Inhibition of the O2−-generating NADPH oxidase of guinea-pig polymorphonuclear leukocytes by rabbit antibody to homologous liver NADPH-cytochrome c (P-450) reductase

Rabbit antibody highly specific for guinea-pig liver NADPH-cytochrome c (P-450) reductase was found to inhibit dose-dependently the O₂^?-generating activity of the membrane fraction isolated from phorbol-myristate acetate-stimulated, homologous polymorphonuclear leukocytes. In addition, the antibody also could inhibit the NADPH-cytochrome c (Nitroblue tetrazolium) reductase from the membrane fractions and phagosomes of leukocytes by polyacrylamide gel electrophoresis or gel filtration on a Sephacryl S-300 column in the presence of 0.2% Triton X-100. These results demonstrate that the NADPH-cytochrome c reductase in the membrane fractions of leukocytes is antigenically cross-reactive with homologous liver NADPH-cytochrome c reductase, and also suggest that the enzyme of leukocytes participates in the respiratory burst.     

Microsomal electron transport. I. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and cytochrome P-450 as electron carriers in microsomal NADPH-peroxidase activity

An electron transport system that catalyzes the oxidation of NADPH by organic, hydroperoxides has been discovered in microsomal fractions. A tissue distribution study revealed that the microsomal fraction of rat liver was particularly effective in catalyzing the NADPH-peroxidase reaction whereas microsomes from adrenal cortex, lung, kidney, and testis were weakly active. The properties of the hepatic microsomal NADPH-peroxidase enzyme system were next examined in detail.The rate of NADPH oxidation by hydroperoxides was first-order with respect to microsomal protein concentration and a K_m value for NADPH of less than 3 μm was obtained. Examination of the hydroperoxide specificity revealed that cumene hydroperoxide and various steroid hydroperoxides were effective substrates for the enzyme system. Using cumene hydroperoxide as substrate, the reaction rate showed saturation kinetics with increasing concentrations of hydroperoxide and an apparent K_m of about 0.4 mm was obtained. The NADPH-peroxidase reaction was inhibited by potassium cyanide, half-maximal inhibition occurring at a cyanide concentration of 2.2 mm. NADH was able to support the NADPH-dependent peroxidase activity synergistically.Evidence compiled for the involvement of NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase, EC 1.6.2.3) in the NADPH-peroxidase reaction included: (1) an identical pH optimum for both activities; (2) stimulation of NADPH-peroxidase activity by increasing ionic strength; (3) inhibition by 0.05 mm, p-hydroxymercuribenzoate with partial protection by NADPH; (4) inhibition by NADP⁺; and (5) inactivation by antiserum to NADPH-cytochrome c reductase. In contrast, antibody to cytochrome b₅ did not inhibit the NADPH-peroxidase activity. Evidence for the participation of cytochrome P-450 in the NADPH-peroxidase reaction included inhibition by compounds forming type I, type II, and modified type II difference spectra with cytochrome P-450; inhibition by reagents converting cytochrome P-450 to cytochrome P-420; and marked stimulation by in vivo phenobarbital administration. The NADPH-reduced form of cytochrome P-450 was oxidized very rapidly by cumene hydroperoxide under a CO atmosphere.It was concluded that the NADPH-peroxidase enzyme system of liver microsomes is composed of the same electron transport components which function in substrate hydroxylation reactions.     

Epoxide hydrase and mixed-function oxidase activities of rat liver nuclear membranes.

Rat liver nuclei have 2 to 12% of the corresponding microsomal aryl hydrocarbon hydroxylase, aminopyrine and benzphetamine N-demethylase, NADPH-cytochrome c reductase, and epoxide hydrase activities. Nuclear membranes were prepared from isolated liver nuclei by a sucrose density centrifugation technique. A 2.5- to 10.2-fold increase in the specific enzyme activities was observed in nuclear membrane as compared to intact nuclei. Several properties of the rat liver nuclear membrane and microsomal epoxide hydrase have been compared. Nuclear epoxide hydrase was similar to the corresponding microsomal enzyme in being induced by phenobarbital whereas 3-methylcholanthrene did not produce any effects. Nuclear membrane and microsomal epoxide hydrase were inhibited to a similar degree by 1,1,1-trichloropropene oxide, cyclohexene oxide, an trans-stilbene oxide. The apparent K_m value of nuclear membrane epoxide hydrase was 20 μm for benzo(a)pyrene 4,5-oxide, which is 5.5-fold lower than the corresponding microsomal K_m value (112 μm). Nuclear membranes were prepared from isolated nuclei of rat kidney, lung, spleen, and heart by the DNase digestion method. Epoxide hydrase activity in intact nuclei was in the following order: kidney > lung ? spleen, or heart. Increases of 2.2- and 2.5-fold in specific epoxide hydrase activity were observed in kidney and lung when nuclear membranes were compared to intact nuclei. DMSO, dimethylsulfoxide     

