Control of the purine nucleotide cycle in extracts of rat skeletal muscle: effects of energy state and concentrations of cycle intermediates

The enzymes of the purine nucleotide cycle-AMP deaminase, adenylosuccinate synthetase, and adenylosuccinate lyase-were examined as a functional unit in an in vitro system which simulates the purine nucleotide composition of sarcoplasm. Activity of each cycle enzyme in extracts of rat skeletal muscle was observed to increase as ATP/ADP, reflecting the energy state of the system, was lowered from approximately 50 to 1. The increase in AMP deaminase activity could be attributed to effects of energy state and factors such as AMP concentration, which are obligatorily coupled to energy state. The increases in synthetase and lyase activities were accounted for by increases in the concentration of IMP and adenylosuccinate, respectively. The inhibitory influence of IMP concentration on synthetase activity reported in other systems was not observed in this system; synthetase activity progressively increased as IMP concentration was raised to approximately 4 mM, and apparent saturation occurred at concentrations above 4 mM. Also, adenylosuccinate was found to be an activator of AMP deaminase. The results of this study document that the activities of the enzymes of the purine nucleotide cycle increase in parallel at low energy states, and the components of the cycle function as a coordinated unit with individual enzyme activities linked via concentrations of cycle intermediates.     

A mathematical model of the Calvin photosynthesis cycle

1. A mathematical model is presented for photosynthetic carbohydrate formation in C3 plants under conditions of light and carbon dioxide saturation. The model considers reactions of the Calvin cycle with triose phosphate export and starch production as main output processes, and treats concentrations of NADPH, NAD+, CO2, and H+ as fixed parameters of the system. Using equilibrium approximations for all reaction steps close to equilibrium steady-state and transient-state relationships are derived which may be used for calculation of reaction fluxes and concentrations of the 13 carbohydrate cycle intermediates, glucose 6-phosphate, glucose 1-phosphate, ATP, ADP, and inorganic (ortho)phosphate. 2. Predictions of the model were examined with the assumption that photosynthate export from the chloroplast occurs to a medium containing orthophosphate as the only exchangeable metabolite. The results indicate that the Calvin cycle may operate in a single dynamically stable steady state when the external concentration of orthophosphate does not exceed 1.9 mM. At higher concentrations of the external metabolite, the reaction system exhibits overload breakdown; the excessive rate of photosynthate export deprives the system of cycle intermediates such that the cycle activity progressively approaches zero. 3. Reactant concentrations calculated for the stable steady state that may obtain are in satisfactory agreement with those observed experimentally, and the model accounts with surprising accuracy for experimentally observed effects of external orthophosphate on the steady-state cycle activity and rate of starch production. 4. Control analyses are reported which show that most of the non-equilibrium enzymes in the system have a strong regulatory influence on the steady-state level of all of the cycle intermediates. Substrate concentration control coefficients for cycle enzymes may be positive, such that an increase in activity of an enzyme may raise the steady-state concentration of the substrate is consumes. 5. Under optimal external conditions (0.15-0.5 mM orthophosphate), reaction flux in the Calvin cycle is controlled mainly by ATP synthetase and sedoheptulose bisphosphatase; the cycle activity approaches the maximum velocity that can be supported by the latter enzyme. At lower concentrations of external orthophosphate the cycle activity is controlled almost exclusively by the phosphate translocator.(ABSTRACT TRUNCATED AT 400 WORDS)     

Urea cycle enzymes in human liver: ontogenesis and interaction with the synthesis of pyrimidines and polyamines

