Intracellular localization and characterization of 3-hydroxykynureninase in human liver

• 1.1. 3-hydroxykynureninase in human liver was present in cytosol and mitoehondria.
• 2.2. The cytosolic enzyme and mitochondrial enzyme had the same physiological and enzymic properties.
• 3.3. The enzyme had a mol. wt of 130,000 by gel filtration and isoelectric point of pH 5.9.
• 4.4. The enzyme was active for 3-hydroxykynurenine and kynurenine, and its activity ratio was 15:1. The apparent K_m values of the enzyme were 7.7 × 10⁻⁵M for 3-hydroxykynurenine, 1.0×10⁻³M for kynurenine and 2.5 × 10⁻⁶M for pyridoxal 5'-phosphate with 3-hydroxykynurenine.
• 5.5. Some other properties of purified enzymes are described.

     

Isolation and characterization of lipoamide dehydrogenase from mackerel dark muscle

• 1.1. Lipoamide dehydrogenase was purified 1500-fold from mackerel dark muscle.
• 2.2. The enzyme was homogeneous as judged by acrylamide gel electrophoresis in the presence and absence of SDS.
• 3.3. Molecular weights of 102,000 and 55,000 were estimated for the native and denatured enzyme, respectively.
• 4.4. Optimal activity for the enzyme was obtained at around pH 5.7 and enhanced with citri acid.
• 5.5. Loss of activity was less than 5% by incubating the enzyme at 70°C for 20 min.
• 6.6. An apparent K_m of 3.1 × 10⁻³ M was obtained for dl-lipoic acid and 1.5 × 10⁻⁵ M for NADH.
• 7.7. The properties of lipoamide dehydrogenase from mackerel dark muscle observed in this investigation were very similar to those reported for the enzyme from other sources.

     

α,α-trehalase from the brine shrimp Artemia salina. purification and properties

• 1.1. Trehalase (α,α-trehalose 1-d-glycohydrolase, E.C. 3.2.1.28) from cryptobiotic Artemia salina embryos was purified and characterized.
• 2.2. Most of the enzyme activity was present in an insoluble form and could be solubilized by deoxycholate treatment at high ionic strength and sonication.
• 3.3. The enzyme had a molecular weight of 75,000 and its isoelectric point was 6.2. It was optimally active at pH 5.6 with a K_m = 4.3 × 10⁻³ M for its natural substrate.
• 4.4. The enzyme was highly specific for trehalose, only lactose and cellobiose being hydrolyzed to a limited extent.

     

On the role of fumarate reductase in anaerobic carbohydrate catabolism of Mytilus edulis L

• 1.1. The role of the fumarate:NADH oxidoreduction in the anaerobic glycolysis of the sea mussel is examined and discussed.
• 2.2. Fumarate reductase activity is present in submitochondrial particles especially from adductor muscle, digestive gland and mantle.
• 3.3. The pH optimum of the enzyme complex is 7.9; the approx K_m's for NADH and fumarate are 4.0 × 10⁻⁵ M and 6.3 × 10⁻⁵ M, respectively.
• 4.4. The enzyme complex is inhibitied by amytal, antimycin, ethanol, malonate, phosphate, rotenone, and succinate, and stimulated by Mg²⁺.
• 5.5. It is concluded that part of the mitochondrial respiratory chain is involved in the reduction of fumarate by NADH, comprising site 1 of the oxidative phosphorylation.

     

Purification,characterization and biological activities of phospholipase a from russell's viper (Vipera russelli) venom

• 1.1. The major phospholipase A has been purified to electrophoretic homogeneity from the venom of Vipera russelli (Russell's viper).
• 2.2. The molecular weight of the purified enzyme was estimated to be 31,000 by Sephadex G-75 gel filtration chromatography and 29,000 by SDS-polyacrylamide gel electrophoresis. The enzyme exhibited an apparent K_m value of 2.3 × 10⁻² M.
• 3.3. The phospholipase A showed edema forming, indirect hemolytic and myonecrotic activities but not hemorrhagic activity.

     

Purification and properties of tetralysine endopeptidase from Escherichia coli AJ005

• 1.1. A new tetralysine endopeptidase from Escherichia coli AJ005 has been purified about 135-fold.
• 2.2. The peptidase seems to be specific to tetralysine among lysine homopolymers.
• 3.3. The optimal pH was about 7.5
• 4.4. The activity was inhibited by KCN but not inhibited by soybean trypsin inhibitor.
• 5.5. The apparent K_m value was 2.5 × 1O⁻³ M for tetralysine.

