Yutaro Motokawa

Glycine metabolism in rat liver mitochondria: V. Intramitochondrial localization of the reversible glycine cleavage system and serine hydroxymethyltransferase☆

Abstract Intramitochondrial localization of the reversible glycine cleavage system and serine hydroxymethyltransferase was studied with the digitonin fractionation technique, and it was demonstrated that they are associated with the inner membrane of rat liver mitochondria. The effect of increasing the concentration of digitonin on the distribution pattern of mitochondrial enzymes has indicated that serine hydroxymethyltransferase is solubilized from the inner membrane fraction while the reversible glycine cleavage system is bound to the inner membrane.

Glycine metabolism by rat liver mitochondria: Reconstitution of the reversible glycine cleavage system with partially purified protein components

Abstract An enzyme system which catalyzes the degradation of glycine to one carbon unit, ammonia, and carbon dioxide and the synthesis of glycine from these three substances has been isolated from rat liver mitochondria. The reversible glycine cleavage system is composed of four protein components named as P-, H-, L-, and T-protein, respectively. A procedure is described for the purification of P-protein which catalyzes the decarboxylation of glycine or its reverse reaction in the presence of H-protein, and for T-protein which participates in the formation of one carbon unit and ammonia or the reverse reaction. The procedure described leads to the isolation of a nearly homogeneous form of T-protein but P-protein still is heterogeneous. The molecular weight of T-protein, estimated by molecular sieve chromatography, is 33,000. Properties of the synthesis and cleavage reactions and the exchange of carboxyl group of glycine with bicarbonate are also presented.

Glycine metabolism by rat liver mitochondria: IV. Isolation and characterization of hydrogen carrier protein, an essential factor for glycine metabolism☆

Abstract Two protein fractions were isolated from rat liver mitochondria which, when combined, catalyzed the synthesis of glycine from l -serine, ammonia, and bicarbonate. These fractions also catalyzed the cleavage of glycine and the exchange of the glycine carboxyl group with bicarbonate. One of the protein fractions, tentatively called “carboxylation enzyme”, was purified partially and the other protein, called “hydrogen carrier protein”, was obtained in an apparently pure form. Hydrogen carrier protein obtained was homogeneous on disc electrophoresis and its molecular weight was about 17,000 as judged from Sephadex chromatography. Per molecule it contained one disulfhydryl entity whose sulfhydryl groups were found to participate in electron transport during glycine metabolism.

On cytochrome oxidase as the terminal oxidase of dark respiration of non-sulfur purple bacteria

Abstract Spectroscopic evidence was obtained for the occurrence of a cytochrome of a type in a particulate preparation of dark-aerobically-grown cells of Rhodopseudomonas spheroides. The particulate preparation catalyzed the aerobic oxidation of reduced bovine cytochrome c, yeast cytochrome c and cytochrome c2. The reaction was completely inhibited by KCN, but was far less sensitive to CO as compared with rat-liver mitochondrial cytochrome oxidase (EC 1.9.3.1): when yeast cytochrome c was the substrate, no inhibition was observed. The particles also catalyzed the aerobic oxidation of succinate as well as p-phenylenediamine, and the rate of O2 uptake was increased significantly by the addition of either cytochrome c or cytochrome c2. The oxidation of either substrate was strongly inhibited by KCN. The inhibition by CO of the oxidation of succinate was extremely small (0–6%). The inhibition of p-phenylenediamine oxidation by CO was substantial in the dark and was considerably reversed by illumination. The oxidation of p-phenylenediamine was not inhibited by o-phenanthroline, EDTA, or sodium diethyldithiocarbamate. Similar cytochrome oxidase activity was found to be associated with particles from light-grown Rps. spheroides and from dark-grown and light-grown Rhodospirillum rubrum cells, and the relative oxidase activity of these different cells decreased in that order. It was concluded that a cytochrome oxidase with unusual properties functions as terminal oxidase in the dark respiration of non-sulfur purple bacteria, possibly coupled with cytochrome c2 as the physiological electron carrier.

Glycine metabolism by rat liver mitochondria. Isolation and some properties of the protein-bound intermediate of the reversible glycine cleavage reaction.

