Metabolism of pipecolic acid in a Pseudomonas species. V. Pipecolate oxidase and dehydrogenase.

Oxidation of pipecolate to Delta(1)-piperideine-6-carboxylate is catalyzed by pipecolate oxidase, an inducible, membrane-bound dehydrogenase associated with the electron transport components of Pseudomonas putida P2. From the oxidase, we obtained a smaller particle containing flavine adenine dinucleotide (FAD) and cytochrome b, but no longer able to catalyze electron transfer to oxygen or to cytochrome c. Certain properties of this l-pipecolate dehydrogenase, an FAD-flavoprotein, are reported.

Metabolism of Pipecolic Acid in a Pseudomonas Species IV. Electron Transport Particle of Pseudomonas putida.

Baginsky, Marietta L. (University of California, San Francisco Medical Center, San Francisco), and Victor W. Rodwell. Metabolism of pipecolic acid in a Pseudomonas species. IV. Electron transport particle of Pseudomonas putida. J. Bacteriol. 92:424-432. 1966.-Enzymes of Pseudomonas putida P2 catalyzing oxidation of pipecolate to Delta(1)-piperideine-6-carboxylate are located in a subcellular fraction sedimenting at 105,000 x g. Since this fraction resembles the mammalian electron transport particle in both chemical composition and enzymatic activities, it was termed Pseudomonas P2 electron transport particle (P2-ETP). P2-ETP contains flavin adenine dinucleotide, flavin mononucleotide, iron, copper, and both b- and c-type cytochromes. The reduced type b cytochrome has absorption maxima at 558 to 559, 530, and 427 mmu. Its oxidized pyridine hemochromogen has an absorption maximum at 406 mmu, with a shoulder at 564 mmu. On dithionite reduction, absorption bands with maxima at 556, 522, and 418 mmu are obtained. The reduced type c cytochrome has absorption maxima at 552, 520, and 422 mmu; its reduced pyridine hemochromogen has maxima at 551, 516 to 519, and 418 mmu. No type a cytochrome was detected. P2-ETP catalyzes oxidation of pipecolate and of reduced nicotinamide adenine dinucleotide (NADH(2)) by oxygen. It can also oxidize these compounds, as well as succinate and reduced nicotinamide adenine dinucleotide phosphate, with 2,6-dichlorophenol-indophenol as electron acceptor. Mammalian cytochrome c can be used as an alternate artificial electron acceptor for the oxidation of pipecolate and succinate, but not for oxidation of NADH(2).

A new method for the measurement of lipoprotein lipase in postheparin plasma using sodium dodecyl sulfate for the inactivation of hepatic triglyceride lipase.

Lipoprotein lipase (LPL) and hepatic triglyceride lipase (H-TGL) are lipolytic activities found in postheparin plasma. A simple and precise method for the direct determination of LPL in postheparin plasma is described. Pre-incubations of this plasma (45--60 min at 26 degrees C) with sodium dodecyl sulfate (35--50 mM) in 0.2 M Tris-HCl buffer, pH 8.2, results in the inactivation of H-TGL, while leaving LPL fully active. Direct determination of H-TGL is done in a separate aliquot of the same postheparin plasma sample using previously reported assay conditons that do not measure LPL. The sodium dodecyl sulfate-resistant lipolytic activity has the characteristics of LPL as judged by a) its activation by serum and by apolipoprotein C-II; b) its inactivation (over 90%) by 0.75 M NaCl; and c) its inactivation by a specific antiserum. No sodium dodecyl sulfate-resistant activity was found in postheparin plasma from a patient with LPL deficiency (primary type I hyperlipoproteinemia). An excellent correlation of values was obtained (r = 0.99) for 30 samples assayed after sodium dodecyl sulfate treatment and after immuno-inactivation of H-TGL. The intra-assay coefficient of variation was +/- 11% and 4% before and after normalization of values, respectively.

Effects of monoolein on hydrolysis of triglyceride by lipoprotein lipase in the presence of an inhibitory apolipoprotein.

