Purification and characterization of a NAD+-dependent secondary alcohol dehydrogenase from propane-grown Rhodococcus rhodochrous PNKb1

NAD⁺-linked primary and secondary alcohol dehydrogenase activity was detected in cell-free extracts of propane-grown Rhodococcus rhodochrous PNKb1. One enzyme was purified to homogeneity using a two-step procedure involving DEAE-cellulose and NAD-agarose chromatography and this exhibited both primary and secondary NAD⁺-linked alcohol dehydrogenase activity. The Mr of the enzyme was approximately 86,000 with subunits of Mr 42,000. The enzyme exhibited broad substrate specificity, oxidizing a range of short-chain primary and secondary alcohols (C₂–C₈) and representative cyclic and aromatic alcohols. The pH optimum was 10. At pH 6.5, in the presence of NADH, the enzyme catalysed the reduction of ketones to alcohols. The K _m values for propan-1-ol, propan-2-ol and NAD were 12 mM, 18 mM and 0.057 mM respectively. The enzyme was inhibited by metal-complexing agents and iodoacetate. The properties of this enzyme were compared with similar enzymes in the current literature, and were found to be significantly different from those thus far described. It is likely that this enzyme plays a major role in the assimilation of propane by R. rhodochrous PNKb1.Abbreviations HPLC high performance liquid chromatography - DEAE diethyl amino ethyl - IEF isoelectrofocusing - NTG nitrosoguanidine - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - pI isoelectric point     

Purification and characterization of an ADP-dependent phosphofructokinase from Thermococcus zilligii

The ADP-dependent phosphofructokinase (PFK) from Thermococcus zilligii has been purified 950 fold; it had a specific activity of 190 U mg⁻¹. The enzyme required Mg²⁺ ions for optimal activity and was specific for ADP. The forward reaction kinetics were hyperbolic for both cosubstrates (pH optimum of 6.4), and the apparent K _m values for ADP and fructose-6-phosphate were 0.6 mM (apparent V _max of 243 U mg⁻¹) and 1.47 mM (apparent V _max of 197 U mg⁻¹), respectively. Significantly, the enzyme is indicated to be nonallosteric but was slightly activated by some monovalent cations including Na⁺ and K⁺. The protein had a subunit size of 42.2 kDa and an estimated native molecular weight of 66 kDa (gel filtration). Maximal reaction rates for the reverse reaction were attained at pH 7.5–8.0, and the apparent K _m values for fructose-1,6-bisphosphate and AMP were 0.56 mM (apparent V _max of 2.9 U mg⁻¹) and 12.5 mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared to ATP and pyrophosphate-dependent phosphofructokinases. Received: August 14, 1998 / Accepted: December 2, 1998     

Ibuprofen Inhibits Arylamine N-Acetyltransferase Activity in the Bacteria Klebsiella pneumoniae

Ibuprofen, one of the nonsteroidal anti-inflammatory drugs, inhibited arylamine N-acetyltransferase activity of Klebsiella pneumoniae both in vitro and in vivo. The NAT activities of Klebsiella pneumoniae were inhibited by ibuprofen in a dose-dependent manner both in vitro and in vivo. In vitro, the NAT activity was 0.675 ± 0.028 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was 0.506 ± 0.002 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In vivo, the NAT activity was 0.279 ± 0.016 nmol/min/10¹⁰ colony forming units (CFU) for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was 0.228 ± 0.008 nmol/min/10¹⁰ CFU for the acetylation of 2-aminofluorene. The inhibition of NAT activity by ibuprofen was shown to persist for at least 4 h. For in vitro examination, the values of apparent K _m and V _max were 1.08 ± 0.05 mM and 9.17 ± 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen was added to the reaction mixtures, the values of apparent K _m and V _max were 1.19 ± 0.01 mM and 6.67 ± 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. For in vivo examination, the values of apparent K _m and V _max were 1.24 ± 0.48 mM and 4.18 ± 1.06 nmol/min/10 × 10¹⁰ CFU, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen was added to the culture, the values of apparent K _m and V _max were 0.95 ± 0.29 mM and 2.77 ± 0.37 nmol/min/mg protein, respectively, for 2-aminofluorene, respectively. This report is the first finding of ibuprofen inhibition of arylamine N-acetyltransferase activity in a strain of Klebsiella pneumoniae. Received: 28 January 1997 / Accepted: 12 February 1997     

Horse liver alcohol dehydrogenase SS: purification and characterization of the homogenous isoenzyme.