     

Ouabain sensitive Na+/K+-transporting ATPase from the brain of Poekilocerus bufonius

• 1.1. The properties of Na⁺/K⁺-transporting ATPase in microsomal fractions from the nervous tissue of the grasshopper, Poekilocerus bufonius were investigated.
• 2.2. Two components of ATPase activity are present.
• 3.3. Inclusion of 1 mM ouabain in the incubation media reduced the activity of total and Na⁺/K⁺-ATPase by 57 and 79%, respectively.
• 4.4. The maximum velocity (V_max) was decreased by the addition of 1 mM ouabain, whereas the apparent K_m value was not affected indicating a non-competitive type of inhibition.
• 5.5. The calculated value of the pI₅₀ was 6.4 (I₅₀ = 3.98 × 10⁻⁷M) for ouabain inhibition of the enzyme showing great sensitivity to the cardiac glycoside ouabain.
• 6.6. The present results show that the physicochemical properties of Na⁺/K⁺-transporting ATPase from the brain of P. bufonius are essentially the same as for the enzyme prepared from the excretory system of the insect which has been previously investigated.
• 7.7. Dissimilarities were also observed between these tissues in the way that the enzyme from the brain was sensitive to ouabain inhibition with a non-competitive type rather than a ouabain-resistance and a competitive type of inhibition for the enzyme from the excretory system.
• 8.8. These dissimilarities are probably due to different isoenzyme patterns available in the same insect.

     

l-lysinamidase from Cryptococcus laurenti 112. Purification and properties

• 1.1. The purified enzyme hydrolyzes the linear l-lysinamide and the cycle amide of l-lysine—l-α-amino-ϵ-caprolactam.
• 2.2. The apparent relative molecular mass is 180,000. The enzyme consists of four subunits and the molecular mass of a single subunit was found to be 47,000.
• 3.3. The coefficient of molecular sedimentation equals 8.3 S, the isoelectric point was determined to be pH 4.3
• 4.4. The enzyme is not a glycoprotein. p-Mercuribenzoate binds 10 SH-groups of the native enzyme molecule and 20 SH-groups in the presence of 0.7% SDS.
• 5.5. pH- optimum for the hydrolysis of l-lysine amides was observed to be 7.5–7.7. The enzyme is strictly dependent on Mn²⁺ and Mg²⁺.
• 6.6. The kinetic parameters for the hydrolysis of l-lysinamide where K_m = 3.8 mM and k_cat = 3000 sec⁻¹ For the hydrolysis of cyclic L-lysinamide K_m = 4.8 mM and k_cat = 2600 sec⁻.

     

Purification and some properties of a Mg2+-activated acid phosphatase from rat testis

• 1.1. The acid phosphatase (AcPase, EC 3.1.3.2) IV from rat testicular tissue was purified to apparent homogeneity.
• 2.2. The enzyme displays a native molecular weight of 70 kDa determined on gel permeation chromatography on a Sephadex G-100 column and 68 kDa using linear 5–20% sucrose density gradient centrifugation. The subunit molecular weight on SDS-PAGE analysis is 67 kDa, suggesting that the enzyme is a monomeric protein.
• 3.3. The enzyme does not bind to Concanavaline A-Sepharose 4B column, indicating that it is not a glycoprotein.
• 4.4. The rat testis AcPase IV is a metal activated enzyme in which Mg²⁺ is the metal activating agent with a K_a, = 0.88 × 10⁻³ M. The Michaelis constant for p-nitrophenylphosphate, in the presence of saturating concentrations of Mg²⁺ ions, is 0.23 × 10⁻³ M.
• 5.5. The enzyme preferentially hydrolizes p-nitrophenylphosphate, phenylphosphate and ATP.

     

Soluble cyclic amp-dependent protein kinases from chick liver: Effects of NaCl and heat-stable modulator

• 1.1. Two cyclic AMP-dependent protein kinases—Fraction I and II—have been isolated from chick liver soluble preparation on DEAE-cellulose.
• 2.2. Both fractions have an apparent K_m for ATP of 2 × 10⁻⁶M, are stimulated maximally by 5 × 10⁻⁸ M cyclic AMP and phosphorylate mainly basic proteins—histone and protamine.
• 3.3. They exhibit various pH values for optimal activity and show differences with respect to both sensitivity to NaCl and substrate specificity.
• 4.4. The heat-stable protein modulator inhibits the cyclic AMP-dependent protein kinase activity of both fractions, but with cyclic GMP one kinase is stimulated and the other inhibited.
• 5.5. Slight differences in histone triggered holoenzyme dissociation as well as the lack of difference between their ability for subunit reassociation do not allow to classify these isozymes as protein kinases of Type I and II, according to Corbin et al. (1975).

