METHYLATION OF E. COLI TRANSFER RIBONUCLEIC ACIDS BY A tRNA ADENINE-l-METHYLTRANSFERASE FROM RAT BRAIN CORTEX AND BULK-ISOLATED NEURONS

Abstract— Brain cortices or bulk-isolated neuronal cell bodies prepared from cortices of 8-day old male rats were used as the source of a l-methyl adenine-specific tRNA methyltransferase (tRNA-AMT). Ammonium sulfate fractionation and chromatography on spheroidal hydroxylapatite and Sephadex G-200 yielded an 80-fold purified enzyme, as determined by using E. coli bulk tRNA as substrate. The kinetic parameters of tRNA-AMT for the substrate S -adenosyl- l -methionine (SAM) ( K _m= 6 μM) and the inhibitor, S -adenosyl- l -homocysteine (SAH) ( K _i= 3.4 μ m ) were determined and several SAH analogs tested as inhibitors. S -Adenosyl- l -cysteine (SAC) ( 10 ^-4 m ) and S -adenosyl- d -homocysteine (SADH) (10^-4 m ) produced a 35 and a 21% reduction in enzyme activity, respectively. The effects of Mg²⁺, NH₄⁺ acetate and of the polyamines spermine, putrescine and spermidine on the brain tRNA-AMT mimicked the effects of these agents on hepatic tRNA-AMT (G lick et al , 1975).
Comparing the ability of cerebral tRNA-AMT to methylate E. coli tRNA^glu2, tRNA^val, tRNA^phe and bulk tRNA revealed tRNA^glu2 as the best and tRNA^phe as the least effective substrate.
tRNA-AMT prepared from neuronal cell bodies showed closely similar characteristics to the cortical enzyme. A comparison of the activities of tRNA-AMT in neurons and glial cells revealed higher values in the former.     

Evidence for inhibitory substrate interactions during cometabolism of 3,4-dichlorobenzoate by Acinetobacter sp. strain 4-CB1

Abstract Acinetobacter sp. strain 4-CB1 cometabolized 3,4-dichlorobenzoate (3,4-DCB), via 3-chloro-4-hydroxybenzoate (3-C-4-OHB) and 4-carboxy-1,2-benzoquinone, in the presence of 4-chlorobenzoate (4-CB) as a growth substrate. In resting cell incubations, 3,4-DCB acted competitively as an inhibitor with 4-CB metabolism, and as a substrate inhibitor of its own metabolism. The inhibitor constant K _i and the affinity constant K _m were 800 and 181 μM, respectively with a maximal rate of 3,4-DCB disappearance of 18.8 nmol 3,4-DCB min⁻¹ (mg protein)⁻¹, in resting cells incubated solely with 3,4-DCB. Resting cells were less tolerant to 3,4-DCB than growing cells, as noted from the inhibition constants ( K _i). Moreover, 3-C-4-OHB competitively inhibited 4-hydroxybenzoate monooxygenase by acting as a pseudosubstrate ( K _i= 7.3 μ M). The next sequential intermediate, 4-carboxy-1,2-benzoquinone, uncompetitively inhibited 4-CB metabolism in resting cell incubations. Thus, 3,4-DCB inhibited its own cometabolism as well as metabolism of 4-CB in Acinetobacter sp. strain 4-CB1.     

Regionally Distinct N-Methyl-D-Aspartate Receptors Distinguished by Quantitative Autoradiography of [3H]MK-801 Binding in Rat Brain

Abstract: Quantitative autoradiography of [³H]MK-801 binding was used to characterize regional differences in N -methyl- d -aspartate (NMDA) receptor pharmacology in rat CNS. Regionally distinct populations of NMDA receptors were distinguished on the basis of regulation of [³H]MK-801 binding by the NMDA antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). CPP inhibited [³H]MK-801 binding in outer cortex (OC) and medial cortex (MC) with apparent K _i values of 0.32-0.48 μ M , whereas in the medial striatum (MS), lateral striatum (LS), CA1, and dentate gyrus (DG) of hippocampus, apparent K _i values were 1.1-1.6 μ M . In medial thalamus (MT) and lateral thalamus (LT) the apparent K _i values were 0.78 μ M . In the presence of added glutamate (3 μ M ), the relative differences in apparent K _i values between regions maintained a similar relationship with the exception of the OC. Inhibition of [³H]MK-801 binding by the glycine site antagonist 7-chlorokynurenic acid (7-ClKyn) distinguished at least two populations of NMDA receptors that differed from populations defined by CPP displacement. 7-ClKyn inhibited [³H]MK-801 binding in OC, MC, MS, and LS with apparent K _i values of 6.3-8.6 μ M , whereas in CA1, DG, LT, and MT, K _i values were 11.4-13.6 μ M . In the presence of added glycine (1 μ M ), the relative differences in apparent K _i values were maintained. Under conditions of differential receptor activation, regional differences in NMDA receptor pharmacology can be detected using [³H]MK-801 binding.     

