Membrane reconstitution in chl-r mutants of Escherichia coli K 12.: VII. Purification of the soluble ATPase of supernatant extracts and kinetics of incorporation into reconstituted particles

Membrane-bound ATPase (EC 3.6.1.3) of Escherichia coli K 12 is released in a soluble form by the mechanical treatments applied to the cells in order to break them. The purification of the soluble enzyme is described. The purified protein gives a single band in 7.5 % polyacrylamide gel electrophoresis. The molecular weight is estimated to be 350 000. The enzyme is cold-labile, Mg²⁺ dependent, insensitive to inhibition by $\text{N,N′-}\text{dicyclohexylcarbodiimide}$ and specific for ATP and ADP. Membranes depleted of their ATPase activity by dilution in a buffer of low ionic strength and without Mg²⁺ are able to incorporate the purified ATPase only in the presence of 2–6 mM Mg²⁺. ATPase binds to particles formed by complementation between supernatant extracts of chl A and chl B mutants. There are three kinds of particles of different buoyant densities (1.10, 1.18 and 1.23); ATPase binds only to the 1.10 and 1.18 particles. The kinetics of incorporation have been studied. ATPase begins to be incorporated into the 1.10 particles after 10 min of incubation up to a maximum at 20 min: from 30 min, ATPase is incorporated only into 1.18 particles and the amount of incorporated ATPase increases in proportion with the peak of 1.18 particles. These kinetics have a hyperbolic pattern. In order to explain the mechanism of assembly involved in complementation, two hypotheses are proposed.     

Membrane reconstitution in CHL-R mutants of Escherichia coli K 12 V. ATPase incorporation into particles formed by complementation

Mechanical treatments of cell suspensions of Escherichia coli K 12 strain PA 601, and its two mutants chl A⁻ and chl B⁻, in a buffer without Mg²⁺ lead to partial solubilization of membrane-bound ATPase. After ultracentrifugation of cell-free extracts, ATPase can be recovered in the soluble fraction. Contrary to membrane ATPase, the soluble enzyme has the following properties: (1) it is insensitive to N,N′-dicyclohexylcarbodiimide; (2) heat-inactivation kinetics show a reactivation in the first 3 min and the half-time is 15 min; (3) ADP is a substrate. In the course of complementation between soluble fractions of mutants chl A⁻ and chl B⁻, a part of soluble ATPase is incorporated into the newly formed particles. The specific activity of these particles is nearly the same as that of native particles; the ATPase bound to native membrane and the ATPase bound to the newly-formed particles both have the same biochemical properties.     

Membrane reconstitution in chl-r mutants of Escherichia Coli K 12: VIII. Purification and properties of the FA factor,the product of the chl B gene

The isolation and purification of the product of the chl B gene of Escherichia coli K 12 from the chl A mutant have been attempted. The purified protein gives a single band in 10 % sodium dodecylsulfate/polyacrylamide gel electrophoresis. The molecular weight is estimated to be 35 000. This protein, that we have named “F_A factor”, does not contain any lipid, has a strong tendency to lose its activity by polymerizing but can be kept in an active state when stored in buffer containing NaCl. The addition of purified F_A protein to a soluble extract from the chl B mutant strain grown under anaerobiosis in the presence of nitrate initiates the “complementation reaction”, i.e. the reconstitution of the nitrate reductase activity and the formation of particulate material similar to the native membrane with respect to the structure and enzymatic function. F_A protein acts both on the rate of reconstitution and on the total amount of reconstituted enzyme. The complementation leads to the reconstitution of nonsedimentable nitrate reductase and to the formation of three types of particles of different buoyant densities (1.10, 1.18 and 1.23) the two lightest of which contain nitrate reductase. It is shown that F_A factor is incorporated only into the particles of intermediate density. In vivo, this factor is located in the native membranes of chl A, chl C, chl D and wild-type strains, whatever the growth conditions, aerobiosis or anaerobiosis, and in the presence or absence of nitrate. Protein F_A can be released from either of these membranes (native or reconstituted) by removing Mg²⁺ or by subjecting Kaback's vesicles to mechanical treatments; in the case of 1.18-reconstituted particles and wild-type membranes, the release of F_A protein does not exert any effect on the level of the nitrate reductase activity.     

Studies of soluble rat liver mitochondrial acid ATPases. I. Purification and catalytic properties of ATPase 1.

