Uridine-diphospho-sulfoquinovose: diacylglycerol sulfoquinovosyltransferase activity is concentrated in the inner membrane of chloroplast envelopes

In experiments on the assembly of the sulfolipid sulfoquinovosyl diacylglycerol in envelope membranes of chloroplasts, UDP-sulfoquinovose (UDPS) was used with highest efficiency, and the corresponding enzyme, UDP-sulfoquinovose:diacylglycerol sulfoquinovosyltransferase, was partially characterized (E. Heinz et al., 1989, Eur J Biochem 184: 445–453). Here, we identified ³⁵S- and ³³P-labelled UDPS from various photosynthetically active organisms, suggesting that the sulfosugar nucleotide used for sulfolipid biosynthesis throughout the plant kingdom, including phototrophic bacteria, may indeed be UDPS. For attribution of the sulfolipid synthase to one of the two plastidial envelope membranes, these membranes were isolated from pea and spinach chloroplasts. The sulfoquinovosyltransferase was localized in the inner membrane of envelopes, which also contains the competing UDP-galactose:diacylglycerol galactosyltransferase. In contrast to the sulfoquinovosyltransferase, a substantial proportion of the galactosyltransferase was found in the outer membranes of envelopes from pea chloroplasts. Received: 6 October 1997 / Accepted: 31 January 1998     

High-affinity transport and phosphorylation of 2-deoxy-D-glucose in synaptosomes

2-Deoxy-d -glucose (2 DG) entered synaptosomes (from rat brain) by a high-affinity, Na⁺-independent glucose transport system with a K_m, of 0.24 mM. 3-O-methyl-glucose, D-glucose, and phloretin were competitive inhibitors of 2-DG transport with K_i's of 7 mM, 64 μM, and 0·75 μM, respectively. Insulin was without effect. 2-DG uptake was also saturable at high substrate concentrations with an apparent low affinity K_m, of 75 mM, where the K_l, for glucose was 17.5 mM. We are not certain whether the rate-limiting step for the low-affinity uptake system is attributable to transport or phosphorylation. However, the high-affinity glucose transport system probably is a special property of neuronal cell membranes and could be useful in helping to distinguish separated neurons from glial cells.     

PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM THE NERVOUS SYSTEM OF DIFFERENT INVERTEBRATES

—Choline acetyltransferase has been purified from three invertebrate species, namely snail (Helix aspersa), cockroach (Periplaneta americana) and horse shoe crab (Limulus polyphemus.) All three enzymes followed a Theorell-Chance enzyme mechanism with a sequential addition of the substrates. All three enzymes were activated by sodium and potassium chloride and inhibited by high concentrations of magnesium or calcium chloride. The apparent K_m for choline and acetyl-CoA was for snail: K_mch= 370 μm ,K_mAcetyl-CoA= 51μm ; cockroach:K_mCh= 550 μm , K_mAcely-CoA= 16 μm horse shoe crab:K_mCn= 2700 μm K_mAcctyl-coA= 68 μm CoA inhibited the enzymes competitively with respect to acetyl-CoA and non-competitively with respect to choline. Acetylcholine inhibited the enzymes competitively with respect to choline and non-competitively with respect to acetyl-CoA. All the enzymes were inhibited strongly by 5,5′-dithiobis (2-nitrobenzoate), iodoacetate, acryloylcholine, chloracetylcholine and 3-bromacetonyltrimethyl-ammonium. The enzymes were only weakly inhibited by the styrylpyridine derivatives. The isoelectric points were 5.3 and 5.0 for the horse shoe crab and cockroach enzymes respectively. All three enzymes showed low affinity for a cation-exchanger (CM-Sephadex).     