Characteristics and regulation of ammonium (methylammonium) transport in Rhizobium meliloti

Abstract Rhizobium meliloti can synthesize an ammonium carrier as demonstrated by concentrative methylammonium uptake ( K _m= 2 μ M), which is competitively inhibited by ammonium ( K _i= 1.2 μ M). The carrier is under nitrogen control: its synthesis is repressed by ammonium, glutamine and asparagine. The asparagine effect may be of importance for the transfer of ammonia from the bacterium to the plant in the nodule.     

Rhodopseudomonas cryptolactis, sp. nov., a new thermotolerant species of budding phototrophic purple bacteria

Abstract A proton motive force (Δp) generated by oxidation of CO in membrane vesicles of Clostridium thermoautotrophicum drove active transport of l -alanine, glycine and l -serine. The maximum rate ( V _max) for l -alanine transport was 12 × higher at 50°C than at 25°C. The apparent transport constant ( K _t) for l -alanine uptake was 30–40 μM and independent of the temperature. Glycine was a substrate for the l -alanine transport system as demonstrated by the competitive inhibition of l -alanine uptake by glycine ( K _i= 6 μ M), by the kinetics of glycine uptake ( K _t= 7 μ M) and by the inhibiton of glycine uptake by l -alanine. The uptake kinetics of glycine was biphasic. l -Serine inhibited competitively also l -alanine and glycine transport but it was taken up by a separate transport system. The rate of amino acid transport, but not the K _t, was dependent on the value of the proton motive force.     

An Endogenous Aminoenkephalinase Inhibitor: Purification and Characterization of Arg0-Met5-Enkephalin from Bovine Striatum

Abstract: Arg⁰-Met⁵-enkephalin (Arg⁰-MEK) was isolated from bovine striatum and purified to homogeneity. The peptide was extracted with trichloroacetic acid, followed by column chromatography successively on Bio-Sil C₈, semipreparative HPLC Radial-Pak C₁₈, fast protein liquid chromatography (FPLC) Mono S, HPLC Ultrasphere-ODS, Supelco C₁₈, Lichromsorb C₁₈, and μBondapak C₁₈. The peptide content was followed by radioimmunoassay with an antibody against synthetic Met-enkephalin. For each of the six HPLC and FPLC systems, the elution time of the immunoreactive fractions coincided exactly with that of synthetic Arg⁰-MEK. The purified peptide showed a highly homogeneous profile in three different analytical HPLC systems. Its retention time and three-dimensional UV spectrum were identical to those of the synthetic Arg⁰-MEK. The structure of the purified material was identified by microsequencing as the hexapeptide Arg-Tyr-Gly-Gly-Phe-Met. Ninety percent of the purified peptide was in oxidized form containing equimolar amounts of Met-( R )- and Met-( S )-sulfoxide. The reduced Arg⁰-MEK inhibited aminoenkephalinase with a K _i of 2.2 µ M , and its sulfoxide analogue inhibited it with a K _i of 8.9 µ M . The reduced or oxidized peptide suppressed the electrically induced contraction of rat vas deferens with an ED₅₀ of 5 µ M , and the effect could be reversed by equimolar naloxone. Our data indicate that Arg⁰-MEK is an immediate Met-enkephalin precursor and an endogenous inhibitor.     

STUDIES ON S-ADENOSYLHOMOCYSTEINE INHIBITION OF HISTAMINE TRANSMETHYLATION IN BRAIN

Abstract— The mechanism of histamine methyltransferase (HMT) inhibition by S -adenosylhomocysteine (SAH) has been investigated on a partially purified enzyme from guinea-pig brain. The kinetic data indicated that this inhibition was competitive with respect to S -adenosylmethionine (SAMe) and noncompetitive with respect to histamine. The K _t, values (about 5 ± 10⁻⁶ M in both cases) indicated that SAH had a higher affinity than SAHe or histamine for the enzyme.
In rats, after administration of a small dose of SAH, methylation of intracisternally injected [³H]histamine was not modified.
However, treatment with l -DOPA or pyrogallol induced a decrease in [³H]histamine methylation, presumably due to a modification in the SAMe/SAH ratio in the brain. Hence, histamine methylation in brain could be regulated according to the value of this ratio and thus related to methylation of other biogenic amines.     

