Ecdysone 20-monooxygenase in mitochondria and microsomes of Manduca sexta (L.) Midgut: Is the dual localization real?

The dual localization of ecdysone 20-monooxygenase in mitochondria and microsomes of Manduca sexta larval midgut was investigated. Cosubstrate requirements and response to osmolarity of the microsomal ecdysone 20-monooxygenase system were found to be different from those previously reported for the mitochondrial enzyme system. The microsomal monooxygenase utilized NADPH and, less efficiently, NADH as cosubstrates. NADPH and NADH effects were neither additive nor synergistic. NADPH yielded identical activities in isotonic and hypotonic incubations. Mitochondria and microsomes showed no synergistic interaction for ecdysone 20-hydroxylation. After washing of the mitochondria, a large proportion of their ecdysone 20-monooxygenase activity was lost. The extent of the loss was inversely correlated to the concentration of mitochondria in the incubation mixture. The addition of bovine serum albumin to the incubations (2 mg/ml) largely restored the original activities. The microsomal contamination in mitochondrial pellets after each of three successive washings was determined by measuring the activity of a microsomal marker enzyme, NADPH-cytochrome c reductase. At each step of the purification, the ecdysone 20-monooxgenase activity of the mitochondrial preparations far exceeded the activity attributable to the microsomal contamination. These results confirm the existence of two independent ecdysone 20-monooxygenase systems in the midgut of M. sexta larvae.     

Characterization of ecdysone 20-monooxygenase activity in wandering stage larvae of Drosophilamelanogaster: Evidence for mitochondrial and microsomal cytochrome P-450 dependent systems

Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone to 20-hydroxyecdysone, was characterized in wandering stage larvae of Drosophila melanogaster using an in vitro radioassay in conjunction with analytical thin layer chromatography. 20-Hydroxyecdysone was confirmed to be the product of the enzyme radioassay system by high pressure liquid chromatography. The 20-monooxygenase was found to be most active in a 0.10 M phosphate buffer, pH 7.5, was inhibited by Ca²⁺, Mg²⁺ and Se⁴⁺ and exhibited a temperature optimum at 35°C. Differential centrifugation, sucrose step gradient centrifugation, electron microscopy and organelle-marker enzyme analysis revealed that ecdysone 20-monooxygenase activity is associated with both the mitochondrial and microsomal fractions. Substrate kinetics experiments indicated that the mitochondrial and microsomal monooxygenase systems exhibit apparent K_ms for ecdysone of 6.4 × 10⁻⁸ and 9.9 × 10⁻⁸ M, respectively, with apparent V_maxs of 4.1 and 10.2 pg 20-hydroxyecdysone formed/min per mg tissue equiv., respectively. Both monooxygenase systems were inhibited by their product 20-hydroxyecdysone. The cytochrome P-450 nature of these insect steroid hydoxylases was initially suggested by their requirement for NADPH, NADH was approximately half as effective in supporting the mitochrondrial monooxygenase activity. In addition, both monooxygenase systems were inhibited by carbon monoxide, ellipticine, p-chloromercuribenzoate, metyrapone and p-aminoglutethimide but not by cyanide. Photochemical action spectra of ecdysone 20-monooxygenase activity confirmed the cytochrome P-450 dependency of both the mitochondrial and microsomal ecdysone 20-hydroxylase systems.     

On the intracellular localization of the ecdysone 20-monooxygenase in Musca domestica. L

The cytochrome P-450-dependent 20-monooxygenation of ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica (L.) larvae; however, about 50% of the activity is associated with mitochondria, and 37% is associated with microsomes. Pretreatment of larvae with ecdysone results in an increase in Vmax and a decrease in Km values in mitochondria but not in microsomes. Phenobarbital, a known cytochrome P-450 inducer, increases the cytochrome P-450 levels in microsomes without affecting the 20-monooxygenase activity, but both the cytochrome P-450 levels and monooxygenase activity are depressed in mitochondria from phenobarbital-pretreated larvae. The ecdysone 20-monooxygenase activity is equally distributed between mitochondria and microsomes in adult insects. Pretreatment of the insects with ecdysone does not significantly modify the 20-monooxygenase activity of either mitochondrial or microsomal fractions, but the cytochrome P-450 levels are reduced in mitochondria. Phenobarbital also depresses the mitochondrial cytochrome P-450 levels while markedly increasing the microsomal cytochrome P-450 levels. However, no significant changes in ecdysone 20-monooxygenase activity are produced by phenobarbital pretreatment. The effects of ecdysone on adult cytochrome P-450 are mostly evidenced in mitochondria isolated from females, whereas in males the changes are not statistically significant. It is concluded that the mitochondrial ecdysone 20-monooxygenase is under regulatory control by ecdysone in the larval stage, which suggests that only the mitochondrial activity has a physiological role during insect development in M. domestica. In adults, both the mitochondrial and microsomal ecdysone 20-monooxygenase activities are not responsive to ecdysone, which, coupled to their high Km values, indicates that the reaction may not be of physiological importance in adult insects and that the mitochondrial cytochrome P-450 species being depressed by ecdysone in females are possibly not involved in ecdysone metabolism.     