Alcohol dehydrogenase SS was prepared from horse liver by salt fractionation, ion-exchange chromatography, and affinity chromatography. The purified isoenzyme is free from extraneous protein and other alcohol dehydrogenase isoenzyme contaminants and contains four Zinc atoms per molecule. The substrate specificity with saturated aliphatic alcohols and aldehydes of two to six carbon chain lengths has been investigated. The K_m values and turnover numbers at maximal velocity (k_cat) are presented. Values of k_cat are constant within a substrate category and independent of the substrate chain length, while the K_m values decrease with the increase of the substrate chain length. The K_m values for two- and three-carbon substrates are large, that for ethanol (40 mm) is two orders of magnitude larger than that reported for classical preparations of horse liver alcohol dehydrogenase. At pH 7, the k_cat values for alcohol oxidation are almost 30 times smaller than for aldehyde reduction. The enzyme has been characterized with regard to specific activity with several nonsteroidal substrates and with two steroids: 3-oxo-5β-androstan-17β-ol and 5β-pregnan-21-ol-3,20-dione hemisuccinate. NAD(H) is the preferred coenzyme. Values of K_m for NADH with constant steroidal substrates are an order of magnitude smaller than the corresponding K_m values with nonsteroidal substrates. A possible explanation for this observation is presented.     

Mutants of the Cyanobacterium Anabaena sp. PCC 7120 Altered in Nitrate Transport and Reduction

Nitrate assimilation-defective mutants SP7, SP9, and SP17 of the cyanobacterium Anabaena sp. PCC 7120 were isolated by use of transposon mutagenesis and screened on medium containing chlorate. SP7 and SP17 represented nitrate reductase-defective nature, while mutant SP9 appeared to be a regulatory mutant exhibiting pleiotropic behavior. Kinetics of nitrate uptake system exhibited K _s values of 31–38 μM for parent, SP7, and SP17 strains; however, mutant SP9 exhibited a high K _s value of 109.5 μM. Defective nitrate reductase was apparent in mutant SP7 and SP9, while mutant SP17 exhibited partial defective nature. Methyl viologen-dependent NR activity in parent strain presented a biphasic nature with K _m values of 0.13 and 2.47 mM, whereas a single K _m value (2.96 mM) was observed for mutant SP17. Mutant SP9 was also defective in nitrite uptake and reduction. Mutant strains exhibited derepressed nitrogenase activity in the presence of nitrate, while glutamine synthetase activity remained unaltered. Received: 20 April 1999 / Accepted: 22 May 1999     

Kinetic Analysis in Mixed Micelles of Partially Purified Rat Brain Phospholipase D Activity and its Activation by Phosphatidylinositol 4,5-Bisphosphate

