Calcium pump activity of the renal plasma membrane and renal microsomes

ATP-dependent Ca²⁺ uptake distinct from that of the mitochondria is found in both plasma membrane and microsomal membranes of rat kidney. Activity attributed to these fractions is enhanced by ammonium oxalate and is apparently insensitive to NaN₃. In contrast, rat kidney mitochondrial Ca²⁺ uptake is blocked by NaN₃. The pH of optimal activity is significantly higher for the mitochondrial fraction. Microsomal membrane Ca²⁺ uptake differs from that of the plasma membrane. Microsomal membranes are four times as active as the plasma membrane at high (5 mM) ATP levels. Apparent K_m values for Mg²⁺-ATP differ in the two preparations with a higher affinity for Mg²⁺-ATP found in the plasma membrane Ca²⁺ uptake activity of the plasma membrane preparation is readily inhibited by Na⁺. Sucrose gradient density fractionation indicates that the observed microsomal membrane Ca²⁺ pump activity is associated with membrane vesicles derived from the endoplasmic reticulum. Ca²⁺ pump activity of both plasma membrane and microsomal fraction is depressed din the adrenalectomized rat. This activity is not restored by a single natriuretic dose of aldosterone.     

Osmotic stress-induced changes in cell wall peroxidase activity and hydrogen peroxide level in roots of rice seedlings

The changes in activity of peroxidase (POD) extracted from the cellwalls and the level of H₂O₂ in rice seedling rootstreatedwith mannitol and their correlation with root growth were investigated.Increasing concentrations of mannitol from 92 to 276 mM, which isiso-osmotic with 50 to 150 mM NaCl, progressively reduced rootgrowth and increased POD activities extracted from the cell walls of riceroots.The reduction of growth was also correlated with an increase inH₂O₂ level. Both diamine oxidase (DAO) and NADHperoxidase(NADH-POD) are known to be responsible for the generation ofH₂O₂. Mannitol treatment increased DAO but not NADH-PODactivities in roots of rice seedlings, suggesting that DAO contributes to thegeneration of H₂O₂ in the cell walls of mannitol-treatedroots. An increase in the level of H₂O₂ and the activityof POD extracted from the cell walls of rice roots preceded root growthreduction caused by mannitol. An increase in DAO activity coincided with anincrease in H₂O₂ in roots caused by mannitol. Since DAOcatalyses the oxidation of putrescine, the demonstration that mannitolincreasesthe activity of DAO in roots is consistent with those that mannitol decreasesthe level of putrescine. In conclusion, cell-wall stiffening catalysed by PODispossibly involved in the regulation of root growth reduction caused bymannitol.     

Effects of prostaglandins on peripheral progesterone and uterine diamine oxidase in the pregnant hamster

Serum progesterone and uterine levels of diamine oxidase (DAO) activity were determined during pregnancy in hamsters. Progesterone was elevated on Day 1 of pregnancy, had a transient peak on Day 5, remained relatively constant on Days 6–10, and then increased on Days 13 and 14. Uterine DAO activity could not be detected until Day 7 of pregnancy, approximately 1 $\text{1}\text{2}$ days after the initiation of implantation. DAO activity was associated with placental tissue, and more than 90% of the activity was localized in the maternal placenta. The temporal relationship between changes in serum concentrations of progesterone and uterine levels of DAO activity following PG administration also was studied. Serum progesterone was significantly depressed by 6 hr after treatment with PGs on Day 7 of pregnancy. However, uterine levels of DAO activity at 6 hr in the treated animals were not different from those in control animals. In contrast, both the serum progesterone concentrations and uterine levels of DAO activity were significantly lower at 24 hr after PG treatment. The effects of PG treatment on uterine DAO activity were completely blocked by concomitant administration of progesterone. However, concomitant administration of Provera® only blocked the effect of one PG analog that was tested (9-deoxo-9-methylene-16,16-dimethyl0-PGE₂). The data indicate that changes in uterine DAO activity following treatment with the PGs used here are primarily a consequence of a decrease in peripheral progesterone (i.e. a luteolytic effect of the PG).     