A method for isolation of a soluble ATPase from rat liver mitochondria after freeze thaw cycling is described. Two enzymatically active fractions were separated by DEAE-cellulose chromatography (ATPase 1 and ATPase 2). ATPase 1 has been purified 300 fold. ATPase 1 was homogenous as judged by polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 5.8-6.0 and the optimum temperature was 45 degrees C. The enzyme follows Michaelis-Menten kinetics: Km (9 X 10(-4) M), Vmax (23,6 mumoles Pi released X min -1 X mg protein -1). The enzyme hydrolysed nucleoside triphosphates, but was inactive upon nucleoside di and monophosphates, glucose 6-phosphate, phosphoserine, pyrophosphate and glycerol 2-phosphate. In contrast to membrane bound ATPase, cations have no effect on the enzyme activity. Nucleoside di and mono phosphates and glycerol 2 phosphate inhibited competitively the enzyme. The enzyme was not affected by oligomycin, but was stimulated by lactate, 2-mercaptoethanol and dithiothreitol.     

Purification and properties of the adenosine triphosphatase released from the liver mitochondrial membrane by chloroform

1. Soluble ATPase (adenosine triphosphatase) activity is released when rat liver submitochondrial particles are shaken with chloroform, provided that ATP or glycerol is present in the suspending medium. The extraction is very rapid and appears to be complete. 2. The ATPase of the chloroform extract is about 50% pure and can be readily purified to a specific activity of 60-70mumol/min per mg of protein by (NH(4))(2)SO(4) fractionation and column chromatography on Sephadex G-200. 3. The particulate and soluble ATPases have many similar properties, including their K(m) values for ATP, activation by various metal ions, hydrolytic activity with other nucleotides and stimulation by bicarbonate ions. 4. Unlike the particulate enzyme, the soluble enzyme is cold-labile and insensitive to oligomycin. 5. The molecular weight indicated by the mobility of the soluble ATPase on Sepharose 6B is 360000. 6. The soluble ATPase combines very readily with liver submitochondrial particles depleted of ATPase by salt extraction, and oligomycin-sensitivity is restored. Very little recombination of the enzyme occurs with chloroform-extracted particles. 7. The soluble enzyme contains orcinol-reactive material, suggesting that it may be a glycoprotein. The carbohydrate content was estimated to be 1-2% by weight. 8. It is concluded that the liver ATPase obtained by the chloroform extraction method of Beechey, Hubbard, Linnett, Mitchell & Munn [(1975) Biochem. J.148, 533-537] is similar to other preparations described previously and that this method is superior in simplicity and speed.     

The proton-translocating adenosine triphosphatase of the obligately anaerobic bacterium Clostridium pasteurianum. 1. ATP phosphohydrolase activity.

1. The cell-membrane ATP phosphohydrolase of vegetatively grown Clostridium pasteurianum was specifically Mg2+-dependent, but demonstrated significant activity with GTP, CTP and UTP. It displayed approximate Michaelis-Menten kinetics only in the presence of certain effectors (e.g. phosphoenolpyruvate, fructose 1,6-bis-phosphate) which decreased the Km for ATP (to below 2 mM) but also V, whilst extending to pH 5.8 the effective pH range of activity of the enzyme. 2. ATP phosphohydrolase activity of the membrane ATPase (BF0F1) was inhibited by N,N'-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan, efrapeptin, leucinostatin and quercetin, and to a lesser degree by aurovertin and citreoviridin. The enzyme was not inhibited by oligomycin, spegazzinine, tributyl tin, triethyl tin or venturicidin. The soluble ATPase (BF1) component differed in not being inhibited by N,N'-dicyclohexylcarbodiimide, butyricin 7423 or leucinostatin. 3. The ATPase (BF0F1) complex and its soluble (BF1) component were separately purified. 4. Dodecylsulphate/polyacrylamide gel electrophoresis separated only four polypeptide components in the purified ATPase (BF0F1), with approximate molecular weights (+/- 10%) as follows: subunit a, 65 500; subunit c, 57 500; subunit da, 43 000; subunit fa, 15 000. The soluble (BF1 component contained only the three polypeptide subunits a, c and da. These were present in the BF0F1 preparation in the ratio 2 : 1 : 2; the contribution of subunit fa could not satisfactorily be quantified. 5. Subunit a was identified as the component binding 4-chloro-7-nitrobenzofurazan and subunit fa as the component binding N,N'-dicyclohexylcarbodiimide. The ATP phosphohydrolase activity of the membrane ATPase was not activated by trypsin treatment and the ATPase (BF0F1) contained no trypsin-sensitive inhibitor protein subunit. 6. Purified ATPase (BF0F1) was incorporated into artificial proteoliposomes which demonstrated ATP-dependent enhancement of 8-anilinonaphthalene-1-sulphonate fluorescence and ATP-dependent proton influx. These reactions were abolished by proton conductors (e.g. carbonylcyanide m-chlorophenylhydrazone) by valinomycin in the presence of a high external concentration of K+, or by N,N'-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan or leucinostatin. Oligomycin, tributyl tin, triethyl tin and venturicidin were not inhibitory. 7. When stripped of the soluble BF1 component, such ATPase-proteoliposomes demonstrated nil ATP phosphohydrolase activity and did not display ATP-dependent enhancement of 8-anilino-naphthalene-1-sulphonate fluorescence or ATP-dependent protein influx. All of these activities were restored by incubation of the BF1-depleted proteoliposomes with a purified preparation of the soluble BF1 component.     

Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver.

The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent Km for GTP of 35 muM. Ca-2+ stimulated the soluble activity in the presence of low concentrations of Mn-2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca-2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg-2+ was less effective than Mn-2+ as a sole cation, all enzyme fractions were markedly stimulated with Mg-2+ in the presence of a low concentration of Mn-2+. Triton X-100 increased the activity of particulate fractions about 3- to 10-fold and increased the soluble activity 50 to 100%.     

Occurrence and significance of oxygen exchange reactions catalyzed by mitochondrial adenosine triphosphatase preparations.

The capacity of various ATPase preparations from beef heart mitochondria to catalyze exchange of phosphate oxygens with water has been evaluated. Oligomycin-sensitive ATPase preparations retain a capacity for considerable intermediate Pi equilibrium HOH exchange per Pi formed during ATP hydrolysis at relatively high ATP concentration (5 mM). Submitochondrial particles prepared by an ammonia-Sephadex procedure with 5 mM ATP showed more rapid ATPase, less oligomycin sensitivity, and less capacity for intermediate exchange. With these particles, intermediate Pi equilibrium HOH exchange per Pi formed was increased as ATP concentration was decreased. The purified, soluble ATPase from mitochondria catalyzed little or no intermediate Pi equilibrium HOH exchange at 5 mM ATP but showed pronounced increase in capacity for such exchange as ATP concentration was lowered. The ATPase also showed a weak catalysis of an ADP-stimulated medium Pi equilibrium HOH exchange. The results support the alternating catalytic site model for ATP synthesis or cleavage. They also demonstrate that a transmembrane protonmotive force is not necessary for oxygen exchange reactions. At lower ATP concentrations, ADP and Pi formed at a catalytic site appear to remain bound and continue to allow exchange of Pi oxygens until ATP binds at another site on the enzyme.     

Purification and properties of a peptidase acting on a synthetic substrate for collagenase from monkey kidney.

A peptidase cleaving a synthetic substrate for collagenase, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (designated as PZ-peptide) has been purified extensively (about 5200-fold) from a soluble extract of monkey kidney with a view of carrying out studies on its possible physiological role. The purified PZ-peptidase appeared essentially free of collagenase, nonspecific protease and di- and tri-peptidase activities. The properties of the purified PZ-peptidase resemble very much the granuloma enzyme. It is optimally active around pH 7.0. Its apparent Km value for PZ-peptide is 0.72 mM and V is 10.1 mumol/mg protein/min. It is reversibly inhibited by p-hydroxymercuribenzoate and HgCl2, whereas iodoactetamide does not affect the enzyme activity. N-Ethylmaleimide inhibited the enzyme partially (50%). Heavy metals like Cu-2+, Cd-2+, Ag+, Pb-2+, Ni-2+, and Zn-2+ completely inhibited the enzyme activity, while the inhibition by Co-2+ was only partial. Fe-2+ did not exert any effect on the activity. The enzyme activity was completely inhibited by EDTA and was restored almost to the original value by metal ions like Mn-2+, Mg-2+, Ca-2+ and Ba-2+. The approximate molecular weight of the purified enzyme was estimated to be 56 000.     

Spermine binding to submitochondrial particles and activation of adenosine triphosphatase.

