Metabolism of glycerate-2,3-P2--II. Enzymes involved in the glycerate-2,3-P2 metabolism in chicken skeletal muscle

1. Four enzyme fractions which may be involved in the synthesis and breakdown of glycerate-2,3-P2 have been isolated from extracted skeletal muscle by gel-filtration and ion-exchange chromatography. 2. One of the fractions, corresponding to the glycerate-2,3-P2 dependent phosphoglycerate mutase, has been purified to homogeneity. In addition to the main enzymatic activity, it shows intrinsic glycerate-2,3-P2 synthase activity and glycerate-2,3-P2 phosphatase activity stimulable by glycolate-2-P. Its synthase activity represents about 10% of the total synthase activity of the tissue, and its phosphatase activity corresponds to about 60% of the total phosphatase activity. 3. Two of the fractions have glycerate-2,3-P2 synthase, glycerate-2,3-P2 phosphatase and phosphoglycerate mutase activities in a ratio similar to that of the glycerate-2,3-P2 synthase described in mammalian skeletal muscle. Their synthase activity corresponds to about 90% of the total synthase activity, and their phosphatase activity represents about 1% of the total phosphatase activity of the tissue. 4. The fourth fraction shows only glycerate-2,3-P2 phosphatase activity and represents about 40% of the total activity of the tissue. 5. It is suggested that in chicken skeletal muscle the metabolism of the glycerate-2,3-P2 is regulated in a way similar to that described in mammalian skeletal muscle.     

Metabolism of glycerate-2,3-P2--IV. Effect of Hg2+ on the enzymes involved in the metabolism of glycerate-2,3-P2 in pig skeletal muscle

Type M phosphoglycerate mutase and skeletal muscle bisphosphoglycerate synthase-phosphatase from pig are similarly affected by Hg2+. Both enzymes lose the phosphoglycerate mutase and the glycerate-2,3-P2 synthase activities, and increase the glycerate-2,3-P2 phosphatase activity upon Hg2+-treatment. In contrast, bisphosphoglycerate phosphatase from pig skeletal muscle is inactivated by Hg2+. These results confirm the similarity between phosphoglycerate mutase and bisphosphoglycerate synthase-phosphatase. In addition they support the existence of separate binding sites for monophosphoglycerates and for bisphosphoglycerates at the phosphoglycerate mutase active site.     

Metabolism of glucose 1,6-P2. I. Enzymes involved in the synthesis of glucose 1,6-P2 in pig tissues

Pig tissues show four enzymatic activities of glucose 1,6-P2 synthesis: (A) 2 [glucose 1-P]----glucose 1,6-P2 + glucose; (B) glucose 1-P + ATP----glucose 1,6-P2 + ADP; (C) glucose 1-P + fructose 1,6-P2----glucose 1,6-P2 + fructose 6-P; (D) glucose 1-P + glycerate 1,3-P2----glucose 1,6-P2 + glycerate 3-P. Brain is the tissue with highest capability of glucose 1,6-P2 synthesis. With the exception of skeletal muscle, activity "D" represents the highest activity of glucose 1,6-P2 synthesis. In muscle, activity "B" is the major activity. The existence of a specific glucose 1,6-P2 synthase which catalyzes reaction "D" is confirmed. Two peaks of such an enzyme are isolated by ion-exchange chromatography. There is an enzyme which specifically catalyzes reaction "C", not previously described. There is a glucose 1-P kinase not identical to phosphofructokinase.     

Metabolism of glycerate 2,3-P2--XI. Essential amino acids of pig phosphoglycerate mutase isozymes

Phosphoglycerate mutase isozymes (types M, B and MB) from pig tissues are inactivated upon treatment with reagents specific for histidyl, arginyl and lysyl residues. Their mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities are concurrently lost, although some differences exist in the rate of inactivation. No significant differences are observed between the isozymes. The reversion of the modifying reactions reactivates the three enzymatic activities. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. Titration with pCMB shows the existence of two essential thiol groups per subunit type M. These results provide evidence of the intrinsic character of the three enzymatic activities, favor their location at the same active site and suggest the existence of separate binding sites for monophosphoglycerates and bisphophoglycerates. Both type M and B subunit from pig phosphoglycerate mutase are similar to type M subunit from rabbit and to the enzyme from yeast.     

Phytase activity in chicken erythrocytes and its control by organic phosphates (glycerate-2,3-P2 and inositol-P5) during avian development

The activity and basic kinetic constants of phytase were studied in chicken erythrocytes during animal development. The regulatory inhibition of phytase by IHP and 2,3-BPG takes place at key stages of the development. As in mammals, there is a specific control of the levels of organic phosphate involved in the oxygenation process of haemoglobin, during animal development.     