Cell wall peroxidase activity,hydrogen peroxide level and NaCl-inhibited root growth of rice seedlings

The changes in cell-wall peroxidase (POD) activity and H₂O₂ level in roots of NaCl-stressed rice seedlings and their correlation with root growth were investigated. Increasing concentrations of NaCl from 50 to 150 mM progressively reduced root growth and increased ionically bound cell-wall POD activity. NaCl had no effect on covalently bound cell-wall POD activities. The reduction of root growth by NaCl is closely correlated with the increase in H₂O₂ level. Exogenous H₂O₂ was found to inhibit root growth of rice seedlings. Since ammonium and proline accumulation are associated with root growth inhibition caused by NaCl, we determined the effects of NH₄Cl or proline on root growth, cell-wall POD activity and H₂O₂level in roots. External application of NH₄Cl or proline markedly inhibited root growth, increased cell-wall POD activity and increased H₂O₂ level in roots of rice seedlings in the absence of NaCl. An increase in cell-wall POD activity and H₂O₂ level preceded inhibition of root growth caused by NaCl, NH₄Cl or proline. NaCl or proline treatment also increased NADH-POD and diamine oxidase (DAO) activities in roots of rice seedlings, suggesting that NADH-POD and DAO contribute to the H₂O₂ generation in the cell wall of NaCl- or proline-treated roots. NH₄Cl treatment increased NADH-POD activity but had no effect on DAO activity, suggesting that NADH-POD but not DAO is responsible for H₂O₂ generation in cell wall of NH₄Cl-treated roots.     

Identity of microsomal glutathione-S-transferases

Summary Mouse liver microsomes were prepared by repeated washing, homogenization, and centrifugation until almost no more soluble enzymes were found in the supernatant of the last centrifugation. About 0.09% of the total glutathione S-transferase activity and comparable amount of soluble enzymes were detected in microsomes solubilized with Emulgen 913. By double immunodiffusion, microsomal glutathione S-transferases were shown to have a complete immunological identity with cytosolic F2 and F3 transferase from mouse liver. By Sephadex gel filtration chromatography in 1% Emulgen 913, part of the microsomal transferase activity (20 to 50%) was shown to be associated with the microsomal membrane protein fraction and appeared in the void volume. Partially purified microsomal transferases were found to have molecular weights, isoelectric points and K_m's for substrate and GSH which are comparable to those of soluble liver transferases. This study seems to suggest that the presence of glutathione S-transferases in microsomes is the result of specific and nonspecific association between the microsomal membrane and soluble liver transferases.     

Comparison of phospholipid effects on insulin-sensitive low Km cyclic AMP phosphodiesterase in adipocyte plasma membranes and microsomes

Both adipocyte plasma membranes and microsomes possess insulin-sensitive low K_m cyclic AMP phosphodiesterase activity. The activity of the enzyme from both sources was susceptible to activation by several anionic phospholipids. Activators of the plasma membrane enzyme were lysophosphatidylglycerol > lysophosphatidylcholine > lysophosphatidylserine > phosphatidylserine > phosphatidylglycerol. These same phospholipids activated the microsomal enzyme but the extent of activation by each phospholipid was reversed. Neutral phospholipids and other anionic phospholipids were without effect. The phospholipids had no effect on high K_m cAMP phosphodiesterase in either membrane. The results suggest that the phospholipid headgroup was an important determinant for enzyme activation by phospholipid. The increased susceptibility of the plasma membrane enzyme to lysophospholipid may be attributed to a difference in the plasma membrane enzyme compared to the microsomal membrane enzyme or to differences in plasma membrane and microsomal membrane phospholipid composition and their ability to regulate low K_m cAMP phosphodiesterase activity.     

