Synthetase Molecules Research Articles

The effect of triiodothyronine on fatty acid synthetase activity and content in differentiating ob 17 preadipocytes

The effect of triiodothyronine on the activity and amount of the key lipogenic enzyme fatty acid synthetase was studied in differentiating preadipocyte cells (ob 17) isolated from ob/ob mouse epididymal fat pad. In the presence of physiological concentrations of insulin, the acquisition of adipose morphology was accompanied by a parallel increase (10–15-fold) in synthetase specific activity and radioimmunoassayable amount relative to soluble cellular proteins. Inclusion of T 3 at confluence significantly enhanced synthetase activity and content, with a maximum of 1.5–2-fold above controls at the physiological 1.5 nM concentration, whether insulin is present or not. During adipose conversion, T 3 increased the development of enzyme activity and, after a longer lag period, the accumulation of the synthetase. Our results suggest that the stimulating effect of T 3 upon synthetase activity could involve as a first step the activation of preexisting inactive synthetase molecules and as a second one an increased accumulation of activable synthetase. After longer culture periods, inactive radioimmunoassayable synthetase accumulated.

Elevated levels of asparagine synthetase activity in physiologically and genetically derepressed Chinese hamster ovary cells are due to increased rates of enzyme synthesis.

The activity of asparagine synthetase in Chinese hamster ovary (CHO) cells is increased in response to asparagine deprivation or decreased aminoacylation of several tRNAs (Andrulis, I. L., Hatfield, G. W., and Arfin, S. M. (1979) J. Biol. Chem. 254, 10629-10633). CHO cells resistant to beta-aspartylhydroxamate have up to 5-fold higher levels of asparagine synthetase than the parental line (Gantt, J. S., Chiang, C. S., Hatfield, G. W., and Arfin, S. M. (1980) J. Biol. Chem. 255, 4808-4813). We have investigated the basis for these differences in enzyme activity by combined radiochemical and immunological techniques. The asparagine synthetase of beef pancreas was purified to apparent homogeneity. Antibodies raised against the purified protein cross-react with the asparagine synthetase of CHO cells. Immunotitrations show that the amount of enzyme protein in physiologically or genetically derepressed CHO strains is proportional to the level of enzyme activity. Measurement of the relative rates of asparagine synthetase synthesis by pulse-labeling experiments demonstrate that the difference in the number of asparagine synthetase molecules is closely correlated with the rate of enzyme synthesis. In contrast, the half-life of asparagine synthetase in wild type cells and in physiologically or genetically derepressed cells is very similar. It appears that the increased levels of asparagine synthetase can be attributed solely to an increased rate of enzyme synthesis.

Anti-AMP antibody precipitation of multiply adenylylated forms of glutamine synthetase from Escherichia coli: a model relating both concentration and density of antigenic sites with the antibody-antigen interaction.

Sheep antibodies directed against an AMP-bovine serum albumin conjugate exhibit highly specific binding toward AMP. These antibodies bind to the AMP moiety of adenylylated glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] from Escherichia coli and to no other antigenic determinant on the protein. E coli glutamine synthetase can exist in variously modified (isomeric) forms that differ with respect to the number (0-12) and the distribution of identically adenylylated subunits [Ciardi, J. E., Cimino, F. & Stadtman, E. R. (1973) biochemistry 12, 4321-4330]. Using this enzyme, together with the AMP-specific antibodies, we have investigated the effects of the total concentration, population density, and topographical distribution of multiple identical antigenic determinants on the antigen-antibody interaction. Stopped flow fluorescence measurements show that the rate and extent of initial binding of the antibodies to the antigen are a function of the total concentration of AMP groups and are independent of the number of AMP groups der dodecamer. However, the rate of lattice formation increases with increasing epitope density, and the maximal amount of glutamine synthetase precipitated is directly proportional to the average number of adenylylated subunits per dodecamer. The data suggest that partially adenylylated enzyme preparations are composed of subpopulations of glutamine synthetase molecules that differ in their tendency to form precipitable aggregates, due presumably to differences in the topographical distribution of antigenic determinants on the surface of the enzyme. The enzyme species that form soluble immune complexes do so possibly due to intramolecular crosslinkage of the bivalent antibodies with adenylylated subunits to the exclusion of intermolecular crosslinkage.

