Activation parameters of the adenosine triphosphatase of Micrococcus lysodeikticus. A comparison of the soluble and membrane-bound forms of the enzyme.

The Arrhenius plots for the membrane-bound ATPase and its soluble form purified from Micrococcus lysodeikticus, presented discontinuities near 30 degrees C at pH 7.5. Glycerol-containing lipids were not responsible for these discontinuities. The values of the enthalpies of activation were 12 (soluble) and 22 (membrane-bound) kcal/mol (50.2 and 92.0 kJ/mol) above 30 degrees C and 42 (soluble) and 29 (membrane-bound) kcal/mol (175.7 and 121.3 kJ/mol) below that temperature. The results suggested that both molecular forms of the ATPase were able to adopt at least two different structures, above and below the critical temperature. Of the two, only the high-temperature structure seemed to be enzymically active. In the case of lipid-dependent ATPases, such as the Escherichia coli enzyme, the transition between both enzyme structures probably occurred with simultaneous "melting" of their lipid microenvironment.     

Membrane adenosine triphosphatase of Micrococcus lysodeikticus: effect of millimolar Mg2+ in modulating the properties of the membrane-bound enzyme

The latency of Micrococcus lysodeikticus membrane-bound Mg(2+)-adenosine triphosphatase (ATPase) is expressed by the ratio of its activity assayed in the presence of trypsin ("total") versus the activity assayed in absence of the protease ("basal"). By isolating membranes in the presence of variable concentrations of Mg(2+) (50 mM, 10 mM, or none) and by washing them with different Mg(2+)- and ethylenediaminetetraacetic acid-containing tris(hydroxymethyl)aminomethane-hydrochloride buffers (pH 7.5), we showed that the enzyme latency was dependent on the environmental concentration of this divalent metal ion. Mg(2+) bound to at least two classes of sites. The binding of Mg(2+) to low-affinity sites (saturation at approximately 40 mM external Mg(2+)) induced a high basal ATPase activity, whereas its binding to medium-affinity sites (saturation at about 2 mM Mg(2+)) correlated with low basal activity and a very high stimulation by trypsin. Membranes with tightly bound Mg(2+) (high affinity?) revealed an intermediate behavior for the latency of M. lysodeikticus ATPase. The Mg(2+)/Ca(2+) antagonism as activators of the membrane ATPase was not directly related to Mg(2+) binding by the membranes. The efficiency of the ATPase release from M. lysodeikticus membrane by 3 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.5) was inversely proportional to the concentration of external and/or bound Mg(2+). Deoxycholate (DOC) (1%) solubilized the ATPase from all types of membrane. All the soluble ATPases behaved as Ca(2+)-ATPases, but the DOC-soluble fractions showed degrees of latency like those of the original membranes. The DOC-soluble ATPase preparation revealed a vesicular structure and complex protein patterns by sodium dodecyl sulfate gel electrophoresis. We propose that ATPase latency is modulated via a Mg(2+)-ATPase-membrane complex.     

Optical properties and denaturation by guanidinium chloride and urea of the adenosine triphosphatase of Micrococcus lysodeikticus. A comparison of four molecular forms of the enzyme.

1. The fluorescence and circular dichroism of four homogeneous preparations of ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus differing in molecular structure and enzymic properties were examined at pH 7.5 and 25 degrees. Emission was maximum at 325 and 335 nm and the relative intensities at these wavelengths may be used to characterize the different ATPase preparations. The circular-dichroism spectra exhibited negative extrema at 208 and 220 nm, and the relative value of the molar ellipticity at these wavelengths was also different for each molecular form of the enzyme. 2. The four preparations undergo two consecutive major unfolding transitions in guanidinium chloride (midpoints at 0.94 and 1.5 M denaturant), with concomitant destruction of the quaternary structure of the protein. A comparatively minor alteration in the ATPase structure also occurred in 0.05-0.2M-guanidine and led to complete inactivation of the enzyme. The inactivation and the first unfolding transition were reversible by dilution of the denaturant; the transition with midpoint at 1.5M-guanidine was irreversible. 3. Similar results were obtained in urea, except that the successive transitions had midpoints at concentrations of denaturant of 0.4, 2.0 and 4.5M. Low concentrations of urea caused a noticeable activation of the enzyme activity and alterations of the electrophoretic mobility of the ATPase. 4. A model is proposed in which one of the major subunits, alpha, is first dissociated and unfolded reversibly by the denaturants, followed by the irreversible unfolding and dissociation of the other major subunit, beta, from subunit delta and/or the components of relative mobility 1.0 in dodecyl sulphate/polyacrylamide-gel electrophoresis (rho).     