Summary All the five enzymes of urea synthesis and the formation of urea in vitro can already be demonstrated in human liver as early as the 9th week of fetal development. At this stage the activity of carbamoyl phosphate synthetase is the highest, whereas that of ornithine carbamoyltransferase is the lowest as compared to those in the adult. The kinetic parameters of the urea cycle enzymes are the same in fetal liver as in adult liver, except that the Km values of ornithine carbamoyltransferase for L-ornithine are 3.5 mM and 0.42 mM in the fetus and in adult liver, respectively.Urea formation in vivo seems to begin in the second half of fetal life, and a gradual increase can be detected in the activity of the enzymes of urea synthesis. The activity of ortnithine decarboxylase, the glutamine-dependent carbamoyl phosphate synthetase and aspartate carbamoyltransferase, however, changes in the opposite direction.The concentration of carbamoyl phosphate and aspartate remains constant, but that of ornithine gradually decreases during ontogenesis. The ornithine, carbamoyl phosphate and aspartate pools are probably utilized in the polyamine, pyrimidine and urea syntheses at varying rates.     

Isolation, characterization, and structural organization of 10-formyltetrahydrofolate synthetase from spinach leaves

One-carbon metabolism mediated by folate coenzymes plays an essential role in several major cellular processes. In the prokaryotes studied, three folate-dependent enzymes, 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) generally exist as monofunctional or bifunctional proteins, whereas in eukaryotes the three activities are present on one polypeptide. The structural organization of these enzymes in plants had not previously been examined. We have purified the 10-formyltetrahydrofolate synthetase activity from spinach leaves to homogeneity and raised antibodies to it. The protein was a dimer with a subunit molecular weight of Mr = 67,000. The Km values for the three substrates, (6R)-tetrahydrofolate, ATP, and formate were 0.94, 0.043, and 21.9 mM, respectively. The enzyme required both monovalent and divalent cations for maximum activity. The 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase activities of spinach coeluted separately from the 10-formyltetrahydrofolate synthetase activity on a Matrex Green-A column. On the same column, the activities of the yeast trifunctional C1-tetrahydrofolate synthase coeluted. In addition, antibodies raised to the purified spinach protein immunoinactivated and immunoprecipitated only the 10-formyltetrahydrofolate synthetase activity in a crude extract of spinach leaves. These results suggest that unlike the trifunctional form of C1-tetrahydrofolate synthase in the other eukaryotes examined, 10-formyltetrahydrofolate synthetase in spinach leaves is monofunctional and 5,10-methyl-enetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase appear to be bifunctional. Although structurally dissimilar to the other eukaryotic trifunctional enzymes, the 35 amino-terminal residues of spinach 10-formyltetrahydrofolate synthetase showed 35% identity with six other tetrahydrofolate synthetases.     

In Situ Glutamine Synthetase Activity in a Marine Unicellular Alga (Development of a Sensitive Colorimetric Assay and the Effects of Nitrogen Status on Enzyme Activity)

A malachite green colorimetric assay for glutamine synthetase is described. Glutamine synthetase activity was determined in situ in the marine diatom Phaeodactylum tricornutum Bohlin using cells permeabilized by freeze/thawing. Higher activities were obtained with cells permeabilized in N-2-hydroxyethylpiperazine-N[prime]-2-ethanesulfonic acid compared with N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, tris(hydroxymethyl)aminomethane, or imidazole, and the optimum pH was 7.9. Activities were higher in cells permeabilized in the presence of reductant, particularly dithiothreitol. Glutamine synthetase activities were markedly decreased in the presence of methionine sulfoximine. In the presence of saturating concentrations of glutamate and ATP, the apparent Km for ammonia was 320 [mu]M, but this value decreased to 110 [mu]M with subsaturating concentrations of glutamate and ATP. The apparent Km values for glutamate and ATP, in the presence of saturating concentrations of ammonia, were 9.7 and 2.9 mM, respectively. Ammonia-grown cells had lower glutamine synthetase activities than did nitrate-grown cells. During nitrogen starvation of both ammonia- and nitrate-grown cells, glutamine synthetase activities increased rapidly during the first 8 h, reaching maximum values after 24 to 48 h. Moreover, the time course for the increases in glutamine synthetase activities and rate of methylamine uptake following the transfer of nitrate-grown cells to nitrogen-deficient medium were very similar. In nitrate-grown cells and cells deprived of combined nitrogen, glutamine synthetase activities and maximum rates of ammonia uptake gave comparable values when measured at the same temperature (20[deg]C).     