A partially purified rat brain membrane phospholipase D (PLD) activity was characterized in a mixed micellar system consisting of l-palmitoyl-2-[6-N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)-amino]caproyl-phosphatidylcholine (NBD-PC) and Triton X-100, under conditions where Triton X-100 has a surface dilution effect on PLD activity and the catalytic rate is dependent on the surface concentration (expressed in terms of molar ratio) of NBD-PC. PLD activity was specifically activated by phosphatidylinositol 4,5-bisphosphate (PIP₂), and the curve of activation versus PIP₂ molar ratio fitted a Michaelis-Menten equation with a K_act value between molar ratios of 0.001–0.002. Maximal activation was observed at a PIP₂ molar ratio of 0.01. Similar values were obtained when activities of partially purified PLD as well as membrane-bound PLD were determined towards pure NBD-PC micelles. In the mixed micellar system PIP₂ was shown to elevate by 6–22 fold the specificity constant of PLD towards NBD-PC (K_A, which is proportional to V_max/K_m). Kinetic analysis of PLD trans-phosphatidylation activity towards ethanol, 1-propanol and 1-butanol revealed a Michaelis-Menten type dependence on alcohol concentration up to 1000, 200 and 80 mM, respectively. While V_max values were similar towards all three alcohols, enzyme affinity increased as the alcohol was longer, and K_m values for ethanol, 1-propanol and 1-butanol were 291, 75 and 16 mM (respectively). PLD specificity constants (K_A) towards ethanol, 1-propanol and 1-butanol were shown to be respectively 260, 940 and 5,920 times higher than to water, the competing substrate. 1-Propanol and 1-butanol inhibited PLD activity above 400 and 100 mM, respectively. The present results indicate that partially purified PLD obeys surface dilution kinetics with regard to its phospholipid substrate PC and its cofactor PIP₂, and that in the presence of alcohols, its transphosphatidylation activity may be analyzed as a competitive reaction to the hydrolysis reaction.     

Some properties of choline acetyltransferase isolated from the electric organ ofTorpedo marmorata: Specificity for choline analogues and inhibition by certain amines and amino acids

(1) Choline acetyltransferase ofTorpedo marmorata electric organ was studied by using soluble tissue extracts partially purified by (NH₄)₂SO₄ fractionation. (2) Linear enzymatic rates were observed at 30°C, in the presence of 350 M acetyl-CoA and 50 mM choline, over a 30–40 min incubation period. (3) A number of analogues of choline, including mono-, di-, and triethylcholine and pyrrolcholine were synthesized and theK _m (apparent) andV (maximum velocity) values determined. TheK _m (apparent) for choline (11.5 mM), with theTorpedo enzyme, was high in comparison to values reported for mammalian or invertebrate nervous tissue. TheTorpedo enzyme was also not so specific for choline in comparison with the other choline analogues (based onK _m values) as were other sources of the enzyme. TheV values for choline and mono-, di-, and triethylcholine with theTorpedo enzyme indicated a direct relationship between enzyme activity andN-alkyl substitution. (4) Several amines and amino acids inhibited choline acetyltransferase fromTorpedo. Histamine (15 mM) brought about a 60% inhibition and was found to be a noncompetitive inhibitor with respect to choline.     

The activity of yeast ADH I and ADH II with long-chain alcohols and diols

The activities of yeast ADH I and ADH II towards long chain alcohols and diols were studied using rather unusual conditions (1.0 M Tris pH 8.75, approximately 0.3 mg/ml enzyme and [S]< < <K_m ) where the alcohols are oxidised quantitatively in a first-order manner. Plots of the apparent first-order rate constant versus primary alcohol chain length show double peaks with similar values for ethanol and 1-decanol and relatively low values for 1-butanol through to 1-octanol. With the α,ω diols only one peak of activity was observed with 1,14-tetradecanediol, the preferred substrate, being oxidised about the same rate as ethanol. Both enzymes were essentially inactive with short-chain diols (C₂–C₈). For all of these assays normalised rates with ADH II were about threefold faster than with ADH I.     

Transport kinetics of maltotriose in strains ofSaccharomyces

Summary Maltotriose transport was studied in two brewer's yeast strains, an ale strain 3001 and a lager strain 3021, using laboratory-synthesized¹⁴C-maltotriose. The maltotriose transport systems preferred a lower pH (pH 4.3) to a higher pH (pH 6.6). Two maltotriose transport affinity systems have been indentified. The high affinity system hasK _m values of 1.3 mM for strain 3021 and 1.4 mM for strain 3001. The low affinity competitively inhibited by maltose and glucose withK _i values of 58 mM and 177 mM. respectively, for strain 3021, and 55 mM and 147 mM, respectively, for strain 3001. Cells grown in maltotriose and maltose had higher maltotriose and maltose transport rates, and cells grown in glucose had lower maltortriose and maltose transport rates. Early-logarithmic phase cells transported glucose faster than either maltose or maltotriose. Cells harvested later in the growth phase had increased maltotriose and maltose transport activity. Neither strain exhibited significant differences with respect to maltose and maltotriose transport activity.     

