dynein, ATP-binding subunits in preparations of each of the enzymes were identified using a photoaffinity probe of ATP, 8-azido-ATP (8-N3ATP), and three high molecular weight (HMW) polypeptide components of the two enzymes were compared by one-dimensional peptide mapping. Two heavy chains (A and B) of both the flagellar and egg ATPases bound [alpha-32P]8-N3ATP. The labeling of the HMW bands was specifically inhibited by ATP or ADP. Both the cytoplasmic ATPase and flagellar dynein utilized 8-N3ATP as a substrate indicating that the reagent binds to the active site. The two HMW ATP-binding polypeptides and one other HMW component of the egg ATPase were compared to flagellar dynein heavy chains by peptide mapping. Digestion of the egg versus flagellar HMW polypeptides with Staphylococcus V8 protease or alpha-chymotrypsin produced a highly similar group of peptides, and each pair of heavy chains was qualitatively estimated to be over 85% homologous. These data support the identification of the egg ATPase heavy chains as components of a cytoplasmic dynein and suggest that the HMW polypeptides form active enzymatic sites in flagellar and egg dynein which are substantially homologous.     

Calmodulin confers calcium sensitivity on ciliary dynein ATPase

Extraction of demembranated cilia of Tetrahymena by Tris-EDTA (denoted by the suffix E) yields 14S-E and 30S-E dyneins with ATPase activities that are slightly increased by Ca++. This effect is moderately potentiated when bovine brain calmodulin is added to the assay mixture. Extraction with 0.5 M KCl (denoted by the suffix K) yeilds a 14S-K dynein with a low basal ATPase activity in the presence of Ca++. Subsequent addition of calmodulin causes marked activation (up to 10- fold) of ATPase activity. Although 14S-K and 14S-E dyneins have Ca++- dependent ATPase activities that differ markedly in the degree of activation, the concentration of calmodulin required for half-maximal saturation is similar for both, approximately 0.1 microM. Both 30S-K and 30S-E dyneins, however, require approximately 0.7 microM bovine brain calmodulin to reach half-maximal activation of their Ca++- dependent ATPase activities. Tetrahymena calmodulin is as effective as bovine brain calmodulin in activating 30S dynein , but may be slightly less effective than the brain calmodulin in activating 14S dynein. Rabbit skeletal muscle troponin C also activates the Ca++-dependent ATPase activity of 30S dynein and, to a lesser extent, that of 14S dynein, but in both cases is less effective than calmodulin. The interaction of calmodulin with dynein that results in ATPase activation is largely complete in less than 1 min, and is prevented by the presence of low concentrations of ATP. Adenylyl imidodiphosphate can partially prevent activation of dynein ATPase by calmodulin plus Ca++, but at much higher concentrations than required for prevention by ATP. beta, gamma-methyl-adenosine triphosphate appears not to prevent this activation. The presence of Ca++-dependent calmodulin-binding sites on 14S and 30S dyneins was demonstrated by the Ca++-dependent retention of the dyneins on a calmodulin-Sepharose-4B column. Gel electrophoresis of 14S dynein that had been purified by the affinity-chromatography procedure showed that presence of two major and one minor high molecular weight components. Similar analysis of 30S dynein purified by this procedure also revealed on major and one minor high molecular weight components that were different from the major components of 14S dynein. Ca++-dependent binding sites for calmodulin were shown to be present on axonemes that had been extracted twice with Tris-EDTA or with 0.5 M KCl by the use of 35S-labeled Tetrahymena calmodulin. It is concluded that the 14S and 30S dyneins of Tetrahymena contain Ca++- dependent binding sites for calmodulin and the calmodulin mediates the Ca++-regulation of the dynein ATPases of Tetrahymena cilia.     

