Purification and properties of several transfer RNA methyltransferases fromS. typhimurium

Summary A fast method for a single-step fractionation of a number of tRNA methyltransferases fromSalmonella typhimurium is described. The method basically consists of ion-exchange chromatography on a phosphocellulose column and permits the separation of the enzymes forming mt⁶A, m¹G, m⁵U, m⁷G. The enzyme fractions appear sufficiently purified to allow the estimation of some molecular and kinetic properties. The apparent K_M for adenosylmethionine range between 1.5 to 3.2×10⁻⁵ M, whereas K_M for undermethylated tRNA range between 3.1×10⁻⁵ M to 3.1×10⁻⁴ M. Glycerol gradient determination indicates the following M_r for the native proteins: 25×10³, 40×10³, 50×10³ and 65×10³ for m⁷G-, mt⁶A-, m¹G- and m⁵U-forming enzymes, respectively. A complete analysis of methylated nucleosides formedin vivo inS. typhimurium has been obtained: it also allowed us to infer the pattern of the various tRNA methyltransferases for this prokaryote. The tRNA methyltransferase forming mt⁶A has been isolated for the first time from any type of cell.     

Evaluation of kinetic data: What the numbers tell us about PRMTs

Protein arginine N-methyltransferase (PRMT) kinetic parameters have been catalogued over the past fifteen years for eight of the nine mammalian enzyme family members. Like the majority of methyltransferases, these enzymes employ the highly ubiquitous cofactor S-adenosyl-l-methionine as a co-substrate to methylate arginine residues in peptidic substrates with an approximately 4-μM median K_M. The median values for PRMT turnover number (k_cat) and catalytic efficiency (k_cat/K_M) are 0.0051 s⁻¹ and 708 M⁻¹ s⁻¹, respectively. When comparing PRMT metrics to entries found in the BRENDA database, we find that while PRMTs exhibit high substrate affinity relative to other enzyme-substrate pairs, PRMTs display largely lower k_cat and k_cat/K_M values. We observe that kinetic parameters for PRMTs and arginine demethylase activity from dual-functioning lysine demethylases are statistically similar, paralleling what the broader enzyme families in which they belong reveal, and adding to the evidence in support of arginine methylation reversibility.     

The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons. I. Effects of conditioned medium

S-Adenosylmethionine:homocysteine methyltransferase activity was monitored during embryogenesis of the housefly, Musca domestica. A rapid decrease in the activity of S-adenosylmethionine:homocysteine methyltransferase was observed during the first 3 hr of embryogenesis. Activity continued to decline less rapidly until hatching at 12 hr. An inverse relationship was found to exist between the activities of S-adenosylmethionine:homocysteine methyltransferase and the tRNA methyltransferases during Musca embryogenesis.     

Some properties of γ-tocopherol methyltransferase solubilized from spinach chloroplasts

γ-Tocopherol methyltransferase occurs in the chloroplast fraction of spinach leaves. Its specific activity with γ-tocopherol and S-adenosyl-l-methionine was 3.91 nmol hr⁻¹ mg⁻¹ protein. The enzyme was effectively solubilized by 6 mM sodium deoxycholate from the membrane fraction of chloroplasts. The activity was maximum at pH 7.5 and 35°. γ-Tocopherol was preferred to β-tocopherol (25:7). The K_m value for S-adenosyl-l-methionine as methyl donor was 9.1 μM.     

Amdxidation and demethylation of N,N-dimethylaniline by rabbit liver and lung microsomes effects of age and metals

