Purification and kinetic characterization of γ-aminobutyraldehyde dehydrogenase from rat liver

Oxidative deamination of putrescine, the precursor of polyamines, gives rise to γ-aminobutyraldehyde (ABAL). In this study an aldehyde dehydrogenase, active on ABAL, has been purified to electrophoretic homogeneity from rat liver cytoplasm and its kinetic behaviour investigated. The enzyme is a dimer with a subunit molecular weight of 51,000. It is NAD⁺-dependent, active only in the presence of sulphhydryl compounds and has a pH optimum in the range 7.3–8.4. Temperatures higher than 28°C promote slow activation and the process is favoured by the presence of at least one substrate. K_m for aliphatic aldehydes decreases from 110 μM for ABAL and acetaldehyde to 2–3 μM for capronaldehyde. The highest relative V-values have been observed with ABAL (100) and isobutyraldehyde (64), and the lowest with acetaldehyde (14). Affinity for NAD⁺ is affected by the aldehyde present at the active site: K_m for NAD⁺ is 70 μM with ABAL, 200 μM with isobutyraldehyde and capronaldehyde, and>800 μM with acetaldehyde. The kinetic behaviour at 37°C is quite complex; according to enzymatic models, NAD⁺ activates the enzyme (K_act 500 μM) while NADH competes for the regulatory site (K_in 70 μM). In the presence of high NAD⁺ concentrations (4 mM), ABAL promotes further activation by binding to a low-affinity regulatory site (K_act 10 mM). The data show that the enzyme is probably an E₃ aldehyde dehydrogenase, and suggest that it can effectively metabolize aldehydes arising from biogenic amines.     

Purification, Characterization, and Crystallization of the Components of the Nitrobenzene and 2-Nitrotoluene Dioxygenase Enzyme Systems

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an α₃β₃ hexamer. The apparent K_m of 2-nitrotoluene dioxygenase for 2NT was 20 μM, and that for naphthalene was 121 μM. The specificity constants were 7.0 μM⁻¹ min⁻¹ for 2NT and 1.2 μM⁻¹ min⁻¹ for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.     

Properties and purification of the catalytic subunit of cyclic AMP-dependent protein kinase of adipose tissue

The catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6–8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (<5 μg/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent K_m of 60 μM and V_max of 20 μmol·min⁻¹·mg⁻¹, corresponding values with mixed histones were 12 μM and 1.2 μmol·min⁻¹·mg⁻¹. With both protein substrates the apparent K_m for ATP was 11 μM. Concentrations of Mg²⁺ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7–2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated ³²P_i from [γ-³²P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.     

Kinetic and immunological differences between the retinal specific C4- and B4-lactate dehydrogenase of the cichlid fish Oreochromis mossambicus

The eye-specific C₄-lactate dehydrogenase (LDH) and the widely distributed B₄-LDH isozymes from the fish Oreochromis mossambicus were purified to homogeneity using DEAE Sepharose ion-exchange chromatography and oxamate-linked sepharose affinity-chromatography. Kinetic analysis was performed on pure B₄- and C₄-LDH. The Michaelis-Menten constant (K_m) for B₄-LDH and pyruvate was 32.3μM, for B₄-LDH and lactate 717 μM for C₄-LDH and pyruvate 14.1 μM and for C₄-LDH and lactate 1898 μM. The pure C₄-isozyme was subjected to SDS-polyacrylamide gel electrophoresis and the Coomassie Brilliant Blue-stained band injected into a rat to produce antiserum. The antiserum proved to be C₄-LDH monospecific, which will allow using it to localize the isozyme in the retina at a light and electron microscopical level.     

4,4,14α-trimethyl 9β,19-cyclo-5α-26-homocholesta-24,26-dien-3β-ol: a potent mechanism-based inactivator of Δ- to Δ-sterol methyl transferase

The title compound (4A) was synthesized and tested as a mechanism-based inactivator of the sterol methyl transferase (SMT) enzyme from Prototheca wickerhamii. Using cycloartenol as substrate, 4A was found to exhibit time-dependent inactivation kinetics, generating a K_i value of 30 μM and K_inact value of 0.30 min⁻¹.     