Substructure of sea urchin egg cytoplasmic dynein

The substructure of the cytoplasmic dynein molecule was studied using the quick-freeze, deep-etch technique. Cytoplasmic dynein purified as a 12 S form from the eggs of the sea urchin Hemicentrotus pulcherrimus was composed of a single high molecular weight polypeptide. Rotary shadowing images of cytoplasmic dynein either sprayed on to a mica surface or quick-frozen on mica flakes demonstrated a single-headed molecule, in contrast to the two-headed molecule of sea urchin sperm flagellar 21 S dynein. More detailed substructure was visualized by rotary shadowing after quick-freeze deep-etching. Cytoplasmic dynein consisted of a head and a stem. The head was pear-shaped (16 nm X 11 nm) and a little smaller than the pear-shaped head of 21 S dynein (18 nm X 14 nm). The form of the stem was irregular, and its apparent length varied from 0 to 32 nm. Binding of cytoplasmic dynein to brain microtubule in the solution was observed by negative staining, and that in the precipitate was examined by the quick-freeze, deep-etch method as well. Both methods revealed the presence of two kinds of microtubules, one a fully decorated microtubule and the other a non-decorated microtubule. Cytoplasmic dynein bound to microtubule also appeared as a globular particle. Neither the periodic binding nor the crossbridges that were observed with 21 S dynein were formed by cytoplasmic dynein, although cytoplasmic dynein appeared to bind to microtubules co-operatively.     

Preparation of antiserum against a tryptic fragment (fragment A) of dynein and an immunological approach to the subunit composition of dynein.

An improved method for purifying the tryptic fragment (Fragment A) of flagellar ATPase (dynein) from sea urchin spermatozoa is described. The preparation appears homogeneous as judged by ultracentrifugation, electrophoresis on polyacrylamide gels, and immunological techniques. The molecular weight of undenatured Fragment A was determined to be 400,000 and 370,000 by the two methods of disc electrophoresis on polyacrylamide gel and sedimentation equilibrium, respectively. The fragment dissociated into two principal polypeptide chains with molecular weights of 190,000 and 135,000 when heated in the presence of sodium dodecyl sulfate. Antiserum against dynein was prepared in rabbits using purified Fragment A from the sea urchin Anthocidaris crassispina as an antigen. The specificity of this serum toward Fragment A and toward dynein was determined by double diffusion in agarose, by inhibition of ATPase activity, and by sodium dodecyl sulfate-electrophoresis of the antigen-antibody complex. This antiserum also reacted with the enzymes from two other species of sea urchin, Pseudocentrotus depressus and Hemicentrotus pulcherrimus. Analysis of the precipitated antigen-antibody complex showed that the antiserum reacted specifically with the "high molecular weight" polypeptide seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude dynein fractions. This finding supports previous reports that this band derives from dynein ATPase. In our preparations, this "high molecular weight" dynein band appeared single.     

Quantitation of the dynein pool in unfertilized sea urchin eggs

A dynein-like ATPase activity has been isolated previously from soluble extracts of unfertilized sea urchin eggs. However, the use of non-quantitative isolation techniques, in particular affinity for microtubules or Ca2+/calmodulin, has precluded accurate estimates of dynein pool size. We have taken the unique approach of using dynein-like ATPase activity to quantitate the egg dynein pool. This approach is based on the isolation by anion-exchange chromatography on DEAE-Sephacel of a peak of dynein-like ATPase activity comprising 65% of soluble ATPase activity in the cytosolic extract. Identification of cytoplasmic dynein was based on dose-dependent inhibition by erythro-9-[3-(2-hydroxynonyl)]adenine and orthovanadate, low GTPase activity and a sedimentation coefficient of 12 S. Two high molecular weight polypeptides corresponding to the A- and D-bands of axonemal dynein were shown to copurify with dynein-like ATPase activity and to undergo specific photocrosslinking with [alpha-32P]ATP, suggesting that they were egg dynein catalytic polypeptides. The specific ATPase activity of these putative catalytic polypeptides was determined to be 1.2 mumol.min-1.mg-1. The specific dynein-like ATPase activity of the crude soluble extract of unfertilized sea urchin eggs was determined to be 0.004 mumol.min-1.mg-1. The concentration of putative dynein catalytic polypeptides was therefore determined from the ratio of the specific activities of crude to pure cytoplasmic dynein catalytic polypeptide to be 0.33% of soluble protein, or 99 pg per egg. This is approximately 3-fold greater than the mass of dynein catalytic polypeptides estimated to be present in cilia at the blastula stage of sea urchin embryonic development. The large amount of cytoplasmic dynein in unfertilized eggs suggests that it could act as a precursor of embryonic ciliary dynein. Three minor peaks of ATPase activity were also resolved from cytosolic extracts and shown to be dynein-like. However, their GTPase activities were 2-4-fold higher than that of cytoplasmic dynein, raising the possibility that egg cytoplasm may contain several isoforms of dynein.     