Dibutyryl cyclic AMP decreases glutamine synthetase in cultured 3T3-L1 adipocytes.

Glutamine synthetase specific activity increases greater than 100-fold during the insulin-mediated differentiation of confluent 3T3-L1 cells into adipocytes. Incubation of the adipocytes for 22 h with 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline, 0.2 mM 8-bromo-cyclic AMP, 10 micro M epinephrine, or 1 microgram of alpha 1-24 adrenocorticotropic hormone/ml decreased glutamine synthetase by greater than 60%. During the same incubation period, there was no effect of these compounds on protein or on the specific activities of glucose-6-P dehydrogenase or hexokinase. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase activity was half-maximal at 50 micro M dibutyryl cyclic AMP. Furthermore, between 10 micro M and 5 mM dibutyryl cyclic AMP, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase was similar in the absence or presence of 1 microgram of insulin/ml. Immunotitration of glutamine synthetase activity from 3T3 adipocytes indicates that the dibutyryl cyclic AMP-mediated decrease in the activity is due to a decrease in the cellular content of glutamine synthetase molecules. We studied the effects of dibutyryl cyclic AMP on the synthesis and degradation of glutamine synthetase. Synthesis rate was estimated from the incorporation of L-[35S]methionine into glutamine synthetase during a 60-min incubation period. Degradation rate was estimated from the first order disappearance of radioactivity from glutamine synthetase in 3T3 adipocytes previously incubated with L-[35S]methionine. Glutamine synthetase was isolated by immunoprecipitation followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Incubation of 3T3 adipocytes with dibutyrl cyclic AMP resulted in a rapid decline in the apparent synthesis rate of glutamine synthetase. In addition, dibutyryl cyclic AMP treatment increased the initial rate of glutamine synthetase degradation. The half-life of glutamine synthetase was 24.5 h in control cultures and 16 h in dibutyryl cyclic AMP-treated cultures. In contrast, dibutyryl cyclic AMP had little effect on the synthesis or degradation of soluble protein. Our data indicate that the dibutyryl cyclic AMP-mediated decrease in 3T3 adipocyte glutamine synthetase activity results from a decrease in the synthesis rate and an increase in the initial degradation rate of the enzyme.

Affinity purification of anti-pigeon liver fatty acid synthetase immunoglobulin and comparative immunoreactivity of the catalytic reactions

Rabbit anti-pigeon liver fatty acid synthetase antibody was prepared by affinity chromatography on Sepharose-fatty acid synthetase to near monospecificity (98% or more) as shown by immunodiffusion plates and rocket immunoelectrophoresis. Immunotitrations of the highly purified monospecific antibody against the overall activity and partial activities of fatty acid synthetase were then carried out. Only 6 mol of antibody/mol of enzyme was required to inactivate overall fatty acid synthetase activity and the condensation reaction, while 12 to 18 mol were required to partially inactivate the β-ketoacyl reductase and the malonyl- and acetyl-CoA transferases. Palmitoyl-CoA thioesterase (deacylase) activity was not inhibited by the antibody. The degree of inactivation of the partial reactions by antibody was not affected by dissociation of the fatty acid synthetase. Immunoprecipitation of the enzyme indicated that there are approximately 35 immunoreactive sites on the fatty acid synthetase molecule. The possible implications of these results to an understanding of the structural organization of pigeon liver fatty acid synthetase and its antigenic determinants are discussed.

Averages of glutamine synthetase molecules as obtained with various skin and electron dose conditions.

Averaged projections of individual glutamine synthetase molecules have been obtained by using electron microscopy and image processing. The methodology of correlation averaging under low dose conditions is described in detail. Because of their low signal-to-noise ratio, images made under low dose conditions cannot be directly interpreted in terms of high resolution features. Computer averaging of these images reveals a division of the subunit projection into two domains whose sizes agree with results of Lei et al [2] limited proteolysis experiments.

