The enzymatic transformation of water-insoluble reactants in nonaqueous solvents. Conversion of cholesterol to cholest-4-ene-3-one by a Ncardia sp.

The rapid conversion of cholesterol to cholestenone by Nocardia in the presence of high proportions of water-immiscible solvent has been demonstrated. At high agitator speeds, the reaction rate was not limited by the rates of transfer of oxygen or cholesterol to the microorganisms. Using 100 g of thawed cells in 200 ml of carbon tetrachloride containing 16% (w/v) cholesterol, at 20°C cholestenone was formed at 7 g/hr. Cells could be separated easily from the organic solvent and reused. After 7 runs (69 hr) the reaction rate had fallen only to half the value for the first run.     

Production of androsta-1,4-diene-3,17-dione from cholesterol using two-step microbial transformation

A novel two-step transformation process for the production of androsta-l by microorganisms-diene-3,17-dione (ADD) from a high concetration of cholesterol by microorganisms is proposed. Cholesterol (20 g/l) was initially converted to cholest-4-en-3-one (cholestenone) by an inducible cholesterol oxidase-producing bacterium, Arthrobacter simplex U-S-A-18. The maximum productivity of cholestenone was 8 g/l per day and the molar conversion rate was 80%. Subsequently, a fine suspension of cholestenone (50 g/l), which was prepared directly from the fermentation broth of A. simplex, was converted to ADD by Mycobacterium sp. NRRL B-3683 in the presence of an androstenone adsorbent, Amberlite XAD-7. The maximum productivity of ADD was 0.91 g/l per day and the molar conversion rate was 35%. Correspondence to: W.-H. Liu     

Enzyme-catalyzed oxidation of cholesterol in pure monolayers at the air/water interface.

Mean molecular area vs. lateral surface pressure isotherms were determined for monolayers containing cholesterol, 4-cholesten-3-one (cholestenone), or binary mixtures of the two. At all lateral surface pressures examined, cholestenone had a larger mean molecular area requirement than cholesterol. Results with the binary mixtures of cholesterol and cholestenone suggested that the sterols did not mix ideally (non additive mean molecular area) with each other in the monolayer; the observed mean molecular area for mixtures was less than would be expected based on ideal mixing. The mixed sterol monolayers also displayed a reduction in the lateral collapse pressure which appeared to be a linear function of the mole fraction of cholestenone in the monolayer, suggesting that cholesterol and cholestenone were completely miscible in the mixed monolayer. The pure cholesterol monolayer was next used to examine the cholesterol oxidase-catalyzed (Brevibacterium sp.) oxidation of cholesterol to cholestenone at different lateral surface pressures at 22 degrees C. The difference in mean molecular area requirements of cholesterol and cholestenone was directly used to convert monolayer area changes (at constant lateral surface pressure) into average reaction rates. It was observed that the average catalytic activity of cholesterol oxidase increased linearly with increased lateral surface pressure in the range of 1 to 20 mN/m. In addition, the enzyme was capable to oxidize cholesterol in monolayers with a lateral surface pressure close to the collapse pressure of cholesterol monolayers (collapse pressure 45 mN/m; oxidation was observed at 40 mN/m). The adsorption of cholesterol oxidase to an inert sterol monolayer film at low surface pressures (around 9 mN/m) was marginal, although clearly detectable at very low (0.5-4 mN/m) lateral surface pressures, suggesting that the enzyme did not penetrate deeply into the monolayer in order to reach the 3 beta-hydroxy group of cholesterol. This interpretation is further supported by the finding that a maximally compressed cholesterol monolayer (40 mN/m) was readily susceptible to enzyme-catalyzed oxidation. It is concluded that cholesterol oxidase is capable of oxidizing cholesterol in laterally expanded monolayers as well as in tightly packed monolayers, where the lateral surface pressure is close to the collapse pressure. The kinetic results suggested that the rate-limiting step in the overall process was the substrate availability per surface area (or surface concentration) at the water/lipid interface.     

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in hepatoma tissue culture cells by pure cholesterol and several cholesterol derivatives. Evidence supporting two distinct mechanisms.20l.