Hepatic microsomal Ca2+-dependent ATPase. Calmodulin-dependence and partial purification.

The hepatic microsomal fraction contains tightly bound calmodulin as demonstrated by affinity chromatography. When this calmodulin was partially removed by EGTA treatment (0.5 mM-EGTA), the uptake of 45Ca2+ by the microsomal vesicles was stimulated by added calmodulin and inhibited by trifluoperazine (TFP). The Ca2+-dependent ATPase was partially purified on a calmodulin column. This partial purification resulted in a 500-fold increase in the specific activity of the enzyme when measured in the presence of added calmodulin. Antibodies prepared against calmodulin prevented this stimulatory effect. The fraction eluted from the calmodulin column contained several protein bands indicating that the specific activity of the Ca2+-dependent ATPase is probably still underestimated. There are likely to be other calmodulin-sensitive processes present in the hepatic microsomal fraction.     

Identity and origin of the ATPase activity associated with neuronal microtubules. II. Identification of a 50,000-dalton polypeptide with ATPase activity similar to F-1 ATPase from mitochondria

We determined that the ATPase activity contained in preparations of neuronal microtubules is associated with a 50,000-dalton polypeptide by four different methods: (a) photoaffinity labeling of the pelletable ATPase fraction with [gamma-32P]-8-azido-ATP; (b) analysis of two- dimensional gels (native gel X SDS slab gel) of an ATPase fraction solubilized by treatment with dichloromethane; (c) ATPase purification by glycerol gradient sedimentation and gel filtration chromatography of a solvent-released ATPase fraction, (d) demonstration of the binding of affinity-purified antibody to the 50-kdalton polypeptide to ATPase activity in vitro. Beginning with preparations of microtubules we have purified the ATPase activity greater than 700-fold and estimate that the purified enzyme has a specific activity of 20 mumol Pi x mg-1 x min- 1 and comprises 80-90% of the total ATPase activity associated with neuronal microtubules. With affinity-purified antibody we also demonstrate cross-reactivity to the 50-kdalton subunits of mitochondrial F-1 ATPase and show that the antibody specifically labels mitochondria in PtK-2 cells. Biochemical comparisons of the enzymes reveal similar but not identical subunit composition and sensitivity to mitochondrial ATPase inhibitors. These studies indicate that the principal ATPase activity associated with microtubules is not contained in high molecular weight proteins such as dynein or MAPs and support the hypothesis that the 50-kdalton ATPase is a membrane protein and may be derived from mitochondria or membrane vesicles with F-1-like ATPase activity.     

Interrelationships between thermal-, N-ethylmaleimide-, and Ca2+-calmodulin-mediated activation/inactivation of dynein ATPase activities

Interactive effects between calmodulin activation of 30 S dynein ATPase activity and activation by heat or N-ethylmaleimide (NEM) have been studied. Addition of calmodulin during the heat treatment caused a larger increment in ATPase activity (above that caused by heating alone) than did addition of calmodulin after the heat treatment. Similar results were obtained in experiments where activation was caused by NEM treatment. For both the heat and NEM treatments, the synergistic effect of calmodulin when present during the treatment was Ca²⁺ dependent although activation of ATPase activity by either treatment alone was not Ca²⁺ dependent. Heating 14 S dynein inhibited its ATPase activity and reduced the effectiveness of calmodulin as an activator. The activating effect of calmodulin added after heat or NEM treatments was about the same as if the calmodulin was present during the treatment, i.e., interactive effects were minimal. Concentrations of NEM that had little effect on the ATPase activity of 14 S dynein largely eliminated the ability of calmodulin to activate its ATPase activity. Chromatography of the heat-treated 14 S dynein on calmodulin-Sepharose 4B indicated that the loss of sensitivity of 14 S dynein ATPase to calmodulin was not due to loss of ability of the dynein to bind to calmodulin. Retention of calmodulin binding ability was also shown for heat-treated 30 S dynein. These results suggest that calmodulin and heat/NEM activate solubilized 30 S dynein ATPase by separate mechanisms which may include a common process.     

Dynein 2. A new adenosine triphosphatase from sea urchin sperm flagella.