Midgut and fatbody mitochondrial transhydrogenase activities during larval-pupal development of the tobacco hornworm, Manduca sexta

Midgut and fatbody mitochondria from fifth larval instar Manduca sexta display a membrane-associated transhydrogenase that catalyzes a reversible hydride ion transfer between NADP(H) and NAD(H). The NADPH-forming activity occurs as a nonenergy- or energy-linked activity with energy for the latter derived from either electron transport-dependent NADH or succinate utilization, or ATP hydrolysis by Mg⁺⁺-dependent ATPase. During the ten-day developmental period preceding the larval-pupal molt (fifth larval instar), significant peaks in the mitochondrial transhydrogenase activities of midgut and fatbody tissues were noted and these peaks were coincident with the onset of wandering behavior and with the fifty-fold increase in ecdysone 20-monooxygenase (E20-M) activity previously reported for M. sexta midgut. Since E20-M preferentially uses NADPH in catalyzing ecdysone conversion to the physiologically active molting hormone, 20-hydroxyecdysone, the physiological and developmental significance of the mitochondrial, NADPH-forming energy-linked transhydrogenations were made apparent. Moreover, that the increases in all transhydrogenase activities resulted from de novo enzyme synthesis were indicated by the cycloheximide-dependent reductions in these activities.     

Allelochemical stimulation of ecdysone 20-monooxygenase in fall armyworm larvae

The influence of dietary allelochemical on ecdysone 20-monooxygenase activity was studied in the fall armyworm, Spodoptera frugiperda (J.E. Smith). Feeding the indoles (indole-3-carbinol, indole-3-acetonitrile), flavonoids (flavone, β-naphthoflavone), monoterpenes (menthol, menthone, peppermint oil), and a coumarin (xanthotoxin) to the larvae stimulated midgut microsomal ecdysone 20-monooxygenase activity from 28 to 200% as compared with the controls. β-Naphthoflavone was the most potent inducer among those tested. Phenobarbital, a well-known cytochrome P450 inducer, also caused a 2-fold increase in the microsomal ecdysone 20-monooxygenase activity. Ecdysone 20-monooxygenase activity was 2.7-fold higher in the microsomal fraction than in the mitochondrial fraction isolated from larval midguts. Microsomal ecdysone 20-monooxygenase activity was highest in the fat body, followed by the midgut and Malpighian tubules. Tissue localization and enzyme inducibility were different between ecdysone 20-monooxygenase and xenobiotic-metabolizing cytochrome P450 monooxygenases, including aldrin epoxidase, biphenyl hydroxylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, p-chloro-N-methylaniline N-demethylase, and phorate sulfoxidase in fall armyworm larvae. © 1995 Wiley-Liss, Inc.     

Ecdysone 20-monooxygenase in a cricket,Gryllus bimaculatus (Ensifera,Gryllidae)—characterization of the microsomal midgut steroid hydroxylase in adult females

