Goro Kikuchi

The glycine cleavage system: Composition, reaction mechanism, and physiological significance

SummaryThe glycine cleavage system catalyzes the following reversible reaction: Glycine + THF + NAD+ ⇌ 5,10-methylene-THF + + CO2 + NH3 + NADHReversibility of the overall reaction was established through the studies with the enzymes prepared from liver mitochondria of rat and cock and from extracts ofArthrobacter globiformis grown on glycine. The glycine cleavage system is composed from four protein components. The four proteins were revealed to exist originally as an enzyme complex in the liver mitochondria. Partial reactions of glycine cleavage and glycine synthesis were studied in detail with partially purified individual protein components. Particularly a protein-bound intermediate of glycine metabolism could be isolated and its nature and role were clarified. A tentative scheme was presented to explain the whole process of the reversible glycine cleavage.The glycine cleavage system was shown to represent the major pathway of catabolism of both glycine and serine in vertebrates, including mammals, birds, reptiles, amphibians, and fishes. Serine catabolism in these animals proceeds mainly by way of the cleavage of serine to form methylene-THF and glycine rather than deamination by serine dehydratase. In ureotelic and ammonotelic animals methylene-THF formed from the α-carbon of glycine as well as theβ-carbon of serine could be further oxidized to CO2 in either the mitochondria or the soluble tissue fractions, while in uricotelic animals methylene-THF could hardly be oxidized to CO2 and instead, was utilized mostly for purine synthesis. Glycine synthesis by the glycine cleavage system did not appear to have appreciable physiological significance in animals.

The mitochondrial glycine cleavage system

SummaryThe glycine cleavage enzyme system is composed of four different proteins tentatively called P-protein, H-protein, T-protein and L-protein, and catalyzes the following reaction reversibly: Glycine + tetrahydrofolate + NAD+ ⇋ 5, 10-methylene-tetrahydrofolate + NH3 + CO2 + NADH + H− Glycine decarboxylase, tentatively called P-protein, is able by itself to catalyze glycine decarboxylation, yielding methylamine as product, but at an extremely low rate. P-Protein alone is also able to catalyze slightly the exchange of carboxyl carbon of glycine with CO2. However, the rates of the P-protein-catalyzed reactions are greatly increased by the co-existence of aminomethyl carrier protein, a lipoic acid-containing enzyme tentatively called H-protein. Several lines of evidence suggest that H-protein brings about a conformational change of P-protein which may be relevant to the expression of the decarboxylase activity of P-protein and that the functional glycine decarboxylase may be an enzyme complex composed of both P-protein and H-protein. H-Protein seems to play a dual role in the glycine decarboxylation; the one as a regulatory protein of P-protein, and the other as an electron-pulling agent and concomitantly as a carrier of the aminomethyl moiety derived from glycine. The idea that H-protein functions as a modulator of P-protein was further supported by the study of a patient with nonketotic hyperglycinemia. The primary lesion in this patient appeared to consist in structural abnormality in H-protein; the H-protein purified from the liver of this patient was apparently devoid of functional lipoic acid. Nevertheless, H-protein from the patient could stimulate the P-protein-catalyzed exchange of the carboxyl carbon of glycine and CO2, although only to a limited extent. The observed activity should be independent of the functioning of lipoic acid and would be a reflection of a conformational change in P-protein brought about by H-protein.P-Protein was inactivated when it was incubated with glycine in the presence of II-protein, and the inactivation was completely prevented when bicarbonate was further added so as to allow the glycine-CO2 exchange to proceed. The inactivation was accompanied by a spectral change of P-protein. The inactivation of P-protein seemed to take place as a side reaction of the glycine decarboxylation and to reflect the formation of a ternary complex of P-protein, H-protein and aminomethyl moiety of glycine through a Schiff base linkage of the H-protein-bound aminomethyl moiety with the pyridoxal phosphate of P-protein.

