Volume 9 1, number 1 FEBS LETTERS July 1978 SPECIFIC IMMUNOPRECIPITATION OF ATPase FROM ESCHERICHIA COLI Dina RALT, Nathan NELSON+ and David GUTNICK Department of Microbiology, George S. Wise Center for Life Sciences, Tel-Aviv University, Tel-Aviv and +Department of Biology, Technion-Israel, institute of Technology, Haifa, Israel Received 23 April 1978 1. Introduction Energy-transducing membrane ATPases in mito- chondria [l] , chloroplasts [2] and bacteria [3,4] exhibit a large degree of similarity with respect to function, subunit composition and stoichiometry, although some variability exists with different systems. It might be expected, therefore, that among bacterial mutants defective in ATPase activity, certain classes would exhibit alterations in the latter two properties. In order to examine the possibility of such modifications in mutants, it is necessary to isolate the defective ATPase using a procedure which does not depend on assay of catalytic activity. One assay frequently employed in the characterization of mutant protein involves the isolation of material which cross-reacts with specific antibody prepared against the purified parental protein [5]. This report describes a relatively simple immunological procedure designed to selectively precipitate active or defective ATPase from a crude Triton extract of E. coli mem- branes using antibody prepared against the purified wild-type enzyme. The cross-reacting material in the immuno-precipitate is subsequently analysed by SDS- gel electrophoresis. Abbreviations: Ab, antibody; ATPase, Mg*+-Ca*+ adenosine triphosphatase; BSA, bovine serum albumin; cpm, counts per minute; CRM, cross-reacting material; EDTA, ethylenediamine tetrasodiumacetate; IgG, gamma globulin; OVA, ovalbumin; PMSF, phenyl methyl sulfonyl fluoride; POP, 2, Sdiphenyl- oxazol;POPOP, 2,2-p-phenylen-bis-(4-methyl-5-phenyloxazol); SDS, sodium dodecyl sulfate 2. Materials and methods 2.1. Strains Parental strains A428 [6], ML 308-225 [7], S7 [8] and the ATPase-defective mutant NR70 [8] . 2.2. Materials Rabbit antiserum was prepared as in [6]. Two kinds of antisera were prepared using: (i) Purified ATPase from A428, anti BF I ; (ii) A trypsin-treated preparation of purified ATPase consisting exclusively of Q: + fl subunits; this preparation retains cataIytic activity [6], anti (a t/J). 14C- and 3H-labeled L-amino acid mixtures with spec. act. 0.1 mCi/ml in 0.1 N HCl were purchased from knew England Nuclear. Soluene-350 was purchased from Packard. All other reagents were of the highest quality available. 2.3. Growth of cells Cells were grown on Davis minimal medium sup- plemented with 0.5% glucose 50 pg/ml each of L- proline and L-histidine and 1 pg/ml vitamin B1. For labeling experiments 0.1 mCi 14C or 0.4 mCi 3H of an L-amino acid mixture was added, per liter of culture. Cells, 500 ml, at late log phase were harvested in a Sorvall RC2B centrifuge and washed once with STNT buffer (0.04 M tricine, 8 mM NaCl, 0.3 M sucrose, pH 8 .O). 2.4. Isolation of cross-reacting material About 0.8 g (wet wt) of washed cells were sus- ElsevierfNorth-Holland Biomedical Press 85