Metabolism of glycerate 2,3-P2--XII. Characterization of the 2,3-bisphosphoglycerate synthase-phosphatase and of the hybrid phosphoglycerate mutase/2,3-bisphosphoglycerate synthase-phosphatase from pig brain

2,3-Bisphosphoglycerate synthase-phosphatase and the hybrid phosphoglycerate mutase/2,3-bisphosphoglycerate synthase-phosphatase have been partially purified from pig brain. Their 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities are concurrently lost upon heating and treatment with reagents specific for histidyl, arginyl and lysyl residues. The two enzymes differ in their thermal stability and sensitivity to tetrathionate. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. The synthase activity of both enzymes shows a nonhyperbolic pattern which fits to a second degree polynomial. The Km, Ki and optimum pH values are similar to those of the 2,3-bisphosphoglycerate synthase-phosphatase from erythrocytes and the hybrid enzyme from skeletal muscle. The synthase activity is inhibited by inorganic phosphate and it is stimulated by glycolyate 2-P.     

Metabolism of glucose 1,6-P2--II. Glucose 1,6-P2 phosphatase in pig muscle

Most of the glucose 1,6-P2 phosphatase activity of pig skeletal muscle is present in the cytosolic fraction. Four peaks of glucose 1,6-P2 phosphatase activity are obtained when the cytosolic fraction from pig muscle is subjected to DE-cellulose chromatography. All the peaks hydrolyze other phosphocompounds in addition to glucose 1,6-P2. The glucose 1,6-P2 phosphatase activity of the main peak shows an optimal neutral pH. It is activated by divalent cations, Mg2+ being more effective than Mn2+. The addition of Ca2+ or EGTA does not affect the enzymatic activity. IMP does not possess any effect. It is concluded that this enzyme is different from the glucose 1,6-P2 phosphatases found in mouse brain cytosol and rat skeletal muscle.     

Enzymes involved in l-lactate metabolism in humans

l-lactate formation occurs via the reduction of pyruvate catalyzed by lactate dehydrogenase. l-lactate removal takes place via its oxidation into pyruvate, which may be oxidized or converted into glucose. Pyruvate oxidation involves the cooperative effort of pyruvate dehydrogenase, the tricarboxylic acid cycle, and the mitochondrial respiratory chain. Enzymes of the gluconeogenesis pathway sequentially convert pyruvate into glucose. In addition, pyruvate may undergo reversible transamination to alanine by alanine aminotransferase. Enzymes involved in l-lactate metabolism are crucial to diabetes pathophysiology and therapy. Elevated plasma alanine aminotransferase concentration has been associated with insulin resistance. Polymorphisms in the G6PC2 gene have been associated with fasting glucose concentration and insulin secretion. In diabetes patients, pyruvate dehydrogenase is down-regulated and the activity of pyruvate carboxylase is diminished in the pancreatic islets. Inhibitors of fructose 1,6-bisphosphatase are being investigated as potential therapy for type 2 diabetes. In addition, enzymes implicated in l-lactate metabolism have revealed to be important in cancer cell homeostasis. Many human tumors have higher LDH5 levels than normal tissues. The LDHC gene is expressed in a broad range of tumors. The activation of PDH is a potential mediator in the body response that protects against cancer and PDH activation has been observed to reduce glioblastoma growth. The expression of PDK1 may serve as a biomarker of poor prognosis in gastric cancer. Mitochondrial DNA mutations have been detected in a number of human cancers. Genes encoding succinate dehydrogenase have tumor suppressor functions and consequently mutations in these genes may cause a variety of tumors.     

Enzymes involved in hepatic acylglycerol metabolism in the chicken

In laying hens, massive hepatic mobilization of fatty acids is required for the synthesis of oocyte-targeted very-low density lipoproteins (VLDL). The current study aims at identification of enzymes that hydrolyze hepatic acylglycerol stores regulated in a fashion compatible with supporting enhanced VLDL synthesis. We show that unlike mammals, chickens express adipose triglyceride lipase (ATGL) also in liver, where it is upregulated by fasting, while the enzyme patatin-like phospholipase domain-containing lipase 3 (PNPLA3) is suppressed. For the first time in any system, we show that hepatic arylacetamide deacetylase (AADA) is upregulated by fasting, and that its affinity for an insoluble carboxylester substrate is compatible with an in-vivo function similar to that of ATGL. Unknown heretofore, hepatic expression of chicken AADA is estrogen-responsive, and is induced to the same degree as the stimulation of VLDL-production by estrogen. These observations support roles of chicken ATGL, PNPLA3, and AADA in acylglycerol metabolism related to the high rates of VLDL synthesis that are essential for reproduction.     

