John W. Porter

A modification of the Ellman procedure for the estimation of protein sulfhydryl groups

The reaction of the Ellman reagent [5,5′-dithiobis-(2-nitrobenzoic acid)] with a protein sulfhydryl group yields 1 mole of thiophenylated protein and 1 mole of thiophenylate anion, when the reaction is carried out above pH 7.0. The quantity of thiophenylate anion liberated in this reaction is then determined by light absorption measurement at 412 mμ. In the present paper, this technique is modified so that the thiophenylated protein is isolated from the reaction mixture. The amount of thionitrobenzoic acid bound to the protein is then determined. Using this principle, sulfhydryl analyses have been successfully made on (1) a sulfhydryl-containing protein in the presence of a reduced thiol; (2) heme proteins; (3) an insoluble protein; and (4) a protein rendered insoluble by denaturation. The classical Ellman procedure cannot be used for assays of free sulfhydryl groups on any of these proteins.

Stimulation by insulin of rat liver β-hydroxy-β-methylglutaryl coenzyme A reductase and cholesterol-synthesizing activities

Abstract β-Hydroxy-β-methylglutaryl coenzyme A reductase activity in rat liver increased 2 to 7-fold after subcutaneous administration of insulin into normal or diabetic animals. Reductase activity began increasing after one hour, rose to a maximum in two to three hours, and then declined to the control level after six hours. This response was elicited during the time of day when the normal diurnal variation in reductase activity approached a minimum. It was also elicited when animals did not have access to food. This stimulation of reductase activity was completely blocked when glucagon was administered in conjunction with insulin. The increase in reductase activity after insulin administration was accompanied by a proportionate increase in activity for the conversion of acetate to cholesterol.

Synthesis of long-chain fatty acids by microsomes of pigeon liver☆

Abstract Results are presented which show that pigeon liver microsomes contain an enzyme system capable of synthesizing saturated and unsaturated C-18 and C-20 fatty acids by the addition of malonyl-CoA to shorter chain fatty acids. Since no malonyl-CoA was incorporated into the first five “two carbon” units of synthesized oleic acid (pelargonic acid), it is concluded that the shortest chain fatty acid elongated by this system is decanoic acid. This conclusion is supported by the relatively high specific radioactivity of the carboxyl group in stearic and oleic acids synthesized from 1,3- 14 C-malonyl-CoA and by the failure of octanoyl-CoA to stimulate the incorporation of malonyl-CoA into fatty acids. Requirements for ATP and a reduced nucleotide for microsomal fatty acid synthesis are also reported. The role of ATP in this system is discussed, and it is concluded that ATP probably functions in the activation of primer fatty acid units and in the synthesis of phospholipids. The latter are major end products of microsomal fatty acid synthesis.

The partial purification and properties of a phytoene synthesizing enzyme system

An enzyme system catalyzing the synthesis of phytoene from isopentenyl pyrophosphate has been isolated from tomato fruit plastids and purified approximately 350-fold in specific activity. This enzyme system has a molecular weight of approximately 200,000. The rate of phytoene formation is maximal at pH 7.0 and 23 °C and the apparent Km for isopentenyl pyrophosphate is 10 μm The rates of phytoene synthesis when geranylgeranyl pyrophosphate and isopentenyl pyrophosphate were used as substrates were 0.08 and 0.17 nmol of phytoene/mg of protein/h, respectively. The enzyme complex showed an absolute requirement for Mn2+, but not for NADP+. At a concentration of 2 mm, NADP+ produced only a 1.5- to 3-fold stimulation, and this effect varied from preparation to preparation. The addition of NADPH to the incubation mixture produced inhibition of phytoene synthesis and there was no evidence for the concomitant accumulation of lycopersene. The acid labiles produced on acid treatment of the incubation mixture indicated that geranylgeranyl pyrophosphate was formed by the enzyme complex. The enzyme system is stabilized in the presence of 30% glycerol and 10 mm dithiothreitol and it can be stored at −20 °C for over 1 month without significant loss of activity. However, the enzyme activity for phytoene formation is heat labile, and it is not stable when attempts are made to purify it further by ion-exchange chromatography.

Biosynthesis of phytoene from isopentenyl and farnesyl pyrophosphates by a partially purified tomato enzyme system

Abstract The solubilization, stabilization, and partial purification of an enzyme system that synthesizes phytoene from isopentenyl pyrophosphate is reported. Solubilization and partial purification of the enzyme system was achieved through the preparation of an acetone powder of tomato plastids, extraction of the powder with buffer, fractionation of this extract with ammonium sulfate, and passage of the enzyme through a column of Sephadex G-200. Stabilization was effected by storage in the presence of dithiothreitol. Either isopentenyl pyrophosphate, or farnesyl pyrophosphate plus isopentenyl pyrophosphate, serve as substrate for phytoene synthesis. The presence of isopentenyl pyrophosphate is required for the incorporation of farnesyl pyrophosphate into phytoene. Incorporation of substrate into phytoene is maximum at pH 8.0 and 20 °, and the Km value for isopentenyl pyrophosphate is 1.33 × 10−6 m . Mn++ and Mg++ are required for phytoene synthesis, but pyridine nucleotides are not. Synthesis of phytoene is inhibited by -SH inhibitors and it is stimulated by dithiothreitol. The enzyme system also synthesizes acid-labile terpenyl pyrophosphates from isopentenyl pyrophosphate. Cleavage of these compounds with alkaline phosphatase yields a mixture of C15 and C20 terpenols. The enzyme system also effects the condensation of isopentenyl and farnesyl pyrophosphate. C20 terpenols are liberated on alkaline phosphatase treatment of the products.