Pure cholesterol associated in complexes with lipoproteins (whole serum and human low density lipoproteins) or esterified with succinic acid (cholesteryl succinate) and bound to albumin effectively suppresses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in hepatoma tissue culture (HTC) cells grown in lipoprotein-poor serum medium during short 4-hour) incubation periods. Simultaneous measurments of the kinetics of uptake of radioactive unesterified cholesterol of whole serum and cholesteryl succinate, their conversion to lipid products, and the decay in enzyme activity, suggest that the cholesterol-induced suppression is mediated by the sterol itself rather than by inhibitory lipid products derived from its metabolism. Several cholesterol derivatives such as cholestenone, 7-ketocholesterol, and 7alpha-and 25-hydroxycholesterol also suppress reductase activiy in HTC cells and are significantly more inhibitory than the pure cholesterol preparations. The decrease in enzyme activity produced by cholesterol and its derivatives is concentration-dependent and specific. [1-14C]Oleate incorporation experiments indicate that cholesterol ester formation in HTC cells is not increased at inhibitory concentrations of the steroids. These data suggest that sterol ester formation is not an obligatory process in the feedback control of HMG-CoA reductase activity. The half-life of the reductase (3 to 4 hours) is not significantly changed by cycloheximide, plus or minus whole serum, and cholesteryl succinate. In contrast, the half-life is strongly reduced when HTC cells are incubated with cycloheximide plus maximal concentrations of 25-hydroxycholesterol, 7-ketocholesterol, or cholestenone, resulting in t1/2 values of 24, 36, and 60 min, respectively. Increasing concentrations of whole serum and cholesteryl succinate have no significant effect on the apparent rate constant of inactivation of the enzyme, whereas its apparent rate of synthesis is decreased 3- and 10-fold, respectively. These results are reversed with oxygenated steroid inhibitors. The rate of synthesis of reductase is essentially unchanged as the concentrations of 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone are increased in the culture medium, whereas the apparent rate constant for degradation is increased 9-, 7-, and 3-fold, respectively. HMG-CoA reductase activity in HTC cells thus appears to be modulated by two different mechanisms in which steroid structure is important. Whole serum and cholesteryl succinate specifically decrease the rate of enzyme synthesis, while 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone increase the rate of inactivation of the reductase.     

Transbilayer movement of cholesterol in the human erythrocyte membrane

The rate of transbilayer movement of cholesterol was measured in intact human erythrocytes. Suspended erythrocytes were incubated briefly with [3H]cholesterol in ethanol at 4 degrees C, or with liposomes containing [3H]cholesterol over 6 hr at 4 degrees C to incorporate the tracer into the outer leaflet of erythrocyte plasma membranes. The erythrocytes were then incubated at 37 degrees C to allow diffusion of cholesterol across the membrane bilayer. Cells were treated briefly with cholesterol oxidase to convert a portion of the outer leaflet cholesterol to cholestenone, and the specific radioactivity of cholestenone was determined over the time of tracer equilibration. The decrease in specific radioactivity of cholestenone reflected transbilayer movement of [3H]cholesterol. The transbilayer movement of cholesterol had a mean half-time of 50 min at 37 degrees C in cells labeled with [3H]cholesterol in ethanol, and 130 min at 37 degrees C in cells labeled with [3H]cholesterol exchanged from liposomes. The cells were shown, by the absence of hemolysis, to remain intact throughout the assay. The presence of 1 mM Mg2+ in the assay buffer was essential to prevent hemolysis of cells treated with cholesterol oxidase perturbed the cells, resulting in an accelerated rate of apparent transbilayer movement. Our data are also consistent with an asymmetric distribution of cholesterol in erythrocyte membranes, with the majority of cholesterol in the inner leaflet.     

Ultrasound enhanced bioprocesses: cholesterol oxidation by Rhodococcus erythropolis

Cholesterol oxidation to cholestenone by resting cells of Rhodococcus erythropolis ATCC25544 was investigated under a computer-controlled ultrasonic irradiation at a frequency of 20 kHz. The optimization of the ultrasound intensity and its mode of application to a stirred bioreactor was first established at a level which preserved the structural integrity of the cells and enabled their metabolic activity. A significant enhancement in the kinetic rates of the biotransformation was observed in microbial slurries of 1.0 and 2.5 g/L cholesterol when sonicated for 5 s every 10 min with a power output of 2.2 W/cm(2). In contrast, ultrasound had no effect on the enzymatic oxidation of cholesterol (2.5 g/L) by cholesterol oxidase. A high loading of cholesterol (5.0 g/L) in sonicated microbial systems had, however, an adverse effect. The ultrasound enhancement is discussed in terms of an increased dissolution rate of the sustrate crystals and more importantly, in terms of the uniquely ultrasound-induced enhancement of mass transfer inside and outside a cell.     