Energy cost of proofreading to increase fidelity of transfer ribonucleic acid aminoacylation

The paradox of relatively error free function in biological systems composed of relatively error prone components has recently come under intensive investigation. In the case of tRNA aminoacylation, aminoacyl-tRNA synthetases were discovered to have a separate function that allows misacylated molecules to be hydrolyzed more rapidly than correctly acylated molecules. This additional function of the synthetases provides a proofreading or verification mechanism that is believed to improve significantly the overall accuracy of tRNA aminoacylation. In this paper we provide an explicit relationship between the accuracy achieved by proofreading and the energy cost. Experimental data available in the literature are examined in light of this relationship. The following are the principal conclusions from our study: (1) high-accuracy proofreading of tRNA aminoacylation has a high energy cost, as much as 100 times greater than indications from early experimental work; (2) the minimum net error derived in previous theoretical studies is never actually reached; (3) mechanisms in which misacylation and subsequent proofreading occur on the surface of the same synthetase molecule achieve a much higher accuracy than mechanisms in which these functions occur on the surface of different synthetase molecules.

The site of β-chitin fibril formation in centric diatoms. II. The chitin-forming cytoplasmic structures

β-Chitin fibril formation was investigated in the centric diatoms Cyclotella cryptica, Cyclotella nana, Cyclotella meneghiniana, and Thalassiosira fluviatilis with transmission electron microscopy. The pores in the silica valves from which the fibrils originate were examined in serial sectioning to identify the underlying cytoplasmic structures involved in chitin formation. In all species synthesizing chitin, the fibrils originate from special invaginations of the plasma membrane. These invaginations are conical and bear a special coat on their cytoplasmic face. This coat shows a hexagonal arrangement. It is assumed that this coat plays a role in chitin fibril formation, which seems to be a transmembrane event. Kinetic data on fibril formation and calculations on the package density of synthetase molecules which should be localized in the conical plasma membrane invagination are discussed in relation to the special coat subunits. A relationship between the conical invaginations and a chitosome-like type of vesicles was observed only in Thalassiosira.

Limited proteolysis of glutamine synthetase is inhibited by glutamate and by feedback inhibitors.

Limited proteolysis of glutamine synthetase from Escherichia coli has been studied under nondenaturing conditions (pH 7.6, 20 degrees C). Trypsin cleaves the polypeptide chain of glutamine synthetase into two principal fragments, Mr = about 32,000 and 18,000. The covalently bound AMP group is attached to the larger fragment and its presence does not affect cleavage. Although the cleaved polypeptide chain does not dissociate under nondenaturing conditions, catalytic activity is lost. Chymotrypsin and Staphylococcus aureus protease produce similar cleavages in glutamine synthetase. The substrate L-glutamate retards tryptic as well as chymotryptic digestion. Tryptic digestion is also retarded by some of the feedback inhibitors of glutamine synthetase including CTP, L-alanine, L-serine, L-histidine, and glucosamine 6-phosphate. An implication of these findings is that there is a region of the glutamine synthetase polypeptide chain that is particularly susceptible to proteolysis. Either the glutamate and inhibitor sites are formed partly by this suceptible peptide or the binding of glutamate and some inhibitors induces conformational changes within the E. coli glutamine synthetase molecule in the region of the susceptible peptide.

Ultracentrifugation studies of yeast valyl-tRNA synthetase and of its interaction with tRNA Val

Yeast valyl-tRNA synthetase and its complexes with yeast tRNA Val were investigated by means of analytical ultracentrifugation. A molecular weight of 125 700 ± 1500 and a sedimentation coefficient ( S 0 20,w) of 6.3 ± 0.3 were found for the native enzyme. When the enzyme (3–60 μM) was mixed with its cognate tRNA, several types of complex were observed, depending on the relative amount of the two macromolecules. In the presence of equimolecular amounts of tRNA and enzyme, a complex formed by the association of one of each molecule was observed with a sedimentation coefficient of about 7.3 S. However, for tRNA enzyme stoichiometries lower than one, beside the 1 : 1 complex, a complex of higher molecular weight was observed, with a sedimentation coefficient of about 10.0 S which fits with the association of two valyl-tRNA synthetase molecules with one tRNA molecule. This 2 : 1 complex was predominant for tRNA enzyme stoichiometries lower than 0.3. It dissociated into the 1 : 1 complex upon addition of monovalent salts or MgCl 2, suggesting the electrostatic nature of the interaction in this association. All these association and dissociation phenomena were detected over a large range of pH (6.0–7.5) and in various buffers.

Distribution of yeast fatty acid synthetase subunits: three-dimensional model of the enzyme.