N 5, N10-Methylenetetrahydromethanopterin reductase from Methanosarcina barkeri

N ⁵ N ¹⁰-Methylenetetrahydromethanopterin reductase was purified 13-fold to apparent homogeneity from methanol grown Methanosarcina barkeri . The colourless enzyme was found to be composed of four identical subunits of apparent molecular mass 36 kDa. It catalysed the reduction of methylenetetrahydromethanopterin ( K _m=15 μM) to methyltetrahydromethanopterin with reduced coenzyme F₄₂₀ ( K _m=12 μM) at a specific rate ( V _max) of 2200 μmol min⁻¹· mg protein⁻¹ ( K _cat=1320 s⁻¹). With respect to coenzyme specificity, molecular properties and catalytic mechanism the enzyme was found to be similar to CH₂=H₄MPT reductase of Methanobacterium thermoautotrophicum which phylogenetically is only distantly related to M. barkeri .     

Triiodothyronine Is a High-Affinity Inhibitor of Amino Acid Transport System L1 in Cultured Astrocytes

Abstract: The relationship between the transport of thyroid hormones and that of amino acids was examined by measuring the uptake of amino acids that are characteristic substrates of systems L, A, and N, and the effect of 3,3',5-triiodo-L-thyronine (T₃) on this uptake, in cultured astrocytes. Tryptophan and leucine uptakes were rapid, Na⁺-independent, and efficiently inhibited by T₃ (half-inhibition at ∼ 2 μ M ). Two Na⁺-independent L-like systems (L₁ and L₂), common to leucine and aromatic amino acids, were characterized kinetically. System L₂ had a low affinity for leucine and tryptophan ( K _m= 0.3–0.9 m M ). The high-affinity system L₁ ( K _m∼ 10 μ M for both amino acids) was competitively inhibited by T₃ with a K _i of 2–3 μ M (close to the T₃ transport K _m). Several T₃ analogues inhibited system L₁ and the T₃ transport system similarly. Glutamine uptake and α-(methylamino)isobutyric acid uptake were, respectively, two and 200 times lower than tryptophan and leucine uptakes. T₃ had little effect on the uptakes of glutamine and α-(methylamino)isobutyric acid. The results indicate that the T₃ transport system and system L₁ are related.     

The Uptake of Carnitine by Slices of Rat Cerebral Cortex

Abstract: The properties of carnitine transport were studied in rat brain slices. A rapid uptake system for carnitine was observed, with tissue-medium gradients of 38 ± 3 for L-[¹⁴CH₃]carnitine and 27 ± 3 for D-[¹⁴CH₃]carnitine after 180 min incubation at 37°C in 0.64 mM substrate. Uptake of L- and D-carnitine showed saturability. The estimated values of K _m for L- and D-carnitine were 2.85 mM and 10.0 mM, respectively; but values of V _max (1 μmol/min/ml in-tracellular fluid) were the same for the two isomers. The transport system showed stereospecificity for L-carnitine. Carnitine uptake was inhibited by structurally related compounds with a four-carbon backbone containing a terminal carboxyl group. L-Carnitine uptake was competitively inhibited by γ-butyrobetaine ( K _i= 3.22 mM), acetylcarnitine ( K _i= 6.36 mM), and γ-aminobutyric acid ( K _i= 0.63 mM). The data suggest that carnitine and γ-aminobutyric acid interact at a common carrier site. Transport was not significantly reduced by choline or lysine. Carnitine uptake was inhibited by an N₂ atmosphere, 2,4-dinitrophenol, carbonylcyanide- N -chlorophenylhydrazone, potassium cyanide, n-ethylmaleimide, and ouabain. Transport was abolished by low temperature (4°C) and absence of glucose from the medium. Carnitine uptake was Na⁺-dependent, but did not require K⁺ or Ca²⁺.     