Studies on the mechanism of action of ACTH on triglyceride synthesis in fat cells.

It was found that ACTH greatly reduced lipogenesis in fat cells in the presence of calcium ion, but not in the absence of calcium ion. Of the enzymes involved in triglyceride synthesis from fatty acid in lipid micelle membranes, only acyl-CoA synthetase was inhibited by calcium ion, the apparent Ki value of calcium ion being 4.2 X 10(-4) M. The Km values of the enzyme for palmitate and ATP were 2.0 X 10(-4) M and 2.5 X 10(-4) M, respectively and calcium ion caused non-competitive inhibition with both palmitate and ATP. The acyl-CoA synthetase activity of lipid micelle membranes was inhibited by treatment with phospholipase A or C, but not by treatment with phospholipase D. The mechanism of inhibition of triglyceride synthesis by ACTH is discussed on the basis of these results.     

The riboflavin/FAD cycle in rat liver mitochondria.

Here we provide evidence that mitochondria isolated from rat liver can synthesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake takes place via a carrier-mediated process, as shown by the inverse relationship between fold accumulation and riboflavin concentration, the saturation kinetics [riboflavin Km and Vmax values were 4.4+/-1.3 microM and 35+/-5 pmol x min(-1) (mg protein)(-1), respectively] and the inhibition shown by the thiol reagent mersalyl, which cannot enter the mitochondria. FAD synthesis is due to the existence of FAD synthetase (EC 2.7.7.2), localized in the matrix, which has as a substrate pair mitochondrial ATP and FMN synthesized from taken up riboflavin via the putative mitochondrial riboflavin kinase. In the light of certain features, including the protein thermal stability and molecular mass, mitochondrial FAD synthetase differs from the cytosolic isoenzyme. Apparent Km and apparent Vmax values for FMN were 5.4+/-0.9 microM and 22.9+/-1.4 pmol x min(-1) x (mg matrix protein)(-1), respectively. Newly synthesized FAD inside the mitochondria can be exported from the mitochondria in a manner sensitive to atractyloside but insensitive to mersalyl. The occurrence of the riboflavin/FAD cycle is proposed to account for riboflavin uptake in mitochondria biogenesis and riboflavin recovery in mitochondrial flavoprotein degradation; both are prerequisites for the synthesis of mitochondrial flavin cofactors.     

o-Phosphotyrosyl glutamine synthetase: modification of the nucleotide ligation site of adenylylated glutamine synthetase

The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes. The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase. The Km for this macromolecular substrate with the nuclease was 40 microM, at pH 8.9. The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased. The Km for the native adenylylated glutamine synthetase with the SVPDE was 36 microM, i.e., similar to that for the nuclease. When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme. Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme. The optimum pH, apparent Km for each of three substrates, glutamate, ATP, and NH3, and Vmax were in good agreement, as to either Mg2+- or Mn2+-dependent biosynthetic activity. From these results we can conclude that the regulation of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation.     

Stringent response of Bacillus stearothermophilus: evidence for the existence of two distinct guanosine 3',5'-polyphosphate synthetases.

Bacillus stearothermophilus reacted to pseudomonic acid-induced inhibition of isoleucine-transfer ribonucleic acid (RNA) acylation and to energy downshift caused by alpha-methylglucoside addition with accumulation of guanosine 3',5'-polyphosphates [(p)ppGpp] and restriction of RNA synthesis. In vitro studies indicated that (p)ppGpp was synthesized by two different enzymes. One enzyme, (p)ppGpp synthetase I, was present in the ribosomal fraction, required the addition of a ribosome-messenger RNA-transfer RNA complex for activation, and was inhibited by tetracycline and thiostrepton. It is suggested that (p)ppGpp synthetase I is comparable to the relA gene product from Escherichia coli and is responsible for (p)ppGpp accumulation during amino acid starvation. The other enzyme, (p)ppGpp synthetase II, was found in the high-speed supernatant fraction (S100). It functioned independently of ribosomes, transfer RNA, and messenger RNA and was not inhibited by the above-mentioned antibiotics. (p)ppGpp synthetase II is thought to be responsible for (p)ppGpp accumulation during carbon source downshift. The two enzymes differ in their Km values for adenosine triphosphate (ATP):2mM ATP for synthetase I and 0.05 mM ATP for synthetase II. They also have different molecular weights: apparent Mr of 86,000 (+/- 5,000) for synthetase I and 74,000 (+/- 5,000) for synthetase II.     