Isolation of plasma membrane and binding of the Ca2+ antagonist nimodipine in Chlamydomonas reinhardtii

Chlorophyll-free plasma membranes of the unicellular green alga Chlamydomonas reinhardtii Dangeard were purified from a microsomal fraction using an aqueous polymer two-phase system of 6.5% (w/w) dextran T500, 6·5% (w/w) polyethylene glycol 3350, 60 mM NaCI, 0 33 M sucrose and 5 mM potassium phosphate (pH 7·8). The plasma membrane fraction contained only 2·4% of the microsomal membrane protein. Specific activity of the plasma membrane marker enzyme, K*, Mg²⁺-ATPase (EC 3.6.1.3). was enriched 9-fold over the microsomal fraction, and 22% of total activity was recovered in the upper, polyethylene glycol-rich phase. Contamination from intracellular membranes was minimal. K*, Mg²⁺-ATPase showed a pH optimum at about 6·5, and addition of 0·05% (w/v) Triton X-100 stimulated the activity 3-fold. [³H]-Nimodipinc was employed to characterize 1,4-dihydropyridine-specific membrane receptors. Two apparent binding sites with different affinities to nimodipine were found in the crude microsomal fraction. The separation of plasma membranes from intracellular membranes revealed that one binding site with higher affinity (K_D= 9 nM) was located on the plasma membrane and a second binding site with lower affinity (K_D= 36 nM) on an intracellular membrane The apparent dissociation constants determined from the association and dissociation rate constants in kinetic experiments were comparable to those determined by equilibrium experiments. The maximum number of binding sites of the plasma membrane fraction and the intracellular membrane fraction was B_max= 440 and 470 fmol (mg protein)^-1, respectively. [³H]-Nimodipinc binding was inhibited by (±) verapamil and stimulated by D-cis-diltiazem in both fractions. Moreover, ethyle-neglycol-bis(2-aminoethylcther)-N, N'-tetraacctic acid (EGTA) inhibited [³H]-nimo-dipinc binding in the plasma membrane fraction but not in the intracellular membrane fraction This effect was cancelled by the addition of CaCl₂.     

Cadmium-induced microsomal membrane-bound peroxidases mediated hydrogen peroxide production in barley roots

The effect of cadmium on microsomal membrane-bound peroxidases and their involvement in hydrogen peroxide production was studied in barley roots. One anionic and two cationic peroxidases were detected, which were strongly activated by Cd treatment. Positive correlation was found between root growth inhibition and increased peroxidase, NADH oxidase activity and H₂O₂ generation in root microsomal membrane fraction of Cd-treated barley roots.     

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     

Immunological and biochemical characterization of nuclear envelope reduced nicotinamide adenine dinucleotide phosphate-cytochrome c oxidoreductase.

NADPH-cytochrome c oxidoreductase (EC 1.6.99.2) activity innate to rat liver nuclear envelope displays antigenic identity with the corresponding microsomal enzyme in a standard Ouchterlony double immunodiffusion test. As with the microsomal enzyme, the nuclear envelope enzyme is selectively released by restricted proteolysis and may be quantitatively isolated from the supernatant phase of the digest by immunoprecipitation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the immunoprecipitates reveals that the oxidoreductase has a molecular weight of 72,000 regardless of its membrane of origin. Radial immunodiffusion titration demonstrates that nuclear envelope contains about one-third the level of NADPH-cytochrome c oxidoreductase (0.21%) as compared to microsomal membrane (0.71%) on a weight basis. By comparison, the specific activity of the nuclear envelope enzyme was half that of the microsomal enzyme. Turnover studies employing NaH¹⁴CO₃ indicate that the half-lives for the nuclear envelope and microsomal enzymes are indistinguishable, each being approximately 55 h.     