Summary Ecdysone 20-monooxygenase, the enzyme system which converts ecdysone into 20-hydroxyecdysone, was characterized in the midgut of 4-day-old female adult Gryllus bimaculatus using an in vitro radioassay. Differential centrifugation and sucrose gradient centrifugation revealed that ecdysone 20-monooxygenase activity is associated with the microsomal fractions. The 20-monooxygenase was found to be most active in potassium phosphate buffer, pH 7.8, at an osmolarity of 100 mOsm and at 39 °C assay temperature. The conversion of ecdysone into 20-hydroxyecdysone was linear over an incubation period of 12 min and with respect to a protein concentration of 3 mg·ml^–1. K⁺ and Na⁺ (10^–3–10^–1 M), Ca²⁺ (2.3 mM), and EDTA (1–5 mM) did not affect monooxygenase activity, whereas Mg²⁺ (2.3–10 mM) slightly inhibited enzyme activity. The enzyme complex has an apparent K_m for ecdysone of 3.7·10^–7 M and is competitively inhibited by its product, 20-hydroxyecdysone, with an apparent K_i of 4·10^–6 M. The cytochrome P-450 nature of the steroid hydroxylase was shown by its obligate requirement for NADPH and its inhibition by carbon monoxide, metyrapone, and p-chloromercuribenzoate, but not by cyanide. The insect systemic growth disruptor, azadirachtin, was found to inhibit ecdysone 20-monooxygenase activity with a I₅₀ of 8·10^–4 M. From the CO-difference spectrum, a cytochrome P-450 content of 285 pmol·mg protein^–1 was calculated for midgut microsomes of 4-day-old females.Abbreviations GO carbon monoxide - EDTA ethylenediamine tetraacetic acid - HPLC high performance liquid chromatography - I ₅₀ concentration for 50% inhibition - KCN potassium cyanide - K ₁ inhibition constant - K _m Michaelis-Menten constant - MOPS 3-morpholinopropanesulfonic acid - NADH/NAD ⁺ nicotinamide adenine dinucleotide reduced/oxidized - NADPH/NADP ⁺ nicotinamide adenine dinucleotide phosphate reduced/oxidized - Na ₂ S ₂ O ₄ sodium dithionite - SEM Standard error of mean - TLC thin-layer chromatography - TRIS 2-amino 2-hydroxymethyl-1,3-propanediol (trishydroxymethyl aminomethane) - V _max maximal reaction velocity     

Calcium alginate-Immobilized hepatic microsomes: Effect of NADPH cofactor on oxidation rates

An important function of the liver is detoxification of drugs, toxins and foreign compounds. Within the liver cell, the endoplasmic reticulum, isolated as the microsomal fraction, is especially active. Microsomal oxidation is the major oxidation pathway for many compounds, and the requirement for NADPH, an expensive cofactor, is an important consideration in bioreactor design. This paper presents design information for NADPH- and substrate-dependent oxidation rates for free and immobilized microsomes. The primary goal of this paper is determining NADPH requirements for oxidation. The effect of various initial levels of nicotinamide adenine dinucleotide phosphate (NADPH) on chlorpromazine oxidation rate has been studied for a crude hepatic microsomal fraction immobilized in calcium alginate gel. At an initial NADPH concentration of 600 nmoles/ml, immobilized microsomes accelerate to a maximal velocity of ≈ 240 nmoles min⁻¹ ml⁻¹ of oxygen consumption. In comparison, free microsomes reach a maximal velocity of approximately 150 nmoles min⁻¹ ml⁻¹ at an initial NADPH concentration of 220 nmoles/ml. By fitting the “initial” rate as a function of NADPH concentration to Michaelis-Menten kinetics, the apparent half-saturation coefficients (K_m)app are 3.5 nmole/ml for free microsomes and 134.4 nmole/ml for immobilized microsomes, however the maximum reaction velocity, V_max, for immobilized microsomes is calculated to be 322 nmoles min⁻¹ ml⁻¹ compared with 145 nmols min⁻¹ ml⁻¹ for free microsomes. Preliminary studies indicate that is is possible to obtain significant reaction rates using calcium alginate immobilized microsomes and that this system may offer advantages due to its simplicity and lower cost.     

In vivo pharmacokinetics and in vitro production of cocaethylene in pregnant guinea pigs

Simultaneous exposure to cocaine and ethanol results in the formation of cocaethylene, an active metabolite of cocaine. The concurrent abuse of both cocaine and ethanol is common during human pregnancy, but the kinetics of elimination and formation of this ethyl ester of cocaine have not been studied during pregnancy in any species. In the late gestation guinea pig (61 to 63 days), cocaethylene, at doses of 2 to 4 mg · kg⁻¹, is rapidly eliminated with a half-life of 29 min and a total body clearance of 77 ml · min⁻¹ · kg⁻¹. It is formed enzymatically by hepatic microsomal preparations from fetal, neonatal and maternal guinea pigs. The maximum rate of cocaethylene production (apparent V_max) when either ethanol or cocaine are varied while the other substrate is held constant, increases with age, from the late fetal period (65 days gestation, term 70 days) to adulthood. However, the Michaelis-Menten constant (apparent K_M) does not change with age. The rapid elimination of cocaethylene, coupled with the slow rate of formation (apparent V_max of 140 pmol · min⁻¹ · mg microsomal protein⁻¹) and the small amount of plasma analyzed most likely explains the inability to detect cocaethylene in vivo after concomitant cocaine and ethanol administration.     