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.     

Purification and Properties of Primary and Secondary Alcohol Dehydrogenases from Thermoanaerobacter ethanolicus

Thermoanaerobacter ethanolicus (ATCC 31550) has primary and secondary alcohol dehydrogenases. The two enzymes were purified to homogeneity as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The apparent M_rs of the primary and secondary alcohol dehydrogenases are 184,000 and 172,000, respectively. Both enzymes have high thermostability. They are tetrameric with apparently identical subunits and contain from 3.2 to 5.5 atoms of Zn per subunit. The two dehydrogenases are NADP dependent and reversibly convert ethanol and 1-propanol to the respective aldehydes. The V_m values with ethanol as a substrate are 45.6 μmol/min per mg for the primary alcohol dehydrogenase and 13 μmol/min per mg for the secondary alcohol dehydrogenase at pH 8.9 and 60°C. The primary enzyme oxidizes primary alcohols, including up to heptanol, at rates similar to that of ethanol. It is inactive with secondary alcohols. The secondary enzyme is inactive with 1-pentanol or longer chain alcohols. Its best substrate is 2-propanol, which is oxidized 15 times faster than ethanol. The secondary alcohol dehydrogenase is formed early during the growth cycle. It is stimulated by pyruvate and has a low K_m for acetaldehyde (44.8 mM) in comparison to that of the primary alcohol dehydrogenase (210 mM). The latter enzyme is formed late in the growth cycle. It is postulated that the secondary alcohol dehydrogenase is largely responsible for the formation of ethanol in fermentations of carbohydrates by T. ethanolicus.     

Hepatic and extra-hepatic glutathione-S-transferase activity in wild pigeons (Columba livia)

Glutathione-S-transferase (EC 2.5.1.18) activity was assayed in hepatic and extra-hepatic tissues of pigeons using l-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates. Gluthathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene in pigeon was in the order: kidney > liver > testes > brain > lung> heart. The enzyme activity with 1-chloro-2,4-dinitrobenzene as substrate was 40–44 times higher in pigeon liver and kidney than that observed with 1,2-dichloro-4-dinitrobenzene as substrate.K _m values of hepatic and renal glutathione transferase with l-chloro-2,4-dinitrobenzene as substrate were 2.5 and 3 mM respectively. Double reciprocal plots with varying reduced gluthathione concentrations resulted in biphasic curves with twoK _m values (liver 0.31 mM and 4mM; kidney 0.36 mM and 1.3 mM). The enzyme activity was inhibited by oxidized gluthathione in a dose-dependent pattern. 3-Methylcholanthrene elicited about 50% induction of hepatic glutathione transferase activity whereas phénobarbital was ineffective.     

Adaptation of Pseudomonas sp. GJ1 to 2-bromoethanol caused by overexpression of an NAD-dependent aldehyde dehydrogenase with low affinity for halogenated aldehydes