Disposition of intracellular cholesterol in human fibroblasts

We have examined the intracellular distribution of unesterified cholesterol in cultured human fibroblasts. Intact cells were treated with cholesterol oxidase to selectively transform cell surface cholesterol to cholestenone. Isopycnic centrifugation of homogenates showed that the cholestenone had a peak buoyant density of 1.13 g/cm3. The approximately 10% of total cholesterol which remained unoxidized was distributed in two peaks of roughly equal size: a sharp peak at approximately 1.09 g/cm3 and a broad peak centered at 1.18 g/cm3. When intact cells were incubated with exogenous [3H]cholesterol, the radiolabel entered the nonoxidizable pool in a temperature-dependent fashion with a half time of 3 h at 37 degrees C. This label initially was associated with the dense but not the buoyant peak of nonoxidized cholesterol. After 40 h, the buoyant peak also became labeled; both peaks then had a specific activity slightly less than the surface cholestenone. The buoyant density of the unoxidized cholesterol did not coincide with markers for the Golgi apparatus, endoplasmic reticulum, or lysosomes. However, two ingested markers of pinocytosis, calcein and horseradish peroxidase, comigrated with the dense peak of unoxidized cholesterol. That the size of the unoxidized cholesterol pool was greater in cells deprived of serum lipoproteins than in fed cells suggested that none of the intracellular cholesterol need be ascribed to ingested sterols. The mass of unoxidizable cholesterol was not diminished when cholesterol biosynthesis was inhibited by lovastatin in lipoprotein-deprived cells. Furthermore, the newly synthesized radiolabeled cholesterol resistant to cholesterol oxidase did not migrate with intracellular cholesterol mass on sucrose density gradients. The newly synthesized cholesterol amounted to about 10% of the total unoxidized sterol. These data indicate that most of the intracellular cholesterol was not newly synthesized. We conclude that a) approximately 90% of fibroblast cholesterol is associated with the cell surface; b) the bulk of intracellular cholesterol, approximately 10% of total, is derived from internalized (endocytic) plasma membrane; and c) the most recently synthesized cholesterol, approximately 1% of the total, is in a discrete organelle.     

Detergentless microemulsions as media for enzymatic reactions. Cholesterol oxidation catalyzed by cholesterol oxidase

Catalytic activity and stability of cholesterol oxidase dissolved in ternary systems composed of n-hexane, isopropanol, and water were studied. The dependence of catalytic activity on the composition of the system revealed two maxima, in contrast to the behaviour of previously studied enzymes where a single maximum has been observed. The stability profile of cholesterol oxidase showed a single sharp maximum coinciding with the microemulsion region of the phase diagram. Both catalytic activity and the first-order inactivation rate constant of cholesterol oxidase dissolved in n-hexane/isopropanol/water ternary systems were found to decrease with decreasing temperature. This decrease was more rapid for the inactivation rate constant than for catalytic activity, the activation energies being 200 and 60 kJ.mol-1, respectively. Preparative conversion of cholesterol to cholestenone catalyzed by cholesterol oxidase in n-hexane/isopropanol/water ternary systems was carried out with 100% yield. Decreased temperature and the presence of catalase were required to achieve high degrees of cholesterol conversion. A simple procedure suitable for rapid separation of the reaction product and recovery of the enzyme was developed.     

Initial step in the catabolism of cholesterol by Mycobacterium smegmatis mc2 155

The first step in the catabolism of cholesterol, i.e. the transformation of cholesterol into cholestenone, has been investigated in Mycobacterium smegmatis. In silico analysis identified the MSMEG_1604 gene encoding a putative protein similar to the ChoD cholesterol oxidase of M. tuberculosis H37Rv (Rv3409c) and the MSMEG_5228 gene coding for a protein similar to the NAD(P)-dependent cholesterol dehydrogenase/isomerase of Nocardia sp. The expression of the MSMEG_5228 gene was inducible by cholesterol whereas the expression of MSMEG_1604 gene was constitutive. When both genes were expressed in Escherichia coli only the MSMEG_5228 protein was active on cholesterol. The function of ChoD-like MSMEG_1604 protein remains to be elucidated, but it does not appear to play a critical role in the mineralization of cholesterol as a MSMEG_1604(-) mutant was not affected in the production of cholestenone. However, a MSMEG_5228(-) mutant showed a drastic reduction in the synthesis of cholestenone. The finding that this mutant was still able to grow in cholesterol, allowed us to demonstrate that the cholesterol-inducible MSMEG_5233 gene encodes an additional cholesterol dehydrogenase/isomerase similar to the AcmA dehydrogenase of Sterolibacterium denitrificans. The observation that the double MSMEG_5228-5233(-) mutant was able to grow in cholesterol suggests that in addition to these enzymes other dehydrogenase/isomerases can also catalyse the first reaction of the cholesterol degradation pathway in M. smegmatis, which is not the limiting step of the process.     

