Synthetase In Extracts Research Articles

Proteolytic Inactivation of Glutamine Synthetase in Extracts From Wheat Leaves: Effects of pH, Inorganic Ions and Metabolites

Glutamine synthetase in extracts from young wheat (Triticum aestivum L.) leaves was quite stable at pH 7.0-9.0, whereas it was remarkably more labile below and above this range. Added extract from senescing wheat leaves accelerated the inactivation over the whole investigated pH range (6.0-10.5) and was most effective around pH 8.5-9.0. At pH 7.5, glutamine synthetase inactivation by endogenous or other supplied endopeptidases was delayed in the presence of magnesium sulfate, magnesium chloride and L-lysine, while potassium chloride stabilised only slightly. No major effect was caused by the addition of sucrose, L-alanine, L-serine or glycine. These results, and the fact that the stabilities of various enzymes are affected differently by the same solutes, suggest stabilising interactions with the substrate protein (glutamine synthetase) rather than effects on the inactivating endopeptidases. From immunoblots, it became evident that the inactivation of glutamine synthetase was paralleled by the degradation of the intact subunit. A smaller fragment was detected on immunoblots during the catabolism of this enzyme. Stabilising solutes retarded the loss of the intact subunit and the formation of the fragment. Solute concentrations must be considered as factors regulating the catabolism of a particular protein by given proteolytic activities.

Quinolinate synthetase: The oxygen-sensitive site of de novo NAD(P) + biosynthesis

The ability of niacin to relieve the growth-inhibiting effect of hyperoxia on Escherichia coli can be attributed to the dioxygen sensitivity of quinolinate synthetase. The activity of this enzyme within E. coli was diminished by exposure of the cells to 4.2 atm O 2, while the activity in extracts was rapidly decreased by 0.2 atm O 2. Neither catalase nor superoxide dismutase afforded detectable protection against the inactivating effect of O 2, indicating that H 2O 2 and O 2 − were not significant intermediates in this process. Nevertheless, H 2O 2 at 1.0 m m did inactivate quinolinate synthetase, even under anaerobic conditions and in the absence of catalatic activity which might have generated O 2. Addition of paraquat to aerobic cultures of E. coli caused an inactivation of quinolinate synthetase, which may be explained in terms of an increase in the production of H 2O 2. The O 2-dependent inactivation of quinolinate synthetase in extracts was gradually reversed during anaerobic incubation and this reactivation was blocked by α,α′-dipyridyl or by 1,10-phenanthroline. The sequence of the quinolinate synthetase “A” protein contains a—cys-w-x-cys-y-z-cys—sequence, which is characteristic of (FeS) 4-containing proteins. This sequence, together with the effect of the Fe(II)-chelating agents, suggests that the O 2-sensitive site of quinolinate synthetase is an iron-sulfur cluster which is essential for the dehydration reaction catalyzed by the A protein.

Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes

When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged (a) by the incorporation of [ 14C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, (b) by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incroporation of [ 14C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and (c) by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogeneously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn 2+, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe 2+ (or Ni 2+ together with ATP (or other nucleoside di- and triphosphates. After 40–60 h dialysis Fe 3+ together with NADH (but not ATP) are required for glutamine synthetase inactivation. The results suggest that accelerated protein degradation in cells exposed to nitrogen-limited conditions reflects the differential destruction of some proteins, including aspartokinases I and III, in order to sustain the biosynthesis of others such as glutamine synthetase. The loss of glutamine synthetase activity in cell-free extracts is likely mediated in part by mixed-function oxidation systems and could represent a ‘marking’ step in protein turnover.