New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes

The synthesis of fluorescent derivatives of nucleosides and nucleotides, by reaction with isatoic anhydride in aqueous solution at mild pH and temperature, yielding their 3'-O-anthraniloyl derivatives, is here described. The N-methylanthraniloyl derivatives were also synthesized by reaction with N-methylisatoic anhydride. Upon excitation at 330-350 nm these derivatives exhibited maximum fluorescence emission at 430-445 nm in aqueous solution with quantum yields of 0.12-0.24. Their fluorescence was sensitive to the polarity of the solvent; in N,N-dimethylformamide the quantum yields were 0.83-0.93. The major differences between the two fluorophores were the longer wavelength of the emission maximum of the N-methylanthraniloyl group and its greater quantum yield in water. All anthraniloyl derivatives, as well as the N-methylanthraniloyl ones, had virtually identical fluorescent properties, regardless of their base structures. The ATP derivatives showed considerable substrate activity as a replacement of ATP with adenylate kinase, guanylate kinase, glutamine synthetase, myosin ATPase and sodium-potassium transport ATPase. The ADP derivatives were good substrates for creatine kinase and glutamine synthetase (gamma-glutamyl transfer activity). The GMP and adenosine derivatives were substrates for guanylate kinase and adenosine deaminase, respectively. All derivatives had only slightly altered Km values for these enzymes. While more fluorescent in water, the N-methylanthraniloyl derivatives were found to show relatively low substrate activities against some of these enzymes. The results indicate that these ribose-modified nucleosides and nucleotides can be versatile fluorescent substrate analogs for various enzymes.     

Mitochondrial carbamoyl phosphate synthetase activity in the absence of N-acetyl-L-glutamate. Mechanism of activation by this cofactor

Rat liver carbamoyl-phosphate synthetase I is shown to have synthetase and ATPase activity in the absence of acetylglutamate. Km values for ATP, Mg2+ and K+ are greatly increased, the Km for HCO-3 is not changed much, and the Km for NH+4 is markedly reduced. Vmax for the synthetase reaction is less than 20% of that of the acetylglutamate-activated enzyme whereas Vmax for the ATPase activity is greater than 40% of that with acetylglutamate. Pulse-chase experiments with H14CO-3 show formation of less "active CO2" (the central intermediate) than with acetylglutamate; ATPase activity is reduced in proportion, but the synthetase activity is much smaller. Binding of one ATP molecule with high affinity (Kd = 20-30 microM) is shown in the absence of acetylglutamate. This appears to be the molecule of ATPB (ATPB provides the phosphoryl group of carbamoyl phosphate). In contrast, the affinity for ATPA (ATPA yields Pi) is much reduced. Initial velocity measurements without acetylglutamate show a time lag before reaching a constant velocity. At 50 microM acetylglutamate the lag is much longer, but at 10 mM acetylglutamate it is shorter. Activation by acetylglutamate requires ATP at concentrations sufficient to occupy the ATPA and the ATPB binding sites. Preincubation with 10 mM acetylglutamate alone shortens the activation time. From these findings we propose an allosteric model for activation of carbamoyl-phosphate synthetase in which there are two active states, R and R . AcGlu. Binding of ATPA is associated with the conversion of T to R. R . AcGlu differs from R in that transfer to carbamate of the gamma-phosphoryl group of ATPB appears to be facilitated.     