Quantification of gut injury with diamine oxidase activity: development of a fission neutron RBE and measurements with combined injury in mouse models

Plasma and small intestine diamine oxidase (DAO) activities were measured on Days 2, 4, and 6 following irradiation of mice with a range of doses of fission neutrons and 60Co. With increasing doses of radiation, plasma DAO activity increased on Day 2 and intestinal DAO activity decreased on Day 4; moreover, the approximate relative biological effectiveness values for these changes in activity were 5.81 for plasma DAO activity on Day 2 and 3.88 for intestinal DAO activity on Day 4. On Day 6 relatively high levels of radiation caused DAO activity in the small intestine to remain depressed whereas low levels resulted in recovery with activities at or near controls. In animals with combined injury (radiation plus 30% surface burn or wound), changes in DAO activity in the intestine were similar to those with radiation alone; plasma DAO activity, in contrast to radiation alone, did not show an increase at the 2-day mark. These dose-dependent relationships should provide a basis for using DAO as a potential indicator of biological damage from radiation exposure within the lethal range.     

Modulation of biosynthesis of phosphatidylcholine via CDP-choline in rat liver: Influence of ethanol on the microsomal cholinephosphotransferase activity

We have studied in vitro the effects of ethanol on the different enzymes involved in the biosynthesis of phosphatidylcholine (PC) via CDP-choline. Ethanol alters neither choline kinase (CK) nor CTP:phosphocholine cytidylyltransferase (CT) activities but, at levels higher than 50 mM, it does significantly inhibit microsomal cholinephosphotransferase (CPT) activity concomitantly with an increase in the ethanol concentration. A study of the kinetics of the reaction catalysed by CPT shows that ethanol decreases V_max without altering K_m, indicating a non-competitive inhibitory effect. An analysis of the thermodependence of CPT activity in the absence of ethanol reveals a break in the Arrhenius plot and thus a straight relationship between enzyme activity and the physico-chemical state of the microsomal membrane. Incubation of microsomes in the presence of ethanol increased the transition temperature from 25.8–28.2°C. Microsomes were also incubated with n-alkanols with chain-lengths of fewer than five carbon atoms at concentrations which, according to their partition coefficients, produce equimolar levels in the membrane. Under these conditions all the alkanols caused the same inhibitory effect. All these results demonstrate that ethanol modulate the PC biosynthesis at the level of CPT activity and does not affect the CT enzyme. The inhibition found on CPT is clearly dependent on the alteration produced by ethanol on the hepatic microsomal membrane.     

NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b₅ reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b₅ reductase).     

ROS generation and proline metabolism in calli of halophyte Nitraria tangutorum Bobr. to sodium nitroprusside treatment

Nitric oxide (NO) is a stress factor or a signal molecule involved in various plant physiological and developmental processes. In the present study, the generation of reactive oxygen species and the metabolism of proline due to different sodium nitroprusside (SNP, an NO donor) concentrations were investigated in callus from halophyte Nitraria tangutorum Bobr. Treatment with SNP led to significant increases of hydrogen peroxide (H₂O₂) content and cell viability but notable reductions in hydrogen radical level and lipid peroxidation degree, and superoxide onion (O₂ ^?) content also enhanced in 100 μM SNP-treated calli. Using a chemical inhibitor for plasma membrane (PM) NADPH oxidase diphenylene iodonium (DPI), we found low O₂ ^? generation in untreated and 25 μM SNP-treated calli, whereas in those treated with 100 μM SNP O₂ ^? level exhibited a very little alteration, comparable to the absence of DPI. These suggest a high activity of PM NADPH oxidase in untreated calli. H₂O₂ scavenging enzymes (catalase, peroxidase [POD] and ascorbate peroxidase) and H₂O₂ forming enzymes (superoxide dismutase [SOD], cell wall-POD and diamine oxidase [DAO]) stimulated significantly in calli treated with different SNP concentrations while glutathione reductase activity decreased. In addition, a reduction in proline content was observed in SNP-treated calli. Moreover, different SNP concentrations stimulated proline dehydrogenase (PDH) and ornithine δ-aminotransferase but inhibited r-glutamyl kinase (GK). In conclusion, our results suggest that the increasing H₂O₂ generation was associated with the stimulation of SOD, cell wall-POD and DAO, and that the reduction of proline content might be the consequence of increased PDH activity and decreased GK activity in N. tangutorum Bobr. calli under SNP treatment.     