A new ATPase electrophoretically and immunologically distinct from the dynein ATPase studied previously has been solublized and purified from sea urchin sperm flagella. This ATPase has properties similar to those of dynein ATPase. Therefore, we propose that the two ATPases be considered as dynein isoenzymes, with previously studied dynein being known as dynein 1, and the newly discovered ATPase as dynein 2. Some physicochemical and enzymatic properties of dynein 2 have been determined. The molecular weight calculated from the sedimentation coefficient (12.3 "/- 1 S) and Stokes radius (12.8 "/- 0.4 nm) is 690,000 +/- 70,000. The molecular weight of the high molecular weight subunit of dynein 2 has been determined to be 325,000 +/- 40,000 by Na dodecyl-SO4-polyacrylamide gel electrophoresis. The enzymatic properties of dynein 1 and dynein 2 are similar in substrate specificity, pH optimum, and Mg2+ requirement for ATPase activity, but they differ in their Michaelis constant and in their dependence of ATPase activity upon salt concentration. Digestion of dynein 2 with trypsin yields an ATPase-containing protein fragment, similar to Fragment A obtained from dynein 1. An antiserum prepared against Fragment A from dynein 1 did not precipitate dynein 2 or inhibit its ATPase activity.     

Calmodulin affinity chromatography yields a functional purified erythrocyte (Ca+ + Mg2+)-dependent adenosine triphosphatase.

The (Ca2+ + Mg2+)-dependent ATPase of human erythrocyte membranes was solubilized with deoxycholate and purified by calmodulin affinity chromatography to yield a functional enzyme. The method gave an enzyme purified 207-fold as compared with that of the erythrocyte membranes. The molecular weight of the ATPase was in the range 135 000-150 000, as revealed by a single major band after electrophoresis on dodecyl sulphate/polyacrylamide gels. The isolated enzyme was highly sensitive to calmodulin, since the activity was increased about 9-fold. At 37 degrees C and in the presence of calmodulin the purified ATPase had a specific activity of 10.1 mumol/min per mg of protein. Triton X-100 or deoxycholate stimulated the calmodulin-deficient enzyme in a concentration-dependent fashion whereby the calmodulin-sensitivity was lost. The purification method is suitable for studying the lipid-sensitivity of the ATPase, since the lipids can easily be exchanged without a significant loss of activity. A purification procedure described by Niggli, Penniston & Carafoli [(1979) J. Biol. Chem. 254, 9955-9958] resulted in an enzyme that indeed was pure but was lacking a predominant feature, namely the modulation by calmodulin.     

Partial purification and characterization of dynein adenosine triphosphatase from bovine sperm

Outer dynein arm polypeptides that possess Mg+2-adenosine triphosphatase (ATPase) activity have been extracted from the flagellar axonemes of demembranated bovine sperm. Electron microscopy of intact and salt-extracted sperm demonstrates a relatively selective removal of the outer dynein arms. The salt extract contains a specific ATPase activity of 55 nmoles inorganic phosphate (Pi)/min/mg protein. Sucrose density gradient centrifugation of this extract results in a 6-fold increase in specific activity of ATPase (333 nmole/Pi/min/mg protein), which sediments as a single 13S peak. Concomitant with the increase in specific activity, there is enrichment of three high molecular weight polypeptides (Mr greater than 300,000) characteristic of dynein heavy chains. ATPase activities in the initial extract and in the 13S peak are inhibited by concentrations of vanadate and erythro-9-[3-2-(hydroxynonyl)]adenine similar to those that inhibit ATPase activity in sea urchin sperm dynein. These findings indicate that outer arm dynein ATPase can be extracted and partially purified from bovine sperm.     

Lectins induce the redistribution and internalization of receptors on the surface of cultured neurons

A cytoplasmic dynein ATPase has been identified in three species of unfertilized sea urchin eggs, Strongylocentrotus droebachiensis, S. purpuratus, and Arbacia punctulata. The enzyme was partially purified by sucrose gradient density centrifugation, and its polypeptide chain weight and composition were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein has enzymatic characteristics in common with flagellar dynein. It is activated nearly equally by Mg²⁺ and Ca²⁺, has no activity in the presence of K⁺ and EDTA, shows a specificity for ATP over other nucleoside triphosphates, and is inhibited by vanadate anion. On SDS-PAGE, the enzyme shows two major bands at 320,000 and 385,000 daltons, comigrating with certain ciliary and flagellar dynein polypeptides. The enzyme, given the name “egg dynein,” constitutes 2 to 4% of the total cell protein in the unfertilized egg and maintains this high value from fertilization through the late blastula stage. It appears to be equally distributed throughout the embryo at the 16-cell stage. Possible functions of egg dynein are discussed and models for dynein-microtubule mediated movements within the cytoplasm are presented.     