The configuration of phytoene

Abstract The configuration of natural phytoene is established as 15,15′ cis -7,8,7′,8′,11,12, 11′,12′-octahydrolycopene through ultraviolet, infrared, and nuclear magnetic resonance spectral analyses and through catalytic isomerization with iodine. The configurations of naturally occurring cis -phytofluene and ζ-carotene are established as 15,15′ cis -7,8,7′,8′,11′,12′-hexahydrolycopene and all- trans -7,8,7′,8′-tetrahydrolycopene, respectively, through ultraviolet, visible, and infrared spectral analyses and through catalytic isomerization with iodine. A small amount of all- trans -phytofluene is also reported to be a normal constituent of some tomato varieties.

The enzymatic synthesis of geranyl geranyl pyrophosphate by enzymes of carrot root and pig liver

Abstract The isolation of geranyl geranyl pyrophosphate synthetases from carrot root and pig liver and the properties of these enzymes are reported. Each of the enzymes catalyzes the formation of geranyl geranyl pyrophosphate from isopentenyl-4-C 14 and farnesyl pyrophosphates. The product of the reaction has been characterized by gas-liquid and paper chromatography and derivative formation. An absolute metal requirement has been demonstrated for the reaction and Mn ++ is the most effective metal in stimulating activity. The pH optimum of the reaction is 6.7–6.8 and 6.9–7.0, respectively, for the carrot root and pig liver enzymes. K m values calculated for farnesyl pyrophosphate and isopentenyl pyrophosphate are 1 × 10 −4 M and 1 × 10 −5 M respectively, for the carrot root enzyme, and 7.6 × 10 −5 M and 6.6 × 10 −6 M , respectively, for the pig liver enzyme.

Separation of water-soluble steroid and carotenoid precursors by DEAE-cellulose column chromatography

Abstract A mixture of water-soluble steroid or carotenoid precursors was separated on a DEAE-cellulose column through stepwise elution with solutions of (NH4)2CO3 or K2HPO4. This method separates the compounds in the metabolic sequence between mevalonic acid and farnesyl or geranylgeranyl pyrophosphate. Good resolution between compounds was obtained except for isopentenyl pyrophosphate and dimethylallyl pyrophosphate. Some overlap in retention time of these compounds with mevalonic acid phosphate also occurred. Since a basic solvent system is used, the DEAE-cellulose chromatographic method provides an excellent means of purifying the acid-labile as well as the acid-stable intermediates from a biosynthetic mixture.

Studies on a protein isolated from livers of diabetic and fasted rats

Abstract A protein that is synthesized by livers of diabetic and fasting rats has been isolated and purified. This protein is either not synthesized or it is synthesized in trace amounts by livers of normal or fasted and refed animals. This component, designated as “7S” protein, accompanies the fatty acid synthetase through all steps of purification. However, it can be separated from the fatty acid synthetase by two successive sucrose density gradient centrifugations. When separated in this way it is nearly homogenous in size and charge (disk gel electrophoresis). The 7S proteins obtained from diabetic and fasted livers are identical with respect to size, charge, and immunochemical properties. This protein does not catalyze any of the partial reactions of fatty acid synthesis, does not bind acetyl or malonyl groups, and it does not exhibit acetyl-CoA carboxylase activity. Immunological studies show it to be unrelated to the fatty acid synthetase. Finally, it has no effect on the rate of fatty acid synthesis or on acetyl-CoA carboxylase activity. Thus, on the basis of these properties, the 7S protein is not related to the fatty acid synthetase and probably not to acetyl-CoA carboxylase. The function of this protein remains to be determined.

Properties of farnesyl pyrophosphate synthetase of pig liver

Abstract The isolation and partial purification of farnesyl pyrophosphate synthetase of pig liver are described. This enzyme does not have isopentenyl pyrophosphate isomerase, geranyl pyrophosphate synthetase, or geranyl geranyl pyrophosphate synthetase activities. Farnesyl pyrophosphate synthetase catalyzes the formation of farnesyl pyrophosphate through the condensation of geranyl and isopentenyl pyrophosphates. The product of this reaction has been characterized by paper and gas-liquid chromatography. A pH of 7.0 is optimum for the reaction, and Km values for geranyl and isopentenyl pyrophosphates are 4.0 × 10−6 and 2.0 × 10−6M, respectively. An absolute metal requirement has been established for this reaction and Mn++ is approximately 40 times more effective than Mg++ at low ion concentrations. At pH 7.0, 2 × 10−3M iodoacetamide does not inhibit the reaction, but 1 × 10−5M p-hydroxy mercuribenzoate completely inactivates the enzyme.