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.     

Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein.

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.     

Conformational and molecular responses to pH variation of the purified membrane adenosine triphosphatase of Micrococcus lysodeikticus.

A preparation of ATPase from the membranes of Micrococcus lysodeikticus, solubilized and more than 95% pure, showed two main bands in analytical polyacrylamide gel electrophoresis. They did not correspond to isoenzymes because one band could be converted into the other by exposure to a mildly alkaline pH value. The conversion was paralleled by changes in molecular weight, circular dichroism and catalytic properties. Denaturation by pH at 25 degrees C was followed by means of circular dichroism, ultracentrifugation and polyacrylamide gel electrophoresis. A large conformational transition took place in the acid range with midpoints at about pH = 3.6 (I = 10(-4) M), 4.3 (I = 0.03 M) and 5.3 (I = 0.1 M). The transition was irreversible. Strong aggregation of the protein occurred in this range of pH. The final product was largely random coil, but even at pH 1.5 dissociation into individual subunits was not complete. However, partial dissociation took place at pH 5 (I = 0.028 M). At this pH value the enzyme was inactive, but 20-30% of the activity could be recovered when the pH was returned to 7.5. In the alkaline region the midpoint of the transition occurred near pH = 11 (I = 0.028 M). The pK of most of the tyrosine residues of the protein was about 10.9. The unfolding was irreversible and the protein was soon converted into peptide species with molecular weights lower than those determined for the subunits by gel electrophoresis in the presence of sodium dodecyl sulphate. Conventional proteolysis did not account for the transformation.     

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.     

MgATP-induced inhibition of the adenosine triphosphatase activity of the chloroform-released mitochondrial adenosine triphosphatase.

1. Preincubation of the ox heart chloroform-released mitochondrial ATPase with MgATP results in a time-dependent inhibition of ATPase activity. No re-activation occurs when MgATP remains in the preincubation medium. The enzyme activity returns when all the MgATP in the preincubation system has been hydrolysed. 2. The mechanism of the MgATP-induced inhibition was examined. Inhibition occurs on incubation with MgATP or other hydrolysable nucleotides. Incubation with MgADP or Pi does not cause any inhibition. Neither freshly bound adenine nucleotide nor Pi is associated with inhibited enzyme. The rate of MgATP-induced inhibition correlates with the rate of ATP hydrolysis in the preincubation medium. Changing the rate of ATP hydrolysis at a fixed concentration of ATP also changes the rate of MgATP-induced inhibition by the same proportion. The inhibition is thus related to the ATP-hydrolysis process itself. 3. We propose that intermediate enzyme species of the ATP-hydrolytic sequence can undergo a conformational change to form inhibited species. The kinetics of the inhibition suggest that a substrate-activation step is involved in ATP hydrolysis and MgATP-induced inhibition. 4. The effects of the nature of the preincubation medium on the process of MgATP-induced inhibition and its reversal were examined.     

D-alanine carboxypeptidase activity of Micrococcus lysodeikticus released into the protoplasting medium.

Conversion of whole cells of Micrococcus lysodeikticus to protoplasts allowed the release of a soluble form of a D-alanine carboxypeptidase into the protoplasting medium. The enzyme cleaves the terminal D-alanine from the radioactively labelled UDP-N-acetylmuramyl-pentapeptide containing L-lysine as the diamino acid. However, the enzyme is only minimally active in this fraction so that it had to be enriched and partially purified before its properties could be studied. Chromatography on carboxymethyl-Sephadex removed the lysozyme used in the protoplasting of the cells. The material which was unadsorbed to the column was applied to an affinity chromatography column of Ampicillin-Sepharose. Most of the contaminating protein was washed from the column while the D-alanine carboxypeptidase adhered to the resin and could be eluted with 0.5 M Tris-HCl buffer pH 8.6. Some of the properties of the enzymic activity were studied using this preparation. The enzyme was activated by Mg2+ ions with a broad optimum from 15--35 mM. It was maximally active when NaCl at a concentrations of 0.06--0.08 M was added to the assay, and the pH curve was biphasic with an alkaline optimum. The Km for substrate was found to be 0.118 mM. Enzymic activity was completely inhibited by low concentrations of Ampicillin and penicillin G.