Autotrophic CO2 fixation via the reductive tricarboxylic acid cycle in different lineages within the phylum Aquificae: evidence for two ways of citrate cleavage

Autotrophic carbon fixation was characterized in representative members of the three lineages of the bacterial phylum Aquificae. Enzyme activity measurements and the detection of key genes demonstrated that Aquificae use the reductive tricarboxylic acid (TCA) cycle for autotrophic CO(2) fixation. This is the first time that strains of the Hydrogenothermaceae and 'Desulfurobacteriaceae' have been investigated for enzymes of autotrophic carbon fixation. Unexpectedly, two different mechanisms of citrate cleavage could be identified within the Aquificae. Aquificaceae use citryl-CoA synthetase and citryl-CoA lyase, whereas Hydrogenothermaceae and 'Desulfurobacteriaceae' use ATP citrate lyase. The first mechanism is likely to represent the ancestral version of the reductive TCA cycle. Sequence analyses further suggest that ATP citrate lyase formed by a gene fusion of citryl-CoA synthetase and citryl-CoA lyase and subsequently became involved in a modified version of this pathway. However, rather than having evolved within the Aquificae, our phylogenetic analyses indicate that Aquificae obtained their ATP citrate lyase through lateral gene transfer. Aquificae play an important role in biogeochemical processes in a variety of high-temperature habitats. Thus, these findings substantiate the hypothesis that autotrophic carbon fixation through the reductive TCA cycle is widespread and contributes significantly to biomass production particularly in hydrothermal habitats.     

A kinetic study of a ternary cycle between adenine nucleotides

In the present paper, a kinetic study is made of the behavior of a moiety-conserved ternary cycle between the adenine nucleotides. The system contains the enzymes S-acetyl coenzyme A synthetase, adenylate kinase and pyruvate kinase, and converts ATP into AMP, then into ADP and finally back to ATP. L-Lactate dehydrogenase is added to the system to enable continuous monitoring of the progress of the reaction. The cycle cannot work when the only recycling substrate in the reaction medium is AMP. A mathematical model is proposed whose kinetic behavior has been analyzed both numerically by integration of the nonlinear differential equations describing the kinetics of the reactions involved, and analytically under steady-state conditions, with good agreement with the experimental results being obtained. The data obtained showed that there is a threshold value of the S-acetyl coenzyme A synthetase/adenylate kinase ratio, above which the cycle stops because all the recycling substrate has been accumulated as AMP, never reaching the steady state. In addition, the concept of adenylate energy charge has been applied to the system, obtaining the enabled values of the rate constants for a fixed adenylate energy charge value and vice versa.     

Regulation of the mammalian carbamoyl-phosphate synthetase II by effectors and phosphorylation. Altered affinity for ATP and magnesium ions measured using the ammonia-dependent part reaction.

We have measured the 'core' mammalian carbamoyl-phosphate synthetase II (CPSII) activity, using NH4Cl as the nitrogen-donating substrate and trapping carbamoyl phosphate as urea through its reaction with ammonium ions. When ATP and magnesium ion concentrations are close to those found in the cell, the substrate saturation curves for ammonia and bicarbonate are hyperbolic, giving Km (NH3) values of 166 microM at high ATP concentrations and 26 microM at low ATP concentrations, while the Km (bicarbonate) is 1.4 mM at both ATP concentrations used. These values for the Km (NH3) are lower than previously reported for CPS II, and closer to the values for the mitochondrial counterpart. The Km for ammonia and bicarbonate are not altered by phosphorylation of the multienzyme polypeptide CAD, which contains the first three enzyme activities of pyrimidine biosynthesis. The CPS II activity is lower with an excess of either ATP or magnesium ions, causing the apparently sigmoid dependence of activity upon ATP concentration to be enhanced at low concentrations of free magnesium ions. The feedback inhibitor, UTP, acts by stabilising a state with a low affinity for magnesium ions and for ATP. In the presence of the activator, 5-phosphoribosyl diphosphate (PRibPP), the enzyme has a higher affinity for magnesium ions and thus the ATP dependence of the activity is hyperbolic. Phosphorylation of CAD similarly activates the CPS II enzyme by increasing the affinity for magnesium ions and by pushing the equilibrium away from the low-affinity UTP-stabilised state. Using our improved assay procedure, we observe a very large activation by PRibPP of carbamoylphosphate synthesis at low concentrations of magnesium ions, and we find that unlike UTP, the activator PRibPP is able to act on the phosphorylated enzyme.     