     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

Kinetic and regulatory properties of phenylalanine ammonia-lyase from Citrus sinensis

• 1.1. The kinetic and regulatory properties of phenylalanine ammonia-lyase from Citrus sinensis fruit tissue were investigated. The substrate specificity of the enzyme was determined as well as the effects of pH and temperature on the catalytic activity.
• 2.2. The enzyme exhibits negative homotropic effects between the substrate binding centra.
• 3.3. Binding of l-phenylalanine to the enzyme is characterized by two K_m-values; K_m^L = 13 μM and K_m^H = 52 μM; with a Hill-interaction coefficient of 0.75.
• 4.4. The enzyme is subject to product inhibition by trans-cinnamate, but the effects of allosteric effectors and inhibitors seem to be of much greater importance in the short-term regulation of phenylpropanoid metabolism in Citrus sinensis.
• 5.5. The enzyme activity was found to be modulated by end-products of diverging metabolic pathways, viz. umbelliferone, scopoletin, naringenin, quercetin, kaempferol, benzoic acid and gallic acid.

     

Kinetic analysis of rat liver sorbitol dehydrogenase

• 1.1. A steady state kinetic investigation was performed on an improved preparation of rat-liver sorbitol dehydrogenase (l-iditol: NAD-oxidoreductase, EC 1.1.1.14).
• 2.2. Data analyses indicate the enzyme follows a rapid equilibrium random mechanism in the direction of sorbitol oxidation and a random mechanism in the direction of fructose reduction.
• 3.3. Kinetic constants were: K_m^NAD 0.082 mM; K_m^sorbitol 0.38 mM; K_m^NADH 67 μm; K_m^fructose 136 μM.
• 4.4. Evidence is adduced to indicate the more rapid reverse (fructose reduction) reaction is susceptible to metabolic control by formation of abortive enzyme-fructose-NAD and enzyme-NADH-sorbitol complexes.

     

Ostrich fructose-1,6-bisphosphatase: Kinetic characterization of the liver isoenzyme

• 1.1. Purified ostrich (Struthio camelus) liver fructose-1,6-bisphosphatase exhibited an absolute requirement for Mg²⁺.
• 2.2. The enzyme catalyzed the hydrolysis of fructose-1,6-bisphosphate, sedoheptulose-l,7-bisphosphate and ribulose-l,5-bisphosphate.
• 3.3. S_0.5 for substrate was 1.4 μM.
• 4.4. AMP was a potent non-competitive inhibitor with respect to substrate (K_i of 25 μM).
• 5.5. Fructose-2,6-bisphosphate was a potent competitive inhibitor of the enzyme (K_i of 4.8 μM).

     

A fluorescence study of substrate and inhibitor binding to bovine liver dihydrofolate reductase

• 1.1. Covalent coupling of fluorescein to methotrexate (MTX) by a 5-carbon spacer yields a dihydrofolate reductase (DHFR) inhibitor (FMTX) with K_i = 11 nM.
• 2.2. FMTX shows a fluorescence quenching with respect to fluorescein which is relieved by binding to the enzyme.
• 3.3. The dissociation constants (K_d) of MTX, FMTX, NADPH and 7,8-dihydrofolate (DHF) from bovine liver DHFR have been determined by fluorometric titrations.
• 4.4. The K_d values for NADPH, MTX and FMTX from the complementary binary complexes (MTX·DHFR, FMTX·DHFR and NADPH·DHFR) were also obtained; these show a 2- to 4-fold decrease with respect to those obtained by titration of the free enzyme.
• 5.5. A competitive assay for MTX has been developed by exploiting the fluorescence enhancement of DHFR-bound FMTX. This assay may be useful for the routine determination of MTX in the concentration range from 10⁻⁹ to 10⁻⁷ M.