Enzymes involved in crotonate metabolism in Syntrophomonas wolfei

Cell-free extracts of Syntrophomonas wolfei subsp. wolfei grown with crotonate in pure culture or in coculture with Methanospirillum hungatei contained crotonyl-coenzyme A (CoA): acetate CoA-transferase activity. This activity was not detected in cell-free extracts from the butyrate-grown coculture which suggests that the long lag times observed before S. wolfei grew with crotonate were initially due to the inability to activate crotonate. Cell-free extracts of S. wolfei grown in pure culture contained high specific activities of hydrogenase and very low levels of formate dehydrogenase. The low levels suggest a biosynthethic rather than a catabolic role for the latter enzyme when S. wolfei is grown in pure culture. CO dehydrogenase activity was not detected. S. wolfei can form butyrate using a CoA transferase activity, but not by a phosphotransbutyrylase or enoate reductase activity. A c-type cytochrome was detected in S. wolfei grown in pure culture or in coculture indicating the presence of an electron transport system. This is a characteristic which separates S. wolfei from other known crotonate-using bacteria.     

Enzymes involved in acetoacetate formation in various bovine tissues

1. The activities of acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase and lyase and acetoacetyl-CoA deacylase were measured in homogenates of samples of liver, rumen epithelium (long papillae), kidney and lactating mammary gland derived from slaughtered cows. 2. The activities of the four enzymes in bovine liver were similar to the activities previously reported for the corresponding enzymes in rat liver. 3. Acetoacetyl-CoA thiolase and hydroxymethylglutaryl-CoA synthase and lyase were present in rumen epithelium. The activities of the enzymes were all lower on a wet weight basis than in liver. Only very slight deacylase activity was detected. 4. Kidney contained acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA lyase and acetoacetyl-CoA deacylase, but only trace amounts of hydroxymethylglutaryl-CoA synthase. 5. Mammary gland contained acetoacetyl-CoA thiolase and some hydroxymethylglutaryl-CoA lyase, but virtually no hydroxymethylglutaryl-CoA synthase or acetoacetyl-CoA deacylase. 6. Since physiologically significant ketogenesis probably occurs solely via the hydroxymethylglutaryl-CoA pathway, it is evident that, of the four tissues examined, such ketogenesis must be restricted to the liver and the rumen epithelium. 7. All the enzymes except hydroxymethylglutaryl-CoA lyase were also assayed in the four tissues derived from cows suffering from bovine lactational ketosis. Ketosis did not cause a statistically significant change in the activity of any of the enzymes measured. 8. Hepatic hydroxymethylglutaryl-CoA synthase and lyase were found to be associated mainly with the particulate fraction, as in the rat. A considerably greater proportion of these enzymes was found to be present in the cytoplasmic fraction from rumen epithelium, although it was not excluded that this was due to mitochondrial damage during homogenization. 9. Appreciable hydroxymethylglutaryl-CoA synthase was also present in epithelium from the dorsal region of the rumen, from the reticulum and from the omasum, but not from the abomasum.     

Levels of glycerate 2,3-P2, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities in rat tissues. A method to quantify blood contamination of tissue extracts

The levels of glycerate 2,3-P2 and of 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities have been determined in isolated rat hepatocytes and adipocytes and in perfused rat tissues to discard blood contamination. The values obtained are much lower than those previously reported, ranging 0.50-40 nmol/g tissue. No relationship appears to exist between glycerate 2,3-P2 concentration and the levels of the enzymatic activities involved in glycerate 2,3-P2 metabolism. Assay of glycerate 2,3-P2 in tissue extracts constitute a very useful way to quantify blood contamination.     

Non-cyclic photoreductive carbon fixation in photosynthesis. Light and dark transients of the glycerate-3-P special pair

It is demonstrated that carbon fixation in photosynthesis is regulated in two kinetically coupled pathways involving the specialized pair of non-equivalent, enzyme-bound glycerate-3-P (3-PGA) molecules obtained from ribulose 1,5-bisphosphate (RuBP) carboxylation in the light. A non-cyclic pathway is suggested (reaction 2) for the direct biosynthesis of sucrose from the 3-PGA obtained from C-3, C-4 and C-5 of the six-carbon carboxylation adduct. Concomitant to the appearance of sucrose as the principal product, the Mg2+-bound 3-PGA molecule formed from C-1, C-2 and C-2' of the C6 intermediate is released and subsequently reduced in regenerating the RuBP. It is proposed that the nocturnal inhibitor, 2-carboxyarabinitol-1-phosphate (1-PCA) is obtained from a condensation of 3-PGA and glyceraldehyde.     

2,3-Diphosphoglycerate affects Enzymes of Glucose Metabolism in Red Blood Cells

THE bulk of organic phosphate in the erythrocyte exists in the form of 2,3-diphosphoglycerate (2,3-DPG), which is known to determine the position of the oxygen dissociation curve^1,2 as well as to influence the glycolytic pathway as a cofactor of the monophosphoglyceromutase (MPGM) reaction³ and as an inhibitor of its own formation in the diphosphoglyceromutase reaction⁴. But whether 2,3-DPG inhibits the hexokinase reaction is unclear and reports are contradictory^5–7.     