Nucleotide-metabolizing enzymes in Chlamydomonas flagella.

Nucleotides have at least two functions in eukaryotic cilia and flagella. ATP, originating in the cells, is utilized for motility by energy-transducing protein(s) called dynein, and the binding of guanine nucleotides to tubulin, and probably certain transformations of the bound nucleotides, are prerequisites for the assembly of microtubules. Besides dynein, which can be solubulized from Chlamydomonas flagella as a heterogeneous, Mg2+ or Ca2+-activated ATPase, we have purified and characterized five other flagellar enzymes involved in nucleotide transformations. A homogeneous, low molecular weight, Ca2+-specific adenosine triphosphatase was isolated, which was inhibited by Mg2+ and was not specific for ATP. This enzyme was not formed by treating purified dynein with proteases. It was absent from extracts of Tetrahymena cilia. Its function might be an auxiliary energy transducer, or in steering or tactic responses. Two species of adenylate kinase were isolated, one of which was much elevated in regenerating flagella; the latter was also present in cell bodies. A large part of flagellar nucleoside diphosphokinase activity could not be solubilized. Two soluble enzyme species were identified, one of which was also present in cell bodies. Since these enzymes are of interest because they might function in microtubule assembly, we studied the extent to which brain nucleoside diphosphokinase co-polymerizes with tubulin purified by repeated cycles of polymerization. Arginine kinase was not detected in Chlamydomonas flagellar extracts.     

Interaction of calmodulin with myosin light chain kinase and cAMP-dependent protein kinase in bovine brain

Myosin light chain kinase and a fraction of type II cAMP-dependent protein kinase have been partially purified from bovine brain by affinity chromatography on calmodulin-Sepharose. The myosin kinase was purified approximately 3700-fold and has an estimated molecular weight of 130,000 +/- 10,000 by sodium dodecyl sulfate gel electrophoresis. A fraction of soluble cAMP-dependent protein kinase also bound to calmodulin-Sepharose and was purified 2300-fold. A fraction of this cAMP-dependent protein kinase after purification by glycerol gradient centrifugation was shown to contain the two subunits of calcineurin, a major calmodulin-binding protein in brain, and the two subunits of type II cAMP-dependent protein kinase in a ratio of 1:1:2:2. Its sedimentation coefficient was 8.1 S and 9.0 S when centrifuged in the absence or presence of calmodulin, suggesting the formation of a complex between calmodulin and protein kinase. Our results suggest the possibility that calcineurin may be involved in the interaction between the protein kinase and calmodulin. Furthermore, our studies imply that the regulatory subunit of the cAMP-dependent protein kinase, but not the catalytic subunit, is the site of interaction with calmodulin since the catalytic subunit of protein kinase was partially resolved from the complex by cAMP.     

Two approaches to isolate cytoplasmic dynein ATPase from Neurospora crassa

Cytoplasmic dynein is a force-producing enzyme that, in association with dynactin, conducts minus-end directed transport of various organelles along microtubules. Biochemical analyses of cytoplasmic dynein and dynactin have been conducted primarily in vertebrate systems, whereas genetic analyses have been explored mainly in yeast and the filamentous fungi. To provide a complementary biochemical approach for the study of fungal dynein, we isolated/partially purified cytoplasmic dynein ATPase from the filamentous fungus Neurospora crassa. N. crassa dynein was partially purified by slightly modifying the existing procedures, described for mammalian cytoplasmic dynein that uses dynein-microtubule binding, followed by release with ATP and sucrose gradient fractionation. A novel approach was also used to isolate dynein-specific ATPase by gel filtration (Sepharose CL-4B). The K(m), ATP obtained by isolating dynein ATPase using gel filtration was similar to that obtained by using conventional method, suggests that contaminant proteins do not interfere with the dynein ATPase activity. Like vertebrate dynein, N. crassa dynein is a general NTPase with highest activity toward ATP, and only the ATPase activity is stimulated by microtubules. The K(m), ATP for N. crassa cytoplasmic dynein is 10- to 15-fold higher than that of the vertebrate enzyme.     