Function and induction of the microsomal heme oxygenase

SummaryThe microsomal heme oxygenase system consists of heme oxygenase and NADPH-cytochrome P-450 reductase, and is considered to play a key role in the physiological heme catabolism to yield biliverdin in animals. Heme oxygenase purified from either pig spleen or rat liver has a minimum molecular weight of 32 000, and binds heme to form a l:1 complex which exhibits properties resembled to those of hemoglobin and myoglobin. Heme degradation in the heme oxygenase reaction proceeds essentially as a series of autocatalytic oxidation of heme which is bound to heme oxygenase. The possible mechanism of heme degradation in the heme oxygenase reaction was presented.Heme oxygenase can be induced by heme in various tissues such as liver, kidney and macrophages, possibly in a substrate-mediated induction. Heme oxygenase, especially in the liver, has also been shown to be inducible to various extents by a number of non-heme substances including insulin, epinephrine, endotoxin, carbon disulfide, certain metal ions, diethylmaleate, bromobenzene, chlorinated benzenes, and interferon-inducing agents, and some of those non-heme substances appear to induce heme oxygenase independently of the mediation by heme. Some principal features of heme oxygenase induction by hemin and several non-heme inducers were examined comparatively mainly in pig alveolar macrophages and in rat liver, especially taking the degree of heme saturation of tryptophan pyrrolase as a probe for estimating the intracellular heme concentration in the liver. Inductions by carbon disulfide, endotoxin, insulin, and epinephrine are likely to be mediated by heme, whereas inductions by metal ions, diethylmaleate, and bromobenzene appear to be caused by some unknown mechanism unrelated to heme. The induction by apparently heme-independent inducers has some organ specificity and perhaps species specificity. In the rat, however, the heme oxygenase induced by either hemin or non-heme substances and in either liver or kidney were immunochemically identical.Cell-free synthesis of heme oxygenase directed by polysomes isolated from either pig alveolar macrophages or livers of rats treated with various inducers were examined by a combined use of [14C] or [3H]-labeled leucine and antibodies (IgG) specific to pig spleen heme oxygenase and rat liver heme oxygenase, respectively. In both macrophage and rat liver, free polysomes were the major site of heme oxygenase synthesis and the ability of polysomes to direct synthesis of heme oxygenase was greatly increased in the induced systems. Moreover, the abilities of polysomes isolated from livers of rats treated with hemin, Cd2+, and bromobenzene were proportional to the heme oxygenase activities in respective livers from which polysomes were prepared, indicating that all these inducers enhanced the synthesis of mRNA for heme oxygenase, giving rise to increased synthesis of heme oxygenase in the liver.

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.

Induction of heme oxygenase by hemin in cultured pig alveolar macrophages.

Heme oxygenase was induced in cultured pig alveolar macrophages by several inducers, such as heterologous rat erythrocytes, hemoglobin, hemin particles, and hemin solution, and hemin solution was found to be the most effective in inducing the enzyme. Under the standard conditions, where 1.5×10 8 alveolar macrophages were incubated with 60 nmol of hemin (final concentration in the medium, 3 μM), there was an initial lag phase of about 1 h and then the heme oxygenase activity increased almost linearly for about 5 h. The maximum activity attained was about 20-fold higher than the control values before the induction or in the cells incubated without hemin. Actinomycin D prevented the increase in heme oxygenase activity when added at the start of incubation, but when the drug was added after the induction had commenced, the heme oxygenase activity continued to increase for about 3 h after the addition of actinomycin D. The addition of cycloheximide at any time during incubation resulted in an immediate cessation of induction. Neither cobalt chloride nor cobalt-protoporphyrin induced heme oxygenase activity appreciably in the alveolar macrophage system. Also, hydrocortisone, insulin, dibutyryl cyclic AMP, theophylline, isoproterenol, and cyclic GMP had no remarkable effect on the heme oxygenase induction by hemin in cultured pig alveolar macrophages.

Mechanism of allylisopropylacetamide-induced increase of δ-aminolevulinate synthetase in liver mitochondria: IV. Accumulation of the enzyme in the soluble fraction of rat liver

Abstract When the synthesis of hepatic ALA synthetase was induced in rats by the administration of allylisopropylacetamide, high activities of ALA synthetase accumulated not only in the mitochondrial fraction but also in the soluble fraction. Cycloheximide inhibited the synthesis of both ALA synthetases in mitochondrial and extramito-chondrial fractions, while the enzyme synthesis was not affected by chloramphenicol. The induction of ALA synthetase in either fraction was also suppressed by the administration of mitomycin C, actinomycin D, hemin, or bilirubin. The apparent halflife time of ALA synthetase in the soluble fraction was approximately 20 min, while that of the mitochondrial enzyme was about 68 min. Furthermore, a definite difference between these two enzymes was observed on fractionation by ammonium sulfate. The possibility was suggested that ALA synthetase is synthesized originally in the microsomal system and subsequently is transferred into mitochondria and settles there, and the enzyme is probably modified to some extent before or after entering the mitochondria.

Mechanism of allylisopropylacetamide-induced increase of δ-aminolevulinate synthetase in liver mitochondria: V. Mechanism of regulation by hemin of the level of δ-aminolevulinate synthetase in rat liver mitochondria