Elevated Levels of Interferon-Induced 2'-5' Oligoadenylate Synthetase in Generalized Persistent Lymphadenopathy and the Acquired Immunodeficiency Syndrome

The levels of the 2'-5' oligoadenylate enzyme synthetase in extracts of peripheral blood mononuclear cells from individuals with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) were measured and compared with synthetase levels in peripheral blood mononuclear cells (PBMs) from healthy heterosexual and homosexual controls. The mean basal synthetase level in heterosexual and homosexual controls was 14 +/- 13 and 12 +/- 9 pmol per hr/10(5) PBMs, respectively. Thirteen individuals with AIDS had a mean basal level of 129 +/- 75 pmol. Serial levels were persistently elevated in six of these individuals over a one- to 10-month period. Twelve of the 13 individuals had antibodies to human T cell lymphotrophic virus-III/lymphadenopathy-associated virus (HTLV-III/LAV). Thirty-three individuals with ARC had a mean basal synthetase level of 68 +/- 84 pmol. Thirty-two of the 33 had antibodies to HTLV-III/LAV. Eleven (33%) have had consistently normal synthetase levels (less than 2 SD above the mean for the homosexual controls, i.e., 30 pmol) over a three- to nine-month follow-up period. Fourteen (42%) had persistently elevated levels over the same period; four (29%) of these developed AIDS during the follow-up period. Eight have had fluctuating levels but have remained clinically well. These studies suggest that persistently elevated synthetase levels in individuals with ARC and antibodies to HTLV-III/LAV indicate progressive virus-induced disease activity. Elevated synthetase levels may be an important prognostic indicator of increased risk of progression to AIDS.

Enzyme markers for the presence of circulating interferon: 2-5A synthetase in blood lymphocytes and protein kinase in platelet-rich plasma.

The level of 2-5A synthetase in extracts of peripheral blood lymphocytes and a specific protein kinase activity in platelet-rich plasma were measured in normal individuals and in patients suffering from viral or bacterial infections. The level of these enzymes was tested at different times during the disease. The level of 2-5A synthetase and the protein kinase activity was enhanced by several-fold during viral and bacterial infections and decreased during the course of the disease in parallel with clinical ameliorations and reversal of clinical symptoms. Among the different types of infections studied, higher levels of these enzymes were observed during viral than bacterial infections. Our results emphasize the use of these enzymes as markers to evaluate the state of the disease and recovery. Furthermore, they provide evidence for the production of interferon during different types of infection.

Assignment of structural gene for asparagine synthetase to human chromosome 7.

Somatic cell hybrids obtained from the fusion of human B lymphocytes and an asparagine synthetase-deficient Chinese hamster ovary cell line were isolated after growth in asparagine-free medium. The human and hamster forms of asparagine synthetase differ significantly in their rate of inactivation at 47.5 degrees C. The asparagine synthetase activity expressed in the hybrids was inactivated at 47.5 degrees C at the same rate as the human form of the enzyme. Karyotypic analysis and analysis for chromosome-specific enzyme markers showed that the structural gene for asparagine synthetase is located on chromosome 7 in humans. The heat-inactivation profile for asparagine synthetase in extracts of hybrids formed between human peripheral leukocytes and a hamster cell line expressing asparagine synthetase activity was intermediate between the two parental types when human chromosome 7 was present, but was identical to the hamster parent when chromosome 7 was absent.

Apparent synergism between amino donors for CTP synthesis in Chinese hamster fibroblasts.

The synergistic effects of potential amino donors were studied in the assay of CTP synthetase in extracts of Chinese hamster fibroblasts. We found that L-glutamine was not effective as the sole amino donor, but combinations of L-glutamine with NH4HCO3, L-arginine or potassium phosphate did result in the conversion of UTP to CTP. L-arginine or potassium phosphate were also not effective when used alone, and NH4HCO3 was only slightly effective. Our studies demonstrate that the individual synergistic combinations were not additive; multiple combinations of components decreased rather than increased the formation of CTP. The synergistic combinations of L-glutamine with either NH4HCO3 or L-arginine had an absolute requirement for ATP; when ATP and PEP were absent no conversion of UTP to CTP occurred. The presence of GTP in a reaction mixture slightly increased the formation of CTP when L-glutamine and NH4HCO3 were used and substantially increased CTP formation when L-glutamine and L-arginine were used. De novo CTP synthesis was greatly reduced when nonradioactive CTP was added to an assay mixture, suggesting feedback inhibition. A TLC procedure has been developed that allows for the direct separation of UTP and CTP without requiring prior conversion to the mononucleotide or nucleoside level.