Different pathways of iodothyronine 5′-deiodination in rat cerebral cortex

Microsomal fractions of rat cerebral cortex catalyze the 5′-deiodination of 3,3′,5′-triiodothyronine (rT₃) in the presence of thiols such as dithiothreitol. Evidence is presented that two different enzymatic pathways are involved. One of these has a low apparent K_m (2.7 nM) for rT₃, is inhibited by nanomolar concentrations of thyroxine (T₄), but not by up to 1 mM 6-propyl-2-thiouracil (PTU). The other pathway has a high apparent K_m (31 nM) for rT₃, is inhibited by PTU, but not by <1 μM T₄. The relative proportion of rT₃ 5′-deiodination via either pathway depends on thyroid status, with increased contributions from the low-K_m system especially in short-term hypothyroidism.     

Stachyose synthesis in leaves of Cucurbita pepo

An enzyme synthesizing stachyose, galactinol-raffinose galactosyltransferase (EC2.4.1.67), has been purified ca 40-fold from mature leaves of Cucurbita pepo using ammonium sulphate precipitation, Sephadex gel filtration and DEAE-Sephadex gel chromatography. The purified enzyme fraction was separated from all but 2 % of the total,α-galactosidase activity extracted from the tissue. The enzyme was optimally active at pH 6.9 and was stable for at least a month at 4° in the presence of 20 mM 2-mercaptoethanol. The enzyme displayed high specificity for the donor galactinol (K_m 7.7 mM) and the acceptor raffinose (K_m 4.6 mM) and was unable to effect synthesis of any other member of the raffinose series of galactosyl-sucrose oligosaccharides. Co²⁺, Hg²⁺, Mn²⁺ and Ni²⁺ ions were particularly inhibitory; no metal ion promotion was observed and 5 mM EDTA was ineffective. Myo-inositol was strongly inhibitory (K_i 2 mM), melibiose weakly so. Tris buffer (0. 1 M) was also inhibitory. Galactinol hydrolysis occurred in the absence of the acceptor raffinose but there was no hydrolysis of either raffinose or stachyose in the absence of the donor galactinol. The reaction was readily reversible and exchange reactions were detected between substrates and products. It is proposed that the synthesis of stachyose in mature leaves ofC. pepo proceeds via this galactosyltransferase and not via α-galactosidase.     

Galactolipid synthesis in chromoplasts in vitro

Isolated chromoplasts from Narcissus pseudonarcissus flowers contain: a fatty acid synthesizing system; acyl-CoA synthetase (EC 6.2.1.3); glycero-phosphate acyltransferase (EC 2.3.1.15); acylglycero-phosphate acyltransferase; phosphatidate phosphatase (EC 3.1.3.4); diacylglycerol galactosyltransferase (EC 2.4.1.46); and diacylgalactosylglycerol galactosyltransferase, i.e. all enzymatic activities necessary for the synthesis of diacylgalactosylglycerol and diacylgalabiosylglycerol from acetate, HCO _- ³ , sn-glycerol 3-phosphate, and UDP-d-galactose. Diacylgalactosylglycerol and diacylgalabiosylglycerol, however, are synthesized from these precursors to only a very low extent in an in vitro system. This is attributed to a specificity of diacylglycerol galactosyltransferase for highly unsaturated diacylglycerols. Specificities of acyltransferase reactions were also found.     

Towards a phylogeny of phototrophic purple sulfur bacteria — the genus Ectothiorhodospira

While most strains of heterofermentative lactobacilli and strains of Leuconostoc species contained only traces of a dehydratase reacting with glycerol or propanediol-1,2, three strains of Lactobacillus brevis and one strain of L. buchneri that metabolized glycerol readily in the presence of glucose, contained propanediol-1,2 dehydratase (EC 4.2.1.28). This cobamide requiring enzyme from L. brevis B 18 was partially purified. It reacts with the substrates propanediol-1,2, glycerol and ethanediol-1,2 with the relative activities of about 3:2:1. This ratio remained unchanged throughout the purification procedure. The substrate affinities were measured: propanediol-1,2 K _m=0.6 mM, glycerol K _m=4 mM, ethanediol-1,2 K _m=5.3 mM coenzyme B₁₂ (substrate glycerol) K _m=0.007 mM. The activity of the dehydratase was promoted by potassium or ammonium ions and inhibited by sodium, lithium, magnesium or specially manganese. The apparent molecular weight of propanediol-1,2 dehydratase was determined as M_r=180,000.Dedicated to Prof. Dr. H. G. Schlegel on behalf of his 60th birthday     