Enzymes involved in adenine nucleotide metabolism of developing chick embryo liver.

1. This paper describes the changes in the activity of adenylate deaminase, adenylate and inosinate phosphatase, and adenosine deaminase in the developing chick embryo liver. 2. The adenylate and inosinate phosphatase and adenosine deaminase activity appears considerably higher in chick embryo liver with respect to other chick embryo tissues previously examined. 3. During development the control exerted by ATP on AMP breakdown undergoes variations. Consequently, in the first period of incubation AMP is degraded by the direct pathway (AMP-IMP) and in the last period of incubation by the indirect pathway (AMP-adenosine). In the intermediate period (from the 12th to the 15th day of incubation) both pathways may be followed. 4. The ability to synthesize purine nucleotides through "salvage pathway" seems to be acquired by embryonic liver at least at the 15th day.     

Enzymes involved in the metabolism of flavin nucleotides in Streptomyces olivaceus

The work was aimed at studying enzymes involved in the metabolism of flavin nucleotides, namely, riboflavin kinase (EC 2.7.1.26) and FAD pyrophosphorylase (EC 2.7.7.2), as well as flavin mononucleotide hydrolysis by acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) in Streptomyces olivaceus actively producing vitamin B12. No correlation could be established between changes in the activity of the above enzymes during the culture growth and the qualitative composition of flavins. The enzyme activity was assayed using, as an enzyme preparation, both intact cells and a cell-free extract obtained by disintegrating the mycelium with different techniques. The screening effect of phosphatases exerted when the activity of riboflavin kinase was assayed could be partly eliminated by adding sodium fluoride to the incubation medium. The localisation of the above enzymes in the cytoplasm is discussed.     

Enzymes of glycerol metabolism in the storage tissues of Fatty seedlings

Various enzymes of glycerol metabolism in the extracts of 5-day-old eastor bean (Ricinus communis L. var. Hale) endosperm and 4-day-old peanut (Archis hypogaea L.) cotyledon were studied. NAD-glycerol dehydrogenase and NAD-α-glycerolphosphate dehydrogenase were not detected. Glycerol kinase was detected in the soluble fractions and an α-glycerolphosphate oxidoreductase was found in the particulate fractions. The particulate fractions were separated into various organelle fractions by sucrose gradient centrifugation and the α-glycerolphosphate oxidoreductase was shown to be present in the mitochondria. The properties of the castor bean mitochondrial α-glycerolphosphate oxidoreductase resembled those of a similar enzyme present in the mitochondria of many animal tissues. A survey showed that the α-glycerolphosphate oxidoreductase was present in great amount only in the storage tissues of fatty seedlings but not in other nonfatty plant tissues. It is concluded that in the storage tissues of fatty seedlings, the soluble glycerol kinase and the mitochondrial cytochrome-linked α-glycerolphosphate oxidoreductase are the two enzymes responsible for the initial conversion of glycerol to hexose.     

Enzymes of serine metabolism in normal and neoplastic rat tissues

Enzymes involved in the pathway of de novo serine biosynthesis (L-phosphoserine aminotransferase) and in alternative pathways of serine utilization (L-serine hydroxymethyltransferase, L-serine dehydratase and L-serine aminotransferase) were assayed in normal adult and fetal rat tissues and in a range of transplantable sat tumors. Serine dehydratase and serine aminotransferase activities were essentially confined to normal adult liver and kidney, whereas phosphoserine aminotransferase and serine hydroxymethyltransferase activities showed a more ubiquitous tissue distribution. In particular, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were appreciable in neoplastic tissues, in the absence of the other enzymes of serine utilization. The pattern of enzyme distribution suggests that the synthesis of serine de novo is metabolically coupled to its utilization for nucleotide biosynthesis in tumors of differing tissue origins.     

Enzymes of serine metabolism in normal and neoplastic rat tissues

Enzymes involved in the pathway of de novo serine biosynthesis (L-phosphoserine aminotransferase) and in alternative pathways of serine utilization (L-serine hydroxymethyltransferase, L-serine dehydratase and L-serine aminotransferase) were assayed in normal adult and fetal rat tissues and in a range of transplantable rat tumors. Serine dehydratase and serine aminotransferase activities were essentially confined to normal adult liver and kidney, whereas phosphoserine aminotransferase and serine hydroxymethyltransferase activities showed a more ubiquitous tissue distribution. In particular, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were appreciable in neoplastic tissues, in the absence of the other enzymes of serine utilization. The pattern of enzyme distribution suggests that the synthesis of serine de novo is metabolically coupled to its utilization for nucleotide biosynthesis in tumors of differing tissue origins.