The birA gene of Escherichia coli encodes a biotin holoenzyme synthetase.

Mutations in the birA gene of Escherichia coli cause defects in biotin operon repression, biotin uptake and retention of intracellular biotin (Campbell et al., 1972: Barker &, Campbell, 1980). We report here that the birA gene encodes the major biotin-fixing enzyme of this organism, the acetyl-CoA carboxylase biotin holoenzyme synthetase (EC 6.3.4.15). Unlike the situation in wild-type E. coli extracts, measurements of labeled biotin incorporation into protein in sonicated extracts reveal no in vitro activity. Three different mutants exhibit altered holoenzyme synthetase activity, including one clear instance of a thermolabile activity specified by birA361. Amplification of birA gene expression by infection of cells with a λ phage bearing an EcoRI fragment of the E. coli chromosome which includes the gene results in a 20- to 40-fold increase in specific activity. When the λbirA phage carries the birA85 mutation, no activity increase is observed. Infection of cells with a λbirA361 transducing phage results in a 20- to 40-fold increase in temperature-sensitive activity. We have purified the activity specified by birA361 approximately 1000-fold and have shown that the purified enzyme is more thermolabile than similarly purified wild-type enzyme. Measurements of holoenzyme synthetase in extracts and biotin uptake by whole cells indicate that certain mutations located at the same chromosomal position as birA mutations but initially characterized as defective only in bio repression are also deficient in biotin holoenzyme synthetase and biotin uptake. This result indicates that all mutations at this location affect the same enzyme, and we have redesignated these “bioR” mutations as birA. Results of complementation analysis of birA mutations and biochemical characterization of the gene and its product, presented in the accompanying paper, support the view that the birA product functions both as the bio repressor and biotin holoenzyme synthetase.

Separation of Two Forms of Anthranilate Synthetase from 5‐Methyltryptophar Susceptible and ‐Resistant Cultured Solarium tuberosumCells

AbstractPotato cell suspension cultures (Solanum tuberosumL. cv. Merrimack) have been selected which are resistant to growth inhibition by D,L‐5‐methyltryptophan. Anthranilate synthetase activity in crude extracts from resistant cells was less sensitive to feedback inhibition by L‐tryptophan and D,L‐5‐methyltryptophan than the activity from the sensitive line. This altered feedback control apparently accounts for the cell's resistance to growth inhibition since there is a 48‐fold increase in free tryptophan in one of the resistant cell lines. Preparative polyacrylamide gel electro‐phoresis separated feedback‐sensitive and ‐resistant forms of anthranilate synthetase in extracts from both 5‐methyltryptophan‐susceptible and ‐resistant cells, with a predominance of the corresponding form in the respective cell type. The anthranilate synthetase activity from the 5‐methyltryptophan‐resistant line was inactivated more slowly by incubation of crude extracts at 50°C than the activity from the sensitive line. These results suggest the presence of two isoenzymes of anthranilate synthetase in cultured potato cells.

Methionyl-tRNA synthetase from sheep liver. Purification of a fully active monomer derived from high-molecular-weight complexes by trypsin treatment. Evidence for immunological cross-reaction with the corresponding enzyme from sheep mammary gland.