Activation parameters and molecular changes induced by substrate hydrolysis of the adenosine triphosphatase of Micrococcus lysodeikticus. A comparison of three different soluble forms of the enzyme

Summary The Arrhenius plots for the active and low activity soluble forms of the ATPase purified from the membranes ofMicrococcus lysodeikticus grown at 30°C presented discontinuities at 30 and 33°C, respectively. Their activation parameters differed, being highest for the low activity form of the enzyme.Both forms underwent changes in their molecular properties as a consequence of being enzymically active, i.e., upon incubation with substrates at an adequate temperature. These changes consisted of a decrease in the relative mobilities of some of their subunits in dodecyl sulphate polyacrylamide gel electrophoresis, and the temperature at which they occurred depended on the energy of activation of the particular form of the ATPase used. The low activity form required an incubation temperature of 50°C, whereas for an active form 37°C was sufficient.     

Role of the subunits of the energy-transducing adenosine triphosphatase from Micrococcus lysodeikticus membranes studied by proteolytic digestion and immunological approaches.

An energy-transducing adenosine triphosphatase (ATPase, EC 3.6.1.3) that contains an extra polypeptide (delta) as well as three intrinsic subunits (alpha, beta, gamma) was purified from Micrococcus lysodeikticus membranes. The apparent subunit stoichiometry of this soluble ATPase complex is alpha 3 beta 3 gamma delta. The functional role of the subunits was studied by correlating subunit sensitivity to trypsin and effect of antibodies raised against holo-ATPase and its alpha, beta and gamma subunits with changes in ATPase activity and ATPase rebinding to membranes. A form of the ATPase with the subunit proportions 1.67(alpha):3.00(beta:0.17(gamma) was isolated after trypsin treatment of purified ATPase. This form has more than twice the specific activity of native enzyme. Other forms with less relative proportion of alpha subunits and absence of gamma subunit are not active. Of the antisera to subunits, only anti-(beta-subunit) serum shows a slight inhibitory effect on ATPase activity, but its combination with either anti-(alpha-subunit) or anti-(gamma-subunit) serum increases the effect. The results suggest that beta subunit is required for full ATPase activity, although a minor proportion of alpha and perhaps gamma subunit(s) is also required, probably to impart an active conformation to the protein. The additional polypeptide not hitherto described in Micrococcus lysodeikticus ATPase had a molecular weight of 20 000 and was found to be involved in ATPase binding to membranes. This 20 000-dalton component can be equated with the delta subunit of other energy-transducing ATPases and its association with the (alpha, beta, gamma) M. lysodeikticus ATPase complex appears to be dependent on bivalent cations. The present results do not preclude the possibility that the gamma subunit also plays a role in ATPase binding, in which, however, the major subunits do not seem to play a role.     

Membrane protein synthesis in Micrococcus lysodeikticus and selective effect of chloramphenicol.

Micrococcus lysodeikticus cytoplasmic membranes labeled with ]-14C]arginine plus [-14C]-threonine were prepared and subjected to mild washing treatments to fractionate membrane proteins. Polyacrylamide gel electrophoresis of total membranes, in the presence of sodium dodecyl sulfate, results in the separation of 28-30 bands of labeled protein. Three peaks of protein show higher specific radioactivity than the others. Chloramphenicol at 100 mug/ml inhibits the incorporation of labeled precursors into membrane proteins by 45-70 percent, some of them being more affected by the antibiotic. From all available results, we suggest that the partial inhibitory effect shown by this antibiotic could be due to the existence of specific biosynthetic sites for some membrane proteins, which are differently affected by chloramphenicol.     