Abstract Increased synthesis of hepatic ALA 2 synthetase was induced in rats by administration of AIA 2 and effects of injection of hemin on the level of hepatic ALA synthetase in different subcellular fractions of rat liver were studied at various stages of induction. When hemin was injected to rats at an initial stage of induction, AIA-induced increase of ALA synthetase was strongly suppressed in either mitochondrial or extramitochondrial fraction. However, when hydrocortisone was administered to animals together with AIA, induction of ALA synthetase was not appreciably affected by hemin. On the other hand, when hemin was injected to animals at a later stage of induction, ALA synthetase level in the extramitochondrial fraction increased markedly, whereas the level in mitochondria decreased sharply and therefore the total activity of ALA synthetase in the liver was not significantly decreased. ALA synthetase accumulating in the soluble fraction after hemin injection was of the “soluble form”. Unlike hemin, neither actinomycin D nor cycloheximide brought about the increase of level of extramitochondrial ALA synthetase. Further, when hemin was injected to AIA-treated rats, the apparent half-life time of ALA synthetase in the soluble fraction was markedly lengthened. Bilirubin and ALA exhibited essentially similar effects to those of hemin. It was assumed that hemin controls the level of ALA synthetase in liver mitochondria firstly by suppressing the initiation of induction of ALA synthetase and secondly by inhibiting the conversion of the “soluble form” of ALA synthetase into the “mitochondrial” ALA synthetase.

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.

Regulation by heme of synthesis and intracellular translocation of δ-aminolevulinate synthase in the liver

SummaryThe primary factor regulating the overall activity of heme biosynthesis in animals is supposed to be the level of ALA synthase in mitochondria. In animals with chemically induced hepatic porphyria, however, a considerable amount of ALA synthase also accumulates in the liver cytosol fraction, although the extent of accumulation is variable according to the species of animals and drugs used. Kinetic studies using a combination of [3H]leucine and an anti-ALA synthase IgG showed that the ALA synthase accumulating in the cytosol is a precursor in transit to mitochondria; the enzyme is transferred into mitochondria at a half-disappearance time of about 20 min. Kinetic studies also revealed that the transfer of ALA synthase from the liver cytosol into mitochondria is strongly inhibited by heme. This inhibition would represent a new mechanism of feedback regulation of metabolism in animal in the sence that the inhibition of intracellular translocation of ALA synthase would bring about reduction of the activity of porphyrin biosynthesis. Taking advantage of the inhibition by heme of the intracellular enzyme translocation, the real half-life of ALA synthase in the rat liver mitochondria was estimated to be about 35 min.There is evidence that synthesis of ALA synthase is subject to feedback regulation by heme. In mammalian liver, the inhibition by heme appeared to occur mainly at a posttranscriptional step, although the data obtained did not necessarily exclude the possibility that heme also interferes with the transcriptional step.Comparative study of the effects of administration of hemin on the degree of heme saturation of tryptophan pyrrolase and the extent of inhibition of synthesis and intracellular translocation of ALA synthase revealed a close correlation between the extent of those three events, suggesting that both the synthesis and the intracellular translocation of ALA synthase may be regulated by the variation of ‘regulatory heme’ pool at a physiological range in the liver cell.ALA synthase in rat liver is synthesized almost exclusively on free polyribosomes and the transfer of the enzyme from the liver cytosol into mitochondria appears to be accompanied with processing of the enzyme protein.

Major pathways of glycine and serine catabolism in rat liver

The metabolism of glycine and serine in rat liver was studied with mitochondria, cell homogenates and liver slices. It was shown that the most significant pathway of glycine catabolism in rat liver is the direct cleavage of glycine to form methylene-THF, CO2and ammonia, followed by the further oxidation of methylene-THF to CO2, possibly by the sequential action of methylene-THF dehydrogenase, cyclohydrolase and 10-formyl-THF: NADP+ oxidoreductase in liver mitochondria. Liver mitochondria, as well as liver homogenates and liver slices, actively catabolized serine and the patterns of serine catabolism in the liver homogenate and liver slice systems were essentially similar to those observed with liver mitochondria. A greater amount of 14CO2 was obtained from serine-3-14C than from serine-1-14C in any system tested, and this difference was more distinct when relatively low concentrations of serine were employed. In the liver homogenate system, as well as in the liver slice system, the rate of 14CO2 formation from serine-3-14C was much higher than that from pyruvate-3-14C. However, the activity of serine catabolism in the homogenate system was somewhat higher than in the mitochondrial system, although the soluble fraction alone catalyzed very little 14CO2 formation from either serine-1-14C or -3-14C. In the homogenate system, the β-carbon of serine was converted to CO2 even more rapidly than the carboxyl carbon of glycine. This seemed to be due to the participation of serine hydroxymethyltransferase, methylene-THF dehydrogenase and other enzymes related to one-carbon metabolism in the soluble liver fraction; these enzymes would increase the production of glycine, as well as the conversion of the β-carbon of serine to some one-carbon compound which may be oxidized to CO2 in mitochondria. It was assumed that serine catabolism in liver under physiological conditions would proceed mainly by way of preliminary cleavage to methylene-THF and glycine and their subsequent oxidation to yield CO2 as the final product in mitochondria. Serine can also be catabolized by serine dehydratase in the soluble liver fraction and through other minor pathways.