The size distribution of methionyl-tRNA synthetase in extracts from sheep liver is compared to that of lysyl-tRNA, isoleucyl-tRNA, leucyl-tRNA and seryl-tRNA synthetases by gel filtration on Biogel A-5m. Extraction conditions are described which lead to isolation of methionyl-tRNA synthetase exclusively in the form of complexes of molecular weight close to 10(6). Limited trypsin treatment of these aggregates releases a fully active low-molecular-weight form of methionyl-tRNA synthetase which was purified to a specific activity of 674 units/mg at 25 degrees C with a yield of 40%. The homogeneous enzyme appears to be undistinguishable from the corresponding enzyme derived from sheep lactating mammary gland, as judged by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by titration with antibodies raised against the enzyme purified from liver.

Dihydrosphingosine growth inhibition and repression of 3-ketodihydrosphingosine synthetase activity in [formula omitted

Addition of dihydrosphingosine or 3-ketodihydrosphingosine to growing cultures of Bacteroides melaninogenicus markedly reduced the activity of 3-ketodihydrosphingosine synthetase in extracts. Neither compound reduced the activity of a previously solubilized preparation of the enzyme. Dihydrosphingosine and synthetic acetyl ceramides inhibited the growth of the microorganism; 3-ketodihydrosphingosine was not inhibitory and reversed the growth inhibition caused by dihydrosphingosine. This reversal may indicate a preferential utilization of 3-ketodihydrosphingosine over dihydrosphingosine by the cells for the biosynthesis of complex sphingolipids.

The mode of action of bacilysin and anticapsin and biochemical properties of bacilysin-resistant mutants.

Bacilysin is hydrolysed to L-alanine and anticapsin by suspensions of a bacilysin-sensitive strain of Staphylococcus aureus but not by those of a resistant strain derived from it. In contrast, it is hydrolysed by extracts of both strains. Anticapsin is a powerful inhibitor of glucosamine synthetase in extracts of both the bacilysin-sensitive and -resistant strains of Staph. aureus. Bacilysin, by comparison, is a relatively poor inhibitor of glucosamine synthetase in crude extracts when its hydrolysis is inhibited by EDTA. A phenylalanine auxotroph of Staph. aureus readily uses L-analyl-L-phenylalanine for growth, but a bacilysin-resistant mutant of this strain does not. It is suggested that the antibacterial activity of bacilysin depends on its transport into the organism, its hydrolysis to anticapsin and on inhibition by the latter of glucosamine synthetase, and that bacilysin-resistant mutants are defective in a transport system.

Developmental pattern of thymidylate synthetase activity in the silkworm, Bombyx mori

Thymidylate synthetase activity (as expressed per mg of protein and per the whole insect) in extracts from Bombyx mori changes considerably during development. The enzyme is present in unfertilized eggs. After fertilization its activity initially increases but then decreases in parallel with the beginning of diapause. During postdiapause embryonic development the level of the enzyme quickly increases, but decreases again after hatching. In larvae the activity is low during the first instars but rises dramatically after the 4th ecdysis. The whole period of pharate pupal, pupal, and pharate adult development is characterized by a U-shaped pattern of the thymidylate synthetase activity. In order to explain some aspects of this pattern the activity of the thymidylate synthetase in extracts from some organs of the silkworm was also examined.

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides a role for trisulphides.