Pseudomonas sp. GJ1 is able to grow with 2-chloroethanol as the sole carbon and energy source, but not with 2-bromoethanol, which is toxic at low concentrations (1 mM). A muatnt that could grow on 2-bromoethanol with a growth rate of 0.034 h^–1 at concentrations up to 5 mM was isolated and designated strain GJ1M9. Measurement of enzyme activities showed that mutant and wild-type strains contained a PMS-linked alcohol dehydrogenase that was active with halogenated alcohols and that was threefold overexpressed in the mutant when grown on 2-chloroethanol, but only slightly overproduced when grown on 2-bromoethanol. Both strains also contained an NAD-dependent alcohol dehydrogenase that had no activity with halogenated alcohols. Haloacetate dehalogenase levels were similar in the wild-type and the mutant. Activities of NAD-dependent aldehyde dehydrogenase were only slightly higher in extracts of the mutant grown with 2-bromoethanol than in those of the wild-type grown with 2-chloroethanol. SDS-PAGE, however, showed that this enzyme amounted to more than 50% of the total cellular protein in extracts of the mutant from 2-bromoethanol-grown cells, which was fourfold higher than in extracts of the wild-type strain grown on 2-chloroethanol. The enzyme was purified and shown to be a tetrameric protein consisting of subunits of 55 kDa. The enzyme had low K_m values for acetaldehyde and other non-halogenated aldehydes (0.8–4 μM), but much higher K_m values for chloroacetaldehyde (1.7 mM) and bromoacetaldehyde (10.5 mM), while V_max values were similar for halogenated and non-halogenated aldehydes. Cultures that were pregrown on 2-chloroethanol rapidly lost aldehyde dehydrogenase activity after addition of 2-bromoethanol and chloroamphenicol, which indicates that bromoacetaldehyde inactivates the enzyme. To achieve growth with 2-bromoethanol, the high expression of the enzyme thus appears to be necessary in order to compensate for the high K_m for bromoacetaldehyde and for inactivation of the enzyme by bromoacetaldehyde. Received: 31 August 1995 / Accepted: 4 December 1995     

A new enzymatic activity from elicitor‐treated pear cell cultures converting trans‐cinnamic acid to benzaldehyde

Cell cultures of Asian pear (Pyrus pyrifolia) are known to produce benzoate‐derived biphenyl phytoalexins upon elicitor treatment. Although the downstream pathway for biphenyl phytoalexin biosynthesis is almost known, the upstream route of benzoic acid biosynthesis in pear has not been completely elucidated. In the present work, we report benzaldehyde synthase (BS) activity from yeast extract‐treated cell suspension cultures of P. pyrifolia. BS catalyzes the in vitro conversion of trans‐cinnamic acid to benzaldehyde using a non‐oxidative C₂‐side chain cleavage mechanism. The enzyme activity was strictly dependent on the presence of a reducing agent, dithiothreitol being preferred. C₂‐side chain shortening of the cinnamic acid backbone resembled the mechanisms catalyzed by 4‐hydroxybenzaldehyde synthase (HBS) activity in Vanilla planifolia and salicylaldehyde synthase (SAS) activity in tobacco and apple cell cultures. A basal BS activity was also observed in the non‐elicited cell cultures. Upon yeast extract‐treatment, a 13‐fold increase in BS activity was observed when compared to the non‐treated control cells. Moreover, feeding of the cell cultures with trans‐cinnamic acid, the substrate for BS, resulted in an enhanced level of noraucuparin, a biphenyl phytoalexin. Comparable accumulation of noraucuparin was observed upon feeding of benzaldehyde, the BS product. The preferred substrate for BS was found to be trans‐cinnamic acid, for which the apparent K_m and V_max values were 0.5 mM and 50.7 pkat mg^?1 protein, respectively. Our observations indicate the contribution of BS to benzoic acid biosynthesis in Asian pear via the CoA‐independent and non‐β‐oxidative route.     

Acetate oxidation to CO2 via a citric acid cycle involving an ATP-citrate lyase: a mechanism for the synthesis of ATP via substrate level phosphorylation in Desulfobacter postgatei growing on acetate and sulfate