Surface-enhanced Raman spectroscopy combined with atomic force microscopy for ultrasensitive detection of thrombin

Indole-3-acetic acid (IAA) amide conjugates play an important role in balancing levels of free IAA in plant cells. The GH3 family of proteins conjugates free IAA with various amino acids. For example, auxin levels modulate expression of the Oryza sativa (rice) GH3-8 protein, which acts to prevent IAA accumulation by coupling the hormone to aspartate. To examine the kinetic properties of the enzyme, we developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay system. Bacterially expressed OsGH3-8 was purified to homogeneity and used to establish the assay system. Monitoring of the reaction confirms the reaction product as IAA–Asp and demonstrates that production of the conjugate increases proportionally with both time and enzyme amount. Steady-state kinetic analysis using the LC–MS/MS-based assay yields the following parameters: V/E_t^IAA = 20.3 min⁻¹, K_m^IAA = 123 μM, V/E_t^ATP = 14.1 min⁻¹, K_m^ATP = 50 μM, V/E_t^Asp = 28.8 min⁻¹, K_m^Asp = 1580 μM. This is the first assignment of kinetic values for any IAA–amido synthetase from plants. Compared with previously described LC- and thin-layer chromatography (TLC)-based assays, this LC–MS/MS method provides a robust and sensitive means for performing direct kinetic studies on a range of IAA-conjugating enzymes.     

The Metabolic Pathway of 4-Aminophenol in Burkholderia sp. Strain AK-5 Differs from That of Aniline and Aniline with C-4 Substituents

Burkholderia sp. strain AK-5 utilized 4-aminophenol as the sole carbon, nitrogen, and energy source. A pathway for the metabolism of 4-aminophenol in strain AK-5 was proposed based on the identification of three key metabolites by gas chromatography-mass spectrometry analysis. Strain AK-5 converted 4-aminophenol to 1,2,4-trihydroxybenzene via 1,4-benzenediol. 1,2,4-Trihydroxybenzene 1,2-dioxygenase cleaved the benzene ring of 1,2,4-trihydroxybenzene to form maleylacetic acid. The enzyme showed a high dioxygenase activity only for 1,2,4-trihydroxybenzene, with K_m and V_max values of 9.6 μM and 6.8 μmol min⁻¹ mg of protein⁻¹, respectively.     

Purification and substrate characterization of α-ketobutyrate decarboxylase from Pseudomonas putida

α-Ketobutyrate decarboxylase encoded in the -methionine catabolism operon of Pseudomonas putida is homologous with the E1 component of pyruvate dehydrogenase complex from gram-negative bacteria. The enzyme was purified to homogeneity from the cell extract of an Escherichia coli transformant. The purified enzyme was homodimeric with a subunit of M_r 93,000 on SDS-PAGE. The enzyme activity was activated by the addition of both thiamine pyrophosphate (TPP) and a divalent cation, such as Mg²⁺, Mn²⁺, and Co²⁺. The enzyme showed high activity for α-ketobutyrate and α-keto-n-valerate rather than pyruvate, but the α-keto acids with increasing length of the side chain as well as branching, such as α-keto-n-caproate and α-keto-3-methylvalerate, were not used by the enzyme. The K_m values for α-ketobutyrate and pyruvate were 0.016 and 0.147 mM, respectively, and the k_cat/K_m value (10.69 s⁻¹ mM⁻¹) for α-ketobutyrate was 29-fold greater than that for pyruvate. Thus, α-ketobutyrate decarboxylase is distinguished from the pyruvate dehydrogenase E1 component with respect to the substrate specificity, although their structural and enzymological properties were similar. These results suggest that the unique substrate specificity of α-ketobutyrate decarboxylase is due to a slight difference in the highly conserved active sites of both enzymes.     

Cyclobranol: a substrate for C25-methyl sterol side chains and potent mechanism-based inactivator of plant sterol methyltransferase

Cyclobranol 8A, an analog of the cycloartenol substrate 1A for the plant sterol C24-methyltransferase (SMT), was shown to be an acceptor of the soybean SMT1 as well as an inhibitor of enzyme action. The K_m and k_cat for 8A was 37 μM and 0.006 min⁻¹, respectively. The enzyme-generated product was identified by MS and ¹H NMR to be a C24, C25-doubly alkylated Δ²⁴⁽²⁸⁾-olefin 10A. Inhibitor treatment was concentration and time-dependent affording an apparent K_i of 25 μM, a maximum rate of inactivation of 0.15 min⁻¹ and a partition ratio (k_cat/k_inact) calculated to be 0.04.     

