Wilhelm R. Frisell

Purification and characterization of the flavin prosthetic group of sarcosine dehydrogenase

Abstract The flavin prosthetic group of sarcosine dehydrogenase from rat liver mitochondria has been separated from other mitochondrial acid-extractable and acid-non-extractable flavins and purified by chromatography on Florisil, Dowex 1, CM Sephadex, and DEAE-Sephadex. The flavin is similar to that isolated previously from succinate dehydrogenase of beef and pig heart mitochondria. Characteristically, the flavin cannot be released from the apoenzyme by precipitation of the protein with trichloracetic acid, but can be solubilized by hydrolysis of the protein with trypsin, α-chymotrypsin, and other proteases. The oxidized mononucleotide form of the flavin exhibits an absorption spectrum almost identical with that of the flavin of succinate dehydrogenase. Maxima occur at 351 and 445 mμ with a 351:445 mμ ratio of 0.77. A maximum is found at 268 mμ with a 268:445 mμ ratio of 2.9. The fluorescence emission of the flavin occurs at approximately 523 mμ and exhibits a maximum fluorescence at pH 3, even when isolated as a mononucleotide. The data support the conclusion that the native state of the flavin is a dinucleotide. The midpoint reduction potential at pH 7.0 of the flavin is approximately −167 mV at 26 °C.

One-carbon metabolism in microorganisms. I. Oxidative demethylation in a sarcosine-utilizing bacterium.

Abstract A bacterial strain utilizing sarcosine as its sole course of C, N, and energy has been isolated in pure culture by the soil enrichment technique. The organism is a straight, Gram-negative rod, 0.7 × 1.25 μ, and is motile with a polar flagellum 80 A × 5 μ. When cultured in the absence of Fe 2+ or Fe 3+ , the cells produce a blue-fluorescent pigment whose absorption spectrum has a single peak at 400 mμ (pH 8.30). Demethylation is the major dehydrogenation reaction which sarcosine undergoes as a substrate. The oxidation level reached by the methyl carbon is determined by the degree of cell integrity and/or the age of the culture. In unfractionated preparations of cells isolated in log phase, the N-CH 3 group can be oxidized to carbon dioxide. With older cultures or with (NH 4 ) 2 SO 4 fractions of the supernatant of sonically irradiated cells, the methyl carbon is oxidized only to the level of formaldehyde. In all cases, glycine also accumulates as a reaction product and is the only amino acid which can be detected after all of the sarcosine has been oxidized. Flavin and also cytochromes of the c and b (or o ) types function as electron carriers in the sarcosine oxidase system. Preparations lacking the b (or o ) cytochrome have oxidase activity only in the presence of phenazine methosulfate. The oxidase flavin is peptide bound, or acid nonextractable, and its concentration in the bacteria parallels the level of sarcosine oxidase.

Inhibition by formaldehyde of energy transfer and related processes in rat-liver mitochondria

Abstract 1. Exogenous formaldehyde inhibits phosphorylating oxidation of succinate in coupled mitochondria in the presence of ADP. The inhibition of the oxidation can be released by dinitrophenol. Formaldehyde also prevents the stimulation of succinate respiration by arsenate. 2. Formaldehyde has little or no effect on respiration in the absence of ADP. Neither does it greatly affect the respiration of mitochondria which have been uncoupled by osmotic disruption or pre-treatment with dinitrophenol. 3. Formaldehyde interferes with the endergonic, anaerobic reduction of DPN + by succinate in phosphorylating particles of rat-liver mitochondria. Moreover, the ATP-induced contraction of intact mitochondria can be inhibited by formaldehyde or by oligomycin under the same conditions.

[141] Sarcosine dehydrogenase and dimethylglycine dehydrogenase (rat liver; monkey liver)☆

Publisher Summary This chapter discusses the assay, purification, and properties of sarcosine dehydrogenase and dimethylglycine dehydrogenase. Assay of the sarcosine and dimethylglycine dehydrogenase employs phenazine methosulfate and 2, 6-dichlorophenolindophenol (DCPIP). Sarcosine dehydrogenase and dimethylglycine dehydrogenase are found in the liver mitochondria of the rat, pig, rabbit, guinea pig, pigeon, and chicken. These enzyme activities are also present in microorganisms. Sarcosine dehydrogenase does not oxidize glycine, serine, monomethylaminoethanol, dimethylaminoethanol, choline, betaine, and threonine. Sarcosine dehydrogenase, but not dimethylglycine dehydrogenase, from mammalian liver mitochondria is inhibited reversibly and competitively by methoxyacetate and related acetates. The absorption spectra of the oxidized form of the purified enzymes show a major peak at 405–415 m μ and a broad shoulder in the region of 430–460 m μ . With sodium phosphate or pyrophosphate buffers of ionic strength 0.1, the pH optima of the sarcosine dehydrogenase and dimethylglycine dehydrogenase in the phenazine methosulphate (PMS)–DCPIP assay system are 8 and 8.5–9, respectively. The purified preparations of both the sarcosine and dimethylglycine dehydrogenases can be lyophilized, and the dry powders are stable for at least a week when stored at -4°.

