Synthesis of citrate from phosphoenolpyruvate and acetylcarnitine by mitochondria from rabbit, pigeon and rat liver: Implications for lipogenesis

Rabbit, pigeon and rat liver mitochondria convert exogenous phosphoenolpyruvate and acetylcarnitine to citrate at rates of 14, 74 and 8 nmol/15 min/mg protein. Citrate formation is dependent on exogenous HCO3, is increased consistently by exogenous nucleotides (GDP, IDP, GTP, ADP, ATP) and inhibited strongly by 3-mercaptopicolinate and 1,2,3-benzenetricar☐ylate. Citrate is not made from pyruvate alone or combined with acetylcarnitine. Pigeon and rat liver mitochondria make large amounts of citrate from exogenous succinate, suggesting the presence of an endogenous source of acetyl units or a means of converting oxalacetate to acetyl units. Citrate synthesis from succinate by pigeon and rabbit mitochondria is increased significantly by exogenous acetylcarnitine. Pigeon and rat liver contain 80 and 15 times, respectively, more ATP:citrate lyase activity than does rabbit liver. Data suggest that mitochondrial phosphoenolpyruvate car☐ykinasein vivo could convert glycolysis-derived phosphoenolpyruvate to oxalacetate that, with acetyl CoA, could form citrate for export to support cytosolic lipogenesis as an activator of acetyl CoA car☐ylase, a carbon source via ATP:citrate lyase and NADPH via NADP: malate dehydrogenase or NADP: isocitrate dehydrogenase.     

Rapid changes in the concentration of phosphoenolpyruvate carboxykinase mRNA in rat liver and kidney. Effects of insulin and cyclic AMP

Starvation and diabetes both caused a marked increase in the concentration of hepatic phosphoenolpyruvate caroboxykinase mRNA while the administration of insulin to diabetic rats or refeeding glucose to starved animals caused a marked reduction in the levels of enzyme mRNA as measured by hybridization using a cDNA probe.l The Administration of dibutyryl cAMP to a starved-refed cat caused an 8-fold induction of phosphoenolpyruvate carboxykinase mRNA in 1 h. Triamcinolone plus acidosis induced the levels of enzyme mRNA in kidney 3-fold within 6 h, however, starvation for 24h had only marginal effects. In all of the above conditions, the levels of phosphoenolpyruvate carboxykinase mRNA measured by hybridization assay agreed well with the relative levels of translatable mRNA for the enzyme. The half-time of phosphoenolpyruvate carboxykinase mRNA, determined after the administration of either alpha-amanitin or cordycepin to starved animals, was approximately 40 min. However, cycloheximide either alone or together with cordycepin, not only prevented the decrease in phosphoenolpyruvate carboxykinase mRNA sequence abundance, but induced it 2-fold. Cycloheximide itself, when injected into 21-day fetal rats in utero caused an induction of enzyme mRNA equal to that noted when dibutyryl cAMP was administered. The mRNA for phosphoenolpyruvate carboxykinase is approximately 2.8 kb in length, but nuclei from the livers of diabetic rats contain a number of putative precursor RNA species for the enzyme, up to 6.5 kb in size, all containing a poly(A) tail. Two hours after refeedng glucose to a starved rat, these nuclear RNA species could no longer be detected by hybridization to our cDNA probe.     

Acetate-nonutilizing Mutants of Neurospora crassa II. Biochemical Deficiencies and the Roles of Certain Enzymes

The levels of Krebs cycle, glyoxylate cycle, and certain other enzymes were measured in a wild-type strain and in seven groups of acetate-nonutilizing (acu) mutants of Neurospora crassa, both after growth on a medium containing sucrose and after a subsequent 6-hr incubation in a similar medium, containing acetate as the sole source of carbon. In the wild strain, incubation in acetate medium caused a rise in the levels of isocitrate lyase, malate synthase, phosphoenolpyruvate carboxykinase, acetyl-coenzyme A synthetase, nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, citrate synthase, and fumarate hydratase. Isocitrate lyase activity was absent in acu-3 mutants; acu-5 mutants lacked acetyl-coenzyme A synthetase activity; and no oxoglutarate dehydrogenase activity (or only low levels) could be detected in acu-2 and acu-7 mutants. In acu-6 mutants, phosphoenolpyruvate carboxykinase activity was either very low or absent. No specific biochemical deficiencies could be attributed to the acu-1 and acu-4 mutations. The role of several of these enzymes during growth on acetate is discussed.     