Purification and properties of human erythrocyte membrane NADH-cytochrome b5 reductase

An NADH:(acceptor) oxidoreductase (EC 1.6.99.3) of human erythrocyte membrane was purified by DEAE-cellulose anion exchange, hydroxyapatite adsorption, and 5′-ADP-hexane-agarose affinity chromatographies after solubilization with Triton X-100. The purified reductase preparation was homogeneous and estimated to have an apparent molecular weight of 36,000 on SDS-polyacrylamide slab gel electrophoresis and of 144,000 on Sephadex G-200 gel filtration in the presence of 0.2% Triton X-100, whereas a soluble NADH-cytochrome b₅ reductase of human erythrocyte had a molecular weight of 32,000 by both methods, indicating the existence of a distinct membrane reductase. Digestion of the membrane reductase with cathepsin D yielded a new polypeptide chain which gave the same relative mobility as the soluble reductase on SDS-polyacrylamide slab gel electrophoresis. The membrane enzyme, the cathepsin-digested enzyme, and the soluble enzyme all cross-reacted with the antibody to rat liver microsomal NADH-cytochrome b₅ reductase. The enzyme had one mole FAD per 36,000 as a prosthetic group and could reduce K₃Fe(CN)₆, 2,6-dichlorophenolindophenol, cytochrome c, methemoglobin-ferrocyanide complex, cytochrome b₅ and methemoglobin via cytochrome b₅ when NADH was used as an electron donor. NADPH was less effective as an electron donor than NADH. The specific activity of the purified enzyme was 790 μmol ferricyanide reduced min^?1 mg^?1 and the turnover number was 40,600 mol ferricyanide reduced min^?1 mol^?1 FAD at 25 °C. The apparent K_m values for NADH and cytochrome b₅ were 0.6 and 20 μm, respectively, and the apparent V value was 270 μmol cytochrome b₅ reduced min^?1 mg^?1. These kinetic properties were similar to those of the soluble NADH-cytochrome b₅ reductase. The results indicate that the NADH:(acceptor) oxidoreductase of human erythrocyte membrane could be characterized as a membrane NADH-cytochrome b₅ reductase.     

Purification of a flavoprotein having NADPH-cytochrome c reductase and transhydrogenase activities from Nitrobacter winogradskyi and its molecular and enzymatic properties

A flavoenzyme which showed NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase EC 1.6.2.4) and transhydrogenase (NADPH-NAD⁺ oxidoreductase, EC 1.6.1.1) activities was purified to an electrophoretically homogeneous state from Nitrobacter winogradskyi. The reductase was a flavoprotein which contained one FAD per molecule but no FMN. The oxidized form of the enzyme showed absorption maxima at 272, 375 and 459 nm with a shoulder at 490 nm, its molecular weight was estimated to be 36,000 by SDS polyacrylamide gel electrophoresis, and the enzyme seemed to exist as a dimer in aqueous solution. The enzyme catalyzed reduction of cytochrome c, DCIP and benzylviologen by NADPH, oxidation of NADPH with menadione and duroquinone, and showed transhydrogenase activity. NADH was less effective than NADPH as the electron donor in the reactions catalyzed by the enzyme. The NADPH-reduction catalyzed by the enzyme of N. winogradskyi cytochrome c-550 and horse cytochrome c was stimulated by spinach ferredoxin. The enzyme reduced NADP⁺ with reduced spinach ferredoxin and benzylviologen radical.Abbreviations DCIP dichlorophenolindophenol - Tris trishydroxy-methylaminomethane - Mops 3-(N-morpholino) propanesulfonic acid - SDS sodium dodecylsufate     