1. The aminolaevulinate synthetase activator of fresh extracts of semi-anaerobically grown Rhodopseudomonas spheroids was resolved into two fractions by ion-exchange chromatography. One fraction was identified as cystine trisulphide (CySSSCy). Analysis of the other fraction indicated the presence of about equal amounts of glutathione trisulphide (GSSSG) and the mixed trisulphide of glutathione and cystine (GSSSCy). 2. Four further fractions with activator activity were observed on ion-exchange chromatography of extracts prepared by methods similar to those described earlier [Neuberger et al. (1973)Biochem. J. 136,491-499]. These activators were generated by the extraction procedure. Two of them have been identified as trisulphanedisulphonate (S5O62-) and additional cystine trisulphide. 3. For the series of polysulphanedisulphonates (-O3S-Sn-SO3-, n greater than or equal to 1), activator activity at muM concentrations was exhibited only by compounds with n greater than 3. This, together with the results described above, indicates that for a compound R-Sn-R' (where R and R' are organic or inorganic groups) the only structural requirement for activity is n greater than or equal to 3. 4. Oxygenation of a semipanaerobic culture of R. spheroids for 1.5h before harvesting the cells produced a decrease of more than 90% in the cellular content of cystine trisulphide and glutathione trisulphides. 5. Chromatography on DEAE-Sephadex confirmed the presence of multiple forms of aminolaevulinate synthetase in extracts of R. spheroides [Tuboi et al. (1970) Arch.Biochem. Biophys. 138,147-154]. Oxygenation of a semi-anaerobic culture resulted in the disappearance of high-activity enzyme (a-form) and the accumulation of low-activity enzyme (b-form) in the cell. Spontaneous activation [Marriott et al. (1969) Biochem. J. 111,385-394] And activation by cystine trisulphide both resulted in the almost complete conversion of the b-form into the a-form.

Allostery of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase in Clostridium: another conserved generic characteristic.

Enzymological studies were done to characterize the allosteric control of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase in three species of Clostridium. Allosteric control was identified as feedback inhibition by phenylalanine and was qualitatively similar for the DAHP synthetases of C. butyricum, C. acetobutylicum, and C. tetanomorphum. Quantitative differences in the enzymology and kinetics of allosteric control distinguished C. tetanomorphum from C. butyricum and C. acetobutylicum. Crude extracts contained apparent proteolytic activity which could be fractionated from DAHP synthetase. The proteolytic activity was more labile than DAHP synthetase in extracts and was progressively inactivated by serial freeze-thaw treatments. Protease activity was at least partially inhibited by phenylmethylsulfonyl-fluoride. The method of comparative allostery of DAHP synthetase distinguishes the genera Bacillus and Clostridium, each having a strongly conserved pattern of regulation for DAHP synthetase. The data reinforce previous conclusions that allosteric control patterns governing the activity of DAHP synthetase are stable, reliable generic characteristics.

δ-aminolaevulinate synthetase in extracts of cultured soybean cells

1. 1. δ-Aminolaevulinate synthetase has been detected in extracts of soybean callus tissues and the enzyme activity reached its maximum when callus were 11 days old. 2. 2. The presence of a compound which seems to control δ-aminolaevulinate synthetase activity was demonstrated. The enzyme was present in the soluble fraction and was very labile. 3. 3. When crude extracts or 500 × g supernatant were stored at 4−6°, the apparent activity of δ-aminolaevulinate synthetase increased by as much as 3–6 times, while the activities of δ-aminolaevulinate dehydratase and succinyl-CoA synthetase did not significantly change during the storage. Activation was dependent on concentrations of cells suspensions during disruption and aging. 4. 4. Gel filtration with Sephadex G-25 of 2000 × g supernatants produced an enzyme fraction 30% more active. An increase in enzyme activity was observed when dark-grown callus were exposed to light. 5. 5. The addition of ATP, gibberellic acid and δ-aminolaevulinate to the culture media diminished activity; iron deficiency also produced an δ-aminolaevulinate synthetase less active.

Activation of delta-aminolaevulate synthetase in extracts of Rhodopseudomonas spheroides.

1. The spontaneous activation of delta-aminolaevulate synthetase in extracts from Rhodopseudomonas spheroides grown semi-anaerobically in the light requires oxygen and does not take place anaerobically in the dark. 2. Activation is completely prevented by azide or cyanide and is partially inhibited by chlorpromazine. These compounds inhibit markedly the succinoxidase activity of extracts. 3. NADH delays activation, but when it has been oxidized by the extract activation begins at the normal rate and complete activation occurs. By contrast both the rate and the extent of activation are markedly decreased by even small amounts of carboxylic acids. 4. The inhibitory effects of succinate and citrate on activation can be prevented by malonate and fluorocitrate respectively. 5. These results suggest that for activation to occur some endogenous compound has to be oxidized via the electron transport chain. 6. Activation occurs under anaerobic conditions in the light, probably by photo-oxidation.