Activity and androgenic control of enzymes associated with the tricarboxylic acid cycle, lipid oxidation and mitochondrial shuttles in the epididymis and epididymal spermatozoa of the rat.

1. Enzyme activities (units/g wet wt.) were determined in the caput and cauda epididymidis and in epididymal spermatozoa of the rat. 2. The activity of most enzymes in the cauda was between 50 and 100% of that in the caput, except that ATP citrate lyase was barely detectable in the cauda. 3. Spermatozoa, unlike epididymal tissue, contained sorbitol dehydrogenase but lacked ATP citrate lyase. NADP+-malate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, succinate dehydrogenase, carnitine acetyltransferase and citrate synthase were 5 to 400 times as active in spermatozoa as in epididymal tissue. 4. 2-Oxoglutarate dehydrogenase was the least active member of the tricarboxylic acid cycle in all tissues and most closely matched the measured flux through the cycle. 5. The concentrations of hydroxyacyl-CoA dehydrogenase and carnitine palmitoyltransferase were equivalent to the more active enzymes of the tricarboxylic acid cycle, indicating the capacity for extensive lipid oxidation, and the presence of 3-hydroxybutyrate dehydrogenase suggests that these tissues can also oxidize ketone bodies. 6. Transfer of reducing equivalents from cytoplasm to mitochondrion is unlikely to occur by means of the glycerol phosphate cycle because mitochondrial glycerol 3-phosphate dehydrogenase is relatively inactive in epididymal tissue, whereas the cytoplasmic enzyme has little activity in spermatozoa, but transfer may be accomplished by the malate-aspartate shuttle. 7. Transfer of acetyl units from mitochondrion to cytoplasm could be effected by the pyruvate-malate cycle in the caput of androgen-maintained rats, but not in the other tissues because of the low activity of ATP citrate lyase. Acetyl unit transfer could take place via acetylcarnitine, mediated by carnitine acetyltransferase. 8. Castration resulted in a decrease in the concentration of nearly all enzymes, although subsequent administration of testosterone restored concentrations to values similar to those in animals maintained by endogenous androgen. The extent to which enzyme concentration was changed by an alteration in androgen status was highly variable, but was most marked in the case of pyruvate carboxylase.     

Phosphoenolpyruvate carboxykinase and gluconeogenesis in cotyledons of Cucurbita pepo

1. The aim of this work was to investigate the role of phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) in the conversion of fat to sugar by the cotyledons of seedlings of Cucurbita pepo. 2. The enzyme was partially purified from the cotyledons of 5-day-old seedlings. The Michaelis constants for oxaloacetate and ATP were 56 and 119 micron, respectively. The decarboxylation reaction was optimum at pH 7.4. A range of intermediary metabolites did not affect the activity of the enzyme, but 3-mercaptopicolinic acid at micron concentrations was an effective inhibitor. 3. Centrifugation of extracts of 5-day-old cotyledons sedimented appreciable proportions of the ribuloseibisphosphate carboxylase, isocitrate lyase and fumarate hydratase present but very little of the phosphoenolpyruvate carboxykinase. 4. Measurements of phosphoenolpyruvate carboxykinase of cotyledons during germination showed that the maximum catalytic activity exceeded, and changed coincidently with, the rate of gluconeogenesis. 5. 3-Mercaptopicolinic acid inhibited gluconeogenesis from [1-14C]- and [2-14C]acetate supplied to excised cotyledons. The detailed distribution of 14C indicated inhibition of the conversion of oxaloacetate to phosphoenolpyruvate. 6. It is concluded that in marrow cotyledons phosphoenolpyruvate carboxykinase is in the soluble phase of the cytoplasm and catalyses a component reaction of gluconeogenesis.     