Studies on the effects of polyamines on oligomycin-sensitive ATPase activity of ox heart submitochondrial particles showed that, of the polyamines tested, only spermine affected the enzyme activity. Spermine within the physiological concentration range increased the Vmax. of the enzyme, but the Km for ATP was virtually unaffected. Binding studies of [14C]spermine to submitochondrial particles, under the same conditions as used for the ATPase assay, showed that the spermine binds to submitochondrial particles in a co-operative way; Hill plots of the data gave a Hill coefficient of 2 and a Kd of 8 microM. When submitochondrial particles were treated with trypsin, ATPase was not stimulated by spermine and the amount of spermine bound concomitantly was drastically decreased. The ATPase activity of isolated F1-ATPase was not affected by spermine. Removal of the natural protein ATPase inhibitor did not suppress either the stimulation of the ATPase activity by spermine or the spermine binding to the particles. The results obtained suggested that the polyamine binds and acts at the level of the liaison between the coupling factor F1 and the membrane sector F0 of the ATPase complex.     

Cold lability of membrane-bound F1-ATPase.

1. Preincubation of MgATP submitochondrial particles with EDTA or Tris.HCl liberated a measurable amount of ATPase inhibitor that could be rapidly purified using only trichloroacetic acid precipitation and heat treatment. 2. In spite of the emergence of high ATPase activity, a considerable amount of ATPase inhibitor was left in the particles. Comparative analysis of other submitochondrial preparations indicated that only AS-particles were effectively depleted. 3. The high ATPase activity of inhibitor-deficient particles, was labile at low temperature provided that the exposure to cold was done in the presence of MgATP. Other nucleotides could not substitute for ATP. Glycerol inhibited and salts enhanced the cold inactivation of membrane-bound F1-ATPase. Isolation of F1-ATPase from cold-inactivated particles yielded a soluble preparation of correspondingly lower activity. 4. It is concluded that together with the increase of ATPase activity, the ATP-dependent cold lability of membrane-bound F1-ATPase and the dislocation of ATPase inhibitor at non operative sites reveal the extent of ATPase complex disorganization.     

ATPase inhibitor from yeast mitochondria. Purification and properties.

1. Mitochondria from Candida utilis CBS 1516 and Sacchromyces cerevisiae JB 65 possess an ATPase-inhibitor activity. The inhibitor activity depends on the growth conditions of the yeast cells. It is markedly decreased when the cells are grown in the presence of a high concentration of glucose, which suggests that glucose represses the synthesis of the ATPase inhibitor or of a protein required for the insertion of the inhibitor into the inner mitochondrial membrane. 2. The ATPase inhibitor has been isolated from D. utilis mitochondria and purified to homogeneity. The minimal molecular weight calculated from amino acid composition is close to 7500. Dtermination of the molecular weight by sokium dodecylsulfate-polyacrylamide gel electrophoresis gives a value close to 6000. 3. The ATPas inhibitor of C. utilis mitochondria differs from the beef heart ATPase inhibitor by a number of properties. It has a lower molecular weight (6000-7500 vs 10500), a different amino acid composition, and a more acidic isoelectric point 5, 6 vs 7, 6). In spite of these differences, the C. utilis inhibitor cross-reacts with the ATPase of beef heart submitochondrial inhibitor-depleted particles. 4. The interaction of the C. utilis inhibitor with the ATPase of inhibitor-depleted particles requires the addition of Mg-2+-ATP or ATP in the incubation medium. 5. 14-C labelling of the C.utilis inhibitor has been achieved by growing C. utilis in a medium supplemented with [14-C]leucine. It has been found by titration experiments that the C. utilis 14-C-labelled inhibitor binds to the homologous submitochondrial inhibitor-depleted particles with a KD of about 10- minus 7 M. The number of binding sites is of the order of 0.1 nmol/mg protein.     

Purification and characterization of DNA-dependent ATPase II from Escherichia coli.

A new DNA-dependent ATPase was isolated and purified from soluble extracts of Escherichia coli. This enzyme, called ATPase II, has a molecular weight of 86,000 and exists in a monomeric state. It degrades ATP (or dATP) to ADP (or dADP) and Pi in the presence of magnesium and requires a double-stranded polynucleotide as cofactor. A correlation between the efficiency as cofactor and the melting point of the polynucleotide has been found; the lower the melting temperature, the higher the stimulation of ATPase II. The enzyme binds to single-stranded DNA and poly[d(A-T)] copolymer, but not to the double-stranded circular DNA (Form I) of simian virus 40.     