Specific cation modulation of anion transport across the human erythrocyte membrane

The specific modulation by three cations, Ca²⁺, Mg²⁺, and tetracaine of the equilibrium exchange of SO₄²⁻ across the erythrocyte membrane was investigated. While external calcium had no effect on SO₄²⁻ exchange, internal calcium, and external calcium in the presence of 10 μM A23187 were found to be potent inhibitors of the exchange reaction. The apparent inhibition constants (K₁) for Ca²⁺ were calculated to be 6.1 μM and 5 μM for the above two conditions, respectively.Unlike Ca²⁺, Mg²⁺ was shown to be a weak activator of SO₄²⁻ exchange with an apparent dissociation constant of 3.6 μM. Competition experiments demonstrated that the Ca²⁺ and Mg²⁺ sites associated with anion transport are distinct and noninteracting.Tetracaine, a cation at neutral pH, was also found to be an inhibitor of SO₄²⁻ exchange with an apparent K₁ of 0.8 mM. Although tetracaine was observed to displace calcium from non-specific sites on the erythrocyte membrane, it showed no effect on the apparent inhibition constant of Ca²⁺ for SO₄²⁻ exchange. Thus, the Ca²⁺ and tetracaine sites also appear to be independent. The difficulty of situating three mutually independent sites on a single subunit protein, i.e., band 3, is considered.Using the experimental data obtained from five individuals, the concentration of free calcium in the red cell cytoplasm was calculated to range from 0.2 to 0.7 μM. This concentration was sufficient to reduce SO₄²⁻ exchange only 3–8%. It was concluded that calcium inhibition of anion exchange, and, hence, impairment of CO₂ transport, may be physiologically significant only in senescent cells and in certain types of anemia where calcium concentrations are significantly increased.     

Studies on (NA + K)-activated ATPase XLV. Magnesium induces two low-affinity non-phosphorylating nucleotide binding sites per molecule

ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na⁺ + K⁺)-ATPase in the absence of Mg²⁺ (EDTA present) with a homogeneous but 15-fold different affinity, the K_d values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na⁺ + K⁺)-ATPase, respectively. The K_d value for ATP is equal to the K_m for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na⁺ + K⁺)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg²⁺, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The K_d and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg²⁺ concentration. The K_d increases from 0.06 mM at 0.5 mM Mg²⁺ to a maximum of 0.26 mM at 2 mM Mg²⁺ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg²⁺ to 3.3 nmol/mg protein at 4 mM Mg²⁺. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg²⁺ concentration yields a maximal binding capacity at infinite Mg²⁺ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na⁺ + K⁺)-ATPase. The K_d for Mg²⁺ at the sites, where it exerts this effect, is 0.8 mM. The K_d for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg²⁺ to a maximum of 4.2 μM at 2 mM Mg²⁺ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg²⁺ concentration. Hence, Mg²⁺ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na⁺ + K⁺)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites.     

Synthesis and evaluation of phosphopeptides containing iminodiacetate groups as binding ligands of the Src SH2 domain

Phosphopeptide pTyr-Glu-Glu-Ile (pYEEI) has been introduced as an optimal Src SH2 domain ligand. Peptides, Ac-K(IDA)pYEEIEK(IDA) (1), Ac-KpYEEIEK (2), Ac-K(IDA)pYEEIEK (3), and Ac-KpYEEIEK(IDA) (4), containing 0–2 iminodiacetate (IDA) groups at the N- and C-terminal lysine residues were synthesized and evaluated as the Src SH2 domain binding ligands. Fluorescence polarization assays showed that peptide 1 had a higher binding affinity (K_d = 0.6 μM) to the Src SH2 domain when compared with Ac-pYEEI (K_d = 1.7 μM), an optimal Src SH2 domain ligand, and peptides 2–4 (K_d = 2.9–52.7 μM). The binding affinity of peptide 1 to the SH2 domain was reduced by more than 2-fold (K_d = 1.6 μM) upon addition of Ni²⁺ (300 μM), possibly due to modest structural effect of Ni²⁺ on the protein as shown by circular dichroism experimental results. The binding affinity of 1 was restored in the presence of EDTA (300 μM) (K_d = 0.79 μM). These studies suggest that peptides containing IDA groups may be used for designing novel SH2 domain binding ligands.     