Factors involved in changes in hepatic lipogenesis during development of the rat

1. Changes in the activities of acetyl-CoA carboxylase (EC 6.4.1.2), phosphofructokinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), extramitochondrial aconitate hydratase (EC 4.2.1.3) and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) have been measured in the livers of developing rats from late foetal life to maturity. 2. The effect of altering the weaning time on some enzymes associated with lipogenesis has been studied. Weaning rats at 15 days of age instead of 21 days results in an immediate increase in the activity of ;malic' enzyme (EC 1.1.1.40) whereas the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and ATP citrate lyase (EC 4.1.3.8) did not increase until 4-5 days and acetyl-CoA carboxylase 2-3 days after early weaning. Weaning rats on to an artificial-milk diet led to complete repression of the rise in activity of hepatic enzymes associated with lipogenesis normally found on weaning, except for ;malic' enzyme, which increased in activity after 20 days of age. 3. The effect of intraperitoneal injections of glucagon, cortisol, growth hormone and thyroxine on the same hepatic enzymes has been investigated. Only thyroxine had any effect on enzyme activities and caused a 20-fold increase in ;malic' enzyme activity and a twofold increase in ATP citrate lyase activity. 4. The activities of hepatic glucose 6-phosphate dehydrogenase and ;malic' enzyme are higher in adult female than in adult male rats and it has been shown that this sex difference in enzyme activities is due to both male and female sex hormones. 5. Hepatic malate, citrate, pyruvate, glucose 6-phosphate and phosphoenolpyruvate concentrations have been measured throughout development. 6. The results are discussed in relation to the dietary and hormonal control of hepatic enzyme activities during development.     

High glucose concentration and phosphoenolpyruvate carboxykinase activity in human and rat fetal liver cultures.

In cultures of human and rat fetal liver, phosphoenolpyruvate carboxykinase activity increases during the first 24 hr of culturing. This increase can be suppressed by adding cycloheximide to the culture medium or by adding a high glucose concentration. This, however, applies only to human fetal liver and to fetal liver from rats obtained just before term. In younger rat fetal liver, glucose, on the contrary, increases the activity of phosphoenolpyruvate carboxykinase. A high glucose concentration in the medium also leads to higher citrate cleavage enzyme activity and to lower alpha-glycerolphosphate dehydrogenase (cytoplasmic) activity in rat fetal liver cultures.     

Oxaloacetate Production via Carboxylations in Crithidia fasciculata Preparations

SYNOPSIS. Fractions containing soluble enzymes from Crithidia fasciculata had an ADP-linked phosphoenolpyruvate (PEP) carboxykinase. The enzyme produced ATP and oxaloacetate (OAA) from PEP, ADP and HCO⁻₃. OAA was determined as the endproduct of reactions by forming the 2,4-dinitrophenylhydrazone derivative; the hydrazone was identified by thin-layer chromatography. Approximate Michaelis constants (PEP, Mg, HCO⁻₃, ADP) were determined spectrophotometrically by linking OAA production to malic dehydrogenase. The PEP carboxykinase did not utilize GDP, UDP or IDP as cofactors; the metal requirement was also satisfied by Mn. The enzyme was inhibited by the biotin antagonists avidin and desthiobiotin.
A pyruvate carboxylase was also present in the preparations, generating OAA from pyruvate and ATP. The role of both enzymes in OAA production and subsequent production of succinate is discussed with regard to C. fasciculata and other trypanosomatids.     

The Effect of Light on the Synthesis of Mitochondrial Enzymes in Division-synchronized Euglena Cultures

The development of the mitochondrial enzymes fumarase and succinate dehydrogenase has been followed in Euglena cultures division-synchronized by 14-hour light periods alternating with 12-hour dark periods. The activity of both enzymes was unaltered over the light phase, doubled in early dark phase, and thereafter remained constant over the rest of the cycle. The increase in enzyme activity in early dark phase probably represented de novo enzyme synthesis because it was prevented by the addition of cycloheximide at a concentration known to inhibit protein synthesis on Euglena cytoplasmic ribosomes.     

Metabolic zonation in liver of diabetic rats. Zonal distribution of phosphoenolpyruvate carboxykinase, pyruvate kinase, glucose-6-phosphatase and succinate dehydrogenase

The activities and zonal distribution of key enzymes of carbohydrate metabolism were studied in livers of diabetic rats. 48 h after alloxan treatment the following alterations were observed, intermediate values being reached after 24 h: Blood glucose, acetoacetate and beta-hydroxybutyrate were increased to more than 500%; liver glycogen was reduced to about 10%. Portal vein insulin was reduced to below 10%, portal glucagon was increased to almost 200%. The glucogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were enhanced to 320% and 150%, respectively. The glycolytic enzymes glucokinase and pyruvate kinase L (differentiated from the M2 isoenzyme with a specific anti-L-antibody) were lowered to 50% and 75%, respectively. The citrate cycle enzyme succinate dehydrogenase remained unchanged. The normal periportal to perivenous gradient of phosphoenolpyruvate carboxykinase of about 3:1, as measured in microdissected tissue samples, was enhanced to about 4:1 with activities elevated to 230% and 190%, respectively, in the two zones. The normal periportal to perivenous gradient of pyruvate kinase L of about 1:1.7, as determined with the microdissection technique, was reduced to about 1:1.4 with levels lowered to 55% and 45%, respectively, in the two zones. The even zonal distribution of pyruvate kinase M2 remained unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the mechanism of action of isocitrate lyase.