Steroid sulfotransferase in hamster epididymis

Steroid sulfotransferase activity is present in the cytosol fraction of hamster epididymis. The activity of this enzyme is increased by magnesium ion. Cysteine is essential to assure optimal activity. Adenosine-3′-phosphate-5′-phosphosulfate is required as sulfate donor and an apparent K_m of 62 μM was calculated. Inhibition studies suggest that this enzyme preferentially catalyzes the sulfurylation of the 3β-hydroxyl group of Δ⁵-steroids. An unusual feature of the enzyme is a pH optimum at pH 10.     

Geranylpyrophosphate: p-hydroxybenzoate geranyltransferase activity in extracts of Lithospermum erythrorhizon cell cultures

The enzymatic formation of m-geranyl-p-hydroxybenzoic acid from geranylpyrophosphate and p-hydroxybenzoic acid was investigated in cell-free extracts of Lithospermum erythrorhizon cell cultures. The reaction required the presence of a divalent cation, magnesium being the most effective activator. The enzyme showed a very broad pH optimum between pH 7.1 and 9.3. It was highly specific for both p-hydroxybenzoic acid and geranylpyrophosphate, and the apparent K_m values for these two substrates were 0.014 and 0.56 mM, respectively. The activity was located in the pellet of a 100 000 g centrifugation, showing that the enzyme is bound to membranes or microsomes. Shikonin-producing cultures contained an activity of this enzyme 35 times higher than non-producing cultures, suggesting that this enzyme is of regulatory importance in shikonin biosynthesis.     

Adenosine 5‘-Phosphosulfate Sulfotransferase from Norway Spruce: Biochemical and Physiological Properties*

Biochemical and physiological properties of adenosine 5′-phosphosulfate sulfotransferase, a key enzyme of assimilatory sulfate reduction, from spruce trees growing under field conditions were studied. The apparent K_m for adenosine 5′-phosphosulfate (APS) was 29 ± 5.5μM, its apparent M_r was 115,000. 5′-AMP inhibited the enzyme competitively with a K_i of 1 mM, but also stabilized it. MgS0₄ at 800 mM increased adenosine 5′-phosphosulfate sulfotransferase activity by a factor of 3, concentrations higher than lOOOmM were inhibitory. Treatment of isolated shoots with nutrient solution containing 1 or 2 mM sulfate, and 3 or 10 mM glutathione, respectively, induced a significant decrease in extractable adenosine 5′-phosphosulfate sulfotransferase activity over 24h, whereas GSH as well as S^2- up to 5mM cysteine and up to 200 mM SO₃^2- had no effect on the in vitro activity of the enzyme. As with other enzymes involved in assimilatory sulfate reduction, namely ATP sulfurylase (EC 2.7.7.4), sulfite reductase (EC 1.8.7.1) and O-acetyl-L.-serine sulfhydrylase (EC 4.2.99.8), adenosine 5′-phosphosulfate sulfotransferase was still detected at appreciable activities in 2- and 3-year-old needles. Adenosine 5′-phosphosulfate sulfotransferase activity was low in buds and increased during shoot development, parallel to the chlorophyll content. The enzyme activity was characterized by an annual cycle of seasonal changes with an increase during February and March.     