Enzymatic Oxidation of Cholesterol: Properties and Functional Effects of Cholestenone in Cell Membranes

Bacterial cholesterol oxidase is commonly used as an experimental tool to reduce cellular cholesterol content. That the treatment also generates the poorly degradable metabolite 4-cholesten-3-one (cholestenone) has received less attention. Here, we investigated the membrane partitioning of cholestenone using simulations and cell biological experiments and assessed the functional effects of cholestenone in human cells. Atomistic simulations predicted that cholestenone reduces membrane order, undergoes faster flip-flop and desorbs more readily from membranes than cholesterol. In primary human fibroblasts, cholestenone was released from membranes to physiological extracellular acceptors more avidly than cholesterol, but without acceptors it remained in cells over a day. To address the functional effects of cholestenone, we studied fibroblast migration during wound healing. When cells were either cholesterol oxidase treated or part of cellular cholesterol was exchanged for cholestenone with cyclodextrin, cell migration during 22 h was markedly inhibited. Instead, when a similar fraction of cholesterol was removed using cyclodextrin, cells replenished their cholesterol content in 3 h and migrated similarly to control cells. Thus, cholesterol oxidation produces long-term functional effects in cells and these are in part due to the generated membrane active cholestenone.     

Enzyme-Catalyzed oxidation of cholesterol in physically characterized water-in-oil microemulsions

The enzymatic conversion of cholesterol to cholestenone by cholesterol oxidase (Brevibacterium sp.)in reversed micelles in a system composed of AOT/isooctane/water/cholesterol has been examined. The catalytic activity of the enzyme was correlated with the physicochemical properties of water in water-in-oil (w/o) microemulsion systems. In a system consisting of 3 wt % AOT in isooctane, reversed micelles started to form as the [H(2)O]/[AOT] (e.g., the w(0)) ratio increased above 4-5. The formation of reversed micelles with a core of neat (bulk) water was verified from determinations of both the partial molar volume of water and the scissors vibration of water [with Fourier transform infrared (FTIR) spectroscopy] in the w/o microemulsion systems. A plot of enzyme activity vs. w(0) indicated that the hydration of enzyme molecules per se was not sufficient to give rise to catalytic activity. Instead, it appeared that the formation of an aqueous micellar core was necessary for full activation of the enzyme. Based on micelle size distribution analysis, it was estimated that about one micelle per one thousand contained an enzyme molecule. Since the apparent reaction rate could be markedly enhanced by increasing the enzyme/water ratio, we conclude that the number of enzyme-containing micelles was an important rate-limiting factor in the system.     

Low density lipoprotein modification by cholesterol oxidase induces enhanced uptake and cholesterol accumulation in cells.

Oxidation of low density lipoprotein (LDL) by cells of the arterial wall or in the presence of copper ions was shown to result in the peroxidation of its fatty acids as well as its cholesterol moiety. LDL incubation with cholesterol oxidase (CO) resulted in the conversion of up to 85% of the lipoprotein unesterified cholesterol (cholest-5-en-3-ol) to cholestenone (cholest-4-en-3-one) in a dose- and time-dependent pattern. Plasma very low density lipoprotein (VLDL) and high density lipoprotein (HDL) could be similarly modified by CO. In cholesterol oxidase-modified LDL (CO-LDL), unlike copper ion-induced oxidized LDL (Cu-Ox-LDL), there was no fatty acids peroxidation, and lipoprotein size or charge as well as LDL cholesteryl ester, phospholipids, and triglycerides content were not affected. CO-LDL, however, demonstrated enhanced susceptibility to oxidation by copper ions in comparison to native LDL. Upon incubation of CO-LDL with J-774 A.1 macrophage-like cell line, cellular uptake and degradation of the lipoprotein was increased by up to 62% in comparison to native LDL but was 15% lower than that of Cu-Ox-LDL. Similarly, the binding of CO-LDL to macrophages increased by up to 80%, and cellular cholesterol mass was increased 51% more than the mass obtained with native LDL. Several lines of evidence indicate that CO-LDL was taken up via the LDL receptor: 1) Excess amounts of unlabeled LDL, but not acetyl-LDL (Ac-LDL), effectively competed with 125I-CO-LDL for the uptake by cells. 2) The degradation of CO-LDL by various types of macrophages and by fibroblasts could be dissociated from that of Ac-LDL and was always higher than that of native LDL. 3) A monoclonal antibody to the LDL receptor (IgG-C7) and a monoclonal antibody to the LDL receptor binding domains on apoB-100 (B1B6) inhibited macrophage degradation of CO-LDL. The receptor for Cu-Ox-LDL, which is not shared with Ac-LDL, was also partially involved in macrophage uptake of CO-LDL, since Cu-Ox-LDL demonstrated some competition capability with CO-125I-LDL for its cellular degradation. CO-LDL cellular degradation was inhibited by chloroquine, thus implying lysosomal involvement in the cellular processing of the lipoprotein. Incubation of macrophages with LDL in the presence of increasing concentrations of cholestenone resulted in up to 52% enhanced lipoprotein cellular degradation suggesting that the cholestenone in CO-LDL might be involved in the enhanced cellular uptake of the modified lipoprotein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of cholesterol and 7-beta hydroxycholesterol on glutathione status and nitric oxide production in murine peritoneal macrophages