Rabbit and goat antibodies against the isolated alpha and beta subunits of yeast fatty acid aynthetase were raised and characterized. The purified IgG fractions were studied as to their capability to precipitate their antigens and the holoenzyme and to inhibit the partial reactions involved in overall fatty acyl-CoA synthesis. The specificity of the antibodies was investigated by immunodiffusion and by immunotitration. Native enzyme was crosslinked with each of the antibodies, and dimeric and oligomeric groups of IgG-crosslinked fatty acid synthetase molecules were isolated by sucrose density gradient centrifugation. Electron microscopic investigation of the crosslinked material as well as other data led us to suggest a three-dimensional model of yeast fatty acid synthetase.

The variation of intermolecular contacts in helical aggregates of glutamine synthetase

Glutamine synthetase from E. coli is a large (MW 600,000) oligomeric enzyme composed of twelve identical polypeptide chains of MW 50,000. These twelve subunits are arranged at the vertices of two eclipsed hexagons to produce a structure with symmetry 622 or D6 as shown by electron microscopy and X-ray diffraction. The dodecameric molecule has a hexagonal profile of approximately 140 Å diameter and a thickness of approximately 90 Å. When treated with divalent cations such as Co++, Zn++, Ni++ or Cu++, glutamine synthetase molecules aggregate along their six-fold axes to form long strands and these strands will often wrap around one another to form cables (Fig. 1). The aggregation is completely reversed by the addition of excess Mn++ resulting in regeneration of full enzymatic activjjy. The most regular helical aggregates are formed in the presence of 10 mM Co++ at pH 7.0, and we have used these helical cables to study the three dimensional structure and symmetry of the glutamine synthetase molecule and the types of protein-protein interactions involved in the formation of glutamine synthetase cables.

New crystal forms of glutamine synthetase and implications for the molecular structure

New crystal forms of glutamine synthetase from Escherichia coli are reported. Two of these (II A and II B) demand that the dodecameric molecule contains a 2-fold axis of symmetry perpendicular to the apparent hexagonal face. Whereas forms II A and II B and others reported previously (I and III A) were grown from enzyme containing covalently bound AMP groups, a third new form (III C) was grown from enzyme lacking covalently bound AMP groups. Form III C is isomorphous with form III A. This demonstrates that the addition of AMP groups, which profoundly affect the catalytic and regulatory properties of glutamine synthetase, does not alter the dimensions of the molecular envelope. Thus adenylylation of the enzyme does not seem to cause a quaternary structural transition, though small changes of intensities suggest that there may be tertiary structural changes within the subunits. Other new forms include form III B, a low symmetry polymorph, closely related to form III A, and form IV, a trigonal polymorph with large asymmetric unit. All crystal forms are consistent with a symmetry of 622 for the glutamine synthetase molecule.

The aminoacyl-tRNA synthetase-tRNA complex: Detection by differential labelling of lysine residues involved in complex formation

The interaction of tRNA Tyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by differential acetylation of lysine residues. The synthetase was trace-labelled in the free form and as the synthetase-tRNA Tyr complex with [ 3H]acetic anhydride. In a second step the two 3H-labelled enzyme preparations were fully acetylated with cold reagent under denaturing conditions and were mixed with synthetase that had been homogeneously labelled with excess [ 14C]acetic anhydride. Peptides containing labelled lysine residues were isolated after chymotryptic digestion and their 14C 3H ratios were determined. These ratios reflect the reactivity of primary amino groups towards acetic anhydride. Involvement of lysine side-chains in complex formation with tRNA Tyr was suggested from altered 14C 3H ratios. Out of the 22 primary amino groups of tyrosyl-tRNA synthetase at least three showed reduced reactivities towards acetic anhydride in the synthetase-tRNA Tyr complex by factors of 1.6, 1.9 and 6.8, respectively. The sequences around these lysine residues have been determined enabling their placement when the primary and tertiary structure of the enzyme are available (G. L. E. Koch, to be published). No lysine residue of increased reactivity in the synthetase-tRNA Tyr complex has been detected. Only one molecule of tRNA Tyr binds to the dimeric synthetase molecule under the conditions of the differential labelling. If the binding site for the tRNA is on one of the two identical subunits, any observed decrease in chemical reactivity of a particular lysine residue should not exceed a factor of two. The detection of a lysine residue which reacts about seven times more slowly in the synthetase-tRNA complex could therefore indicate that the single binding site is formed by both enzyme subunits.