Lung N-oxidase enzyme activity was about three times higher than liver N-oxidase at the pH optimum, about pH 8.9, whereas the activities were nearly the same at more physiological ranges of pH. The lung N-oxidase was also stimulated about 2-fold by 100 mM Mg²⁺ and by 0.1 mM Hg²⁺, whereas liver N-oxidase activity was inhibited by these concentrations of ions. The difference in response of liver and lung enzymes to Mg²⁺ and Hg²⁺ was not altered by preparing the microsomes in the presence of 50 mM ethylenediamine tetraacetic acid (EDTA) in 0.1 M Tris (hydroxymethyl) amino methane (Tris) buffer or 50 mM EDTA in 0.1 M KPO₄ buffer, both at pH 7.6, indicating that the differences are probably not due to the presence of endogenous metals. The difference between the liver and lung N-oxidase systems may be due to the tissue environment rather than to the enzyme itself since mercury stimulation of lung N-oxidation began to disappear upon partial purification of the N-oxidase enzymes. In contrast to the effects of Hg²⁺ and Mg²⁺, 1 mM Ni²⁺ enhanced liver N-oxidase activity about 30% and 5 mM Ni²⁺ stimulated lung enzyme activity about 30% whereas concentrations above 10 mM were inhibitory to both N-oxidases. Both liver and lung demethylase activities were inhibited by these concentrations of Mg²⁺, Hg²⁺ and Ni²⁺.Various suifhydryl reagents were also tested for their effects on these enzymes. The mercurials, para-chloromercurybenzoate (pCMB) and phenylmercuryacetate (PMA) at concentrations of 0.1 mM had the same effect as HgCl₂ inhibiting both demethylases and liver N-oxidase, but stimulating lung N-oxidase activity. However, 0.1 mM to 1 mMN-ethylmaleimide (NEM) and iodoacetamide had little if any effect on either liver or lung N-oxidase. It was also shown that Hg²⁺ effects on N-oxidase activity could be overcome by dilution.Changes in N,N-dimethyl aniline (DMA) metabolism with age were followed in rabbits from 4 days old to adult. There was a steady increase in lung demethylase activity and N-oxidase activity in the liver and lung to adult levels. However, the liver demethylase had a sharp increase in activity between 2 weeks and 1 month much like that seen with benzphetamine demethylase in rabbit liver.Activities of N-demethylase in liver and lung, and N-oxidr.se in liver from new-born rabbits were from 10 to 20 % of adult levels. However, in lung, N-oxidase activities in the newborn were about 50 % of adult levels. Microsomal N-oxidation in lungs from 2-day-old rabbits was stimulated by 0.1 mM mercury just as in the adult.     

Increased activity of rat liver N2-guanine tRNA methyltransferase II in response to liver damage

Alterations in rat liver transfer RNA (tRNA) methyltransferase activities have been observed after liver damage by various chemicals or by partial hepatectomy. The qualitative and quantitative nature of these activity changes and the time course for their induction have been studied. Since homologous tRNAs are essentially fully modified in vivo, E. coli tRNAs were used as in vitro substrates for the rat liver enzymes in these studies. Each of the liver-damaging agents tested rapidly caused increases in activities of the enzyme(s) catalyzing methyl group transfer to tRNAs that have an unmodified guanine at position 26 from the 5′ end of the molecule. This group of tRNAs includes E. coli tRNA^Nfmet, tRNA^Ala₁, tRNA^Leu₁, or ^Leu₂, and tRNA^Ser₃ (Group 1). In each case N²-methylguanine and N²,N²-dimethylguanine represented 90% or more of the products of these in vitro methylations. The product and substrate specificity observed are characteristic of N²-guanine methyltransferase II ($\text{S-}\text{adenosyl}\text{-}\text{L}\text{-}\text{methionine:tRNA}$ (guanine-2)-methyltransferase, EC 2.1.1.32). In crude and partially purified preparations derived from livers of both control and treated animals this enzyme activity was not diminished significantly by exposure to 50°C for 10 min. The same liver-damaging agents induced little or no change in the activities of enzymes that catalyze methyl group transfer to various other E. coli tRNAs that do not have guanine at position 26 (Group 2). The results of mixing experiments appear to rule out the likelihood that the observed enzyme activity changes are due to stimulatory or inhibitory materials present in the enzyme preperations from control or treated animals. Thus, our experiments indicate that liver damage by each of several different methods, including surgery or administration of chemicals that are strong carcinogens, hepatotoxins, or cancer-promoting substances, all produce changes in liver tRNA methyltransferase activity that represent a selective increase in activity of N²-guanine tRNA methyltransferase II. It is proposed that the specificity of this change is not fortuitous, but is the manifestation of an as yet unidentified regulatory process.     

