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.

     

Fructose-1,6-bisphosphatase in mantle of the sea mussel Mytilus galloprovincialis Lmk—I. Purification and ion requirements

• 1.1. The enzyme fructose-1,6-bisphosphatase was purified from the mantle of the sea mussel Mytilus galloprovincialis Lmk. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The mol. wt and subunit mol. wt of the enzyme were 105,000 and 27,000, respectively.
• 2.2. Divalent cations are essential for the enzyme activity. In the absence of chelating agents, FBPase 1 exhibits hyperbolic kinetics with respect to Mn²⁺, Zn²⁺ and Mg²⁺. The K_m for Mg²⁺ is lower than the physiological concentration of cation in the tissue, whereas its K_m for Mn²⁺ and Zn²⁺ is greater than the respective in vivo concentrations.
• 3.3. The joint action of Mg²⁺ and Zn²⁺ increases the affinity of the enzyme for the substrate Fru-1,6-P₂, though V_max is reduced.
• 4.4. Na⁺ strongly inhibits the enzyme even at very low concentrations. K⁺ has no effect whatsoever.

     

An alkaline phosphatase from Thermus sp strain Rt41A

• 1.1. A thermostable orthophosphoric monoester phosphohydrolase (EC 3.1.3.1) from Thermus sp strain Rt41A has been purified 400-fold to give a specific activity of 25 U/mg at 60°C in IM diethanolamine (pH 11.1).
• 2.2. The enzyme has a M_r of 160,000 and is trimeric.
• 3.3. The half-life of the enzyme is 5 min at 85°C.
• 4.4. The enzyme has a wide specificity for a number of phosphate monoesters.
• 5.5. The H_m of the enzyme is pH dependent, so the pH optimum of the enzyme is affected by the substrate concentration.
• 6.6. The enzyme is inhibited 50% by 20 mM Ca²⁺ or Mg²⁺.
• 7.7. The K_i for phosphate, EDTA-di sodium salt and arsenate (in 1 M diethanolamine, pH 11.1) is approx 1.2, 1.6 and 4mM respectively.
• 8.8. Urea (200 mM) is not inhibitory.

     

Purification and some properties of apurinic/apyrimidinic dna endonucleases in rat liver

• 1.1. Three kinds of apurinic/apyrimidinic (AP) DNA endonucleases, APcI, APcII, APcIII were purified from rat liver chromatin.
• 2.2. Molecular weights of APcI, APcII and APcIII were 30,000, 42,000 and 13,000 Da, which have isoelectric points of 7.2, 6.3 and 6.2, respectively.
• 3.3. Mg²⁺ was essential for the activities of these 3 enzymes, and sulfhydryl compounds (βercaptoethanol) had a stimulatory effect on the enzyme activities while N-ethylmaleimide and HgCl₂ inhibited the enzyme activity.
• 4.4. K_m values of APcI, APcII and APcIII for AP site of DNA were 0.53, 0.27 and 0.36 μM, respectively, and AMP was the most potent inhibitor to these three enzymes among nucleotides tested.

     

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.

     

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.

     

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 studies of catechol-o-methyltransferase from the brain of the African catfish,Clarias gariepinus

• 1.1. Optimum in vitro conditions, and kinetics of the enzyme catechol-O-methyltransferase from the brain of the male African catfish were studied.
• 2.2. A saturated level for S-adenosylmethionine, as methyldonor, and magnesium as cofactor was reached at 5 μM and 10 mM, respectively.
• 3.3. The addition of ascorbic acid, as an antioxidant, and tranylcypromine, as a MAO inhibitor, was not necessary, during incubations with fore-brain homogenates.
• 4.4. Kinetic analysis of the methylation of catecholestrone, catecholestradiol and dopamine showed K_m values of 1.2, 0.6 and 0.5 μM, respectively.
• 5.5. The affinity of the catecholsubstrates for the enzyme catechol-O-methyltransferase is much higher in the brain of the African catfish than in tissues of mammals.

     

The regulation of glucose 6-phosphate dehydrogenase from Dicentrarchus labrax (bass) liver

• 1.1. Kinetic constant values of the reaction catalyzed by bass liver glucose 6-phosphate dehydrogenase show to be modified between 10 and 40°C.
• 2.2. The Arrhenius plot between 10 and 50°C shows two slopes with different activation energies.
• 3.3. These results suggest a regulation of this enzyme by environmental temperature.
• 4.4. Kinetics of ATP inhibition were examined between pH 6.2 and 7.8: patterns and K_i values obtained are affected by the pH variation.
• 5.5. NADH is an effective inhibitor of bass glucose 6-phosphate dehydrogenase but this enzyme does not show NAD-linked activity.
• 6.6. Kinetics of pyridoxal 5′-phosphate inhibition have indicated the presence of a lysine in the catalytic site for NADP⁺.