Stimulation of rat liver mitochondrial adenosine triphosphatase by anions.

The hydrolysis of MgATP by isolated rat liver mitochondrial ATPase (EC 3.6.1.3) at pH 8.0 was stimulated by various anions. The rate of hydrolysis was increased from 18 to 170 mumol per min per mg, a 9.4-fold stimulation, by HSeO3 at 1 mM MgATP. In the absence of a stimulatory anion, reciprocal plots of initial velocity studies with MgATP as the variable substrate were curved (Hill coefficient approximately 0.5). With the addition of anion, the reciprocal plots became linear. When the substrate was MgITP or MgGTP with the isolated enzyme or MgATP with submitochondrial particles, no curvature of the reciprocal plots was observed. With purified ATPase, anions stimulated the hydrolysis of MgITP, MgGTP, MgUTP or MgCTP only slightly. With submitochondrial particles the stimulation by anions of MgATP hydrolysis was limited to approximately 2-fold. These data are interpreted to indicate the existence of two substrate sites for MgATP and an anion-binding site on the isolated enzyme.     

Characterization of the interaction of parathyroid hormone with the mitochondrial ATPase

Parathyroid hormone (PTH) has been shown to bind specifically to the beta subunit of the mitochondrial ATPase on nitrocellulose blots. We have now examined this interaction further, using intact mitochondria, submitochondrial particles, and the purified F1 ATPase. With intact mitochondria, 1 microM concentrations of PTH and its biologically active 1-34 fragment activate the ATPase about 3-fold. This effect was reduced to a 1.4-fold activation with 3-34 and 7-34 fragments of the hormone, and oxidized PTH gave no detectable activity. Activation could only be observed below pH 7. PTH had no significant effect on the activity of the purified enzyme or on submitochondrial particles. However, specific binding of an iodinated PTH analog, [Nle 8,18-Tyr 34] bPTH (1-34) amide, was found with submitochondrial particles and the purified ATPase. Binding affinity with the purified enzyme was about 10(-3) that of the plasma membrane receptor, and the molar stoichiometry was close to 1:1 (PTH:intact enzyme). With submitochondrial particles the affinity was about 10-fold higher than with the purified enzyme. This binding was further examined with PTH derivatives and fragments, and compared to that seen in the plasma membrane receptor. Oxidation of methionine 18 in PTH reduced the affinity about 50%, oxidation of methionine 8 reduced the affinity 95%, and oxidation of both methionines further decreased affinity in both membranes and submitochondrial particles. However, when compared to the native hormone, the 3-34 and 7-34 PTH fragments had much higher affinity for the submitochondrial particles than for the plasma membranes. PTH also reduced chemical crosslinking of the ATP analog, p-fluorosulfonyl benzoyl 5'-adenosine, to the alpha subunit of this enzyme, but did not alter labeling of the enzyme with 3'-O-(4'-benzoyl) benzoyl ATP, suggesting that the hormone binds near a regulatory nucleotide binding site. Direct chemical crosslinking of PTH to the beta-subunit of the enzyme was attained with a cleavable, photoactivate crosslinker, sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl-1,3-dithiopropionate. The crosslinked protein was cleaved with cyanogen bromide and the labeled fragments were sequenced. The labeled fragments were found to be segments of the protein which have previously been implicated as being close to the noncatalytic ATP binding sites.     

SOME CHARACTERISTICS OF ATPase ACTIVITY IN A BRAIN MICROTUBULE PROTEIN PREPARATION

Abstract— The Mg- and Ca-ATPase activities in a brain tubulin preparation have been measured. The activity of the microtubule protein (MTP) preparation was optimal, 3-4.5 nmol Pi/mg protein/min, at pH 8.0 in the presence of 1-2 m m -Mg²⁺ or Ca²⁺, with a half maximal stimulation at about 0.3 m m concentration of either of the divalent ions.
Phosphocellulose (PC) purified tubulin exhibited no or very low activity (0-2 nmol Pi/mg protein/min).
The majority of ATPase activity was found in the microtubule associated proteins (MAP) fraction. It was stimulated by Mg²⁺ and Ca²⁺, inhibited by NaF or high ionic strength but unaffected by vanadate at 10⁻⁴ m . In decreasing order of effectiveness ATP, GTP, UTP, CTP and ADP were hydrolyzed. p -Npp was a poor substrate. V_max values for Mg- and Ca-ATPase activities were about 15 and 10 nmol Pi/mg protein/min, respectively with a K_m value of about 25 μ m . However, double reciprocal plots disclosed more complicated kinetics, which were not fully resolved.
The activity was largely confined to 30-36S material (i.e.'rings'and 'spirals'). The protein responsible for the ATPase activity is possibly the smaller one of the two (or three) high molecular weight (HMW) proteins of mol wt over 200,000.
There are similarities between this enzyme and both flagellar dynein and myosin. However, the present ATPase differs from myosin in several important aspects (i.e. ionic requirements). Furthermore, no peptides of the myosin type were found upon electrophoretic analysis of the MAP fraction.     

