Characterization of two Arabidopsis thaliana fructokinases

Two different fructokinase isoforms of Arabidopsis thaliana have been identified and characterized by non-denaturing electrophoresis followed by activity-staining. The two fructokinases, fructokinase1 (FRK1) and fructokinase2 (FRK2), showed a high specificity for fructose and did not stain when glucose or mannose were used as substrate. Fructose and ATP at high concentrations (above 5 mM) induced a substrate inhibition of the two enzymatic activities. Arabidopsis FRK1 and FRK2 were capable of employing GTP, CTP, UTP and TTP as phosphate donors, although with a significantly lower efficiency than ATP. The two fructokinase activities were also activated by K⁺, at around 10–20 mM, and inhibited by ADP and AMP at concentrations above 10 mM. Finally, FRK1 and FRK2 showed a different expression pattern in the plant, with FRK1 being more abundant in the roots and FRK2 in the shoots. The results demonstrate a simple technique that provides important information about fructokinase activities in the plants and which can be useful for the analysis of Arabidopsis mutants.     

Hexokinases of spinach leaves

Two soluble hexokinases and a particulate hexokinase have been separated and partially purified from spinach leaves. One of the soluble hexokinases showed a high affinity for glucose (K_m = 63 μM) which was far greater than that for fructose (K_m = 9.1 mM). However, with saturating fructose the activity was twice that with saturating glucose. The particulate hexokinase showed kinetic properties similar to those of this soluble hexokinase. The second soluble hexokinase was distinct in that it was much more active with fructose than with glucose at all concentrations tested, although the K_m values for these hexoses (210 μM and 71 μM respectively) were similar. The activity of this hexokinase was stimulated by the monovalent cations K⁺ and NH₄⁺.     

S-Adenosylmethionine synthetase in bloodstream Trypanosoma brucei

S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10–250 μM the K_m for methionine was 20 μM, and this changed to 200 μM for the range 0.5–5.0 mM. In the range 10–250 μM the K_m for ATP was 53 μM, and this changed to 1.75 mM for the range 0.5–5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with [¹⁴C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (K_i 240 μM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-α-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration ≈ 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism.     

The effects of α- and β-adrenergic agents,Ca2+ and insulin on 2-deoxyglucose uptake and phosphorylation in perfused rat heart

Insulin (0.1 μM) and 1 μM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent V_max without altering the K_m. Isoproterenol (10 μM), 50 μM methoxamine and 10 mM CaCl₂ also increased uptake. Lowering of the perfusate Ca²⁺ concentration from 1.27 to 0.1 mM Ca²⁺, addition of the Ca²⁺ channel blocker nifedipine (1 μM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 μM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 μM insulin was only partly inhibited by Ca²⁺ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca²⁺, respectively. Maximal concentrations of insulin (0.1 μM) and epinephrine (1 μM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 μM but maximal concentrations of epinephrine (e.g., 1 μM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca²⁺ in signal reversal was also studied. Removal of 1 μM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca²⁺ concentration was lowered to 0.1 mM or nifedipine (1 μM) was added. Similarly, removal of 0.1 μM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca²⁺ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca²⁺ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca²⁺-dependent increase in 2-deoxyglucose transport from either α or β receptors. Insulin has both a Ca²⁺-dependent and a Ca²⁺-independent component. Reversal studies suggest an additional role for Ca²⁺ in maintaining the activated transport state when activated by either epinephrine or insulin.     

Adrenaline stimulates H2O2 generation in liver via NADPH oxidase

It is known that adrenaline promotes hydroxyl radical generation in isolated rat hepatocytes. The aim of this work was to investigate a potential role of NADPH oxidase (Nox) isoforms for an oxidative stress signal in response to adrenaline in hepatocytes. Enriched plasma membranes from isolated rat liver cells were prepared for this purpose. These membranes showed catalytic activity of Nox isoforms, probably Nox 2 based on its complete inhibition with specific antibodies. NADPH was oxidized to convert O₂ into superoxide radical, later transformed into H₂O₂. This enzymatic activity requires previous activation with either 3 mM Mn²⁺ or guanosine 5′-0-(3-thiotriphosphate) (GTPγS) plus adrenaline. Experimental conditions for activation and catalytic steps were set up: ATP was not required; S_0.5 for NADPH was 44 μM; S_0.5 for FAD was 8 μM; NADH up to 1 mM was not substrate, and diphenyleneiodonium was inhibitory. Activation with GTPγS plus adrenaline was dose- and Ca²⁺-dependent and proceeded through α₁-adrenergic receptors (AR), whereas β-AR stimulation resulted in inhibition of Nox activity. These results lead us to propose H₂O₂ as additional transduction signal for adrenaline response in hepatic cells.     