     

Malate dehydrogenase isoforms from ribbed mussel (Modiolus demissus) gill tissue

• 1.1. The malate dehydrogenase (MHD) activity from the ribbed mussel gill is polymorphic with two distinct mitochondrial forms (M1 and M2) and five forms that could be resolved from cytosolic extracts (C1 to C5) by DEAE-cellulose chromatography and starch gel electrophoresis.
• 2.2. Two of the cytosolic forms (C3 and C4) may represent interchangeable conformational states.
• 3.3. With kinetic analysis there appear to be three distinct cytosolic forms (C1, C2 and C3–C4), with C2 possibly behaving as a heterodimer.
• 4.4. The identity of C5 is uncertain.
• 5.5. The forms isolated from the mitochondria (M1 and M2) exhibited lower apparent K_ms for oxaloacetate (OAA) than the cytosolic forms.
• 6.6. For all isozymic forms, the apparent K_ms for OAA increased as the pH increased between pH 6 and 9
• 7.7. Increasing the salt concentration raised the K_m for OAA for all forms.
• 8.8. The mMDHs were more sensitive to inhibition by NaCl than the cMDHs.
• 9.9. Representative cMDH (C1) and mMDH (M2) isozymes exhibited substrate inhibition by high concentrations of OAA with the mMDH possessing lower K_is for substrate inhibition than the cMDH at each pH tested.
• 10.10. Differences and similarities in K_m app. for OAA at the different pHs and salt concentrations indicated that C1, C2 and C3–C4 and C5 were distinct forms, that M1 and M2 were distinct but very similar to each other, and that C1, C2, C3–C4 and C5 were distinct from M1 and M2.

     

Purification and characterization of casein kinase II from lactating rabbit mammary gland

• 1.1. Highly purified 200 kDa casein kinase II from rabbit lactating mammary gland (MG-CK II) was obtained by means of a new purification procedure consisting of one phosphocellulose and three Mono Q steps.
• 2.2. Its K_m for ATP was 2.22 μM and 0.57 mg/ml and 0.13 mg/ml for partially dephosphorylated casein and phosvitin respectively. Stathmine was also suitable as substrate. 2-aminopurine and 6-dimethylaminopurine inhibited efficiently MG-CK II K_i = 5 and 1 mM respectively).
• 3.3. MG-CK II autophosphorylated on its α-, α '- and β-subunits. The β-subunit auto-phosporylation was enhanced in presence of exogenous substrate. Its modulation was highly dependent on ATP concentration.
• 4.4. The effects of basic compounds which affected dramatically the phosphorylation of dephosphorylated casein in presence of various ATP concentrations were reported.

     

Identification and characterization of a third form (D3) of cyclic 3'5'-nucleotide phosphodiesterase from rhizobwm fredii

• 1.1. A third form (D3) of cyclic nucleotide phosphodiesterase from Rhizobiumfrediiv/as detected and characterized for the first time.
• 2.2. The enzyme could hydrolyse both cyclic AMP and cyclic GMP with apparent K_m for cyclic AMP of approx. 0.2 μM.
• 3.3. D3 cyclic nucleotide phosphodiesterase had a pH optimum of about 6.0 when hydrolysing cyclic AMP.
• 4.4. The enzyme lost almost all its activity when heated to 60°C for 20 min.
• 5.5. Gel filtration with Sephadex G-100 gave a mol. wt of approx. 42.5 kD for the native enzyme.

     

Purification and some properties of an atypical alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium

• 1.1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium.
• 2.2. The enzyme has an apparent molecular weight of 24,000.
• 3.3. A K_m value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions.
• 4.4. The enzyme is selectively activated and stabilized by Mn²⁺.
• 5.5. It requires high salt concentrations for stability and maximum activity.
• 6.6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.

     

A kinetic study of glycogen phosphorylase b from the mantle tissue of Mytilus galloprovincialis,Lmk

• 1.1. In order to assign a meaningful role to the phosphorolytic pathway in Mytilus glycogen metabolism the kinetic mechanism of phosphorylase b, and its allosteric control, were studied.
• 2.2. The kinetic parameters of phosphorylase b from the mussel Mytilus galloprovincialis were determined. Michaelis constants (K_m or S_0.5) were in the range of 0.32–2.49 mg/ml for glycogen, 7–16 mM for P_i and 114–423 μM for AMP. In the direction of glycogen synthesis, the K_m value for glucose-1-P was approximately 180 mM.
• 3.3. The enzyme displayed homotropic co-operativity towards the binding of co-substrate and AMP (Hill coefficients of 2 and 1.4, respectively) and heterotropic co-operativity between substrates and AMP.
• 4.4. The concentration of glycogen in the Mytilus mantle is between 38- and 125-fold higher than the apparent K_m of phosphorylase b; the concentration of AMP varies throughout the year from 10 to 175 μM, up to a value close to the apparent K_m for the effector.
• 5.5. The apparent K_m for P_i is close to the concentration found in the mantle. This ligand showed more important regulatory effects than the effector AMP.

