Pyruvate dehydrogenase phosphatase was purified to apparent homogeneity from bovine heart and kidney mitochondria. The phosphatase has a sedimentation coefficient (S20,w) of about 7.4 S and a molecular weight (Mr) of about 150 000 as determined by sedimentation equilibrium and by gel-permeation chromatography. The phosphatase consists of two subunits with molecular weights of about 97 000 and 50 000 as estimated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Phosphatase activity resides in the Mr 50 000 subunit, which is sensitive to proteolysis. The phosphatase contains approximately 1 mol of flavin adenine dinucleotide (FAD) per mol of protein of Mr 150 000. FAD is apparently associated with the Mr 97 000 subunit. The function of this subunit remains to be established. The phosphatase binds 1 mol of Ca2+ per mol of enzyme of Mr 150 000 at pH 7.0, with a dissociation constant (Kd) of about 35 microM as determined by flow dialysis. Use of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA) at pH 7.6 in conjunction with flow dialysis gave a Kd value for Ca2+ of about 8 microM. In the presence of both the phosphatase and the dihydrolipoyl transacetylase (E2) core of the pyruvate dehydrogenase complex, two equivalent and apparently non-interacting CA2+-binding sites were detected per unit of Mr 150 000, with a Kd value of about 24 microM in the absence and about 5 microM in the presence of EGTA. In the presence of 0.2 M KCl, which inhibits phosphatase activity about 95%, the phosphatase exhibited only one Ca2+-binding site, even in the presence of E2. The phosphatase apparently possesses an "intrinsic" Ca2+-binding site, and a second Ca2+-binding site is produced in the presence of E2. The second site is apparently altered by increasing the ionic strength. It is proposed that the second site may be at the interface between the phosphatase and E2, with Ca2+ acting as a bridging ligand for specific attachment of the phosphatase to E2.

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A new species, BaciZZus naganoensis, is proposed for an obligately aerobic, moderately acidophilic, endospore-forming bacterium that produces a thermostable, aciduric pullulanase (EC 3.2.1.41). The organism was isolated from soil by selection on solid, pullulan-containing medium at pH 4.0 and 30°C. The isolate required a medium pH of less than 6.5 for growth initiation. Fatty acid composition studies revealed that the major fatty acid of cells grown in nutrient broth supplemented with 1 % starch was 14-methylpentadecanoic acid (iso-C,,) at 45 mol%. The guanine-plus-cytosine content of the DNA of this organism was 45 f 2 mol%. A type culture has been deposited with the American Type Culture Collection, Rockville, Md., as strain ATCC 53909.Pullulanases (EC 3.2.1.41) are enzymes which specifically cleave the alpha-1,6-glycosidic linkages of pullulan, starch, glycogen, and amylopectin. Recently, these enzymes have found commercial utility in glucose-and maltose-manufacturing processes (10). Yeasts, higher plants, and microorganisms are known to be producers of pullulanase (17). Within the genus Bacillus, several species and strains have been reported to make pullulanase, including Bacillus sp. strain 202-1 (17), "Bacillus acidopullulyticus" (lo), Bacillus cereus subsp. mycoides (23), Bacillus macerans (l), Bacillus p l ymyxa (9), and Bacillus stearothermopkilus (13,22). Of these, only the "B. acidopullulyticus' ' pullulanase has properties that allow its use in industrial saccharification processes.In our search for a pullulanase that could operate under conventional glucose-manufacturing conditions (i .e., pH 4.3 and 60"C), we assumed that bacteria capable of growth at low pH values or high temperatures or both would produce aciduric or thermostable enzymes. In this paper, we describe the isolation and characterization of a mesophilic, moderately acidophilic bacterium, designated Bacillus naganoensis, that makes a pullulanase with desirable acid and temperature stability characteristics. (20), and 7.5 g of red-colored pullulan. The pH was adjusted with 0.2 N sulfuric acid to pH 4.0. The color of the plates was dark violet. Incorporation of the two colored substrates into the plate medium allowed for preliminary selection of organisms which (i) produced aamylases (enzymes that hydrolyzed the blue-colored starch but not the red-colored pullulan, leaving a red background around a colony); (ii) produced pullulanases (enzymes that hydrolyzed the red-colored pullulan but not the blue-colored starch, leaving a blue background around a colony); or (iii) produced both a-amylases and pullulanases (enzymes that hydrolyzed both the blue-colored starch and the red-colored pullulan, leaving a clear zone of hydrolysis around a colony) * Corresponding author. MATERIALS AND METHODS(12). Red-colored pullulan was prepared by dissolving 100 g of pullulan PFlO (Hayashibara Co., Ltd., Okayama, Japan) in 2 liters of distilled water. The solution temperature was increased to 50"C, and 10 g of Mikacion Brilliant Red 5BS (Mitsubishi Kasei ...

Partial purification of DNA methylase from Novikoff rat hepatoma cells is described. Contamination with other proteins persists although the enzyme preparation has a high specific activity and is purified 980-fold over homogenate activity. Evidence suggests, but does not prove, that there may be more than one species of DNA methylase in these cells. The enzyme has two broad pH optima at pH 7.0 and 7.5 and most readily methylates heterologous denatured DNAs although complex reaction kinetics indicate that native DNAs may eventually be methylated to an equal or greater level. The preparation of undermethylated DNA from Novikoff cells is also described. Undermethylated homologous DNA is an 85-fold greater acceptor of methyl groups than fully methylated Novikoff cell DNA. In contrast to other DNA substrates, the enzyme preparation methylates native undermethylated homologous DNA at a 3.5-fold greater than denatured undermethylated homologous DNA.