Pyruvate carboxylase from rainbow trout liver

Summary A method is described for the partial purification of pyruvate carboxylase from rainbow trout liver. The enzyme has a pH optimum of about 8.0, possesses an absolute requirement for activation by acetylCoA, and prefers MgATP over other nucleoside triphosphates. K⁺ causes a decrease in the apparentK _m for HCO ₃ ^– . AcetylCoA activation shows positive cooperativity withK _a=0.072 mM andn _H=1.78 at pH 7.7, 2.5 mM free Mg²⁺, 100 mM K⁺, and saturating concentrations of substrates. A high acetylCoA concentration causes a decrease in the apparentK _m values for MgATP and HCO ₃ ^– and a biphasic double reciprocal plot with pyruvate as the varied substrate. MgADP and AMP are competitive inhibitors with respect to MgATP. The enzyme shows a U-type response to the adenylate energy charge and retains considerable activity throughout a wide range of energy charge values. It is proposed that intramitochondrial acetylCoA concentration and the adenylate energy charge control the rate of pyruvate carboxylation in vivo.Abbreviations DTT dithiothreitol - PMSF phenylmethylsulfonylfluoride     

Purification and properties of an aminopeptidase from the mid-gut gland of scallop (Patinopecten yessoensis)

An aminopeptidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purified from an acetone-dried preparation by extracting, ammonium sulfate precipitation, Hi-Load Q column chromatography, isoelectric focusing, and POROS HP2 and HQ column chromatography. The molecular weight of the enzyme was estimated to be 61 kDa by SDS-polyacrylamide gel electrophoresis and 59 kDa by gel permeation chromatography. The isoelectric point of the enzyme was 5.2 and the optimum pH was 7.0 toward leucine p-nitroanilide (Leu-pNA). The enzyme was inhibited by o-phenanthroline. The activity of the enzyme treated with o-phenanthroline was completely recovered by adding excess Zn²⁺. Relative hydrolysis rates of amino acid-pNAs and amino acid-4-methylcoumaryl-7-amides (amino acid-MCAs) indicated that the enzyme preferred substrates having Ala or Met as an amino acid residue. The enzyme had a K_m of 32.2 μM and k_cat of 29.5 s⁻¹ with Ala-pNA and a K_m of 11.1 μM and k_cat of 9.49 s⁻¹ with Ala-MCA. The enzyme sequentially liberated amino acids from the amino-termini of Ala–Phe–Tyr–Glu.     

Effects of morphine-3-glucuronide on the antinociceptive activity of peptide and nonpeptide opioid receptor agonists in mice

The effects of morphine-3-glucuronide (M3G), a metabolite of morphine, were determined on the antinociceptive actions, as measured by the tail flick test, of morphine, a μ-opioid receptor agonist, of U-50,488H, a κ-opioid receptor agonist, of [ -Pen², -Pen⁵]enkephalin (DPDPE), a δ₁-opioid receptor agonist, and of [ -Ala²,Glu⁴]deltorphin II (deltorphin II), a δ₂-opioid receptor agonist in mice. Morphine administered ICV (2.5 μg/mouse) or SC (10 mg/kg), U-50,488H (25 mg/kg, IP), DPDPE (15 μg/mouse; ICV), and deltorphin II (15 μg/mouse, ICV) produced antinociception in mice. Intraperitoneal or ICV injections of M3G did not produce any effect on the tail flick latency nor did it affect the antinociception-induced by morphine, U-50,488H, DPDPE, or deltorphin II. Previously M3G has been shown to antagonize the antinociceptive effects of morphine in the rat. It is concluded that in the mouse, M3G neither produces hyperalgesia nor modifies the actions of μ-, κ-, δ₁-, or δ₂-opioid receptor agonists.     