     

S-Adenosyl-l-methionine decarboxylase activities in Mytilus edulis

• 1.1. The activities of S-adenosylmethionine decarboxylase (EC 4.1.1.50) were measured in cell extracts of mantle, hepatopancreas and foot from Mytilus edulis.
• 2.2. The apparent molecular weights of the enzymes estimated by gel filtration chromatography were 65,000 ± 10,000.
• 3.3. The enzymes do not require bivalent cations for catalysis and show optimum pH between 7.0–8.0 in phosphate buffer.
• 4.4. The hepatopancreas enzyme shows different behavior to the other two enzymes against temperature and its activity is strongly inhibited by NH₄⁺.
• 5.5. The apparent K_ms for S-adenosylmethionine were found to be 300, 200 and 250 μM for the hepatopancreas, mantle and foot enzymes, respectively.

     

Gastric acid secretion in the chicken: Effect of histamine H2 antagonists and H+/K+-ATPase inhibitors on gastro-intestinal pH and of sexual maturity calcium carbonate level and particle size on proventricular H+/K+ ATPase activity

• 1.1. Cimetidine was more potent 4hr after a single injection of 25 or lOOmg/kg body wt in increasing gastric pH than other H₂ receptor antagonists, ranitidine and famotidine but was less efficient than H⁺/K⁺-ATPase inhibitors. Omeprazole rose proventricular and gizzard pH at a lower dose than SCH 28080 and Ro 18-5364 (30, 50 and 200 mg/kg body wt, respectively).
• 2.2. Proventricular and gizzard pH values were maximal 1 and 4 hr after a single injection of 7.5 μmol/kg body wt omeprazole. Inhibition of acid secretion was maintained for 24 hr after an injection of 100 μmol/kg.
• 3.3. H⁺/K⁺-ATPase activity in vitro was 10μimol P_i/hr/mg protein in the microsomal fractions of the proventriculus. It was doubled by nigericine and inhibited by SCH 28080. However, western blots by high specific H⁺/K⁺-ATPase monoclonal antibody 95-A3 and 95–111 recognized a 42kDa band but hardly exhibited the specific 95 kDa band recognition.
• 4.4. Chickens and immature pullets showed a higher H⁺/K⁺ -ATPase activity than laying hens. Calcium level of the diet did not affect the enzyme activity but coarse particles of calcium fed to pullets or laying hens enhanced the H⁺/K⁺-ATPase activity when compared with ground particles.

     

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.

     

Kinetic analysis of a cytoplasmic casein kinase II from Artemia sp.

• 1.1. A cytoplasmic casein kinase II (CKII) has been purified more than 10,000-fold from Artemia sp.
• 2.2. The reaction mechanism of the cytoplasmic CKII was determined to be random bi bi, using ATP and casein as substrates, which is in agreement with the results obtained for a DrosophilaCKII [Glover, Shelton and Brutlag (1983) J. biol. Chem.258, 3258–3265]. K_m values for ATP and casein are 8 μM and 0.2 mg/ml respectively. The binding of either substrate lowers the enzyme-affinity for the other by a factor α = 1.65.
• 3.3. In vitro, the enzyme is inhibited by poly(A)²-mRNA and 2,3-diphosphoglyceric acid (2,3-DPG). The inhibition by 2,3-DPG is due to competition with the protein substrate.
• 4.4. The possible in vivo effects of these inhibitors in CKII-mediated translational regulation is discussed.

     

Ionic dependence and vanadate sensitivity of (Na+, K+)-ATPase isolated from branchial sac of ascidians

• 1.1. Ion dependence and vanadium-induced inhibition on branchial sac ATPase in five species of ascidian Phlebobranchiata (vanadium-accumulating) and Stolidobranchiata (iron-accumulating) were studied.
• 2.2. The ATPase was obtained from the microsomal fraction, which was prepared from each ascidian branchial sac.
• 3.3. The ATPase was dependent on Mg²⁺ and activated by exogenous Na⁺ + K⁺.
• 4.4. Ouabain inhibited the ATPase activity in vitro, 10 μM to 100 μM vanadate, in vitro, suppressed the (Na⁺, K⁺)-ATPase.

     

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.