茶多酚对盐胁迫下小麦幼苗叶片生理特性的影响

以春小麦"陇春30号"为实验材料,主要研究了150 mmol/L NaCl和不同浓度(25 mg/L和100 mg/L)茶多酚(tea polyphenols, TP)单独或复合处理对小麦幼苗叶片叶绿素含量、叶绿素荧光参数及过氧化氢(H_2O_2)产生等生理特性的影响。结果表明:(1)150 mmol/L NaCl单独处理导致小麦幼苗叶片叶绿素含量及光适应下实际光量子产量[actual light quantum yield,Y(II)]、光化学淬灭(photochemical quenching, qP)、光合电子传递效率(photosynthetic electron transfer efficiency, ETR)均降低,非光化学淬灭(non-photochemical quenching, NPQ)增大;TP单独处理不影响这些指标。(2)盐胁迫诱导细胞壁过氧化物酶(cell wall-peroxidase, cw-POD)、二胺氧化酶(diamine oxidase, DAO)和多胺氧化酶(polyamine oxidase, PAO)活性显著增高;低浓度TP使cw-POD活性显著增大,而DAO和PAO活性无显著变化;不同的是,高浓度TP不影响cw-POD活性,却使DAO和PAO活性显著减小。(3)与NaCl单独处理相比,TP的添加导致NaCl处理下小麦幼苗叶片叶绿素含量增加,最大光化学效率(maximal photochemical efficiency,F_v/F_m)和ETR值增大,而NPQ值、H_2O_2含量及cw-POD、DAO和PAO三种酶活性均降低。总之,TP有效地缓解了盐胁迫诱导的小麦幼苗叶绿素含量的减少及对PS II光合电子传递效率和光化学反应速率的抑制,增强了植物的光合能力,与此同时降低了cw-POD、DAO和PAO活性,减少了H_2O_2的产生,从而缓解盐胁迫对小麦幼苗造成的伤害,提高小麦幼苗对盐环境的耐受性。     

Is there a plasma-membrane-located anion-sensitive ATPase? II. Further studies on rabbit kidney

A study has been made to determine whether renal plasma membranes contain an HCO₃^? stimulated, ouabain insensitive Mg ATPase. Purified mitochondrial, microsomal and brush border membrane fractions have been isolated from rabbit kidney.The microsomal anion-sensitive ATPase activity appears to be entirely of mitochondrial origin on the basis of the effects of inhibitors of mitochondrial Mg ATPase.The brush border membrane fraction is contaminated with mitochondrial fragments and contains an Mg ATPase activity with low anion-sensitivity. Further purification of this fraction causes parallel decreases in anion-sensitivity of the Mg ATPase activity and in cytochrome $\text{c}$ oxidase activity.These results indicate that conclusions previously reached by other investigators for a role of anion-sensitive Mg ATPase in the bicarbonate reabsorption of the proximal tubule may no longer be tenable.     

Lipoxygenase-mediated production of superoxide anion in senescing plant tissue

Lipoxygenase activity and superoxide (O^.?₂) production by microsomal membranes and cytosol from bean cotyledons increased in parallel as senescence progressed. Superoxide production was heat denaturable and dependent on the availability of linoleate, the substrate for lipoxygenase. The specific inhibitor of lipoxygenase, U28938, caused a parallel reduction in enzyme activity and the formation of O^?₂. These observations demonstrate that lipoxygenase activity mediates the formation of superoxide anion, and support the contention that membrane senescence is attributable to a sequence of reactions in which lipasederived fatty acids are utilized by lipoxygenase to generate O^?₂ and hydroperoxides.     