ATP hydrolysis and synthesis by the membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum.

The membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum showed maximum activity for ATP hydrolysis at pH 8, at temperatures between 65 and 70 degrees C, and at an ATP-Mg2+ ratio of 0.5. Anaerobic conditions were not prerequisite for enzyme activity. The enzyme showed a Km value for ATP of 2 mM, and activity was Mg2+ dependent; Mn2+, Co2+, Ca2+, and Zn2+ could replace Mg2+ to some extent. Other nucleoside triphosphates could be hydrolyzed. N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. A proton-motive force, artificially imposed by a pH shift or valinomycin, resulted in ATP synthesis in whole cells. The ATP synthetase complex of the thermophilic methanogenic bacterium is similar to those described in aerobic and anaerobic microorganisms.     

Phosphorylation of Saccharomyces cerevisiae CTP synthetase at Ser424 by protein kinases A and C regulates phosphatidylcholine synthesis by the CDP-choline pathway

The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinases A and C. Previous studies have revealed that Ser424 is the target site for protein kinase A. Using a purified S424A mutant CTP synthetase enzyme, we examined the effect of Ser424 phosphorylation on protein kinase C phosphorylation. The S424A mutation in CTP synthetase caused a 50% decrease in the phosphorylation of the enzyme by protein kinase C and an 80% decrease in the stimulatory effect on CTP synthetase activity by protein kinase C. The S424A mutation caused increases in the apparent Km values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction. The effect of the S424A mutation on the phosphorylation reaction was dependent on time and protein kinase C concentration. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing Ser424 was a substrate for protein kinase C. Comparison of phosphopeptide maps of the wild type and S424A mutant CTP synthetase enzymes phosphorylated by protein kinases A and C indicated that Ser424 was also a target site for protein kinase C. Phosphorylation of Ser424 accounted for 10% of the total phosphorylation of CTP synthetase by protein kinase C. The incorporation of [methyl-3H]choline into phosphocholine, CDP-choline, and phosphatidylcholine in cells carrying the S424A mutant CTP synthetase enzyme was reduced by 48, 32, and 46%, respectively, when compared with control cells. These data indicated that phosphorylation of Ser424 by protein kinase A or by protein kinase C was required for maximum phosphorylation and stimulation of CTP synthetase and that the phosphorylation of this site played a role in the regulation of phosphatidylcholine synthesis by the CDP-choline pathway.     

Subcellular localization and properties of glyoxylate cycle enzymes in the liver of rats with alloxan diabetes.

Key enzymes of the glyoxylate cycle (isocitrate lyase and malate synthetase) were found in the liver and kidney of rats suffering from alloxan diabetes. The activities of these enzymes in the liver were 0.080 and 0.0430 U/mg protein, respectively. Isocitrate lyase activity in the kidney was 0.030 U/mg protein, and that of the malate synthetase was 0.018 U/mg protein. Peroxisomal localization of the enzymes was shown. A novel malate dehydrogenase isoform was found in a liver of rats suffering from the alloxan diabetes. The isocitrate lyase was isolated by selective (NH4)2SO4 precipitation and DEAE-Toyopearl chromatography. The resulting enzyme preparation had specific activity 6.1 U/mg protein, corresponding to 76.25-fold purification with 32.6% yield. The isocitrate lyase was found to follow the Michaelis--Menten kinetic scheme (Km for isocitrate, 0.08 mM) and to be competitively inhibited by glucose 1-phosphate (Ki = 1. 25 mM), succinate (Ki = 1.75 mM), and citrate (Ki = 1.0 mM); the pH optimum of the enzyme was 7.5 in Tris-HCl buffer.     