Isolation,partial purification,and characterization of the cytochrome P-450-dependent monooxygenase system from the midgut of the earthworm Lumbricus terrestris

1. Cytochrome P-450 was purified from microsomes of the midgut of the earthworm Lumbricus terrestris up to a maximal specific content of 5.5 nmol P-450/mg protein.2. At least 3 different cytochromes P-450 with apparent molecular weights of 48,000, 51,000 and 53,000 were identified by SDS-PAGE.3. Western blot analysis with various polyclonal antibodies did not show structural epitopes common to the cytochromes P-450 of rodents or yeast and L. terrestris.4. The microsomes contained about 43 pmol P-450/mg protein corresponding to 0.51 nmol P-450/g midgut and 64 pmol P-450/g body weight, respectively, and converted benzyloxyresorufin into resorufin with a V_max, of 2.12 pmol resorufin/min.mg protein and a K_m of 770 nM benzyloxyresorufin at 25°C, pH 8.O.5. The microsomes exhibited a NADPH-cytochrome P-450 reductase activity of 9.4 nmol cytochrome c/min.mg protein.6. The apparent molecular weight of the threefold-purified reductase was 63,000.     

The inducible and cytochrome P450-containing dehydroepiandrosterone 7α-hydroxylating enzyme system of Fusarium moniliforme

7α-Hydroxylation of DHEA by Fusarium moniliforme was investigated with regard to inducibility and characterization of the responsible enzyme system. Using GC/MS, the 7-hydroxylated metabolites of DHEA produced after biotransformation by Fusarium moniliforme mycelia were identified. The strain of Fusarium moniliforme hydroxylated DHEA predominantly at the 7α-position, with minor hydroxylation occurring at the 7β-position. Constitutive 7α-hydroxylation activity was low, but DHEA induced the enzyme complex responsible for 7α-hydroxylation via an increase in protein synthesis. DHEA 7α-hydroxylase was found to be mainly microsomal, and the best production yields of 7α-hydroxy-DHEA (28.5 ± 3.51 pmol/min/mg protein) were obtained with microsomes prepared from 18-h-induced mycelia. Kinetic parameters (K_M=1.18 ± 0.035 μM and V_max=909 ± 27 pmol/min/mg protein) were determined. Carbon monoxide inhibited 7α-hydroxylation of DHEA by microsomes of Fusarium moniliforme. Also, exposure of mycelia to DHEA increased microsomal P450 content. These results demonstrated that: (i) DHEA is 7α-hydroxylated by microsomes of Fusarium moniliforme; (ii) DHEA induces Fusarium moniliforme 7α-hydroxylase; (iii) this enzyme complex contains a cytochrome P450.     

In vitro ecdysteroid conjugation by enzymes of Manduca sexta midgut cytosol

In incubations with 80,000g supernatant of Manduca sexta midgut homogenates, [³H]ecdysone was converted to 3-[³H]epiecdysone and tritiumlabeled highly polar metabolites. C₁₈ SEP-PAK cartridges were found suitable for the separation and purification of the free ecdysteroids and of the highly polar metabolites. Eighty to ninety percent of the metabolites were hydrolyzed by enzyme mixtures (mainly β-glucuronidase, sulphatase, and acid phosphatase) from molluscs, even when β-glucuronidase activity was completely inhibited by D-saccharic acid 1,4-lactone, or various human acid phosphatases (free of sulphatase activity). In each experiment, the hydrolysate contained a much higher proportion of 3-epiecydsone than the free (unconjugated) ecdysteroid fraction. [³H]ecdysone was not metabolized in anaerobic incubations of midgut supernatant that had been filtered through Sephadex G-25. Addition of 5 mM ATP and 5 mM Mg²⁺ restored the conjugate formation in incubations of Sephadex-filtered supernatant. Four ecdysone conjugates and two 3-epiecdysone conjugates were resolved by reversedphase ion-pair high-performance liquid chromatography. It is concluded that the midgut cytosol contains several ATP:ecdysteriod phosphotransferases. This is the first demonstration of the formation of ecdysteroid phosphoconjugates in a cell-free system.     