Properties of purified kidney microsomal NADPH-cytochrome c reductase

NADPH-cytochrome c reductase, solubilized by lipase digestion of microsomes prepared from perfused porcine kidney cortex, was purified about 3600-fold to give a turnover number of 1230 nmoles cytochrome c reduced per min per nmole flavin. The kinetic determination of K_m and V with respect to NADPH, cytochrome c, and NADH, resulted in values similar to those obtained with purified liver reductase. The kidney microsomal enzyme also exhibited a ping-pong kinetic mechanism for NADPH-mediated cytochrome c reduction.Spectrofluorometric measurements demonstrated the presence of equimolar amounts of FAD and FMN per mole of reductase. The molecular weight was estimated by Sephadex G-200 gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 68,000 and 71,000 g per mole, respectively.Immunochemical techniques, including Ouchterlony double-diffusion studies and inhibition of catalytic activity by antibody to the liver microsomal NADPH-cytochrome c reductase, established the similarity of the purified liver and kidney reductases.     

Purification and immunochemical characteristics of NADPH-cytochrome P-450 oxidoreductase from tobacco cultured cells

NADPH-cytochrome P-450 oxidoreductase (EC 1.6.2.4) was purified from the microsomal fraction of tobacco (Nicotiana tabacum) BY2 cells by chromatography on two anion-exchange columns and 2′,5′ ADP-Sepharose 4B column. The purified enzyme showed a single protein band with a molecular weight of 79 kDa on SDS-PAGE and exhibited a typical flavoprotein redox spectrum, indicating the presence of an equimolar quantity of FAD and FMN. This enzyme followed Michaelis-Menten Kinetics with K_m values of 24 μM for NADPH and 16 μM for cytochrome c. An in vitro reconstituted system of the purified reductase with a partially purified tobacco cytochrome P-450 preparation showed the cinnamic acid 4-hydroxylase activity at the rate of 14 pmol min ⁻¹nmol⁻¹ P-450 protein and with a purified rabbit P-450_2C14 catalyzed N-demethylation of aminopyrine at the rate of 6 pmol min⁻¹ lnmo⁻¹ P-450 protein. Polyclonal antibodies raised against the purified reductase reacted with tobacco reductase but not with yeast reductase on Western blot analysis. Anti-yeast reductase antibodies did not react with the tobacco reductase. This result indicate that the tobacco reductase was immunochemically different from the yeast reductase. The anti-tobacco reductase antibodies totally inhibited the tobacco reductase activity, but not the yeast reductase. Also, Western blot analyses using the anti-tobacco reductase antibodies revealed that leaves, roots and shoots of Nicotiana tabacum plants contained an equal amount of the reductase protein. From these results, it was suggested that there are different antibody binding sites, which certainly participate in enzyme activity, between tobacco and yeast reductase.     

Localization of nascent NADPH-cytochrome c reductase in rat liver microsomes

Rat liver microsomes incubated with [³H] puromycin in high salt buffer were digested with a mixture of protease, trypsin and chymotrypsin, in both the presence and absence of 1% deoxycholate. Our observations revealed that the proteolysis of peptidyl puromycin labeled with [³H] puromycin was at least partially protected by the presence of microsomal membrane. Immunochemical analyses have further shown that most of the nascent NADPH-cytochrome c reductase in the microsomes was digested with the proteases while serum albumin was effectively protected from the digestion. It is thus proposed that NADPH-cytochrome c reductase synthesized on the membrane bound ribosomes is not transported to the vesicular cavity but directly to the outer surface of the microsomal membrane in a form which is accessible to the proteases.     

ISOLATION AND PROPERTIES OF THE PLASMA MEMBRANE OF KB CELLS

Plasma membranes from KB cells were isolated by the method of latex bead ingestion and were compared with those obtained by the ZnCl₂ method. Optimal conditions for bead uptake and the isolation procedure employing discontinuous sucrose gradient centrifugation are described. All steps of preparative procedure were monitored by electron microscopy and specific enzyme activities. The plasma membrane fraction obtained by both methods is characterized by the presence of the Na⁺ + K⁺-activated ATPase and 5'-nucleotidase, and contains NADPH-cytochrome c reductase and cytochrome b₅. The latter two enzymes are also present in lower concentrations in the microsomal fraction. Unlike microsomes which are devoid of the Na⁺ + K⁺-activated ATPase and which contain only traces of 5'-nucleotidase activity, the plasma membrane fraction contains only trace amounts of the rotenone-insensitive NADH-cytochrome c reductase but no cytochrome P-450, both of which are mainly microsomal components. Morphologically the plasma membrane fraction isolated by the latex bead method is composed of vesicles of 0.1–0.3 µm in diameter. On the basis of the biochemical and morphological criteria presented, it is concluded that the plasma membrane fraction isolated by the above methods are of high degree of purity.     