Abstract Appropriate combinations of purified components of the reversible glycine cleavage system of rat liver catalyze three partial reactions: (1) decarboxylation of glycine or its reverse reaction catalyzed by P- and H-protein, (2) condensation of one carbon substrate and ammonia or its reverse reaction catalyzed by T- and H-protein, and (3) oxidation and reduction of active disulfide of H-protein catalyzed by L-protein. Reactions (1) and (2) give the same product which is bound to H-protein. The protein-bound product was isolated by gel filtration and converted to glycine by incubation with P-protein and CO2 or degraded further to one carbon unit and ammonia by incubation with T-protein and tetrahydrofolate. The data are consistent with the conclusion that the enzyme-bound product is an intermediate in the reversible glycine cleavage reaction. A scheme is presented for the reactions catalyzed by the enzyme system.

Occurrence of both a-type and o-type cytochromes as the functional terminal oxidases in Rhodopseudomonas spheroides

1. n1. The functional terminal oxidase of the light-anaerobically grown Rhodopseudomonas spheroides cells was found to be the o-type cytochrome, whereas that of the dark-aerobically grown cells was the a-type cytochrome. When the dark-aerobically grown cells were further incubated under a semianaerobic condition in the dark, the content of the o-type cytochrome was increased in these cells, while the synthesis of the a-type cytochrome appeared to be repressed. In Rhodospirillum rubrum cells, grown either aerobically in the dark or anaerobically in the light, cytochrome o was the sole functional terminal oxidase. n n2. n2. Reactions with the a-type and o-type cytochromes from Rhodopseudomonas spheroides and also with the o-type cytochrome from Rhodospirillum rubrum were compared using reduced yeast cytochrome c as substrate. The reaction with the a-type cytochrome was far less sensitive to NaN3 and hydroxylamine than those with the o-type cytochromes, whereas all the reactions were inhibited by KCN in apparently the same manner.

Cytochrome systems in dark-aerobically grown Rhodopseudomonas spheroides.

Abstract A soluble cytochrome oxidase preparation was obtained from the particulate preparation of dark-aerobically grown Rhodopseudomonas spheroides cells by treatment with non-ionic detergent. The preparation contained appreciable amounts of a- and c-type cytochromes and a small amount of b-type cytochrome. The oxidase preparation displayed the typical features of a cytochrome oxidase in reactivity. No evidence could be obtained for the occurrence of an o-type of cytochrome in dark-grown Rps. spheroides. The oxidation-reduction potential of the soluble Rps. spheroides cytochrome 550 was found to be +0.34 V, whereas that of the particle-bound c-type cytochrome was approx. +0.25 V. It was concluded that the physiological terminal oxidase of dark-grown Rps. spheroides is the a-type cytochrome, and that particle-bound c-type cytochrome is the physiological electron carrier for it.

ON THE REACTIVITY OF HEMOGLOBIN MIWATE.

Abstract Hemoglobin MIwate, a variant of hemoglobin M found in a Japanese family, was purified by Amberlite XE-64 column chromatography and examined spectrophotometrically. The MIwate molecule was found to contain differently reactive subunits. The α-chain subunits persisted usually in ferric state and reacted none of cyanide, fluoride, azide and hydroxyl ions while could be reduced by sodium dithionite but only very slowly. In contrast, the β-chain subunits appeared to stay in the stable oxyhemoglobin form. Reactivities of the β-chain subunits toward ligands and other reagents as well as the absorption spectra of resulting derivatives appeared to be normal. The absorption spectra of completely reduced hemoglobin MIwate and of its carbonmonoxide complex were indistinguishable from those of respective hemoglobin A derivatives. Also the spectral characteristics of MIwate became less remarkable when the globin moiety was mildly modified. These results are compatible with the assumption that the α-chain of MIwate contains a reactive amino acid side chain which forms an internal complex with the ferric heme.

Evidence for the presence of a protein-bound intermediate in the cleavage and the synthesis of glycine.

Abstract An intermediate bound to a protein called hydrogen carrier protein was isolated from an incubation of the glycine metabolizing system with glycine or methylene tetrahydrofolate followed by filtration on Sephadex G-100. The possible mechanism of the metabolism of glycine was discussed.