A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase—extensive moonlighting in mitochondrial tRNA biogenesis

Transfer RNAs (tRNAs) reach their mature functional form through several steps of processing and modification. Some nucleotide modifications affect the proper folding of tRNAs, and they are crucial in case of the non-canonically structured animal mitochondrial tRNAs, as exemplified by the apparently ubiquitous methylation of purines at position 9. Here, we show that a subcomplex of human mitochondrial RNase P, the endonuclease removing tRNA 5′ extensions, is the methyltransferase responsible for m¹G9 and m¹A9 formation. The ability of the mitochondrial tRNA:m¹R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes. In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein. Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5′ end processing.     

Specific potassium ion interactions facilitate homocysteine binding to betaine‐homocysteine S‐methyltransferase

Betaine‐homocysteine S‐methyltransferase (BHMT) is a zinc‐dependent methyltransferase that uses betaine as the methyl donor for the remethylation of homocysteine to form methionine. This reaction supports S‐adenosylmethionine biosynthesis, which is required for hundreds of methylation reactions in humans. Herein we report that BHMT is activated by potassium ions with an apparent K_M for K⁺ of about 100 µM. The presence of potassium ions lowers the apparent K_M of the enzyme for homocysteine, but it does not affect the apparent K_M for betaine or the apparent k_cat for either substrate. We employed molecular dynamics (MD) simulations to theoretically predict and protein crystallography to experimentally localize the binding site(s) for potassium ion(s). Simulations predicted that K⁺ ion would interact with residues Asp26 and/or Glu159. Our crystal structure of BHMT bound to homocysteine confirms these sites of interaction and reveals further contacts between K⁺ ion and BHMT residues Gly27, Gln72, Gln247, and Gly298. The potassium binding residues in BHMT partially overlap with the previously identified DGG (Asp26‐Gly27‐Gly28) fingerprint in the Pfam 02574 group of methyltransferases. Subsequent biochemical characterization of several site‐specific BHMT mutants confirmed the results obtained by the MD simulations and crystallographic data. Together, the data herein indicate that the role of potassium ions in BHMT is structural and that potassium ion facilitates the specific binding of homocysteine to the active site of the enzyme. Proteins 2014; 82:2552–2564. © 2014 Wiley Periodicals, Inc.     

Studies on the activities of some methyltransferases in the livers and tumor cells from tumor-bearing mice

The activities of $\text{S}$-adenosylmethionine synthetase isozymes and some methyltransferases have been measured in liver and tumor cells of tumor-bearing mice. Following intraperitoneal transplantation of Ehrlich ascites tumor cells into mice, the activity of the β-form of the synthetase isozymes markedly increased, whereas that of the α-form did not increase so much, and the activity of tRNA methyltransferases increased gradually, while that of phospholipid, glycine and guanidoacetate methyltransferases did not. It was shown that tumor cells have only the γ-form of the synthetase and that the activity of tRNA methyltransferases in the tumor cells was very high, while that of other methyltransferases was not detectable.     

Hyperthermophilic DNA Methyltransferase M.PabI from the Archaeon Pyrococcus abyssi

Genome sequence comparisons among multiple species of Pyrococcus, a hyperthermophilic archaeon, revealed a linkage between a putative restriction-modification gene complex and several large genome polymorphisms/rearrangements. From a region apparently inserted into the Pyrococcus abyssi genome, a hyperthermoresistant restriction enzyme [PabI; 5′-(GTA/C)] with a novel structure was discovered. In the present work, the neighboring methyltransferase homologue, M.PabI, was characterized. Its N-terminal half showed high similarities to the M subunit of type I systems and a modification enzyme of an atypical type II system, M.AhdI, while its C-terminal half showed high similarity to the S subunit of type I systems. M.PabI expressed within Escherichia coli protected PabI sites from RsaI, a PabI isoschizomer. M.PabI, purified following overexpression, was shown to generate 5′-GTm6AC, which provides protection against PabI digestion. M.PabI was found to be highly thermophilic; it showed methylation at 95°C and retained at least half the activity after 9 min at 95°C. This hyperthermophilicity allowed us to obtain activation energy and other thermodynamic parameters for the first time for any DNA methyltransferases. We also determined the kinetic parameters of k_cat, K_{m, DNA}, and K_{m, AdoMet}. The activity of M.PabI was optimal at a slightly acidic pH and at an NaCl concentration of 200 to 500 mM and was inhibited by Zn²⁺ but not by Mg²⁺, Ca²⁺, or Mn²⁺. These and previous results suggest that this unique methyltransferase and PabI constitute a type II restriction-modification gene complex that inserted into the P. abyssi genome relatively recently. As the most thermophilic of all the characterized DNA methyltransferases, M.PabI may help in the analysis of DNA methylation and its application to DNA engineering.     