Perinatal development of cytochrome P-450, nadphcytochrome c reductase and ethoxycoumarin deethylase in rat liver nuclear membranes

Perinatal development of rat liver nuclear membrane enzymatic activities was investigated with respect to the metabolism of xenobiotica. The qualitative pattern observed was very close to that reported for microsomal enzymes during development. Cytochrome P-450, NADPH-cytochrome c reductase and ethoxycoumarin deethylase are already present in fetuses at 18 days of gestational age. Phenobarbital pretreatment appears to be effective as an inducing agent for all the enzymes studied, but only after birth. The pattern of induction of cytochrome P-450 showed a peak at the 38th day of life three times higher than basal values at that age. NADPH-cytochrome c reductase presented a constant elevation to about twice basal activity throughout the period taken into consideration. Ethoxycoumarin deethylase activity took only 17 days to reach the basal value observed later in adult animals. This enzyme proved highly inducible by phenobarbital (5-fold) early after birth but the increase dropped to 3-fold from the 24th day of life.     

Ultrastructural and Enzymic Studies of Cell Membranes from Ice-encased and Noniced Winter Wheat Seedlings

A marked increase in the amount of cisternal-like cytoplasmic membranes was observed after ice encasement of winter wheat (Triticum aestivum L.) seedlings. Linear sucrose gradients were employed to separate the various membrane components of the microsomal membrane fraction. NADH- and NADPH-cytochrome c reductase, two specific enzyme markers for plant endoplasmic reticulum (ER) were used to locate the ER in the linear gradients. The identity of the ER fraction was confirmed by determining the effect of EDTA and Mg²⁺ in the preparative media on the distribution of NADH- and NADPH-cytochrome c reductase activity within the gradient. In the presence of EDTA which dissociates ribosomes from ER, peaks of activity for the two enzymes were observed at a density corresponding to that for “smooth” ER. When the media also contained an appropriate concentration of Mg²⁺ to maintain the attachment of ribosomes to the ER, the peaks of activity for the enzymes shifted to a density corresponding to that for “rough” ER. NADH-cytochrome c reductase activity was similar for 24 C-grown and 2 C-grown iced seedlings, but significantly lower for 2 C noniced seedlings. No preferential increase in uptake of radioactive leucine or choline in the ER was observed during ice encasement. The accumulation of electron microscopically visible membrane arrays was not inhibited by the presence of protein synthesis inhibitors at concentrations which severely inhibited incorporation of [1-¹⁴C]leucine into membrane protein, but did not affect survival and growth of the seedlings. These observations indicate that the apparent proliferation of ER during ice encasement does not result from net membrane synthesis, but rather from reorganization of existing membrane elements within the cell.     

NADPH-cytochrome c reductase in outer membrane of kidney mitochondria. Purification and properties.

NADPH-cytochrome c reductase of vitamin D₃-deficient chick kidney mitochondria has been purified approximately 1100-fold to a specific activity of 788 nmol cytochrome c reduced/min/mg protein. Analytical gel electrophoresis of the purified enzyme revealed two bands when stained for protein, but only the more anodic band demonstrated NADPH-tetrazolium reductase activity. The relative ease of solubilization of the reductase without the use of lipases, proteases, or detergents was the first line of evidence that suggested a novel mitochondrial localization for this previously unreported NADPH-linked cytochrome c reductase. The apparent properties of the reductase suggest that the enzyme is a component of kidney mitochondrial outer membrane. The kinetic determination of Michaelis constants with respect to NADPH, cytochrome c, and NADH gave the following values: K_mNADPH = 1.7 μM, K_mcytc = 3.4 μM, and K_mNADH = 20 mM. These constants were different from those of the intact kidney microsomal reductase: K_mNADPH = 5.5 μM, K_mcytc = 10.5 μM, and K_mNADH = 13.3 μM. The mitochondrial as well as the intact microsomal reductases exhibited a ping-pong kinetic mechanism for NADPH-mediated cytochrome c reduction. Spectrofluorometric measurements demonstrated the presence of equimolar amounts of FAD and FMN. The oxidized enzyme has absorption maxima at 280 and 450 nm with a shoulder at 415 nm. Upon reduction with NADPH a distinct loss in the 450-nm absorption was observed. Ouchterlony immunodiffusion studies with rabbit antiserum to chick renal mitochondrial ferredoxin did not reveal cross-reactivity when the purified reductase was the antigen. This excludes the involvement of a ferredoxin-type iron-sulfur protein in the NADPH-mediated reduction of cytochrome c by the purified reductase.     