Purification and properties of ATPase inhibitor from rat liver mitochondria.

(1) The ATPase inhibitior protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9. (2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F1, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria. (3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg2+. ATP at slightly acidic pH. (4) The inhibitor has a minimal effect on Pi-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimules Pi-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

Mixed anhydrides of nucleotides and mesitylenecarboxylic acid as new specific inhibitors of mitochondrial adenosien triphosphatase.

Mixed anhydrides of nucleoside triphosphates and mesitylenecarboxylic acid inhibit soluble mitochondrial ATPase (adenosine triphosphatase), but do not inhibit ATPase of submitochondrial particles. Inhibition of soluble mitochondrial ATPase by the mixed anhydride of epsilon-ATP and mesitylenecarboxylic acid is followed by the covalent binding of one nucleotide residue to a molecule of the protein. It is suggested that this covalent binding occurs in the catalytic site of the mitochondrial ATPase. The mixed anhydride of ADP and mesitylenecarboxylic acid inhibits the ATPase activity of submitochondrial particles and has no effect on the activity of soluble mitochondrial ATPase. After separation of the submitochondrial particles from the mixed anhydride of ADP and mesitylenecarboxylic acid, their ATPase activity is restored to its original value (half-time of reactivation 3--4 min). Incubation of submitochondrial particles or soluble mitochondrial ATPase with the mixed anhydride of ADP and mesitylenecarboxylic acid results in AMP formation.     

Homologous pairing in genetic recombination. Purification and characterization of Escherichia coli recA protein

RecA protein, which is essential for genetic recombination in Escherichia coli, was extensively purified from a strain of E. coli which contained the recA gene cloned in a plasmid (Sancar, A., and Rupp, W. D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3144-3148). Using the DNA-dependent ATPase activity of recA protein as an assay, we obtained about 60 mg of purified recA protein from 100 g of cells. Ten micrograms or 1 microgram of the purified protein exhibited only one detectable band with Mr approximately = 40,000 upon sodium dodecyl sulfate-acrylamide gel electrophoresis. More than 99% of the ATPase activity of purified recA protein was dependent on single-stranded DNA. Purified recA protein had no detectable DNase, topoisomerase, or ligase activities. The enzyme was stable for a least a year when stored at 0-4 degrees C. The half-life of the ATPase activity of 25 microM recA protein was 37 min at 51 degrees C. Purified recA protein binds to single-stranded and double-stranded DNA, unwinds duplex DNA by a mechanism that is stimulated by single-stranded DNA or oligonucleotides, and pairs homologous single strands with duplex DNA.     

Characteristics of subunit composition of H+-ATPase from the anaerobic bacterium Lactobacillus casei

In the presence of Mg2+ or Ca2+ the membranes of the anaerobic glycolytic bacterium Lactobacillus casei hydrolyze 0.1-0.2 mumole ATP/min/mg of protein with a pH optimum 6.4. This activity is inhibited by N,N'-dicyclohexylcarbodiimide and is insensitive to oligomycin, ouabain, vanadate and hydroxylamine. A soluble ATPase was isolated and purified from L. casei membranes. The specific activity of this ATPase is 3.0-4.0 mumole ATP/min/mg of protein. The enzyme homogeneity was established by analytical polyacrylamide gel disc electrophoresis and by analytical centrifugation (S20, omega = 12 +/- 0,5). The molecular weight of the enzyme is 270 000. Polyacrylamide gel electrophoresis of ATPase denaturated by 1% SDS and 8 M urea in the presence of SDS revealed one type of subunits with Mr = 43 000. These subunits could not be separated by isoelectrofocusing in polyacrylamide gel in the presence of 8 M urea and migrated as a single peptide with pI at 4.2. The experimental results suggest that the soluble ATPase from L. casei consists of six identical subunits with Mr of 43 000.