ALBERT

Notes: Summary 1. Treatment of Streptomyces albus G with lysozyme (mg dry weight mycelium/mg lysozyme = 2–3/1) leads to the isolation of membrane fractions whose yields amount to 12–20% of the total celullar protein depending upon the age and state of the bacterial culture. 2. The isolated fractions are composed of 52.7 per cent protein and 41 per cent lipid with minor amounts of hexose (2.3–2.4%), hexosamines (1.6–2.1%), RNA (2%) and DNA (0.45%). They possess a chemical composition similar to that of other membrane systems. 3. NADH oxidase activities are associated with the membrane fractions from cells of 18–20 h of age. These activities are not detected in the membrane fractions from older cells. 4. All membrane fractions shape small vesicles. Differences in size and shape are, however, found between membrane preparations from cells of distinct ages. 5. dd-carboxypeptidase is not selectively localized in a membrane fraction of a certain age. The subcellular distribution of this activity is similar to that of other lytic endopeptidases of S. albus G. 6. The amounts of dd-carboxypeptidase associated with the various membrane fractions are variable and never very high. The specificity of the dd-carboxypeptidase activity associated with membrane fractions differs from that of the soluble enzyme. 7. These results are discussed in relationship with the carboxypeptidase-transpeptidase hypothesis (Leyh-Bouille et al., 1970).

Notes: Summary Dimethyl suberimidate and dithiobis (succinimidyl propionate) have been used to explore the nearest neighbor relationship of the subunits (α, β, γ and δ by decreasing molecular weight) of F1-ATPase or BF1 factor of Micrococcus lysodeikticus. Cross-linking with the two diimido esters inhibited the ATPase activity but this inhibition never exceeded 50% of the initial value. The cross-linking pattern of this BF1 factor, as revealed by sodium dodecyl sulfate gel electrophoresis, shows a relative low proportion of high molecular weight aggregates which move slowly than the heaviest subunit (α). They are resolved as three components of molecular weights 200,000, 130,000 and 100,000 in 5% acrylamide gels, plus an additional component (mol. wt 80,000) identified in 10% acrylamide gels. The other aggregate bands represent cross-linking products of the smaller subunits (γ and δ) that may travel to the conventional position of the heavier subunits. The subunit composition of the aggregate bands has been determined through the reversion of dithiobis (succinimidyl propionate) cross-linking of the BF1 factor by dithiothreitol and analysis in second dimension by gel electrophoresis. The results indicate that γ subunit can cross-link with itself and with each of the other subunits except β. The α subunit is also able to cross-link with itself and with the other subunits although to a minor extent than γ, and that δ2 aggregates are present. These results represent a specific pattern of cross-linking for this BF1 factor as compared to other F1 coupling factors. It suggests a certain asymmetry in the spatial organization of the major subunits of M. lysodeikticus F1-ATPase where the γ subunit must play a central role. A subunit stoichiometry α3 β3 γ2 δ2 is proposed for whole F1-ATPase which leads to a molecular weight 440,000 consistent with the 430,000 value estimated by sedimentation equilibrium at low speed. A tentative structural model of M. lysodeikticus BF1 factor is derived from these data. The significance of the results in relation to the possible generalization of the molecular architecture of F1 factors is discussed.

Notes: Summary Two new forms of the plasma membrane ATP-ase ofMicrococcus lysodeikticus NCTC 2665 were isolated from a sub-strain of the microorganism by polyacrylamide gel electrophoresis. One of them had a mol.wt of 368,000 and a very low specific activity (0.80 µ mol.min−1.mg protein−1) that could not be stimulated by trypsin. This form has been called BI (strain B, inactive). If the electrophoresis was carried out in the presence of reducing agents (i.e., dithiothreitol) and the pH of the effluent maintained at a value of 8.5 another form of the enzyme was obtained. This had a mol.wt of 385,000 and a specific activity of 2.5–5.0 µ mol.min−1.mg protein−1 that could be stimulated by trypsin to 5–10 µ mol.min−1.mg protein−1. This preparation of the ATPase has been called form BA (strain B, enzyme active). The subunit composition of both forms has been studied by sodium dodecyl sulphate and urea gel electrophoresis and compared to that of the enzyme previously purified from the original strain (form A). The three forms of the enzyme had similarβ and δ subunits, with mol.wt of about 50,000 and 30,000 dalton, respectively. They also had in common the component(s) of relative mobility 1.0, whose status as true subunit(s) of the enzyme remains yet to be established. However, subunitα, that had a mol.wt of about a 52,500 in form A (Andreu et al. Eur. J. Biochem. (1973) 37, 505–515), had a mol.wt similar toβ in form BI and about 60,000 in form BA. Furthermore BA usually showed two types of this subunit (α′ andα′′) and an additional peptide chain (ε) with a mol.wt of about 25,000 dalton. This latter subunit seemed to account for the stimulation by trypsin of form BA. Forms BA could be converted to BI by storage and freezing and thawing. Conventional protease activity could not be detected in any of the purified ATPase forms and addition of protease inhibitors to form BA failed to prevent its conversion to form BI. The low activity form (BI) was more stable than the active forms of the enzyme and also differed in its circular dichroism. These results show thatM. lysodeikticus ATPase can be isolated in several forms. Although these variations may be artifacts caused by the purification procedures, they provide model systems for understanding the structural and functional relationships of the enzyme and for drawing some speculations about its functionin vivo.

Notes: Summary Basal and trypsin-stimulated adenosine triphosphatase activities ofEscherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5mm, Vmax (minus trypsin) = 1.6µmol·min−1·mg protein−1, Vmax (plus trypsin) = 2.44µmol·min−1·mg protein−1; for the soluble ATPase: [S0.5] = 1.2mm, Vmax (-trypsin) = 4µmol·min−1·mg protein−1; Vmax (+trypsin) = 6.6µmol·min−1·mg protein−1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4mm) showed two sites (groups) with different K ms: at low ATP the values were 0.38 and 1.4mm for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20mm, respectively. Mg2+ saturation at constant ATP (8mm) revealed michaelian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulatesE. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors ofEscherichia coli ATPase. The Ki values for Pi were 1.6 ± 0.1mm for the membrane-bound ATPase and 1.3 ± 0.1mm for the enzyme in soluble form, the Ki values for ADP being 1.7mm and 0.75mm for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4mm) inhibited the membrane-bound enzyme by 60–70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20µ m) inhibited both states ofE. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.

Notes: 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.