gluconeogenesis, purified preparations2-4 show little activity in the physiological

pH range. We have previously shown that crystalline enzyme preparations from mammalian liver can be modified by dinitrophenylation of specific sulfhydryl groups,5' 6 yielding an enzyme which is equally active at pH 7.5 and 9.2; increases in catalytic activity at neutral pH could also be induced by treating the enzyme, under controlled conditions, with p-mercuribenzoate.7 Evidence for the presence of "neutral FDPase" in crude extracts of mammalian liver has been reported,8 but the nature of this activity was not established. The observations described above suggest that the enzyme may be under metabolic control by virtue of a specific mechanism involving modifications of one or more sulfhydryl groups, resulting in the induction of activity at neutral pH. In the present communication we report the reversible activation of purified rabbit liver FDPase by disulfides, which react with a limited number of cysteine residues in the protein to form mixed disulfides. A number of disulfides, including cystamine, were active, but other naturally occurring disulfides such as oxidized glutathione, oxidized Coenzyme A, and lipoic acid, failed to activate the enzyme. The natural disulfide activator may indeed be cystamine, which has been reported to occur in mammalian liver as a product of pantethine metabolism.9' 10 However, the possibility of exchange with an unknown disulfide activator cannot be excluded. The recent discovery of a volatile mercaptan as a component of a mixed disulfide in a bacterial proteinase is of interest.11 Such compounds, if they were to occur in small amounts in mammalian tissues, would be difficult to detect. Materials and Methods.-FDPase was purified according to the procedure of Pontremoli et al.3 The enzyme activity was measured spectrophotometrically at 340 my by following the rate of production of fructose 6-phosphate in the presence of triphosphopyridine nucleotide (TPN), excess phosphoglucose isomerase, and glucose 6-phosphate dehydrogenase. The assay systems (1 ml) contained 0.1 mM fructose 1,6-diphosphate (FDP), 1 mM MnC12 or 10 mM MgCl2, 0.1 mM TPN, 0.3 units of glucose 6-phosphate dehydrogenase, 2 units of hexosephosphate isomerase, and either 40 mM triethanolamine buffer, pH 7.5, containing 0.1 mM ethylenediaminetetraacetate (EDTA), or 40 mM glycine buffer, pH 9.1. The temperature in the spectrophotometer chamber was 22'. The unit of enzyme activity was defined as the quantity required to produce an absorbance change of 1.0 unit per minute under these conditions. Specific activity is expressed as units per milligram of protein. The protein concentration was determined by the phenol method'2 standardized by dry weight determination of a sample of dialyzed crystalline FDPase. Glucose 6-phosphate dehydrogenase, hexosephosphate isomerase, and glutathione reductase were purchased from Boehringer-Mannheim, Germany. f6-Phosphogluconate dehydrogenase was prepared as previously described.)3 Cystamine dihydrochloride, pantethine, lipoic acid, lipoamide, and Coenzyme A were purchased from Sigma Chemical Corporation; 5,5'-dithio bis (2-nitrobenzoic) acid from K and K Laboratories; and 2-hydroxyethyl disulfide, dithiodiglycollic acid, and 3-carboxypropyl disulfide from Aldrich Chemical Co. Suilfhydryl groups were measured spectrophotometrically by titration with p-mercuribenzoate