Inhibition of Rat Brain Microsomal Na+,K+-ATPase by S-Adenosylmethionine

Abstract: Rat brain microsomes were preincubated with S -adenosylmethionine (SAM), MgCl₂, and CaCl₂, then re-isolated, and the activity of Na⁺,K⁺-ATPase determined. SAM inhibited the Na⁺,K⁺-ATPase activity compared with microsomes subjected to similar treatment in the absence of SAM. A biphasic inhibitory effect was observed with a 50% decrease at a SAM concentration range of 0.4 μ M -3.2 μ M and a 70% reduction at a concentration range above 100 μ M . Inclusion of either S- adenosylhomocysteine or 3-deazaadenosine in the preincubations prevented the SAM inhibition of Na⁺,K⁺-ATPase activity. The inhibition by SAM appeared to be Mg²⁺- or Ca²⁺-dependent.     

Enteric Glia Exhibit P2U Receptors that Increase Cytosolic Calcium by a Phospholipase C-Dependent Mechanism

Abstract: Calcium signaling in fura-2 acetoxymethyl ester-loaded enteric glia was investigated in response to neuroligands; responses to ATP were studied in detail. Carbachol (1 m M ), glutamate (100 µ M ), norepinephrine (10 µ M ), and substance P (1 µ M ) did not increase the intracellular calcium concentration ([Ca²⁺]_i) in cultured enteric glia. An increasing percentage of glia responded to serotonin (4%; 100 µ M ), bradykinin (11%; 10 µ M ), and histamine (31%; 100 µ M ), whereas 100% of glia responded to ATP (100 µ M ). ATP-evoked calcium signaling was concentration dependent in terms of the percentage of glia responding and the peak [Ca²⁺]_i achieved; responses were pertussis toxin insensitive. Based on responsiveness of enteric glia to purinergic agonists and peak [Ca²⁺]_i evoked, ATP = UTP > ADP > β,γ-methyleneadenosine 5'-triphosphate ≫ 2-methylthioadenosine 5'-triphosphate = α,β-methyleneadenosine 5'-triphosphate = AMP = adenosine, suggesting a glial P_2U receptor. Depletion of d - myo -inositol 1,4,5-trisphosphate-sensitive calcium stores by thapsigargin (10 µ M ) abolished glial responses to ATP. Similarly, calcium responses were decreased 92% by U-73122 (10 µ M ), an inhibitor of phospholipase C, and 93% by the phorbol ester phorbol 12-myristate 13-acetate (100 n M ), an activator of protein kinase C. Thus, cultured enteric glia can respond to neurotransmitters with increases in [Ca²⁺]_i. Our data suggest that glial responses to ATP are mediated by a P_2U receptor coupled to activation of phospholipase C and release of intracellular calcium stores.     

OHIO-1 β-lactamase resistant to mechanism-based inactivators

Abstract Spontaneous mutants of OHIO-1 β-lactamase, an SHV-1 family enzyme, resistant to inactivation by clavulanic acid, sulbactam and tazobactam, have been isolated. The resistant mutant (M4) was inhibited by 100 μg/ml ampicillin plus 32 μg/ml clavulanic acid compared to ≤2 μg/ml clavulanic acid required for the parent strain. The pI of the mutant beta-lactamase was 7.0, identical to the parent enzyme. Kinetic parameters showed that the M4 enzyme had an increased V_max/K_m ratio for all beta-lactam substrates compared to the parent enzyme. The apparent K _i for clavulanic acid, sulbactam and tazobactam was 15.1, 182 and 18 μM, respectively, up to 70-fold higher than the parent enzyme. Partial nucleotide sequencing revealed that the mutant enzyme had a predicted methionine₆₉→ isoleucine₆₉ substitution accounting for the observed changes in substrate specificity.     

α-Guanidinoglutaric Acid, an Endogenous Convulsant, as a Novel Nitric Oxide Synthase Inhibitor

Abstract: The effects of α-guanidinoglutaric acid (GGA), the levels of which were increased in the cobalt-induced epileptic focus tissue in the cerebral cortex of cats, on brain nitric oxide synthase (NOS) activity were observed. GGA inhibited NOS activity in a linear mixed manner ( K _i = 2.69 µ M ) and was as effective as N ^G-monomethyl- l -arginine (MeArg; K _i = 3.51 µ M ), a well-known NOS inhibitor. Although MeArg was synthesized by substituting the guanidino nitrogen of l -arginine (Arg), GGA was a non-guanidino nitrogen-substituted guanidino compound. On the other hand, Arg, which is an endogenous NOS substrate, elevates the threshold of seizures induced by GGA. There is evidence that GGA is an endogenous, potent, and non-guanidino nitrogen-substituted NOS inhibitor and that suppression of nitric oxide biosynthesis may be involved in GGA-induced convulsions. Therefore, GGA may be a useful tool in elucidating the chemical nature of NOS and the physiological function of nitric oxide.     