Substrate specifity of the hexose carrier in the plasmalemma of Chenopodium suspension cells probed by transmembrane exchange diffusion

Substrate specifity of the proton-driven hexose cotransport carrier in the plasmalemma of photoautotrophic suspension cells of Chenopodium rubrum L. has been studies through the short-term perturbation of ¹⁴C-labelled efflux of 3-O-methyl-d-glucose. Efflux, occurring exclusively via carrier-mediated exchange diffusion, is trans-stimulated by the substrate and trans-inhibited by the glucose-transport inhibitors phlorizin (K _1/2=7.9 mM) and its aglucon phloretin (K _1/2=84 μM); with both inhibitors, 3-O-methyl-d-glucose efflux may be blocked completely. Trans-stimulation of efflux (up to fourfold) by a variety of the d-enantiomers of neutral hexoses, including glucose (K _1/2=48 μM), 3-O-methyl-d-glucose (K _1/2=139 μM), and fructose (K _1/2=730 μM), but not by, for instance, d-allose, and l-sorbose, shows that carrier-substrate interaction critically involves the axial position at C-1 and C-3, respectively. We suggest that substrate binding by the Chenopodium hexose carrier involves both hydrophobic interaction with the pyran-ring and hydrogen-ion bonding at C-1 and C-3 of the d-glucose conformation.     

Purification and properties of pyruvate kinase type M2 from rat lung

(1) Pyruvate kinase type M₂ from rat lung has been purified 840-fold with an overall yield of 20%. The enzyme gave a single band upon SDS-electrophoresis and isoelectrofocusing and had a specific activity of 1340 U/mg protein. The homotetramer of M_r = 224 000 and an isoelectric point of pH 5.8 had an amino acid composition closely resembling that of other pyruvate kinase isoenzymes type M₂, excepts that of the chicken liver. The enzyme was crystallized. (2) The enzyme has its pH optimum at pH 6.5. The K_0.5 value for phosphoenolpyruvate is 0.26 mM (n_H = 1.81) which decreases in the presence of 0.2 mM fructose 1,6-bisphosphate to 0.056 mM (n_H = 1.06). 1 μM fructose 1,6-bisphosphate activates the enzyme at 0.1 mM phosphoenolpyruvate half-maximally. The K_m value for ADP at 1 mM phosphoenolpyruvate is 0.4 mM. The K_m value for other nucleoside diphosphates increases in the order ADP<GDP<IDP<UDP. (3) No evidence for an interconversion of pyruvate kinase type M₂ from rat or chicken lung was found. The enzyme was neither a substrate for the cAMP-dependent protein kinase from rabbit muscle nor for the cAMP-independent protein kinase from chicken liver. Since pyruvate kinase type M₂ from chicken liver is inactivated by phosphorylation catalyzed by a cAMP-independent protein kinase (Eigenbrodt, E., Abdel-Fattah Mostafa, M. and Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1047–1055) we suggest that the interconvertible form of pyruvate kinase type M₂ may represent a separate form of the pyruvate kinase type M₂ family.     

Purification and characterization of fructokinase from developing tomato (Lycopersicon esculentum Mill.) fruits