Subcellular location of glutamine synthetase and urea cycle enzymes in liver of spiny dogfish (Squalus acanthias)

Glutamine synthetase and glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, both of which are present in high concentrations in liver of urea-retaining elasmobranchs, have been found to be located exclusively in the mitochondria in liver from the representative elasmobranch Squalus acanthias. This observation is consistent with the view that the function of this unique carbamoyl-phosphate synthetase is related to urea synthesis, and that the initial nitrogen-donating substrate for urea synthesis in these species is glutamine rather than ammonia. The urea cycle enzymes, ornithine carbamoyltransferase and arginase, are also located in the mitochondria, whereas argininosuccinate synthetase and argininosuccinate lyase are located in the cytosol. Glutamine synthetase and arginase are mitochondrial enzymes in uricotelic species, but are normally found in the cytoplasm in ureotelic species. the properties of the elasmobranch arginase, however, are characteristic of arginases from ureotelic species (e.g. the Km for arginine is 1.2 mM, and the enzyme has an Mr congruent to 100,000).     

Similarities in properties and a functional difference in purified leucyl-tRNA synthetase isolated from two developmental stages of Tenebrio molitor

In Tenebrio molitor, as well as in other biological systems, there are indications that differences in leucyl-tRNA synthetase activity may play a role in translational control. However, it has not been clear whether the difference in activity is due to the appearance of a multiplicity of enzymes during development or to the alteration of a single enzyme.The purification of leucyl-tRNA synthetase from day 1 and day 7 after the larval pupal molt of Tenebrio molitor is described. The enzyme from both developmental stages was purified over a 1000-fold. The two enzyme preparations are identical in molecular weight (99,000). They show the same characteristics after aging. The pH optimum, heat inactivation behavior, and dependency on divalent cations are the same for both enzymes. They also show identical kinetics with similar values of Km for leucine, ATP, Mg²⁺, and tRNA day 1. However, leucyl-tRNA synthetase purified from day 7 exhibits an additional function in recognizing a new species of isoaccepting tRNA in day 7 tRNA. We have tentatively concluded that the two enzymes are probably different forms of the same enzyme and the additional activity is due to alteration of the enzyme at the macromolecular level during development.     

Characterization of the Acyl-CoA synthetase activity of purified murine fatty acid transport protein 1

Fatty acid transport protein 1 (FATP1) is an approximately 63-kDa plasma membrane protein that facilitates the influx of fatty acids into adipocytes as well as skeletal and cardiac myocytes. Previous studies with FATP1 expressed in COS1 cell extracts suggested that FATP1 exhibits very long chain acyl-CoA synthetase (ACS) activity and that such activity may be linked to fatty acid transport. To address the enzymatic activity of the isolated protein, murine FATP1 and ACS1 were engineered to contain a C-terminal Myc-His tag expressed in COS1 cells via adenoviral-mediated infection and purified to homogeneity using nickel affinity chromatography. Kinetic analysis of the purified enzymes was carried out for long chain palmitic acid (C16:0) and very long chain lignoceric acid (C24:0) as well as for ATP and CoA. FATP1 exhibited similar substrate specificity for fatty acids 16-24 carbons in length, whereas ACS1 was 10-fold more active on long chain fatty acids relative to very long chain fatty acids. The very long chain acyl-CoA synthetase activity of the two enzymes was comparable as were the Km values for both ATP and coenzyme A. Interestingly, FATP1 was insensitive to inhibition by triacsin C, whereas ACS1 was inhibited by micromolar concentrations of the compound. These data represent the first characterization of purified FATP1 and indicate that the enzyme is a broad substrate specificity acyl-CoA synthetase. These findings are consistent with the hypothesis that that fatty acid uptake into cells is linked to their esterification with coenzyme A.