Age-associated changes in glutamine synthetase activity in WI-38 cells

The human fibroblast cell line WI-38 shows a gradual decline in glutamine synthetase specific activity with increasing age in culture. However, the level of functional enzyme per cell does not change with age. Heat inactivation profiles indicate that glutamine synthetase from younger cell cultures is more heat labile than that from older cultures. A possible explanation for these observations is that alterations in the glutamine synthetase molecule occur with increasing age of WI-38 cells in culture.

Reconstruction of glutamine synthetase using computer averaging

The axial projection of the glutamine synthetase molecule has been reconstructed from electron micrographs of a stained preparation by using a new method of correlation search and averaging. The average over 50 individual molecules appears as a radial pattern with sixfold symmetry. The handedness evident in the average is attributed to nonuniformity of the negative stain.

The complexing of yeast transfer-RNA Tyr by tyrosyl-transfer-RNA synthetase of hog pancreas

Summary L-Tyrosyl-transfer-RNA synthetase (AMP) (EC 6.1.1.1) of hog pancreas forms a specific complex with yeast transfer-RNA Tyr , which can be isolated by gel filtration. Stoichiometric analysis of this complex reveals a maximum of one transfer-RNA Tyr per synthetase molecule, even though hog tyrosyl-transfer-RNA synthetase contains two identical protein subunits.

Three-Dimensional Electron Microscopy of Individual Biological Objects Part III. Experimental Results on Yeast Fatty Acid Synthetase

Abstract Experimental results of three-dimensional reconstructions of individual fatty acid synthetase (FAS) molecules are reported. The discussion of the structures has been concentrated on the symmetry relations within the molecules. A molecular structure with 6 copies arranged in point group 32 is probable. The limits of minimal radiation dose reconstruction are estimated.

Glutamine synthetase forms three- and seven-stranded helical cables.

When cobaltous ion is bound to glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2], the two-layered hexagonal molecules polymerize face-to-face, to form long strands. The strands then wind round each other to form three- and seven-stranded cables. The structures of these cables are not immediately evident from electron micrographs because of the confusing superposition of front and back portions of the cables. But optical diffraction and filtering by the procedure of Klug and DeRosier leads to interpretable images of the cables. Because a micrograph of the seven-stranded cable contains 24 views of the glutamine synthetase molecule, it is possible to reconstruct the three-dimensional electron density of a cable and its constituent molecules at a resolution of 30--50 A. This reconstruction confirms that the symmetry of a glutamine synthetase molecule is D6. It suggests that the single subunit is an oblate ellipsoid with its minor axis (about 48 A) roughly parallel to the 6-fold axis of the molecule and its major axis (about 63 A) perpendicular to the 6-fold axis of the molecule. The subunits of the two hexagonal layers of a molecule are eclipsed. Neighboring molecules along a strand also have their hexagonal faces together, but they are rotated about the strand axis by about 7 degrees with respect to one another, rather than being eclipsed. Six outer strands are coiled about a straight central strand, and each forms identical contacts with the central strand. Moreover, these contacts between central and outer strands are apparently similar to the contacts between neighboring outer strands.

The regulation of methionine and s-adenosylmethionine biosynthesis and utilization in mutants of Salmonella typhimurium with defects in s-adenosylmethionine synthetase.

The degree of methionine overproduction by metK mutants defective in S-adenosylmethionine (SAM) synthetase is shown to reflect, with one exception, the loss of function of this enzyme thus giving further support to the hypothesis that SAM is the co-repressor of this system. The exceptional mutant (metKx721) has a high constitutive SAM synthetase activity but its methionine production resembles that of mutants with no measurable SAM synthetase activity (typical metKx mutants) and this and other properties of the mutant suggest that the wild-type SAM synthetase protein has a role in methionine repression. Methionine repression control does not extend to the enzymes of spermidine synthesis from SAM but metKx mutants have a higher rate of utilization of SAM by this pathway than wild-type or other regulatory mutants. This could mean that the wild-type SAM synthetase molecule interacts with an enzyme of this pathway thus lowering its activity.