Effect of Homo-β-Proline and Other Heterocyclic GAB A Analogues on GABA Uptake in Neurons and Astroglial Cells and on GABA Receptor Binding

Abstract: Two groups of GABA (γ-aminobutyric acid) analogues, one comprising derivatives of β-proline and the other compounds structurally related to nipecotic acid, were investigated as potential inhibitors of high-affinity GABA transport in neurons and glial cells, as well as displacers of GABA receptor binding. In addition to cis -4-hydroxynipecotic acid, which is known as a potent inhibitor of GABA uptake, homo-β-proline was the only compound which proved to be a potent inhibitor of glial as well as neuronal GABA uptake. IC₅₀ values for GABA uptake into glial cells and brain cortex "prisms" were 20 and 75 μM, respectively, and the IC₅₀ value obtained for GABA uptake into cultured neurons was 10 μM. A kinetic analysis of the action of homo-β-proline on GABA uptake into cultured astrocytes and neurons showed that this compound acts as a competitive inhibitor of GABA uptake in both cell types. From the apparent K _m values, K _i values for homo-β-proline of 16 and 6 μM could be calculated for glial and neuronal uptake, respectively. This mechanism of action strongly suggests that homo-β-proline interacts with the GABA carriers. Furthermore, homo-β-proline also displaced GABA from its receptor with an IC₅₀ value of 0.3 μM. The cis -4-hydroxynipecotic acid analogues, cis- and trans-4-mercaptonipecotic acid, had no inhibitory effect on glial or neuronal GABA uptake. Other SH reagents, PCMB, NEM and DTNB, were shown to be relatively weak inhibitors of GABA uptake into cultured astrocytes, suggesting that SH groups are not directly involved in the interaction between GABA and its transport carrier.     

Effect of Inhibitors of GABA Transaminase on the Synthesis, Binding, Uptake and Metabolism of GABA

Abstract: Four catalytic inhibitors of GABA aminotransferase (gabaculine, γ-acetylenic GABA, γ-vinyl GABA, ethanolamine O -sulphate) as well as aminooxyacetic acid and valproate were studied for effects on neurochemical assays for GABA synthesis, receptor binding, uptake and metabolism in mouse and rat brain preparations. Gabaculine did not interfere with GABA synthesis as reflected by the activity of glutamate decarboxylase (GAD), it was only a weak inhibitor (IC₅₀= 0.94 mM) of GABA receptor binding sites but was a moderately potent inhibitor of GABA uptake (IC₅₀= 81 μM) and very potent (IC₅₀= 1.8 μM) with respect to inhibition of the GABA-metabolizing enzyme GABA aminotransferase (GABA-T). γ-Acetylenic GABA was a weak inhibitor of GAD and GABA binding (IC₅₀ > 1 mM), but virtually equipotent to inhibit uptake and metabolism of GABA (IC₅₀ 560 and 150 μM, respectively). This was very similar to γ-vinyl GABA, except that this drug did not decrease GAD activity. Ethanolamine O -sulphate was found to show virtually no inhibition of GAD and GABA uptake, but was a fairly potent inhibitor of GABA binding (IC₅₀= 67 μM) and in this respect, 500 times more potent than as an inhibitor of GABA-T. Aminooxyacetic acid was a powerful inhibitor of both GAD and GABA-T (IC₅₀ 14 and 2.7 μM, respectively), but had very little affinity to receptor and uptake sites for GABA. Valproate showed no effects on GABA neurochemical assays which could be related to anticonvulsant action. The present results suggest that the anticonvulsant properties of the four catalytic inhibitors of GABA-T tested are at least in part mediated through a direct influence on GABA receptors and uptake sites.     