O-Methylation of flavonoid substrates by a partially purified enzyme from soybean cell suspension cultures.

A methyltransferase, which catalyzes the methylation of luteolin (K_m, 16 μM) using S-adenosyl-l-methionine as the methyl donor, has been purified about 38-fold from cell suspension cultures of soybean (Glycine max L., var. Mandarin). The following 3,4-dihydroxy phenolic compounds were also methylated: luteolin 7-O-glucoside (K_m, 28 μm), quercetin (K_m, 35 μm), eriodictyol (K_m, 75 μm), 5-hydroxyferulic acid (K_m, 227 μm), dihydroquercetin (K_m, 435 μm), and caffeic acid (K_m, 770 μm). Rutin and quercetin 3-O-glucoside were poor substrates. Methylation proceeded only in the meta position. The enzyme was unable to catalyze the methylation of p-coumaric acid, m-coumaric acid, ferulic acid, isoferulic acid, sinapic acid, apigenin, or naringenin. While the isoflavones biochanin A and daidzein did not serve as substrates, texasin (6,7-dihydroxy-3′-methoxyisoflavone) was methylated (K_m, 35 μm). The methylation of caffeic acid and quercetin showed a pH optimum of 8.6–8.9. The enzyme required Mg²⁺ ions for maximum activity (approximately 1 mm) and could be totally inhibited by EDTA (10 mm). The K_m for S-adenosyl-l-methionine was 11 μm. S-Adenosyl-l-homocysteine inhibited the methylation of luteolin by S-adenosyl-l-methionine.     

Purification and Characterization of S-Adenosyl-l-methionine:6a-Hydroxymaackiain 3-O-Methyltransferase from Pisum sativum

The isoflavonoid phytoalexin pisatin is synthesized by Pisum sativum in response to microbial infection and certain other forms of stress. An enzyme which synthesizes pisatin by methylating the 3-hydroxyl of (+)6a-hydroxymaackiain (HMK) was extracted from CuCl₂-stressed pea seedlings. The enzyme was enriched 370-fold by (NH₄)₂SO₄ precipitation, DEAE chromatography, chromatofocusing, and hydrophobic interaction chromatography (HIC), to a specific activity of 8.2 microkatals per gram protein. Enzyme activity profiles from chromatofocusing and HIC columns suggested the presence of two isozymes, of pl 5.2 and 4.9. Nondenaturing gel filtration of the HIC-purified enzyme gave a single peak of activity at the same elution volume as BSA (66 kilodaltons); the active fractions showed two proteins upon SDS-PAGE, of M_r 66,000 and 43,000. The smaller protein was most abundant in chromatographic fractions containing peak enzyme activity throughout purification. In a partially purified preparation, this 43 kilodalton protein was the only one photoaffinity labelled by [³H]S-adenosyl-l-methionine. The purified enzyme preferred the (+) over the (−) stereoisomer of HMK and other pterocarpans; overall, (+)HMK was the best substrate. K_m values were 2.3 micromolar for (+)HMK and 35 micromolar for S-adenosyl-l-methionine. The methyltransferase had a pH optimum of 7.9 and no apparent divalent cation requirement.     

Properties of Phenylalanyl-tRNA Synthetase and Phenylalanine Ammonia-lyase of Pine Suspension Cultures

Phenylalanyl-tRNA synthetase and phenylalanine ammonia-lyase activities were demonstrated in partially purified extracts of pine (Pinus elliottii) suspension cultures. The optimum pH for the phenylalanyl-tRNA synthetase reaction was 7.5 and the optimum ATP and Mg²⁺ concentrations were 1.0 and 15 mM respectively. Pine, calf liver and yeast tRNA were inadequate substitutes for pea tRNA in the synthetase reaction mixtures. The optimum pH for the phenylalanine ammonia-lyase reaction was 9.0. The K_m for phenylalanine was approximately 6.6 × 10^?5M. The activity of both enzymes in the partially purified extracts was unstable on storage.     