Exopeptidase activity in eggs of Trichostrongylus colubriformis (Nematoda)

Summary

A supernatant from eggs of the ruminant nematode Trichostrongylus colubriformis contained an enzyme that was similar to leucine aminopeptidase (LAP), based on hydrolysis of the substrate L-leucine β-naphthylamide to β-naphthylamine. A Michaelis-Menten constant (K _m) of 0.155 mM was obtained. Rate of hydrolysis of 16 substrates revealed that L-phenylalanine and L-tyrosine β-naphthylamides were hydrolyzed most readily while seven additional substrates were hydrolyzed at lesser rates. The optimum pH for enzymatic activity was 6.75–7.5. Enzymatic activity was lost by heating the egg supernatant to 60°C for 5 min or freezing at 0°C for 28 days. Addition of millimolar concentrations of the chlorides of zinc, manganese and magnesium to the egg supernatant had no stimulatory effect on enzyme activity while 10 and 100 mM concentrations significantly reduced activity. Ethylenediamine tetraacetic acid at 10^?4 M had no effect on enzymatic activity. Activity was inhibited by 10^?4 M 1,10-phenanthroline, but the inhibition was reversed by zinc chloride at 10^?3 M. Di-isopropylphosphofluoridate at 10^?3 M reduced enzymatic activity moderately. Enzyme activity in egg supernatant increased 2.2-fold from 21 days to 60–90 days of a primary infection in the host while a 3.3-fold increase was found in primary versus secondary infections.     

Studies on Metabolic Pathway of NAD in Yeast

Quantitative studies on yeast 5′-nucIeotidase are presented.

K_m values for purine 5′-nucleotides were generally smaller than those for pyrimidine 5′-nucleotides and, among purine series, K_m value for 5′-AMP was the smallest, while their V values were almost same.

The enzyme activity was inhibited in the competitive type by bases, nucleosides, 3′- or 2′-nucleotides, and NMN and in the mixed type by NAD and NADP.

Base-, ribose-, 3′- or 5′-phosphate moiety of nucleoside and nucleotide had some effects on binding with enzyme; especially the structure of base moiety characterizes the K_m or K_i value.

The enzyme activity was accelerated by Ni⁺⁺ or Co⁺⁺, which increases V value but never affects K_m value.

The relationship between the structure of substrate and its affinity towards enzyme is discussed.     

The influence of potassium ion (K+) on digoxin-induced inhibition of porcine cerebral cortex Na+/K+-ATPase

The in vitro influence of potassium ion modulations, in the concentration range 2 mM–500 mM, on digoxin-induced inhibition of porcine cerebral cortex Na⁺/K⁺-ATPase activity was studied. The response of enzymatic activity in the presence of various K⁺ concentrations to digoxin was biphasic, thereby, indicating the existence of two Na⁺/K⁺-ATPase isoforms, differing in the affinity towards the tested drug. Both isoforms showed higher sensitivity to digoxin in the presence of K⁺ ions below 20 mM in the medium assay. The IC₅₀ values for high/low isoforms 2.77 × 10^{? 6} M / 8.56 × 10^{? 5} M and 7.06 × 10^{? 7} M /1.87 × 10^{? 5} M were obtained in the presence of optimal (20 mM) and 2 mM K⁺, respectively. However, preincubation in the presence of elevated K⁺ concentration (50 – 500 mM) in the medium assay prior to Na⁺/K⁺-ATPase exposure to digoxin did not prevent the inhibition, i.e. IC₅₀ values for both isoforms was the same as in the presence of the optimal K⁺ concentration. On the contrary, addition of 200 mM K⁺ into the medium assay after 10 minutes exposure of Na⁺/K⁺-ATPase to digoxin, showed a time-dependent recovery effect on the inhibited enzymatic activity. Kinetic analysis showed that digoxin inhibited Na⁺/K⁺-ATPase by reducing maximum enzymatic velocity (V_max) and K_m, implying an uncompetitive mode of interaction.     

Trichomonas gallinae: charaterization and regulatory properties of lactic dehydrogenase.