MJ0400 from Methanocaldococcus jannaschii exhibits fructose-1,6-bisphosphate aldolase activity

The central carbon metabolism is well investigated in bacteria, but this is not the case for archaea. MJ0400-His₆ from Methanocaldococcus jannaschii catalyzes the cleavage of fructose-1,6-bisphosphate (FBP) to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate with a V _max of 33 mU mg⁻¹ and a K _m of 430 μM at 50 °C. MJ0400-His₆ is inhibited competitively by erythrose-4-phosphate with a K _i of 380 μM and displays heat stability with a half-life of c . 1 h at 100 °C. Hence, MJ0400 is the second gene encoding for an FBP aldolase in M. jannaschii . Previously, MJ0400 was shown to act as an 2-amino-3,7-dideoxy- d - threo -hept-6-ulosonic acid synthase. This indicates that MJ0400 is involved in both the carbon metabolism and the shikimate pathway in M. jannaschii .     

Inhibitors of Histamine Methylation in Brain Promote Formation of Imidazoleacetic Acid, Which Interacts with GABA Receptors

Abstract: In brain, the precursor of imidazoleacetic acid (IAA), a GABA_A agonist but a GABA_C antagonist, is not known. In the periphery, IAA derives from oxidation of histamine. But in brain, histamine is thought to be metabolized solely by histamine methyltransferase (HMT), forming tele -methylhistamine (t-MH) and tele -methylimidazoleacetic acid (t-MIAA). We showed that [³H]histamine (intracerebroventricularly) could be converted to IAA in brains of rats, a process increased by inhibition of HMT. This demonstrated that brain can oxidize histamine and suggested that endogenous histamine might also be oxidized if HMT activity were reduced. We examined, in rat cerebral cortex, effects of the following HMT inhibitors (mg/kg i.p.): metoprine (10), tacrine (10), velnacrine (10, 30), and physostigmine (1, 2). Tacrine was a potent inhibitor ( K _i∼ 22 n M ). To measure histamine in tissue that contained HMT inhibitors, we developed a gas chromatography-mass spectrometry method. After 2 h, all drugs reduced endogenous levels of t-MH and t-MIAA and increased levels of histamine and IAA. Our results show that inhibition of HMT promotes oxidation of histamine in brain, probably by shunting histamine to an alternative metabolic pathway. Formation of IAA provides a novel interaction between histaminergic and GABAergic systems in brain. Accumulation of IAA should be considered when inhibitors of HMT are used to probe brain histamine function.     

Degradation of Dynorphin-Related Peptides by the Puromycin-Sensitive Aminopeptidase and Aminopeptidase M

Abstract: The degradation of dynorphin-related peptides by the puromycin-sensitive aminopeptidase and aminopeptidase M was examined using these peptides as alternate substrate inhibitors. K _i determinations showed that both aminopeptidases exhibit a higher affinity for longer dynorphin-related peptides, i.e., K _i for dynorphin A-17 = 23–30 n M with the K _i increasing to 25–50 µ M for the enkephalin pentapeptides. Binding appears dependent not only on peptide length, but also on its sequence. With aminopeptidase M, as the peptide size increases from five to 10 amino acids, k _cat remains relatively constant; however, as the peptide size increases beyond a decapeptide, k _cat decreases significantly. With the puromycin-sensitive aminopeptidase, similar results were obtained except that k _cat was greatest for the pentapeptide. Thus, if one considers k _cat/ K _m as the relevant kinetic constant for estimating in vivo peptide hydrolysis, these results are consistent with the involvement of aminopeptidase M and the puromycin-sensitive aminopeptidase in the degradation of extended dynorphin-related peptides.     

Indole-3-acetaldehyde reductase in Phycomyces blakesleeanus. Characterization of the enzyme

lndole-3-acetaldehyde reductase (lAAld reductase EC 1.2.3.1) from Phycomyces blakesleeanus Bgff., a 38 kDa polypeptide as determined by gel filtration, is probably localized in the cytoplasm. The formation of indole-3-ethanol (lEt) is dependent on the presence of NAD(P)H. The enzymatic reduction of IAAId shows a pH optimum between 6 and 8 and a temperature optimum at 30°C. Enzyme activity follows Michaelis Menten kinetic (K_m= 200 μ M for IAAId; K_m= 24 μ M for NADPH). The isoelectric point of the IAAId reductase is at pH 5.4. Phenylacetaldehyde and benzaldehyde are competitive substrates. Hydroxymeihylindole promotes the reductive IEt formation, whereas NADP⁺ is a non-competitive inhibitor. Changes in lAAJd reductase activity correlate with certain developmental stages of the fungus.