A procedure is described which allows the purification of fructokinase (EC 2.7.1.4) from young tomato fruit. The procedure yielded a 400-fold purification and two isoenzymes designated fructokinase I and II (FKI and FKII) were separated by anion-exchange chromatography. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the molecular mass was estimated to be 35 kDa. Gel filtration on Sepharose-12 indicated that for both fructokinases the functional form is a dimer. Two dimensional isoelectric focusing/SDS-PAGE combined with immunoblotting showed that FKI has two components with isoelectric points (pIs) of 6.42 and 6.55, while four components with pIs from 6.07 to 6.55 were detected for FKII. A mixture of both fructokinases showed that the components of FKI match the more alkaline components of FKII. The activity of both fructokinases increased with increasing pH to around 8.0 and equal activity was observed from 8.0 to 9.5. Both fructokinases were specific for fructose with K _m values for fructose of 0.131 and 0.201 mM for FKI and FKII, respectively. At high concentrations (> 0.5 mM), fructose was also a strong inhibitor with inhibition constants (K _i) of 1.82 and 1.39 mM for FKI and FKII, respectively. The preferred phosphate donor for both isoforms was ATP, and K _m values of 0.11 and 0.15 mM were observed for FKI and FKII. At low concentrations (0.05–0.2 mM), fructose exhibited noncompetitive inhibition with respect to ATP for both fructokinases. This inhibition pattern changed to uncompetitive when higher fructose concentrations (0.5–10 mM) were used. These data indicated that substrate addition is ordered, with ATP adding first. Inhibition by ADP was also affected by the fructose concentrations. At 0.5 mM fructose, FKI showed non-competitive inhibition by ADP with respect to ATP and this inhibition changed to uncompetitive when 3 mM fructose was used. The isoform FKII showed a competitive inhibition pattern for ADP at 0.5 mM fructose which also changed to uncompetitive when 3 mM fructose was used. The features of the regulation of both fructokinases suggest that this enzyme might have a relevant role in carbon metabolism during tomato fruit development.     

Synthesis of wall glucan from sucrose by enzyme preparations from Pisum sativum

Radioactive sucrose, supplied through the cut base to Pisum sativum epicotyls, was transported to the growing apex (plumule and hook) and used there for the synthesis mainly of uridine diphosphoglucose (UDP- glucose), fructose and cell wall glucan. Enzyme extracts of the apical tissue contained sucrose synthetase activity which was freely reversible, i.e. formed UDP-glucose and fructose from sucrose (pH optimum = 6·6 for the cleavage reaction, K_m for sucrose = 63 mM). Particulate fractions of the same tissue contained a β-glucan synthetase which utilized UDP-glucose for formation of alkali-soluble and -insoluble products (pH optimum = 8·4, K_m for UDP-glucose = 1·9 mM). Values for V_max and yields of these two synthetase activities were sufficient to account for observed rates of cellulose deposition during epicotyl growth (15–25 μg/hr/epicotyl). When soluble pea enzyme was supplied with sucrose and UDP at pH 6·6 and then the preparation was supplemented with particles bearing β-glucan synthetase at pH 8·4, the glucose moiety of sucrose was converted to glucan in vitro. The results indicate that it is feasible for these synthetases to co-operate in vivo to generate β-glucan for expanding cell walls.     

Properties and regulation of a trans-plasma membrane redox system of perfused rat heart

Ferricyanide was reduced to ferrocyanide by the perfused rat heart at a linear rate of 78 nmol/min per g of heart (non-recirculating mode). Ferricyanide was not taken up by the heart and ferrocyanide oxidation was minimal (3 nmol/min per g of heart). Perfusate samples from hearts perfused without ferricyanide did not reduce ferricyanide. A single high-affinity site (apparent K_m=22 μM) appeared to be responsible for the reduction. Perfusion of the heart with physiological medium containing 0.5 mM ferricyanide did not alter contractility, biochemical parameters or energy status of the heart. Perfusate flow rate and perfusate oxygen concentration exerted opposing effects on the rate of ferricyanide reduction. A net decreased reduction rate resulted from a decreased perfusion flow rate. Thus, the rate of supply of ferricyanide dominated over the stimulatory effect of oxygen restriction; the latter effect only becoming apparent when the oxygen concentration was lowered at a high perfusate flow rate. Whereas glucose (5 mM) increased the rate of ferricyanide reduction, pyruvate (2 mM), acetate (2 mM), lactate (2 mM) and 3-hydroxybutyrate (2 mM) each had no effect. Insulin (3 nM), glucagon (0.5 μM), dibutyryl cyclic AMP (0.1 mM) and the β-adrenergic agonist ritodrine (10 μM) also had no effect, however the α₁-adrenergic agonist, methoxamine (10 μM), produced a net increase in the rate of ferricyanide reduction. It is concluded that a trans-plasma membrane electron efflux occurs in perfused rat heart that is sensitive to oxygen supply, glucose, perfusion flow rate, and the α-adrenergic agonist methoxamine.     