The lactic dehydrogenase (l-lactate: NAD oxidoreductase, EC 1.1.1.27, LDH)of Trichomonas gallinae was characterized and some of its regulatory properties studied. Electrophoretic analysis, with specific enzymatic staining of crude and dialyzed cell-free extracts and dialyzed ammonium sulfate fractions, all revealed a single band of enzymatic activity suggesting only one molecular form of the enzyme. The pH optima were found to be the following: 7.0 in the pyruvate to lactate direction and 9.0 in the reverse direction. Thermal inactivation studies showed a narrow temperature optimum peaking at 35 C. The K_m values for all four reaction components were determined and found to be: NADH, 70 μm; pyruvate, 88 μm; NAD, 65 μm; and l-lactate, 4.6 mM. T. gallinae LDH was absolutely specific for NAD, NADH, l-lactate, and pyruvate. The enzyme exhibited negative cooperativity, with both NADH and l-lactate, as evidenced by curvilinear Lineweaver-Burk kinetics and Hill coefficients of less than one. Several glycolytic intermediates lowered the K_m of NADH with variable effects on the K_m of pyruvate. The regulation of LDH by glycolytic intermediates is discussed.     

Effect of Dopamine on Activation of Rat Striatal Adenylate Cyclase by Free Mg2+ and Guanyl Nucleotides1

Abstract: Stimulation of rat striatal adenylate cyclase by guanyl nucleotides was examined utilizing either MgATP or magnesium 5′-adenylylimidodiphos-phate (MgApp(NH) p) as substrate. GTP and 5′- guanylylimidodiphosphate (Gpp(NH) p) stimulate adenylate cyclase under conditions where the guanyl nucleotide is not degraded. The apparent stimulation of adenylate cyclase by GDP is due to an ATP-dependent transphosphorylase present in the tissue which converts GDP to GTP. We conclude that GTP is the physiological guanyl nucleotide responsible for stimulation of striatal adenylate cyclase. Dopamine lowers the K_a for Gpp(NH) p stimulation twofold, from 2.4 μM to 1.2 μM and increases maximal velocity 60%. The kinetics of Gpp(NH) p stimulation indicate no homotropic interactions between Gpp(NH) p sites and are consistent with one nonessential Gpp(NH) p activator site per catalytic site. Double reciprocal plots of the activation by free Mg²⁺ were concave downward, indicating either two sets of sites with different affinities or negative cooperativity (Hill coefficient = 0.3, K_0.5= 23 mM). The data conform well to a model for two sets of independent sites and dopamine lowers the K_a for free Mg²⁺ at the high-affinity site threefold, from 0.21 mM to 0.07 mM. The antipsy-chotic drug fluphenazine blocks this shift in K_a due to dopamine. Dopamine does not appreciably affect the affinity of adenylate cyclase for the substrate, MgApp(NH) p. Therefore, dopamine stimulates striatal adenylate cyclase by increasing the affinity for free Mg²⁺ and guanyl nucleotide and by increasing maximal velocity.     

Properties of larval and imaginal membrane-bound digestive enzymes from Trichosia pubescens

Trichosia pubescens larval midgut ceca cells display in their plasma membranes α-glucosidases (M_r 95,000; pH_o 5.5; K_m 5.7 mM; K_i for TRIS 8.9 mM), trehalases (M_r 69,000; pH_o 5.3; K_m 0.92 mM; K_i for TRIS 57 mM), and aminopeptidases (M_r 95,000; pH_o 8.7; K_m 0.19 mM) which are solubilized by Triton X-100. The enzymes were purified by electrophoresis and used to raise antibodies in a rabbit. T. pubescens imaginal midgut cells display in their plasma membranes an α-glucosidase (M_r 156,000; pH_o 5.8; K_m 2.3 mM; K_i for TRIS 0.2 mM), a trehalase (M_r 93,000; pH_o 5.5; K_m 0.72 mM; K_i for TRIS 45.5 mM), and an aminopeptidase (M_r 210,000; pH_o 9.0; K_m 0.47 mM). Antiserum produced against the larval enzymes shows no precipitation arc when tested by double immunodiffusion or by immunoelectrophoresis with Triton X-100-solubilized membrane proteins from imaginal midguts. Otherwise, a similar test showed that larval midgut cecal enzymes and larval ventriculus enzymes display complete immunological identity. The data suggest that, despite the fact the larval and imaginal aminopeptidase, α-glucosidase, and trehalase probably have similar functions, the genes coding for them in larvae and imagoes must differ.     