Inhibition of basal and calmodulin-activated Ca2+-pump ATPase by fractionated compound 48/80

Compound 48/80 (48/80), a mixture of polycationic compounds was fractionated using affinity chromatography on calmodulin-Sepharose. Unfractionated 48/80 and various fractions were tested for their potential inhibitory effects on ATPase activities of isolated human red blood cell membranes. ATPase activities tested included: Mg2+-ATPase, the Na+/K+-pump ATPase, and the Ca2+-pump ATPase in both its basal (calmodulin-independent) and calmodulin-activated state. Neither 48/80 nor its various fractions were very potent or efficacious inhibitors of the Mg2+-ATPase or the Na+/K+-pump ATPase. In agreement with previous reports, 48/80 was found to be an inhibitor of the calmodulin-activated Ca2+-pump ATPase. By contrast, we found that unfractionated, as well as some fractionated, material inhibited both the basal (calmodulin-independent) and calmodulin-activated Ca2+-pump ATPase activity. A fraction designated as Fraction III bound to calmodulin-Sepharose in the presence of Ca2+ and low salt and was eluted in the absence of Ca2+ and 0.15 M NaCl. By gel filtration, Fraction III had an apparent average molecular weight of 2064 (1320 for unfractionated material). Fraction III was the most potent inhibitor of the Ca2+-pump ATPase with IC50 values for the basal and calmodulin-activated forms of the enzyme of 0.6 and 1.2 micrograms/ml, respectively. Inhibition by Fraction III was cooperative with n apparent values of 2.4 and 5.7, respectively, for the basal and calmodulin-activated forms of the enzyme. Thus, binding of 48/80 constituents to calmodulin can not fully account for the observed data. Direct interaction of 48/80 constituent(s) with the enzyme and/or the lipid portion of the membrane is suggested.     

Purification and some properties of an Mg2+-, Ca2+- and calmodulin-stimulated ATPase from spinach chloroplast envelope membranes

Spinach chloroplasts display an ATPase activity which is associated with the envelope. This envelope-bound activity is stimulated by Ca²⁺, Mg²⁺ and calmodulin (Nguyen, T.D. and Siegenthaler, P.A. (1983) FEBS Lett. 164, 67–70). The Triton X-100-solubilized enzyme was retained specifically on a calmodulin-Sepharose affinity column in the presence of calcium. The fractions eluted by EGTA contained two proteins characterized by pI values of 7.3 and 6.0 (isoelectric focusing). Both proteins, separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-polyacrylamide gel electrophoresis), were resolved into a single polypeptide having and identical apparent M_rmr of 65 000. This suggests that the two initial proteins might be isoelectric variants. However, the amount of the enzyme fraction obtained by the calmodulin-Sepharose column was small and the ATPase activity was very labile. A linear glycerol gradient allowed the recovery of a greater amount of the enzyme which was, however, only partially purified, but the activity of which was much more stable. Electrophoresis of the ATPase-containing fractions in a native polyacrylamide gradient gel permitted the separation of a 260 kDa protein which was resolved by SDS-polyacrylamide gel electrophoresis into a single polypeptide of 65 kDa. Thus, the chloroplast envelope-bound ATPase might be a tetramer (260 kDa) consisting of 4 identical monomers (65 kDa). The purified ATPase had properties similar to that of the envelope-bound enzyme. TheK_m value for ATP was 0.45 mM. The activity was stimulated by Ca²⁺ and Mg²⁺, and further enhanced by calmodulin. The physiological significance of the chloroplast envelope-bound ATPase is discussed.     

Large-scale isolation of human erythrocyte Ca2+-transport ATPase

A rapid procedure for preparing large quantities of purified erythrocyte Ca2+-transport ATPase is presented. The method involves: (1) fast preparation of calmodulin-deficient, essentially haemoglobin-free, erythrocyte membranes by molecular filtration using Pellicon filters; (2) solubilization of membrane proteins by deoxycholate; and (3) a batch procedure using calmodulin-Sepharose 4B gel for purification of Ca2+-transport ATPase.