Amino acid residues of the Escherichia coli tRNA(m5U54)methyltransferase (TrmA) critical for stability, covalent binding of tRNA and enzymatic activity

The Escherichia coli trmA gene encodes the tRNA(m⁵U54)methyltransferase, which catalyses the formation of m⁵U54 in tRNA. During the synthesis of m⁵U54, a covalent 62-kDa TrmA-tRNA intermediate is formed between the amino acid C324 of the enzyme and the 6-carbon of uracil. We have analysed the formation of this TrmA-tRNA intermediate and m⁵U54 in vivo, using mutants with altered TrmA. We show that the amino acids F188, Q190, G220, D299, R302, C324 and E358, conserved in the C-terminal catalytic domain of several RNA(m⁵U)methyltransferases of the COG2265 family, are important for the formation of the TrmA-tRNA intermediate and/or the enzymatic activity. These amino acids seem to have the same function as the ones present in the catalytic domain of RumA, whose structure is known, and which catalyses the formation of m⁵U in position 1939 of E. coli 23S rRNA. We propose that the unusually high in vivo level of the TrmA-tRNA intermediate in wild-type cells may be due to a suboptimal cellular concentration of SAM, which is required to resolve this intermediate. Our results are consistent with the modular evolution of RNA(m⁵U)methyltransferases, in which the specificity of the enzymatic reaction is achieved by combining the conserved catalytic domain with different RNA-binding domains.     

tRNA METHYLTRANSFERASE ACTIVITY IN NEONATAL AND MATURE MAMMALIAN NEURAL TISSUE

Abstract— The activity and kinetic characteristics of tRNA methyltransferases were measured with enzyme preparations obtained from neonatal and adult mouse brain tissue. Both neonatal and mature brain enzyme preparations were shown to contain a considerable amount of protein methylase activity which could interfere with the measurement of the tRNA methyltransferases. When increasing amounts of the unfractionated enzymes from young and adult neural tissue were added to reaction mixtures, the saturation kinetics were found to be considerably different. However, fractionation of the samples by precipitation at pH 5 resulted in an increase in the enzyme activity of preparations obtained from adult brain. Although the precipitation at pH 5 allowed a quantitative recovery of the enzyme activity of immature brain samples, this partial purification step led to an apparent activation of the tRNA methyltransferases in adult preparations. This activation was shown to be independent of differential changes in the thermolability of the enzymes but rather to be associated with an increase in the sites methylated and the measured affinity of the adult enzyme preparations with the tRNA substrate. Nicotinamide, a potent inhibitor of tRNA methyltransferase activity in other tissues, was shown to be ineffective in modulating brain tRNA methyltransferase activity. The results are discussed in light of the possible modulation of the activity of specific enzyme species and the alterations in the synthesis of nucleic acid precursors during neural development.     

Effect of adenosylhomocysteine and other analog thioethers on a prokaryotic tRNA (guanine-7)-methyltransferase

S-Adenosylhomocysteine (SAH), a potent inhibitor of methyltransferases, and several thioethers structurally related to SAH, have been tested as potential inhibitors of tRNA (guanine-7)-methyltransferase from Salmonella typhimurium. The tested compounds are l-, d-, dl-S-adenosylhomocysteine, S-adenosylcysteine, methylthioadenosine, butylthioadenosine, thioethanoladenosine, isobutylthioadenosine, S-inosylhomocysteine, and methylthioinosine. Among them the highest inhibitory activity has been shown by SAH (K_i = 8 μM), whereas butylthioadenosine, isobutylthioadenosine, and thioethanoladenosine are almost inactive as inhibitors. The other compounds inhibit the enzyme with K_i values ranging between 400 and 800 μm. From these data it is possible to evaluate the importance of the -NH₂ and -COOH groups of the substrate in the binding to the enzyme molecule, as well as other features such as the chirality at the α-carbon atom and the length of the hydrocarbon chain connecting the -NH₂ and -COOH groups to the aromatic ring of adenosine. The aminic group of the adenosine is also critical, because S-inosylhornocysteine and methylthioinosine are poorer inhibitors in comparison with SAH and methylthioadenosine.     

Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10

N-1 Methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae, the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m¹G₉ modification in yeast. Although there are 19 unique tRNA species that contain a G at position 9 in yeast, and whose fully modified sequence is known, only 9 of these tRNA species are modified with m¹G₉ in wild-type cells. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known, and sequences that are shared between all substrate or all nonsubstrate tRNAs have not been identified. Here, we demonstrate that the in vitro methylation activity of yeast Trm10 is not sufficient to explain the observed pattern of modification in vivo, as additional tRNA species are substrates for Trm10 m¹G₉ methyltransferase activity. Similarly, overexpression of Trm10 in yeast yields m¹G₉ containing tRNA species that are ordinarily unmodified in vivo. Thus, yeast Trm10 has a significantly broader tRNA substrate specificity than is suggested by the observed pattern of modification in wild-type yeast. These results may shed light onto the suggested involvement of Trm10 in other pathways in other organisms, particularly in higher eukaryotes that contain up to three different genes with sequence similarity to the single TRM10 gene in yeast, and where these other enzymes have been implicated in pathways beyond tRNA processing.     

Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures

N¹-methyladenosine (m¹A) is found at position 58 in the T-loop of many tRNAs. In yeast, the formation of this modified nucleoside is catalyzed by the essential tRNA (m¹A58) methyltransferase, a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p). In this report we describe the cloning, expression and characterization of a Gcd14p homolog from the hyperthermophilic bacterium Thermus thermophilus. The purified recombinant enzyme behaves as a homotetramer of ~150 kDa by gel filtration and catalyzes the site- specific formation of m¹A at position 58 of the T-loop of tRNA in the absence of any other complementary protein. S-adenosylmethionine is used as donor of the methyl group. Thus, we propose to name the bacterial enzyme TrmI and accordingly its structural gene trmI. These results provide a key evolutionary link between the functionally characterized two-component eukaryotic enzyme and the recently described crystal structure of an uncharacterized, putative homotetrameric methyltransferase Rv2118c from Mycobacterium tuberculosis. Interest ingly, inactivation of the T.thermophilus trmI gene results in a thermosensitive phenotype (growth defect at 80°C), which suggests a role of the N¹-methylation of tRNA adenosine-58 in adaptation of life to extreme temperatures.     

Kinetic properties and regulation of glycerol-3-phosphate dehydrogenase from the overwintering, freezing-tolerant gall fly larva, Eurosta solidagenis

The kinetic properties of cytoplasmic glycerol-3-P dehydrogenase from the third instar larva of the gall fly, Eurosta solidaginis, were studied with emphasis on temperature effects on the enzyme and the regulation of enzyme activity during the synthesis of the cryoprotectant, glycerol. Isoelectrofocusing revealed one major and two minor forms of the enzyme with no alteration in the pI's or relative activities of the forms in larvae acclimated to 24 versus ?30 °C. Kinetic properties of the enzyme were also the same in larvae acclimated to high and low temperatures. Arrhenius plots were linear over a 30 to 0 °C range with an activation energy of 12,630 ± 185 cal/mol and a Q₁₀ of 2.16. The K_m for dihydroxyacetone-P was constant, at 50 μM, between 30 and 10 °C but increased by 75% at 0 °C; this increase may be a factor in the cessation of glycerol synthesis which occurs below 5 °C in this species. The K_m(NADH), by contrast, was higher (5–6 μM) at 30 °C but decreased (3 μM) at lower temperatures. In the reverse direction, K_m's were 340 μM for glycerol-3-P and 12 μM for NAD⁺. Effects of most inhibitors (of the forward reaction), glycerol-3-P (K_i = 2.4 mM), NAD⁺ (K_i = 0.2 mM), ATP, Mg·ATP, and P_i, were unaltered by assay temperature but ADP effects were potentiated by low temperature while citrate inhibition was greatest at high temperatures. Glycerol and sorbitol, which accumulate as cryoprotectants in E. solidaginis, had no significant effects on kinetic constants at any temperature but decreased the V_max activity of the enzyme. Thermal inactivation studies showed an increased thermal stability of the larval enzyme compared to the homologous enzyme from rabbit muscle while added polyols stabilized enzyme activity, decreasing the rate of enzyme inactivation at 50 °C.