Desulfobacter postgatei is an acetate-oxidizing, sulfate-reducing bacterium that metabolizes acetate via the citric acid cycle. The organism has been reported to contain a si-citrate synthase (EC 4.1.3.7) which is activated by AMP and inorganic phosphate. It is show now, that the enzyme mediating citrate formation is an ATP-citrate lyase (EC 4.1.3.8) rather than a citrate synthase. Cell extracts (160,000xg supernatant) catalyzed the conversion of oxaloacetate (apparent K _m=0.2 mM), acetyl-CoA (app. K _m=0.1 mM), ADP (app. K _m=0.06 mM) and phosphate (app. K _m=0.7 mM) to citrate, CoA and ATP with a specific activity of 0.3 mol·min^-1·mg^-1 protein. Per mol citrate formed 1 mol of ATP was generated. Cleavage of citrate (app. K _m=0.05 mM; V _max=1.2 mol · min^-1 · mg^-1 protein) was dependent on ATP (app. K _m=0.4 mM) and CoA (app. K _m=0.05 mM) and yielded oxaloacetate, acetyl-CoA, ADP, and phosphate as products in a stoichiometry of citrate:CoA:oxaloacetate:ADP=1:1:1:1. The use of an ATP-citrate lyase in the citric acid cycle enables D. postgatei to couple the oxidation of acetate to 2 CO₂ with the net synthesis of ATP via substrate level phosphorylation.     

Effects of anesthetic alcohols on membrane transport processes in human erythrocytes

1. 1. Anesthetic alcohols (pentanol, hexanol and heptanol) were found to increase the fluidity of red cell membrane lipids as monitored by the fluorescence depolarization of diphenylhexatriene. The relative potency of the alcohols was found to be parallel to their relative membrane/water partition coefficients.

2. 2. Hexanol had biphasic effect on erythritol uptake by simple diffusion by red cells. At concentrations less than 9 mM, hexanol had no significant effect. At concentrations greater than 9 mM, there was an approximately linear increase in erythritol permeability with increasing alcohol concentration.

3. 3. The facilitated transport of uridine was markedly inhibited by hexanol. Hexanol at 6 mM produced a 65% inhibition of uridine (4 mM) uptake. Hexanol decreased both the apparent K_m and V values for the equilibrium exchange of uridine.

4. 4. The facilitated transport of galactose was only slightly inhibited by hexanol.

5. 5. Hexanol was without effect on the passive and active fluxes of Na⁺ and K⁺ in red cells with altered cation contents. Cells that were slightly depleted of K⁺ and cells that were highly K⁺-depleted were both insensitive to hexanol.

Glutamine and glutamic acid uptake by rat renal brushborder membrane vesicles

Summary Glutamine uptake by rat renal brushborder vesicles occurred via two distinct saturable processes withK _m values of 0.145 and 8.5 mM which were stimulated by both ionic and sodium gradients with a pH optimum of 6.8–7.1 Glutamic acid uptake also occurred by a two-component system withK _m values of 0.016 and 3.60 mM. Both components were stimulated specifically by a sodium gradient. The lowK _m system for glutamic acid had a pH optimum of 7.2–7.4. Glutamine entry at 0.06 mM was inhibited by a variety of amino acids at 3 mM, including dibasic amino acids, glycine, valine, and phenylalanine. Glutamic acid entry at 0.06 mM was inhibited 20–30% by 3 mM phenylalanine, valine, -aminoisobutyric acid, and glutamine. No metabolic alteration of glutamic acid was observed on incubation with membrane vesicles, but glutamine was significantly hydrolyzed to glutamic acid upon prolonged incubation. Hydrolysis of glutamine was negligible at 15 sec incubation which was employed for measurement of initial rate of entry. These studies provide support for the existence of an uptake system in the brushborder of the renal proximal tubule cell capable of handling the reabsorption of glutamine normally present in glomerular filtrate.     

Arylsulphatases in human brain: assay, some properties, and distribution

Abstract— Arylsulphatases (aryl-sulphate sulphohydrolases; E.C. 3.1.6.1) in human brain were studied using a highly sensitive fluorometric technique based on the use of 4-methyl-umbelliferone sulphate (MUS) as substrate. In the dialysed homogenate of human brain at least two enzymes could be distinguished on the basis of pH optima and substrate concentration. One MUS-sulphatase, of the ‘insoluble’ type, exhibited a pH optimum of 6–9 and an apparent K_m of 0.05 mM, whereas the second, belonging to the ‘soluble’ type, exhibited pH optimum of 6–0 and an apparent K_m of 6.25 mM. Pronounced activities of the two arylsulphatases were observed in the 18,000 g sediment. About 25 per cent of the total tissue activity of the ‘soluble’-type MUS-sulphatase was found in the soluble subcellular fraction. However, this enzyme was completely solubilized by extraction of acetone-dried human brain with acetate buffer.     