L-Asparaginase activity in Leptosphaeria michotii. Isolation and properties of two forms of the enzyme

The properties of L-asparaginase (EC 3.5.1.1) in Leptosphaeria michotii (West) Sacc., which has previously been shown to have an activity rhythm, were analyzed. Two forms of L-asparaginase were isolated from acetic acid and ammonium sulfate fractionations followed by DEAE-Sephacel chromatography. The activity of L-asparaginase changed rhythmically with the same period as that of crude extracts, but the rhythms of the two enzyme forms were out of phase. The two asparaginase forms differed in their isoelectric points and the substrate concentrations for attaining half-maximal velocity; non-Michaelis-Menten kinetics for hydrolysis of L-asparagine were observed. Analyses of asparaginase form II by polyacrylamide gel electrophoresis showed that four proteins, irrespective of the phase of the activity rhythm at which the enzyme was extracted, could be detected: asparaginase oligomer (M_r 130 000 to 140 000), its dimer, an aggregate (M_r 500 000 to 600 000) having a low asparaginase activity, and a protein (M_r 60 000) without asparaginase activity; the same proteins were found in asparaginase form I. These results indicate that L. michotii asparaginase could be implicated in a protein complex.     

Isolation and characterization of a mannan from mesosomal membrane vesicles of Micrococcus lysodeikticus.

The carbohydrate content of mesosomal membranes of Micrococcus lysodeikticus has been shown to be consistently higher (about four times) than that of corresponding plasma membrane preparations. Analysis of washed membrane fractions by gas-liquid chromatography indicated that mannose was the major neutral sugar of both types of membrane (accounting for 95 and 89%, respectively, of the mesosomal and plasma membrane carbohydrate). Small amounts of inositol, glucose and ribose were also detected. We have shown by polyacrylamide gel electrophoresis in sodium dodecylsulphate and by precipitation and agar gel diffusion experiments with concanavalin A that a mannan is the major carbohydrate component of both types of membrane. This polymer can be selectively released from mesosomal membranes by a simple procedure involving low ionic strength-shock and heating to 80 degrees C for 1 min, and purified by ultrafiltration and ethanol precipitation. The mannan contains mannose as the only neutral carbohydrate, is not phosphorylated and does not contain significant amounts of amino sugars or uronic acids. Agar gel electrophoresis experiments, however, indicate an anionic polymer whose acidic properties are eliminated upon mild base hydrolysis. Analysis of native mannan by infrared spectroscopy reveals absorption bands attributable to ester carbonyl groups and to carboxylate ions, consistent with the presence of succinyl residues in the polymer (Owen, P. and Salton, M.R.J. (1975) Biochem, Biophys. Res. Commun. 63, 875--800). A sedimentation coefficient of 1.39 S was obtained by analytical ultracentrifugation in 1.0 M NaCl and a value of one reducing equivalent per 50 mannose residues by reduction with NaB3H4. The polysaccharide was only slightly degraded (2%) by jack bean alpha-mannosidase and could precipitate 15 times its own weight of concanavalin A. The acidic polymers was also detected in the cell "periplasm" and was secreted from cells grown in defined media during the period of decelerating growth.     

The mitochondrial adenosine triphosphatase of Acanthamoeba castellanii. Oscillatory accumulation of enzyme activity, enzyme protein and F1-inhibitor during the cell cycle.

1. The mitochondrial ATPase of Acanthamoeba castellanii accumulated discontinuously in synchronous cultures prepared by a minimally perturbing size-selection technique. 2. Enzyme activity per ml of culture doubled overall during one cell cycle time of 8 h, but oscillated to give seven maxima during this period. Similar oscillations were observed in the specific activities of ATPase and of the naturally occurring inhibitor protein. 3. These variations in enzyme activity reflected changes in amount of enzyme protein as assayed by an immunological technique. 4. Large variations in I50 values (micrograms of inhibitor/mg of protein necessary for 50% inhibition of inhibitor-sensitive activity) for inhibition of ATPase activity by seven different inhibitors of energy conservation were observed. Activity was more sensitive to inhibition by oligomycin, efrapeptin, citreoviridin and quercetin when values were highest. 5. The results are discussed in relation to the phased organization of biosynthesis and degradation of cellular components known to occur during the cell cycle of this organization.     

The rate-limiting step of the protamine-induced adenosine triphosphatase activity of adenosine triphosphate-G-actin.

The release of Pi from the Pi-G-actin-ADP complex is the rate-limiting step in the ATPase activity that is shown by ATP-G-actin in the presence of protamine.