Presence of an extramitochondrial anion-stimulated ATPase in the rabbit kidney: Localization along the nephron and effect of corticosteroids

Summary To determine whether kidney membrane fractions contain an extramitochondrial anion-stimulated ATPase, we compared the pharmacological and kinetic properties of HCO₃-ATPase activities in mitochondrial and microsomal fractions prepared from rabbit kidney cortex and outer medulla. The results indicated that this activity differed markedly in each type of fraction. Microsomal HCO₃-ATPase was less sensitive than mitochondrial ATPase to azide, oligomycin, DCCD and thiocyanate, but was more sensitive to filipin and displayed different dependency towards ATP, magnesium and pH. Microsomal ATPase activity was stimulated by sulfite much more strongly than by bicarbonate, whereas mitochondrial activity was stimulated by both these anions to a similar extent. These results demonstrate the presence of an extramitochondrial HCO₃-ATPase in kidney membrane fractions. HCO₃-ATPase was also measured in single microdissected segments of the rabbit nephron using a radiochemical microassay previously developed for tubular Na, K-ATPase activity. An enzyme with the pharmacological and kinetic properties of the microsomal enzyme was detected in both proximal tubule, distal convoluted tubule and collecting duct, but the thick ascending limb was devoid of any detectable activity. Long-term DOCA administration markedly increased HCO₃-ATPase activity in the distal convoluted and collecting tubule. The insensitivity of microsomal HCO₃-ATPase to vanadate indicates that it belongs to the F₀–F₁ class of ATPases, and might therefore be involved in proton transport. This hypothesis is also supported by the localization of tubular HCO₃-ATPase activity at the sites of urinary acidification.     

Chlorpromazine oligomer is a potentially active substance that inhibits human D-amino acid oxidase,product of a susceptibility gene for schizophrenia

D-Amino acid oxidase (DAO), a potential risk factor for schizophrenia, has been proposed to be involved in the decreased glutamatergic neurotransmission in schizophrenia. Here we show the inhibitory effect of an antipsychotic drug, chlorpromazine, on human DAO, which is consistent with previous reports using porcine DAO, although human DAO was inhibited to a lesser degree (K_i = 0.7 mM) than porcine DAO. Since chlorpromazine is known to induce phototoxic or photoallergic reactions and also to be transformed into various metabolites, we examined the effects of white light-irradiated chlorpromazine on the enzymatic activity. Analytical methods including high-resolution mass spectrometry revealed that irradiation triggered the oligomerization of chlorpromazine molecules. The oligomerized chlorpromazine showed a mixed type inhibition with inhibition constants of low micromolar range, indicative of enhanced inhibition. Taken together, these results suggest that oligomerized chlorpromazine could act as an active substance that might contribute to the therapeutic effects of this drug.     

Properties of microsomal acyl coenzyme A reductase in mouse preputial glands

Synthesis of long-chain fatty alcohols in preputial glands of mice is catalyzed by an NADPH-dependent acyl coenzyme A (CoA) reductase located in microsomal membranes; sensitivity to trypsin digestion indicates that the reductase is on the cytoplasmic side of the membrane. Results with pyrazole and phenobarbital demonstrate the reaction is not catalyzed by a nonspecific alcohol dehydrogenase or an aldehyde reductase. Acyl-CoA reductase activity is sensitive to sulfhydryl and serine reagent modification, is stimulated by bovine serum albumin, and produces an aldehyde intermediate. The activity is extremely detergent sensitive and cannot be restored even after removal of the detergents. Phospholipase C or asolectin treatment does not release the acyl-CoA reductase from microsomal membranes, but causes a significant decrease in the activity recovered in the membrane pellet. Glycerol does not solubilize the reductase activity, nor does 3.0 m NaCl; however, the combination of glycerol and 3.0 m NaCl did release about 50% of the acyl-CoA reductase from the microsomal pellet. Substrate concentration curves obtained in the presence or absence of bovine serum albumin show significant differences in enzyme activities. The reductase is sensitive to the concentration of palmitoyl-CoA and is progressively inhibited at levels beyond the critical micellar concentration of the substrate. The apparent K_m for acyl-CoA reductase is 14 μm; however, the maximum velocity varies with the concentration of albumin used. Expression of enzyme activity in delipidated microsomes requires specific phospholipids, which suggests that in vivo regulation of acyl-CoA reductase activity could be achieved through modifications in membrane lipid composition.