Purification and some properties of citrate synthase from a nitrite-oxidizing chemoautotroph,Nitrobacter agilis ATCC 14123

Citrate (si)-synthase (citrate oxaloacetate-lyase, EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from a nitrite-oxidizing chemoautotrophic bacterium, Nitrobacter agilis ATCC 14123. The molecular mass (M_r) of the native enzyme was estimated to be about 250,000 by gel filtration, whereas SDS-PAGE gave two bands with M_r values of 45,000 and 80,000, respectively, suggesting that the enzyme is a tetramer consisting of two different subunits (α: 45,000, β: 80,000). The isoelectric point of the enzyme was 5.4. The pH and temperature optima on the citrate synthase activity were about 7.5–8.0 and 30–35°C, respectively. The citrate synthase was stable in the pH range of 6.0–9.0 and up to 55°C. The apparent K_m values for oxaloacetate and acetyl-CoA were about 27 μM and 410 μM, respectively. The activity of citrate synthase was not inhibited by ATP (1 mM), NADH (1 mM) or 2-oxoglutarate (10 mM), but was strongly inhibited by SDS (1 mM). Activation by metal ions was not observed.     

The actions of L-glutamate and its agonists on the ampullary electroreceptor organs of the catfish Ictalurus nebulosus

1. Ampullary electroreceptors of the freshwater catfish, Ictalurus nebulosus, were examined for the effects of bath-applied l-glutamate (l-GLU) and its agonists quisqualate (Q), kainate (KA) and n-methyl-d-aspartate (NMDA). Resting discharge rate and stimulus-evoked activity in single afferent fibers were recorded in an in vitro preparation.2. l-GLU (0.1–1 mM), Q (0.8–40 μM) and KA (10–250 μM) strongly increased both resting activity and stimulus-evoked activity in the afferent fibres. NMDA had no effect, even at a concentration of 5.0 mM.3. The potencies of l-GLU and its agonists, arbitrary defined as the concentrations which gave 50% of maximal frequency increase, were of the order of 7 μM (Q), 25 μM (KA) and 0.6 mM (l-GLU).4. The excitatory effects of l-GLU persisted in receptors suppressed by high Mg²⁺, indicating that l-GLU was acting at the postsynaptic site.5. The data presented are consistent with our current concept that the action of l-GLU in Ictalurus electroreceptors is mediated via Q/AMPA- and KA-types, but not the NMDA-type, of membrane receptors.     

Stimulation of yeast phosphofructokinase activity by Fructose 2,6-bisphosphate

Fructose 2,6-bisphosphate is a powerful activator of yeast phosphofructokinase when assayed at pH levels of ≥7.0. Half maximal stimulation of enzyme activity occurs at 10^?7 M levels of Fru 2,6-P₂ concentration. This stimulating effect by Fru 2,6-P₂ can be synergistic to that exerted by AMP in counteracting the inhibition of phosphofructokinase activity by ATP. The affinity (S_0.5) of the yeast enzyme to fructose 6-phosphate changes from 1.5 mM in the absence of Fru 2,6-P₂ to 40 μM in its presence.     

Vanadium in photosynthesis of Chlorella fusca and higher plants

The influence of vanadium compounds (vanadate, vanadyl citrate) on photosynthesis in Chlorella fusca and in algal and spinach chloroplasts has been investigated. It was found that: 1. At moderately high concentrations (at least 0.1 mM) both vanadate and vanadyl citrate enhance photosynthetic O₂ production in intact C. fusca cells. At lower V concentration (about 2 μM) only vanadate stimulates photosynthesis. The increase is dependent on culture conditions and on light intensity. 2. Up to 1 mM V, neither vanadium compound influences PS II activity, either in intact cells or in algal or spinach chloroplasts. 3. The PS I reaction in algal and spinach chloroplasts is maximally enhanced (3-fold) in presence of vanadium (20 μM). The increase is independent of light intensity. 4. Cr(VI), Mo(VI), and W(VI) (1 mM) stimulate photosynthesis in intact C. fusca cells, but do not influence the photosystems of isolated chloroplasts. Vanadium is suggested to act as a redox catalyst in the electron transport from PS II to PS I.     