GUANYLATE CYCLASES IN CNS: ENZYMATIC CHARACTERISTICS OF SOLUBLE AND PARTICULATE ENZYMES FROM MOUSE CEREBELLUM AND RETINA

Guanylate cyclase activity is present in both soluble and particulate fractions of homogenates of mouse cerebellum and retina. Soluble guanylate cyclases in cerebellum and retina have an apparent K_m for GTP of approx 40 and 70 μM, respectively; are stimulated by Ca²⁺ and Mg²⁺ in the presence of low Mn²⁺; and do not respond to NaN₃, NH₂OH or detergent. The particulate guanylate cyclase found in brain has an apparent K_m GTP of 237 7mu;M, is not stimulated by Ca²⁺ or Mg²⁺ in the presence of low Mn²⁺, but is stimulated by NaN₃, NH₂OH, and detergent. In particulate fractions of normal retina, guanylate cyclase has two apparent K_m GTP values (42 and 225 μM); has higher activity at low concentrations of Mn²⁺ (0.5 mM) than at high concentrations (5.0 mM); is inhibited by Ca²⁺; and does not respond to NaN₃, NH₂OH, or detergent. Retinas essentially devoid of photoreceptor cells (from mice with photoreceptor dystrophy) have soluble guanylate cyclase activity which is similar to that in normal retina, but have only 4% as much particulate guanylate cyclase activity. This residual particulate guanylate cyclase has an apparent K_m GTP value of 392 μM and other properties similar to particulate guanylate cyclase from brain. These data indicate the presence of three distinguishable guanylate cyclases in CNS: (1) a soluble enzyme present in both brain and retina: (2) a particulate enzyme which is also present in brain and in the inner or neural retina: and (3) another particulate enzyme which is apparently unique and confined to retinal photoreceptor cells.     

Activation of aromatic acids and aerobic 2-aminobenzoate metabolism in a denitrifying Pseudomonas strain

The initial reactions possibly involved in the acrobic and anaerobic metabolism of aromatic acids by a denitrifying Pseudomonas strain were studied. Several acyl CoA synthetases were found supporting the view that activation of several aromatic acids preceeds degradation. A benzoyl CoA synthetase activity (AMP forming) (apparent K _m values of the enzyme from nitrate grown cells: 0.01 mM benzoate, 0.2 mM ATP, 0.2 mM coenzyme A) was present in aerobically grown and anaerobically, nitrate grown cells when benzoate or other aromatic acids were present. In addition to benzoate and fluorobenzoates, also 2-amino-benzoate was activated, albeit with unfavorable K _m (0.5 mM 2-aminobenzoate). A 2-aminobenzoyl CoA synthetase (AMP forming) was induced both aerobically and anaerobically with 2-aminobenzoate as growth substrate which had a similar substrate spectrum but a low K _m for 2-aminobenzoate (<0.02 mM). Anaerobic growth on 4-hydroxybenzoate induced a 4-hydroxybenzoyl CoA synthetase, and cyclohexanecarboxylate induced another synthetase. In contrast, 3-hydroxybenzoate and phenyl-acetate grown anaerobic cells appeared not to activate the respective substrates at sufficient rates. Contrary to an earlier report extracts from aerobic and anaerobic 2-aminobenzoate grown cells catalysed a 2-aminobenzoyl CoA-dependent NADH oxidation. This activity was 10–20 times higher in aerobic cells and appeared to be induced by 2-aminobenzoate and oxygen. In vitro, 2-aminobenzoyl CoA reduction was dependent on 2-aminobenzoyl CoA NAD(P)H, and oxygen. A novel mechanism of aerobic 2-aminobenzoate degradation is suggested, which proceeds via 2-aminobenzoyl CoA.