Comparative kinetics of fatty acid-amino acid conjugate elicitor biosynthesis by midgut tissue microsomes of Lepidopterous caterpillar larvae

N‐Linolenoyl‐L ‐glutamine is one of several structurally similar fatty acid–amino acid conjugate (FAC) elicitors found in the oral secretions of Lepidopterous caterpillars and its biosynthesis is catalyzed by membrane‐associated alimentary tissue enzyme(s). FAC elicitors comprise 17‐hydroxylated or non‐hydroxylated linolenic acid coupled with L ‐glutamine or L ‐glutamate by an amide bond. We demonstrate in vitro biosynthesis of N‐linolenoyl‐L ‐glutamine by Manduca sexta, Heliothis virescens, and Helicoverpa zea tissue microsomes. Comparison of N‐linolenoyl‐L ‐glutamine biosynthesis kinetics for these species suggests that concurrent biosynthesis and hydrolysis contribute to proportions of FAC elicitors found in their oral secretions. The apparent K_m values for coupling of sodium linolenate were 8.75±0.79, 14.3±3.7 and 20.7±3.4 mM and V_max values were 2.92±0.14, 6.81±1.2 and 4.95±0.55 nmol/min/mg protein for H. zea, H. virescens and M. sexta, respectively. The K_m values for coupling of L ‐glutamine were 10.5±0.26, 22.3±2.0 and 18.9±2.4 mM and V_max values were 1.78±0.21, 3.71±0.50 and 2.49±0.41 nmol/min/mg of protein for H. zea, H. virescens and M. sexta, respectively. © 2010 Wiley Periodicals, Inc.     

Uptake kinetics of nitrate,ammonia and phosphate by the dinoflagellate Heterocapsa circularisquama isolated from Hiroshima Bay,Japan

Heterocapsa circularisquama is a harmful dinoflagellate whose first bloom in Hiroshima Bay, Japan, appeared in 1992. As suggested by the authors’ group, in the Seto Inland Sea including Hiroshima Bay, oligotrophication particularly the reduction of phosphate starting 1980 is severe. The bloom caused serious damage to the bay's extensive oyster culture. In the present study, the uptake kinetics of nitrate, ammonia, and phosphate by this species were experimentally investigated. The maximum uptake rate (ρ_max) and the half‐saturation constant (Ks) were 0.41 pmol cell^?1 h^?1 and 4.45 μM, respectively, for nitrate, 2.02 pmol cell^?1 h^?1 and 11.1 μM for ammonium, and 0.079 pmol cell^?1 h^?1 and 1.79 μM for phosphate. The maximum specific uptake rates (V_max) for nitrate, ammonia, and phosphate were estimated to be 8.95, 44.1, and 21.3 day^?1, respectively. A comparison of V_max/Ks, which is also an index of affinity to nutrients, between this species and others suggested that H. circularisquama can utilize nitrate and ammonia efficiently, but not phosphate. Considering both reports describing that H. circularisquama has the ability to utilize dissolved organic phosphorus (DOP) and the DOP concentration is higher than phosphate in Hiroshima Bay, it was concluded that H. circularisquama became dominant due to the phosphate reduction measure.     

Uptake of ethanolamine in neuronal and glial cell cultures

The uptake of radioactive ethanolamine has been studied in exclusively neuronal and glial cell cultures from dissociated cerebral hemispheres of chick embryos. Both cell types show saturable kinetics; neurons have an apparentK _m of 6.7 M,V _max 41.4 pmol mg prot.^–1 min^–1 and glial cells aK _m of 119.6 M,V _max 3,917 pmol mg prot^–1 min^–1. The lower affinity of the transport and the 100 fold increase inV _max observed in glial cells correlated with a more important accumulation of free ethanolamine found in glial cells and with a higher degree of phosphorylation of ethanolamine. The uptake appeared to be temperature and Na⁺ ions dependent but was not affected by CN^– or ouabain. Monomethyl-, dimethylethanolamine and choline were effective in inhibiting the uptake. Little or no effect was observed with serine, methionine, carnitine, alanine or glutamate.     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