Characterization of a spontaneous mutant of Azotobacter vinelandii in which vanadium-dependent nitrogen fixation is not inhibited by molybdenum

A spontaneous mutant derivative of Azotobacter vinelandii CA12 (ΔnifHDK), in which vanadium-dependent nitrogen fixation is not inhibited by molybdenum (A. vinelandii CARR), grows profusely on BNF-agar containing 1 μM Na₂MoO₄, alone or supplemented with 1 μM V₂O₅. The expression of A. vinelandii vnfH::lacZ and vnfA::lacZ fusions in A. vinelandii CARR was not inhibited by 1 mM Na₂MoO₄, whereas molybdenum at much lower concentration inhibited the expression of vnfH::lacZ and vnfA::lacZ fusions in A. vinelandii CA12. The mutant also exhibited normal acetylene reduction activity in the presence of 1 μM Na₂MoO₄. The expression of A. vinelandii nifH::lacZ fusion in A. vinelandii CARR was low even though the cells were cultured under non-repressing conditions with urea as nitrogen source in the presence of Na₂MoO₄. The molybdenum content of A. vinelandii CARR cells was found to be about one-fourth that of A. vinelandii CA12. No nitrate reductase activity could be detected in A. vinelandii CARR when the cells were cultured in the presence of 10 μM Na₂MoO₄, whereas A. vinelandii CA12 exhibited some activity even with 100 pM Na₂MoO₄.     

Extracellular potassium controls responsiveness of the noradrenergic cAMP system in the rat retina to fluphenazine

The effects of fluphenazine (FLU) on the noradrenaline (NA) induced cAMP-synthesis in intact rat retinae were studied as a function of extracellular K⁺- and Ca²⁺-ions. Thus NA-induced cAMP levels were measured after incubating intact rat retinae with 50 μM NA in the presence or absence of FLU and in the presence of 1 or 10 mM theophylline. Results were: (1) Experimental condition a: standard NA-responses were measured after incubating retinae at 0.75 mM Ca²⁺, at 10 mM theophylline, at 10 μM FLU and at 2 and 0 mM K⁺. FLU does not affect the NA-response at 2 mM K⁺ significantly; however, it inhibits the NA-response at 0 mM K⁺ in this condition. (2) Experimental condition b: NA-responses were measured after incubating retinae at 0.125 mM Ca²⁺, 10 mM theophylline, 10 μM FLU and at 2 and 0 mM K⁺. At 2 mM K⁺ FLU replaces a Ca²⁺ function probably connected with the synthesis part of the NA-cAMP system and NA-responses in this low Ca²⁺ condition are consequently enhanced by FLU; however, FLU inhibits the NA-response at 0 mM K⁺ in this condition. (3) Experimental condition c: NA-responses were measured after incubating retinae at 0.75 mM Ca²⁺, 1 mM theophylline, 10 μM FLU and at 2 and 0 mM K⁺. At 2 mM K⁺ FLU enhances the NA-response by further inhibition of the degradation part of the NA-cAMP system; FLU inhibits the NA-response at 0 mM K⁺ in this condition. (4) The inhibitions of the NA-responses by FLU at 0 mM K⁺ in all three conditions a, b and c showed an apparent K_m of 1 μM. (5) Low concentrations of K⁺ (0.4–0.8 mM) maintain the property of FLU to enhance the NA-responses at condition b (0.125 mM Ca²⁺) and at condition c (1 mM theophylline). Results suggest that the activation of NA-receptor coupled adenylate cyclases (NA-AC-ases) by NA, resulting in activation of phosphodiesterase activity by the NA-elevated cAMP-levels, is sustained by (a) membraneous factor(s) connected to the NA-receptor. This (these) factor(s) is (are) switched off in the absence of K⁺. Evidence has been presented, that Ca²⁺ and FLU do not have access to this intramembraneous factor-enzyme activating moiety of the NA-cAMP system at 0 mM K⁺. Between 0.4 and 0.8 mM K⁺ the factor-enzyme-NA-receptor complex is still intact.     

Binding and protection of porphyrins by glutathione S-transferases of Zea mays L.

Glutathione S-transferases (GSTs) are multi-functional enzymes, known to conjugate xenobiotics and degrade peroxides. Herein, we report on the potential of four Zea mays GST isoforms (Zm GST I–I, Zm GST I–II, Zm GST II–II and Zm GST III–III) to act as binding and protection proteins. These isoforms bind protoporphyrin IX (PPIX), mesoporphyrin, coproporphyrin, uroporphyrin and Mg-protoporpyhrin, but do not form a glutathione conjugate. The binding is non-covalent and inhibits GSTs enzymatic activity, dependent on the type of the porphyrin and GST isoform tested. I₅₀ values are in the range of 1 to 10 μM for PPIX, the inhibition by mesoporphyrin and Mg-protoporphyrin (Mg-PPIX) is two to five times less. The mode of binding is non-competitive for the hydrophobic substrate and competitive for glutathione. Binding affinities (K_D values) of the GST isoforms are between 0.3 and 0.8 μM for coproporphyrin and about 2 μM for mesoporphyrin.Zm GST III–III prevents the nonenzymatic autoxidation of protoporphyrinogen to the phytotoxic PPIX. Zm GST II–II can reduce the oxidative degradation of hemin. This points to a specific ligand role of distinct GST isoforms to protect tetrapyrroles in the plant cell.     