Localization and Quantitative Determination of Ferredoxin-NADP Oxidoreductase, a Thylakoid-Bound Enzyme in the Cyanobacterium Anabaena sp. Strain 7119

Thylakoid membrane preparations obtained from mechanically disrupted (sonicated) cells of the cyanobacterium Anabaena sp. strain 7119 show a membrane-bound ferredoxin-NADP⁺ oxidoreductase (EC 1.18.1.2) as determined either by specific antibodies or by using the ferredoxin-dependent NADPH-cytochrome c reductase activity, which is a specific test for this enzyme. However, in contrast with higher plant thylakoids, a low yield of the cyanobacterial reductase—only about 20% of the total amount of this protein estimated in whole cell homogenates—was obtained as a membrane-bound form when Mg²⁺ was present during the disruption treatment. It is noteworthy that the addition of water-soluble nonionic polymers, namely polyethylene glycol and polyyinylpyrrolidone, dramatically increased the yield of the thylakoid-bound reductase, reaching values up to 80 to 85% of the total enzyme. Using these thylakoid membrane preparations, a quantitative determination of the reductase has been performed for the first time for cyanobacterial thylakoids. The value determined by immunoelectrophoresis—from 8 to 10 nanomoles per micromole of chlorophyll—is clearly higher than those reported for chloroplast thylakoids.     

DIFFERENCES IN THE SUBCELLULAR AND SUBSYNAPTOSOMAL DISTRIBUTION OF THE PUTATIVE ENDOPLASMIC RETICULUM MARKERS, NADPH-CYTOCHROME c REDUCTASE, ESTRONE SULFATE SULFOHYDROLASE AND CDP-CHOLINE DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE IN RAT BRAIN

Abstract— A comprehensive study has been undertaken on the subcellular and subsynaptosomal distribution of a number of markers for subcellular organelles in preparations from rat brain. Although the activity of most enzymatic markers was decreased by freezing and storage at - 70^oC, no significant changes were noted in the distribution of these activities. This demonstrates that contamination of brain fractions by subcellular organelles can be accurately assessed after freezing and thawing. A marked discrepancy was noted between the distribution of three putative markers for endoplasmic reticulum. CDP-choline-diacylglycerol cholinephosphotransferase (EC 2.7.8.1) activity was mainly limited to the microsomal fraction and was present to a lesser extent in the synaptosomal fraction than the other putative markers for endoplasmic reticulum. Estrone sulfate sulfohydrolase (EC 3.1.6.2) activity demonstrated a bimodal distribution between the crude nuclear and microsomal fractions. However, considerable activity was associated with the synaptosomal fraction. NADPH-cytochrome c reductase (EC 2.3.1.15) activity sedimented in the microsomal and the synaptosomal fractions. Calculations based on the relative specific activities of the microsomal and synaptic plasma membrane fraction indicated that the contamination of the synaptic plasma membranes by endoplasmic reticulum was 44.5% (NADPH-cytochrome c reductase), 38.0% (estrone sulfatase) and 9.0% (cholinephosphotransferase). Since it is believed that virtually all of the synthesis of phosphatidylcholine by cholinephosphotransferase occurs in the neuronal and glial cell bodies, it was concluded that cholinephosphotransferase is a satisfactory marker for the endoplasmic reticulum derived from these sources. The results suggest that NADPH-cytochrome c reductase and estrone sulfatase may be present in the smooth endoplasmic reticulum system responsible for the fast transport of macromolecules along the axon to the nerve endings as well as in the endoplasmic reticulum of the cell bodies. The possible relation between that portion of the smooth endoplasmic reticulum involved in fast axonal transport and the GERL (Golgi, Endoplasmic Reticulum, Lysosomes) complex discovered by Novikoff and his coworkers (Novikoff , 1976) is discussed.     