Purification and characterization fo a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands/ Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 3000 mM, phosphorylated only phosvitin and was not retained on phophocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhbiited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent K_m of 100 μg/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent K_m of 25 and 28 μM, respectively. It had an absolute requirement for Mg²⁺ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecualr weight of 35 000 suggesting a polymeric structure of the enzyme.     

The effects of calcium and lanthanide ions on the activity of bovine heart nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase

(i) The activity of purified NAD-specific isocitrate dehydrogenase from bovine heart was stimulated by free Ca²⁺ in the presence of ADP and subsaturating levels of magnesium isocitrate, but not in absence of ADP. However, Ca²⁺ was not absolutely required for ADP activation. This was particularly apparent when free Mg²⁺ was kept low (0.0024–0.020 mm) and the substrate magnesium dl-isocitrate ranged from 0.07–0.25 mm. When kinetic constants were determined at pH 7.4 under these conditions and in the absence of ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetate, Ca²⁺ had little or no effect on K_m (app) for ADP; the stimulation of rate by Ca²⁺ was mainly due to increased V (app). With subsaturating ADP, there was an interdependence in the interaction of the enzyme with substrate and Ca²⁺. Thus, with ADP constant (0.30 mm) the values of K_m (app) for magnesium dl-isocitrate declined from 0.35 mm at zero Ca²⁺ to 0.19 mm with saturating Ca²⁺ without affecting V; K_m (app) for free Ca²⁺ declined with increasing magnesium isocitrate to a limiting K_m of 0.3 μm. (ii) Ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetate, frequently used as a calcium buffer, inhibited enzyme activity with and without ADP. (iii) The enzyme was not inhibited by the calmodulin inhibitors trifluoperazine and chlorpromazine. Inhibition by lanthanide ions of the isocitrate dehydrogenase was competitive with magnesium isocitrate and not with respect to Ca²⁺. The values of K_is (1.8 to 3.1 μm) for La³⁺, Yb³⁺, Gd³⁺, Eu³⁺, Tb³⁺, and Er³⁺ were about two orders of magnitude smaller than K_m for magnesium dl-isocitrate.     

Effect of cyclic amp on suncus murinus liver serine-aminotransferase and serine dehydratase

• 1.1. Suncus murinus was injected dibutyryl adenosine 3′,5′-cyclic monophosphate (Bt₂cAMP) and assayed serine-glyoxylate aminotransferase (EC 2.6.1.45) and serine dehydratase (EC 4.2.1.13).
• 2.2. Serine dehydratase was induced 4-fold by Bt₂cAMP. The K_m values of the induced enzyme for l-serine and pyridoxal 5′-phosphate was 57 mM and 3.0 μM, respectively. The enzyme had a pH optimum at pH 10.0. These kinetic properties and pH optimum were same as those of the enzyme from the control. Both the holoenzyme and the apoenzyme increased to the same extent by Bt₂cAMP.
• 3.3. Serine-glyoxyate aminotransferase activity was decreased slightly by the Bt₂cAMP injection. The holoenzyme activity was increased, but the apoenzyme decreased. K_m values for l-serine and glyoxylate of this enzyme were 6mM and 0.2 mM, respectively, without change by Bt₂cAMP.

     

S-formylglutathione: The substrate for formate dehydrogenase in methanol-utilizing yeasts

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The K _mvalues of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathine-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a K _mof 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (K _mof 40 mM) but a relatively high affinity for S-formylglutathione (K _mof 1.1 mM). The K _mvalues of formate dehydrogenase in cell-free extracts of methanol-grown Candida boidinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.