Catalytic properties of CYP1A isoforms in the liver of an agnathan (Lampetra fluviatilis) and two species of teleost (Pleuronectes flesus,Anguilla anguilla)

The catalytic activity of CYP1A isoforms and the effect of mammalian CYP1A-specific inhibitors in liver S9 fractions were studied in an agnathan (River lamprey, Lampetra fluviatilis, 30–33 cm) and in two species of teleost fish (European flounder, Pleuronectes flesus, 11–18 cm and common eel, Anguilla anguilla, 31–48 cm). Ethoxyresorufin O-deethylation (EROD), caffeine N-demethylation/C-oxidation and phenacetin O-deethylation (POD) activity increased 3–4-fold in flounders and 17–46-fold in eels, 5 days after fish were injected (i.p.) with 100 mg kg⁻¹ benzo(a)pyrene (B[a]P). In lampreys, basal EROD activity was very low and no increase in activity was observed following exposure to B[a]P. While the apparent Michaelis constant (K_m) for each assay showed only small changes after B[a]P injection, maximum reaction velocity (V_max) values increased by up to 19- and 84-fold for EROD activity, 4- and 35-fold for caffeine-related metabolism and 4- and 19-fold for POD activity in flounders and eels, respectively. The mammalian CYP1A2 inhibitor furafylline (50 μM–1 mM) reduced activity in the EROD, caffeine and POD assays to 65, 21 and 20% of control values in flounders and to 85, 10 and 5% of control values in eels, respectively. By contrast, low concentrations (0.025–0.050 μM) of the mammalian CYP1A1 inhibitor ellipticine completely abolished EROD activity, but had no effect (up to 1 mM) on caffeine metabolism or POD activity in either species. While the inhibitor studies strongly suggest that two separate enzymes are present in flounders and eels, the monophasic Michaelis–Menten kinetics obtained in all the assays imply that only a single CYP1A protein is present that has substrate and inhibitor specificities characteristic of both mammalian CYP1A1 and CYP1A2 isoforms.     

Gut chitin synthase and sterols from larvae of Diaprepes abbreviatus (Coleoptera: Curculionidae)

Gut chitin synthase was characterized and the sterols and ecdysteroids in the sugarcane rootstalk borer weevil, Diaprepes abbreviatus, were identified. An in vitro cell-free chitin synthase assay was developed using larval gut tissues from D. abbreviatus. Subcellular fractionation experiments showed that the majority of chitin synthase activity was located in 10,000g pellets. The gut chitin synthase requires Mg²⁺ to be fully active: 7–8-fold increases in activity were obtained with 10 mM Mg²⁺ present in reaction mixture. Calcium also stimulated activity (4–5-fold with 10 mM Ca²⁺), while Cu⁺² completely inhibited at 1 mM. Other monovalent and divalent cations had little or no effect on activity. The pH and temperature optima were 7 and 25°C, respectively. Gut chitin synthesis was activated ca. 50% by trypsin treatments. GlcNAc stimulated chitin synthase activity, but Glc, GlcN and glycerin did not. Polyoxin D, UDP, and ADP inhibited the chitin synthase reaction with I₅₀'s of 75 μM, 2.3 mM, and 3.6 mM, respectively. Nikkomycin Z was a potent inhibitor of chitin synthase (91% inhibition at 10 μM). Tunicamycin and diflubenzuron had no effect on the enzyme. The apparent Km and V_max for the gut chitin synthase were, respectively, 122.5 ± 7.4 μM and 426 ± 19.7 pmol/h/mg protein utilizing UDP-GlcNAc as the substrate. Sterol analyses indicated that cholesterol was the major dietary and larval sterol. HPLC/RIA data indicated that 20-hydroxyecdysone was the major molting hormone.