The previously demonstrated inhibition of cow's milk lipoprotein lipase by apoLp-Ala and the deinhibition by monoglyceride have been studied in more detail. The apoLp-Ala inhibition is reversible by the addition of monoglyceride before or after enzyme additions. Quantities of monoglyceride accumulate during hydrolysis of triglyceride which are adequate to prevent inhibition by added apoLp-Ala. Accelerating reaction rates observed when the substrate contains the apoprotein at levels producing partial inhibition are also explained by monoglyceride production. These effects were observed with both crude preparations of skim milk and highly pruified lipase. It is suggested that the generation of monoglyceride may be important in facilitating hydrolysis of triglyceride in lipoproteins containing this inhibitory apolipoprotein.

Differential characteristics of purified hepatic triglyceride lipase and lipoprotein lipase from human postheparin plasma.

Evidence is presented that hepatic triglyceride lipase (H-TGL) and lipoprotein lipase (LPL), purified from human postheparin plasma, can each hydrolyze both glyceryl trioleate and palmitoyl-CoA. The average ratio of glyceryl trioleate/palmitoyl-CoA hydrolase activities, obtained with enzyme preparations from 15 human postheparin plasma samples was 1.30 (1.18-1.52) for H-TGL and 8.75 (7.45-10.25) for LPL. Albumin was identified as the serum cofactor required for the hydrolysis of palmitoyl-CoA by H-TGL. It protected this enzyme from inactivation by this substrate. In contrast, palmitoyl-CoA activated and protected LPL from denaturation by dilution and incubation at 25 degrees C. The effects of other detergents were investigated on glyceryl trioleate hydrolase activities of both enzymes. Sodium dodecyl sulfate (0.4 mM) and Trisoleate (0.4 mM), which also effectively activated and protected LPL against inactivation, had only moderate protective effect on H-TGL. Sodium dodecyl sulfate at a higher concentration (1 mM) produced little or no inhibition of LPL, while completely inactivating H-TGL. Conversely, sodium taurodeoxycholate (0.4 mM) protected and activated H-TGL, but had only moderate protective effect on LPL. Triton X-100 (0.1-0.8 mM) and egg lysolecithin (0.05-2 mM) also protected H-TGL, but not LPL. The very dissimilar effects of detergents on preparations on H-TGL and LPL may form the basis for the direct assay of each enzyme in the presence of the other.

Diffraction quality crystals of lysin from spermatozoa of the red abalone (Haliotis rufescens).

Single crystals of the protein lysin (Mr = 16,070) from the spermatozoa of the red abalone (Haliotis rufescens) have been obtained by the vapor diffusion technique, using as precipitants a 32.5% saturated solution of (NH4)2SO4 (incubation at 18 degrees C) or a 5% w/v polyethyleneglycol 8000 solution (incubation at 29 degrees C), both in Bis-Tris-iminodiacetic acid buffers of pH 7.0. The addition to the droplets of EDTA, other carboxylate-containing polyanions, and/or organic solvents improved the size and quality of the crystals and, especially with (NH4)2SO4, addition of EDTA, and/or organic solvents produced a change in crystal habit which resulted in crystals more elongated in the b direction. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with a = 52.3 A, b = 46.0 A, and c = 81.5 A and one molecule per asymmetric unit. The crystals diffract to 2.3 A resolution. The molecular structure of lysin is relevant to the nonenzymatic mechanism by which the protein dissolves a hole in the egg vitelline layer during fertilization.

Quantitative determination of hepatic and lipoprotein lipase activities from human postheparin plasma.

A method was developed to separate and quantitatively determine two different triglyceride lipase activities in human postheparin plasma: hepatic triglyceride lipase (H-TGL) and lipoprotein lipase (LPL). Affinity chromatography on heparin-Sepharose columns was used for the separation. Rechromatography of purified H-TGL on heparin-Sepharose resulted in recoveries of 74 and 97% of these enzyme activities, respectively. The analytical errors for the determinations of the two activities were 11.4 and 9.6%, respectively.