The markers of pig heart mitochondrial sub-fractions: II. — On the association of malate dehydrogenase with inner membrane

Evidence is presented about the dual location of NADPH-cytochrome c reductase in mitochondrial outer membranes as well as in microsomes, from pig heart.A high specific activity, was found in both fractions, even after their purification by washing, digitonin treatments, or passages on sucrose gradients. A large fraction of the total activity was associated with both mitochondria and microsomes.Mitochondrial outer membrane differs from microsomes by a low choline phosphotransferase activity and the absence of cytochrome P-450.The properties of mitochondrial and microsomal rotenone-insensitive NADH- and NADPH-cytochrome c reductases were studied. In microsomes, both activities have the same optimum pH (8.5) ; in contrast, in mitochondria they have a different one. The K_m-NADPH were always much higher than those for NADH. In mitochondria the K_m for NAD(P)H were dependent on cytochrome c concentration.The results show that the rotenone-insensitive NADH- and NADPH-cytochrome c reductases of mitochondria and microsomes have quite different behavior and do not appear to be supported by the same enzyme.     

HETEROGENEITY OF ROUGH-SURFACED LIVER MICROSOMAL MEMBRANES OF ADULT,PHENOBARBITAL-TREATED,AND NEWBORN RATS

Rough microsomes from the livers of adult, phenobarbital-treated, and newborn rats were subfractionated on a continuous sucrose gradient. Among the subfractions a marked heterogeneity in the distribution patterns of some enzyme activities appears. The isopycnic density of the various fractions in aqueous sucrose ranges from 1.17 to 1.25. The sedimentation coefficients (s₀) in 0.25 M sucrose lie between 0.4 x 10³ S and 1.2 x 10³ S. In adult animals, the NADH- and NADPH-cytochrome c reductase as well as the G6Pase activities are much higher in the slower sedimenting fractions than in the pellet. The increase in the level of G6Pase induced by fasting as well as the phenobarbital-induced changes are most prominent in the slowly sedimenting fractions. Three injections of phenobarbital have no effect on the specific NADPH-cytochrome c reductase activity in the pellet, but cause a significant increase of this enzyme activity in the light fractions. In the newborn animal, the NADH-ferricyanide reductase and NADPH-cytochrome c reductase activities are highest in the light fractions. On the other hand, the amount of cytochrome b ₅ is evenly distributed in all cases. Short-term incorporation of leucine-¹⁴C and glycerol-³H in vivo after phenobarbital treatment shows contrasting results, as the former is increased and the latter is decreased in the slowly sedimenting fractions. Leucine-¹⁴C incorporation into isolated, total membrane proteins is greater in both phenobarbital-treated and newborn animals than in untreated adults. The data support a multistep model for membrane biogenesis and indicate dynamic and individual behavior of the different parts of the rough-surfaced endoplasmic reticulum.     

Investigation of the transverse topology of the microsomal membrane using combinations of proteases and the non-penetrating reagent diazobenzene sulfonate

Intact microsomal vesicles from rat liver were subjected to combined treatment with trypsin and an unspecific protease and were also examined after reaction with the chemical probe p-diazobenzene sulfonate. In addition, the latency of various enzymes in intact microsomal vesicles has been investigated. All microsomal electron transport enzymes studied, i.e. NADH-ferricyanide and cytochrome c reductases, cytochrome b₅, NADPH-cytochrome c reductase and cytochrome P-450, were either solubilized or inactivated by one or both treatments. The experimental data indicate that UDPglucuronyl-transferase is also localized at the outer surface of microsomes. In contrast, a number of hydrolytic enzymes are apparently located inside the permeability barrier of the membrane and presumably at the inner surface. Under conditions where the levels of electron transport enzyme activities or amounts are changed, such as in newborn rats and rats treated with phenobarbital or methylcholanthrene, the intramembranous position of these enzymes is the same as in control adult rats. This indicates that the enzyme molecules are not relocated after their insertion into the membrane.     

Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes

An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharose 6B chromatography and Blue Ultrogel chromatography. Of the three peaks of activity eluted from the latter column with a salt gradient, peak 3 had 50% or more of the activity and was almost pure enzyme. The preparation examined in SDS-gel electrophoresis consisted of two types of subunits, a (molecular weight 39,500) and b (37,000) in equal amounts. Peak 2 was less pure but had a similar polypeptide pattern. The active protein is proposed to be a heterotetramer (a₂b₂) having a molecular weight of about 150,000, as found by gel exclusion chromatography. The purified enzyme can reduce several quinones, DCPIP, cytochrome c, and with best efficiency ferricyanide, and is therefore a diaphorase. The kinetics for the substrates are negatively cooperative with Hill coefficients n_H = 0.55 ± 0.05 for NADPH and 0.22 ± 0.04 for duroquinone. A weak inhibition by p-hydroxymercuric benzoate and mersalyl (stronger with microsomal preparations) suggests the presence of essential sulfhydryl group(s). The possibility is discussed that the dehydrogenase is an NAD(P)H-P450 reductase or similar flavoprotein, and that it is responsible for the NADPH-cytochrome c reductase activity of plant microsomes.     

The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria

1. Aerobically grown yeast having a high activity of glyoxylate-cycle, citric acid-cycle and electron-transport enzymes was transferred to a medium containing 10% glucose. After a lag phase of 30min. the yeast grew exponentially with a mean generation time of 94min. 2. The enzymes malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase and NADH–cytochrome c oxidoreductase lost 45%, 17%, 27% and 46% of their activity respectively during the lag phase. 3. When growth commenced pyruvate kinase, pyruvate decarboxylase, alcohol dehydrogenase, glutamate dehydrogenase (NADP⁺-linked) and NADPH–cytochrome c oxidoreductase increased in activity, whereas aconitase, isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), α-oxoglutarate dehydrogenase, fumarase, malate dehydrogenase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase, NADH oxidase, NADPH oxidase, cytochrome c oxidase, glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, isocitrate lyase and glucose 6-phosphate dehydrogenase decreased. 4. During the early stages of growth the loss of activity of aconitase, α-oxoglutarate dehydrogenase, fumarase and glucose 6-phosphate dehydrogenase could be accounted for by dilution by cell division. The lower rate of loss of activity of isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, NADPH oxidase and cytochrome c oxidase implies their continued synthesis, whereas the higher rate of loss of activity of malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase and NADH oxidase means that these enzymes were actively removed. 5. The mechanisms of selective removal of enzyme activity and the control of the residual metabolic pathways are discussed.     

Localization and concentration of hydroxylamine oxidoreductase and cytochromes c-552, c-554, cm-553, cm-552 and a in Nitrosomonas europaea

Two membrane-associated cytochromes, cytochrome c_m-553 and cytochrome c_m-552, were derived from Nitrosomonas europaea. The major c-type cytochrome, cytochrome c_m-553, accounted for 92% of the c heme found in the membrane. It had absorption maxima at 410 nm in the oxidized form and at 417, 523 and 553 nm in the dithionite reduced form. Cytochrome c_m-552 possessed absorption maxima at 409 nm in the oxidized form, at 421, 522 and 552 in the dithionite reduced form, and at 418 in the dithionite reduced plus CO form. The concentration and cellular distribution of the two c-type membrane cytochromes, hydroxylamine oxidoreductase and cytochromes c-552, c-554, and a were determined. Over 95% of the soluble cytochromes (hydroxylamine oxidoreductase cytochromes and c-552 and c-554) were periplasmic, whereas cytochrome c_m-553, cytochrome c_m-552 and cytochrome a were associated with the cell membrane. The outer membrane and cytoplasm were devoid of cytochromes. The extracytoplasmic location of the proton-yielding hydroxylamine oxidizing system (NH₂OH ™ HNO + 2H⁺ + 2e⁻) may contribute to an energy-linked proton gradient. The heme concentrations of hydroxylamine oxidoreductase and cytochromes c-552, c-554, c_m-553, c_m-552 and a were approx. 2.4, 1.2, 0.3, 1.3, 0.1 and 1.1 nmol/mg cell protein, respectively. The corresponding molar ratios of heme were 22:11:2.9:12:1.0:10. The enzyme or cytochrome concentrations for hydroxylamine oxidoreductase and cytochromes c-552, c-554, c_m-553, c_m-552 and a were approx. 0.13, 1.05, 0.09, 0.63, 0.055 and 0.56 nmol/mg cell protein, respectively. The corresponding molar ratios were 0.24:2.2:0.16:1.2:0.1:1.0.