Study of properties of cholinephosphotransferase from fetal guinea pig lung mitochondria and microsomes

Summary We have reported earlier that cholinephosphotransferase (EC 2.7.8.2) is present in both mitochondria and microsomes of fetal guinea pig lung. This study was designed to compare the properties of mitochondrial and microsomal cholinephosphotransferase in fetal guinea pig lung. Various parameters, such as substrate specificity, K_m values, sensitivity to N-ethylmaleimide, dithiothreitol and trypsin were measured. Both showed significant preference for unsaturated diacylglycerols over saturated diacylglycerols. Data on K_m and V_max indicate that the affinity of this enzyme for different diacylglycerols varies between the two forms. The ID₅₀ values for N-ethylmaleimide were 20 mM and 12.5 mM for the mitochondrial and microsomal form of the enzyme, respectively. Dithiothreitol showed an inhibitory effect on both; however, the mitochondrial form was inhibited less than the microsomal form. The effects of N-ethylmaleimide and dithiothreitol on both forms of enzyme indicated that the microsomal cholinephosphotransferase requires a higher concentration of -SH for its activity than the mitochondrial enzyme does. The enzyme was inhibited by trypsin in both mitochondria and microsome under isotonic condition suggesting that this enzyme is on the outside of the membrane in both endoplasmic reticulum and mitochondria.     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum

A study was made of the time course and kinetics of [³H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [³H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent K_m values for GABA neurons for high and low affinity uptake were 0.33 × 10⁻⁶ M and 41.8 × 10⁻⁴ M, respectively. For nonneuronal cells, the apparent K_m for high affinity uptake was 0.29 × 10⁻⁶ M. The apparent V_max values for GABA neurons for high and low affinity uptake were 28.7 × 10⁻⁶ mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent V_max for high affinity uptake was 0.06 × 10⁻⁶ mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 × 10⁻⁹ M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [³H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75–85%) of the [³H]GABA (4.4 × 10⁻⁶ M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.     

Reduction of extracellular dehydroascorbic acid by K562 cells

K562 erythroleukaemic cells produced ascorbate when incubated with dehydroascorbic acid. The reduction depended on the number of cells and on the concentration of dehydroascorbic acid. The observed rate consists of a high affinity (apparent) K_m 7 μM , V_max 3·25 pmol min^?1 (10⁶ cells)^?1 and a low affinity component, which was non-saturable up to 1 mM of DHA (rate increase of 0·1 pmol min^?1 (10⁶ cells)^?1 (1 μM of DHA^?1). The rate was dependent on temperature and was stimulated by glucose and inhibited by phloretin, N-ethylmaleimide, parachloro-mercuribenzoate and thenoyltrifluoroacetone. Although uptake of DHA proceeded at a higher rate than its extracellular reduction, the generation of extracellular ascorbate from DHA cannot be accounted for by intracellular reduction and the release of ascorbate, since the latter was not linear with time and had an initial rate of approximately 3 pmol min^?1 (10⁶ cells^?1). At a concentration of DHA of 100 μM this is 25 per cent of the observed reduction.     

20-Hydroxyecdysone-induced changes in the cell volume of lepidopteran cells associated with population dynamics

Summary The insect cell line MRRL-CH derived from embryos of the lepidopteranManduca sexta responded rapidly, within 15 min, to 20-hydroxyecdysone (20-OH-Ec) (1×10⁻⁸ M) by increasing in cell volume. The induction time for this response contrasts with that for the cell elongation response that appears only after 24 to 48 h. The increase in cell volume represents the most rapid 20-OH-Ec-induced change in cell morphology reported to date. It also has some steroid specificity because no volume change was induced by ecdysone (4×10⁻⁶ M) under the experimental conditions. The magnitude of the cell volume response to 20-OH-Ec varied with the population dynamics of the cell line and was associatated with cell size at the time of treatment. This is a portion of a thesis submitted by L. H. E. in partial fulfillment of the requirements for the Ph.D. degree and published with the approval of the Director of the North Dakota Experiment Station as Journal Article 1130. Mention of a proprietary product or company name in this paper does not imply endorsement by the U.S. Department of Agriculture.