1. The enzymes citrate lyase and isocitrate lyase catalyse similar reactions in the cleavage of citrate to acetate plus oxaloacetate and of isocitrate to succinate plus glyoxylate, respectively. 2. Nevertheless, the mechanism of action of each enzyme appears to be different from each other. Citrate lyase is an acyl carrier protein-containing enzyme complex whereas isocitrate lyase is not. The active form of citrate lyase is an acetyl-S-enzyme but that of isocitrate lyase is not a corresponding succinyl-S-enzyme. 3. In contrast to citrate lyase, the isocitrate enzyme is not inhibited by hydroxylamine nor does it acquire label if treated with appropriately labelled radioactive substrate. 4. Isotopic exchange experiments performed in H18-2O with isocitrate as a substrate produced no labelling in the product succinate. This was shown by mass-spectrometric analysis. 5. The conclusion drawn from these results is that no activation of succinate takes place on the enzyme through transient formation of succinic anhydride or a covalently-linked succinyl-enzyme, derived from this anhydride.     

Intermediary carbohydrate metabolism in the adult filarial worm Setaria digitata.

Several key enzymes related to carbohydrate metabolism were assayed in Setaria digitata. In the cytosolic fraction pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malic enzyme, aspartate transaminase and alanine transaminase were found. Among the TCA cycle enzymes succinate dehydrogenase, fumarate reductase, fumarase (malate dehydration), malate dehydrogenase (malate oxidation and oxaloacetate reduction) and malic enzyme (malate decarboxylation) were detected in the mitochondrial fraction. Only reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase, NADH oxidase and NADH-cytochrome c reductase were found in the mitochondrial fraction. The significance of these results with respect to the metabolic capabilities of the worm are discussed.     

Inhibiton of isocitrate dehydrogenase and isocitrate lyase from Acinetobacter calcoaceticus by acids of the citrate and glyoxylate cycle]

Acinetobacter calcoaceticus contains two forms of NADP+-dependent isocitrate dehydrogenases differing, among others, by their molecular weights and regulatory properties. The regulation of the high-molecular form of isocitrate dehydrogenase and of isocitrate lyase by organic acids, either belonging or related to the citrate and glyoxalate cycle, is investigated. While alpha-ketoglutarate and oxalacetate competitively inhibit the isocitrate dehydrogenase against Ds-isocitrate, glyoxylate and pyruvate were found to increase Vmax and to lower the KM value for Ds-isocitrate and NADP+. Simultaneous addition of oxalacetate and glyoxylate (not, however, addition of the nonenzymatically formed condensation product of both compound) nullified the activation of isocitrate dehydrogenase by glyoxylate, and potentiates the inhibitory effect of oxalacetate. Alpha-ketoglutarate, succinate, and phosphoenolpyruvate inhibit the isocitrate lyase in a noncompetitive fashion against DS-isocitrate; L-malate, oxalacetate and glyoxylate inhibit competitively. The intermediates of the citrate and glyoxylate cycle afford additive inhibition of the isocitrate lyase. The importance of organic acids of the citrate and glyoxylate cycle and of phosphoenolpyruvate for the regulation of the citrate and glyoxylate cycle at the level of isocitrate dehydrogenase and isocitrate lyase is discussed.     

Some enzymes of carbohydrate metabolism in Mesocestoides corti and Heterakis spumosa

The activities of selected enzymes of carbohydrate metabolism were measured in tetrathyridia of Mesocestoides corti and in adult females and males of Heterakis spumosa. When the species were compared, only lactate dehydrogenase and phosphoenolpyruvate carboxykinase activities were considerably higher in M. corti. Activities of other enzymes were higher in H. spumosa, with malate dehydrogenase activity being considerably so. In H. spumosa, enzyme activity was higher, and succinate dehydrogenase markedly so in males, when compared with females. Tetrathyridia aged 170 and 210 days show relatively stable malate and lactate dehydrogenase activities, and mice of ICR and BALB/c strains are suitable for the maintenance of tetrathyridia.