Quantitative studies on the flavins in the soluble fractions of heavy mitochondria of rat liver

Abstract The soluble fraction of heavy mitochondria of rat liver, resulting from sonic irradiation, contains an average of 65% of the acid-extractable and 65% of the acid-nonextractable flavins of the mitochondria. The total flavin of these mitochondria averages 1.42 μmoles/g protein and, of this, 10% is acid-nonextractable. Compared with sonic irradiation, osmotic lysis liberates only four-fifths as much protein and flavin. Using the sarcosine and malate dehydrogenase as markers, evidence is presented that certain soluble constituents in the mitochondria differ in the extent of their release by lysis. However, there are no major differences in the flavoproteins and flavins released by irradiation and lysis since the ratios of both acid-extractable and acid-nonextractable flavins to soluble protein are the same in the two processes. Both fractions contain an average of 0.18 μmole of acid-nonextractable flavin per gram of soluble protein. The major acid-nonextractable flavins of the soluble and membranous fractions of liver mitochondria can be distinguished by chromatography on Dowex 1.

Complexing of riboflavin with the enolate form of barbituric acid

Abstract Riboflavin and the enolate form of barbituric acid form a complex at pH 7.80 as demonstrated by the appearance of two new absorption bands at 400 and 490 nm. The association constant for the complex determined from a Benesi-Hildebrand analysis of absorbances at 490 nm is 30 M−1. The K evaluated from fluorescence quenching at various barbiturate: riboflavin ratios is 33 M−1. The complexing of the barbiturate and riboflavin is accompanied by an inhibition of the photo-oxidation of sarcosine to formaldehyde. Under the same conditions, the barbiturate enhances the rate of photo-oxidation of NADH. The 5,5-disubstituted barbiturates do not give a new absorption band with riboflavin and do not inhibit the photo-oxidation of sarcosine, indicating that an enolate configuration of the β-diketone system is involved in complexing with riboflavin.

N-methyl oxidation in liver mitochondria of triiodothyronine-treated and thyroidectomized rats

Abstract The levels of sarcosine dehydrogenase and acid-nonextractable flavin in the inner matrix of mitochondria of rat liver are decreased in animals treated with triiodothyronine and are elevated in the mitochondria obtained from thyroidectomized animals. Administration of triiodothyronine does not affect the electron-transfer flavoprotein associated with the sarcosine dehydrogenase or the relative amounts of soluble and membrane-bound proteins of the mitochondria. In phosphate-washed mitochondria from either the controls or the triiodothyronine-treated rats, the O 2 uptake equals the total of the [ 14 C]formaldehyde and [β- 14 C]serine isolated as reaction products of the sarcosine-[ 14 C]methyl group. In contrast to its restraint of sarcosine or choline oxidation in preparations capable of oxidative phosphorylation, ADP does not inhibit the oxidation of these substrates in mitochondria of rats given triiodothyronine.

Inhibition by formaldehyde of energy transfer and related processes in rat liver mitochondria: II. Effects on energy-linked reactions in intact mitochondria and phosphorylating particles☆

Abstract Formaldehyde inhibits phosphorylating respiration with either succinate or pyruvate in intact mitochondria. Acetaldehyde inhibits only the oxidation of pyruvate. The effect of formaldehyde can be diminished under conditions which favor its oxidative removal from the mitochondria. The rate of oxidative phosphorylation with succinate, as measured by respiration or phosphate uptake, is more sensitive to formaldehyde than is the efficiency of the process, expressed as P O . Formaldehyde inhibits dinitrophenol-stimulated ATPase of intact mitochondria. Under conditions in which oligomycin has no effect, formaldehyde inhibits the rate of calcium-stimulated respiration in the absence or presence of phosphate. When phosphate is absent, formaldehyde inhibits the rate of cyclic stimulation of oxidation without affecting the ratio of calcium to oxygen uptakes. With inner membrane particles, both phosphorylating respiration of succinate ( P O : 1.5) and endergonic reduction of DPN are inhibited by formaldehyde. The inhibition of DPN reduction can be relieved partially by increasing the ATP concentration. ATPase activity of the particles is not inhibited by formaldehyde. It is proposed that phosphorylation and active accumulation of calcium in mitochondria can be linked to the respiratory chain by different coupling mechanisms.

[55] Electron transfer flavoprotein

Publisher Summary This chapter discusses Electron Transfer Flavoprotein (ETF). The quantitative determination of ETF in the presence of excess sarcosine and sarcosine dehydrogenase is based on the measurement of the rate of reduction of dichlorophenolindophenol (DCPIP). Several chromatographic procedures for purifying the ETF of rat liver mitochondria are presented. The two preparative methods most commonly employed on ETF are those beginning with the osmotic supernatant, followed by chromatography on either equilibrated or unequilibrated DEAE-cellulose. The supernatant fraction obtained from centrifugation of the sonically irradiated mitochondria is referred as the “sonic supernatant.” The ETF in the mitochondria is located in this fraction. The ETF, in the 60-80% ammonium sulfate fractions, is stable for about a week when these fractions are stored as wet precipitates at –18°. The activity of the purified ETF, similar to that of the original 60-80% ammonium sulfate fraction, can be stimulated with the addition of FAD. ETF is located in liver mitochondria, and is absent from other fractions of the liver cell. The richest sources of the enzyme found are rat, pig, and monkey liver. The visible absorption spectrum of rat liver ETF shows a maximum at 410 mμ and a broad shoulder at 450 mμ. The only substrates found for ETF are the substrate-reduced sarcosine and dimethylglycine dehydrogenases and the acyl-eoenzyme A dehydrogenases.