Partial purification of human placental 15-hydroxyprostaglandin dehydrogenase: Kinetic properties

The enzyme system, 15-hydroxyprostaglandin dehydrogenase, which catalyzes the first inactivation step in the catabolism of the prostaglandins has been isolated and purified 107-fold from human placenta. Kinetic studies reveal different Michaelis-Menten constants for most of the naturally occurring prostaglandins. The K_m for PGE₂ was found to be 10 μM, for PGE₁, 27 μM; for PGA₂, 32 μM; for PGA₁, 33 μM; and for PGF_2α 59 μM. The enzyme has a sharp pH-optimum between 7.5 and 8.8. Prostaglandin dehydrogenase appears to be isoenzymic as judged by separation on polyacrylamide disc gel electrophoresis. Inhibition studies with the partially purified enzyme indicate that progesterone and estrogen may influence the conversion of biologically active prostaglandins into the biologically inactive 15-ketoprostaglandins. These findings offer evidence for the control of prostaglandin metabolism in the human placenta.     

Properties of γ-aminobutyraldehyde dehydrogenase from Escherichia coli

γ-Aminobutyraldehyde dehydrogenase from Escherichia coli K-12 has been purified and characterized from cell mutants able to grow in putrescine as the sole carbon and nitrogen source. The enzyme has an M_r of 195 000±10 000 in its dimeric form with an M_r of 95 000±1000 for each subunit, a pH optimum at 5.4 in sodium citrate buffer, and does not require bivalent cations for its activity. K_m values are 31.3±6.8 μM and 53.8±7.4 μM for Δ-1-pyrroline and NAD⁺, respectively. An inhibitory capacity for NADH is also shown using the purified enzyme.     

Mercaptosuccinate Dioxygenase,a Cysteine Dioxygenase Homologue,from Variovorax paradoxus Strain B4 Is the Key Enzyme of Mercaptosuccinate Degradation

The versatile thiol mercaptosuccinate has a wide range of applications, e.g. in quantum dot research or in bioimaging. Its metabolism is investigated in Variovorax paradoxus strain B4, which can utilize this compound as the sole source of carbon and sulfur. Proteomic studies of strain B4 resulted in the identification of a putative mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, possibly representing the key enzyme in the degradation of mercaptosuccinate. Therefore, the putative mercaptosuccinate dioxygenase was heterologously expressed, purified, and characterized in this study. The results clearly demonstrated that the enzyme utilizes mercaptosuccinate with concomitant consumption of oxygen. Thus, the enzyme is designated as mercaptosuccinate dioxygenase. Succinate and sulfite were verified as the final reaction products. The enzyme showed an apparent K_m of 0.4 mm, and a specific activity (V_max) of 20.0 μmol min⁻¹ mg⁻¹ corresponding to a k_cat of 7.7 s⁻¹. Furthermore, the enzyme was highly specific for mercaptosuccinate, no activity was observed with cysteine, dithiothreitol, 2-mercaptoethanol, and 3-mercaptopropionate. These structurally related thiols did not have an inhibitory effect either. Fe(II) could clearly be identified as metal cofactor of the mercaptosuccinate dioxygenase with a content of 0.6 mol of Fe(II)/mol of enzyme. The recently proposed hypothesis for the degradation pathway of mercaptosuccinate based on proteome analyses could be strengthened in the present study. (i) Mercaptosuccinate is first converted to sulfinosuccinate by this mercaptosuccinate dioxygenase; (ii) sulfinosuccinate is spontaneously desulfinated to succinate and sulfite; and (iii) whereas succinate enters the central metabolism, sulfite is detoxified by the previously identified putative molybdopterin oxidoreductase.     

α-cyano-substituted analogoues of decarboxylated S-adenosylmethionine as enzyme activated, irreversible inhibitors of S-adenosylmethionine decarboxylase

A pair of α-cyano analogues of decarboxylated S-adenosylmethionine (2a and 2b) were synthesized as potential enzyme activated, irreversible inhibitors of the[pyruvoyl enzyme S-adenosylmethionine decarboxylase (AdoMet-DC). Each of these analogues acts as an irreversible inactivator for ADoMet-DC from Escherichia coli (IC₅₀ values of 9 and 50 μM, respectively). These analogues also inactivate human AdoMet-DC, with K_I values of 246.6 and 7.2 μM, and k_inact values of 0.29 and 0.03 min⁻¹, respectively.