The reaction of N-ethylmaleimide (NEM) with three different forms of beef heart cytochrome oxidase was studied. Whereas denatured oxidase binds 4–5 mol NEM per heme a, native oxidase and a new form of the oxidase prepared by extensive dialysis against water each binds 0.8–1.0 mol NEM per heme a. Oxidase in the form of coupled vesicles binds only half as much NEM as the native molecule. Six different polypeptides are associated with cytochrome oxidase. They have molecular weights of (A) 45 000; (B) 25 000; (C) 16 000; (D) 12 500; (E) 10 500; and (F) 8500. Four of the six different subunits appear to contain groups that bind NEM. In the native oxidase subunits A, B, D, and F react with NEM, subunit B being the most reactive, subunit F the least. Water-dialyzed oxidase binds NEM to subunits A, B, and F, the binding to D being greatly diminished. Vesicular oxidase binds NEM to only subunit B.
Escherichia coli lipase was found to have a broad pH optimum between pH 8 and 10. Long-chain acyl triacylglycerols such as trioleolglycerol were hydrolysed at a relatively slow rate, whereas, the shorter-chain acyl derivative tracapryloylglycerol was not. Triacylglycerols and diacylglycerols were broken down at a rate 10 to 15 fold greater than that for monoacylglycerol. Simple esters such as methyloleate and cetylpalmitate were hydrolysed at rates greater than that of triacyglycerol. Water-soluble esters such as p-nitrophenylacetate were not attacked. Hydrolysis of lipase substrate occurred more readily in the presence of an anionic detergent such as taurocholate. The enzyme had no marked preference for the 1- or 3-position of triacylglycerols but attacked these positions much more readily than position 2. The enzyme also catalyzed transacylation reaction with simple alcohols such as methanol or ethanol.
An enzyme with phospholipase Al activity was purified some 500-fold from Escherichia coli cell homogenates. Lipase, phospholipase A2, and lysophospholipase copurified with phospholipase A1 and the four activities displayed similar susceptibility to heat treatment. The phospholipase A and lipase activities were recovered in a single band when partially purified preparations were subjected to SDS gel electrophoresis. Phospholipase, lysophospholipase, and lipase all required Ca2+ for activity. Phosphatidylcholine, phosphatidylethanolamine, and their lyso analogues were all hydrolysed at equivalent rates and these were substantially greater than the rate of methylpalmitate or tripalmitoylglycerol hydrolyses under similar incubation conditions. Evidence for a direct but slow hydrolysis of the ester at position 2 of phosphoglyceride was obtained; however, release of fatty acid from this position is mostly indirect involving acyl migration to position 1 and subsequent release of the translocated fatty acid. Escherichia coli, therefore, appears to possess a lipolytic enzyme of broad substrate specificity acting mainly at position 1 but also at position 2 of phosphoglycerides and on triacylglycerols and methyl fatty-acid esters.
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