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Two new forms of the plasma membrane ATP-ase of Micrococcus 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 mumol.min-1.mg protein-1) that could not be stimulated by trypsin. This form has been called B1 (strain B, inactive). If the elctrophoresis 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 mumol.min-1.mg protein-1 that could be stimulated by trypsin to 5-10 mumol.min-1.mg protein-1. This preparation of the ATPase has been called from 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 similar beta and delta 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, subunit alpha, 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 to beta in form B1 and about 60,000 in form BA. Furthermore BA usually showed two types of this subunit (alpha' and alpha") and an additional peptide chain E) 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 B1 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 B1. The low activity form (B1) was more stable than the active forms of the enzyme and also differeed in its circular dichroism. These results show that M. 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 function in vivo.

Micrococcus lysodeikticus ATPase was purified by preparative gel electrophoresis after its "shodk wash" release from the membrane. The method afforded the highest yield of pure protein in the minimum time as compared with former purification procedures. The pure protein had a specific activity of 7 mumol Pi-min- minus 1-mg- minus 1 with incubation times not longer than 3 min, 345 000 mol. wt and was not stimulated by trypsin. By gel electrophoresis at alkaline pH (8.5) in 8 M urea or in sokium dodecylsulfate, the ATPase revealed a complex pattern with two major subunits (alpha and beta) and two minor ones (gamma and delta). The non-identity between the major subunits was demonstrated.

A soluble purified form of Micrococcus lysodeikticus ATPase (form BAT, from strain B, active, trypsin-stimulated) was stimulated 100% by trypsin and this stimulation was inhibited by preincubation of the protease with phenyl methyl sulphonylfluoride. This form of the enzyme was also stimulated 125-150% by filtration on Sephadex G-200. Analysis by sodium dodecyl sulphate-gel electrophoresis showed that stimulation of this form of M. lysodeikticus ATPase was always accompanied by the disappearance of a subunit of mol. wt. 25000 (epsilon subunit). It suggests that this subunit is the natural inhibitor of M. lysodeikticus ATPase. In the case of ATPase stimulation by trypsin, a partial and limited degradation of the alpha subunit was also observed. The interaction between the epsilon subunit and the rest of the ATPase complex was reversibly affected by pH, suggesting its non-covalent nature.

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.