Differentiation in acanthamoeba: Glycogen levels and glycogen synthetase activity during encystment

1. 1. When cells of Acanthamoeba castellanii (Neff) are placed on a salts medium the amoebas encyst over a period of about 30 h with the formation of a cellulose-containing cyst wall 1. The amoebas contain a material which is presumably glycogen because this material and liver glycogen give the same products when incubated with α- and β-amylases. During the first 4 h of encystment, cellular glycogen levels decreased to between one half or one third of that found in amoeboid cells and subsequently decreased to 10–20% of the original values. 2. 2. Cell-free extracts of amoebas contained a synthetase which catalyzed the incorporation of glucose from UDP-[ 14C]glucose into glycogen. CDP-glucose, CDP-glucose, GDP-glucose and ADP-glucose were less effective than UDP-glucose as substrates. The reaction required a glycogen primer and was highly sensitive to the presence of glucose 6-phosphate. 3. 3. The specific activity of the glycogen synthetase in extracts prepared from Acanthamoeba populations at different times of encystment was observed to increased during the first 4–8 h followed by a decrease to values below that found in the initial extracts. The results suggest that the decrease in cellular glycogen levels during the first 4 h of encystment do not involve a diminished capacity of the cells for glycogen synthesis due to a decrease in the activity of the synthetase.

Control of delta-aminolaevulate synthetase activity in Rhodopseudomonas spheroides.

1. delta-Aminolaevulate synthetase from Rhodopseudomonas spheroides grown semi-anaerobically undergoes a spontaneous activation during the first hour after the disruption of cells when homogenates are stored at 4 degrees . 2. After cultures of R. spheroides growing semi-anaerobically are oxygenated no activation of delta-aminolaevulate synthetase occurs in cell extracts. Cessation of activation in extracts is almost complete 10min. after oxygenation of cells has begun. 3. A heat-stable fraction of low molecular weight from semi-anaerobic cells reactivates delta-aminolaevulate synthetase in extracts of oxygenated cells and appears to contain a compound responsible for the spontaneous activation. 4. A heat-stable fraction of low molecular weight from oxygenated cells inhibits the spontaneous activation in extracts of semi-anaerobic cells. 5. The effect of oxygen on the rate of bacteriochlorophyll synthesis in R. spheroides may be mediated through alterations in the concentrations of a low-molecular-weight activator and inhibitor of delta-aminolaevulate synthetase.

Occurrence of a specific and reversible inhibitor of δ-aminolevulinate synthetase in extracts of Rhodopseudomonas spheroides

The presence of a specific and reversible inhibitor of δ-aminolevulinate synthetase in extracts of Rhodopseudomonas spheroides was evidenced. The inhibitor was very labile and lost its inhibitory activity fairly rapidly when the extracts were stored at 0 °, and because of this decrease of active inhibitor, the apparent activity of ALA synthetase in extracts increased by as much as 7–9 times, while the activities of δ-amino-levulinate dehydratase and succinyl-CoA synthetase did not appreciably change during the storage. The inhibitor was also sensitive to light. The inhibitor could be removed from extracts by simply passing the extracts through a column of Dowex-1 and by this way the activity of ALA synthetase in extracts was also increased by about 9 times. The inhibitor could be removed also by dialysis, ultrafiltration and gel filtration with Sephadex G-15 or G-25. Furthermore, fractions having the inhibitor activity could be prepared by fractionating fresh extracts with ethanol or acetic acid. The inhibition was reversible. The inhibitor differs from any of hemin, protoporphyrin and bacteriochlorophyl or its related compounds. It was suggested that the inhibitor may play an important role in tetrapyrrole synthesis in R. spheroides by controling the activity of ALA synthetase.