     

Purification and some properties of NAD+-dependent glutamate dehydrogenase from Halobacterium halobium

• 1.1. A NAD⁺-dependent glutamate dehydrogenase (EC 1.4.1.2.) was purified 126-fold from Halobacterium halobium.
• 2.2. Activity and stability of the enzyme were affected by salt concentration. Maximum activity of the NADH-dependent reductive amination of 2-oxoglutarate occurs at 3.2 M NaCl and 0.8 M KCl, and the NAD⁺-dependent oxidative deamination of l-glutamate occurs at 0.9 M NaCl and 0.4 M KCl. The maximum activity is higher with Na⁺ than with K⁺ in the amination reaction while the reverse is true in the deamination reaction.
• 3.3. The apparent K_m values of the various substrates and coenzymes under optimal conditions were: 2-oxoglutarate, 20.2 mM; ammonium, 0.45 M; NADH, 0.07 mM; l-glutamate, 4.0 mM; NAD⁺, 0.30 mM.
• 4.4. No effect of ADP or GTP on the enzyme activity was found. The purified enzyme was activated by some l-amino acids.

     

Purification and properties of hydroxypyruvate reductase from Lemna minor L

• 1.1. Hydroxypyruvate reductase has been purified 193-fold from Lemna minor L. by affinity chromatography on Blue Sepharose.
• 2.2. The enzyme has activity over a broad pH range (optimum pH 6), a K_m hydroxypyruvate of 59 μ M and K_m NADH of 12μM.
• 3.3. Crude extracts of Lemna exhibit substrate inhibition of activity above 1 mM hydroxypyruvate, a property which is lost on purification.
• 4.4. Oxaloacetate inhibits purified preparations of the enzyme and a possible role for such regulation in vivo is discussed.

     

The dynamics of the permeation of an analogue of insect juvenile hormone into nematodes

• 1.1. ¹⁴C-dichlorofarnesoate permeated rapidly into Haemonchus contortus (infective juveniles) and Panagrellus redivivus (mixed cultures) and was strongly bound by hydrophobic association (K_s > 10⁻⁴M).
• 2.2. Uptake rose linearly with increases in temperature (5–38°C) and external concentration (C₀; 0.07–2.15 × 10⁻⁴ M). Within 1 hr the internal concentration, C₁ was >C C₀.
• 3.3. The pH of the medium (6–8) did not affect uptake.
• 4.4. Efflux of dichlorofarnesoate was low: the half-time of release was > 18 hr.
• 5.5. The uptake curve approximated to the expression C₁/C₀ = a(1 − e^−bt) with a and b as constants and t in hr.
• 6.6. These results clarify previous work on the inhibitory action of juvenile hormone on the development of nematodes.

     

Lysosomal acid deoxyribonuclease from vervet monkey livers—II: Enzymic properties

• 1.1. Primate liver lysosomal acid DNase is an endonucleolytic enzyme.
• 2.2. The enzyme has both 3'- and 5'-nucleotidohydrolase activities.
• 3.3. The oligonucleotides produced by DNase are polymers mainly about 30 mononucleotides long.
• 4.4. The Arrhenius plot shows a discontinuity with a transition temperature at 47°C, with an activation energy of 107 kJ/mol below and 67 kJ/mol above this temperature.
• 5.5. The activation enthalpy is 104kJ/mol and the entropy −0.498 kJ/mol/K.
• 6.6. The enzyme is subject to substrate inhibition and the K_m value is 159 × 10⁻³mM DNA-P.

     

A peptide inhibitor for S-adenosyl-l-methionine-dependent transmethylation reactions: Purification and characterization

• 1.1. A proteinaceous inhibitor for S-adenosyl-l-methionine (AdoMet)-dependent transmethylation reactions has been purified to apparent homogeneity from rat liver cytosolic fraction.
• 2.2. The peptide was made up of 29 amino acid residues with a molecular weight of 2,584. Glycine accounted for 52% of the total amino acids.
• 3.3. Employing AdoMet: protein-carboxyl O-methyltransferase (Protein methylase II) and bovine serum γ-globulin as in vitro substrate, the mode of inhibition was found to be non-competitive with K_i value of 1.9 × 10⁻⁸ M.
• 4.4. When the inhibitor was present in the reaction mixture together with S-adenosyl-l-homocysteine (AdoHcy), which is a competitive inhibitor for AdoMet, the extent of inhibition exceeded that exerted by each individual inhibitor alone, suggesting that the sites of the inhibitors on the enzyme molecule are different.
• 5.5. Almost a stoichiometric relationship exists between the enzyme and the inhibitor molecule, the ratio being approx one.