Present study was conducted to observe the effect of cholesterol and oxidized cholesterol (7beta-hydroxycholesterol,7beta-OH) on the nitric oxide (NO) production and the redox ratio by lipopolysaccharide-stimulated macrophages. Dose-dependent decrease in NO levels was seen with both cholesterol and 7beta-OH at different incubation intervals (6,12,18,24 hr) and concentrations (2.5,5,7.5microg/ml). On comparison, a significant decrease in the NO was observed at 24 hr interval in 7beta-OH exposed cells with all respective concentrations of cholesterol. Incubation with 7beta-OH also resulted in significant increase in levels of oxidized glutathione (GSSG) and decrease in reduced glutathione (GSH), while cholesterol showed no effect on GSSG levels. Moreover, GSH levels were lowered only at highest concentration (7.5microg/ml), and at longer incubation intervals (18,24 hr) with cholesterol exposure. This altered the redox status in both cholesterol/7beta-OH treated macrophages. Increased redox ratio and decreased NO levels indicated increased oxidative stress and decreased vasodilation by 7beta-OH compared to cholesterol.     

Acyl coenzyme A:cholesterol acyl transferase in macrophages utilizes a cellular pool of cholesterol oxidase-accessible cholesterol as substrate

Cholesterol esterification by acyl CoA:cholesterol acyl transferase (ACAT) in macrophages is a key process in atheroma foam cell formation. However, the process of cholesterol substrate delivery to ACAT is not well defined. In this study, J774 macrophages, which form foam cells with native low density lipoprotein (LDL), were labeled with [3H]cholesterol-containing liposomes. Most (80-90%) of the cholesterol label could be converted by cholesterol oxidase to cholestenone, suggesting plasma membrane localization; only 0.6% of the label was in cholesteryl ester (CE). In cells chased for 6 h in medium lacking LDL, the distribution of label was essentially unchanged, whereas in cells chased with LDL, 28% of the label was incorporated into CE concomitant with a decrease in cholestenone label to 50%. [3H]Cholesterol-labeled mouse peritoneal macrophages incubated with acetyl-LDL, and both J774 and mouse peritoneal macrophages incubated with 25-hydroxy-cholesterol, also showed a shift of label from cholestenone to CE. Similar results were found when cellular cholesterol was biosynthetically labeled with [3H]mevalonate. The percentage of cholesterol substrate for ACAT in LDL-treated J774 macrophages which originates from endogenous cellular pools (versus that originating from LDL itself) is approximately 50%. We conclude that upon activation of ACAT in macrophages, there is a novel process whereby a cholesterol oxidase-accessible pool of cellular cholesterol, presumably plasma membrane cholesterol, is translocated to ACAT in the endoplasmic reticulum.     

Application of chitosan beads immobilized Rhodococcus sp. NCIM 2891 cholesterol oxidase for cholestenone production

The cell-bound cholesterol oxidase from the Rhodococcus sp. NCIM 2891 was purified three fold by diethylaminoethyl–sepharose chromatography. The estimated molecular mass (SDS-PAGE) and K_m of the enzyme were ∼55.0 kDa and 151 μM, respectively. The purified cholesterol oxidase was immobilized on chitosan beads by glutaraldehyde cross-linking reaction and immobilization was confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. The optimum temperature (45 °C, 5 min) for activity of the enzyme was increased by 5 °C after immobilization. Both the free and immobilized cholesterol oxidases were found to be stable in many organic solvents except for acetone. Fe²⁺ and Pb²⁺ at 0.1 mM of each acted as inhibitors, while Ag⁺, Ca²⁺, Ni²⁺ and Zn²⁺ activated the enzyme at similar concentration. The biotransformation of cholesterol (3.75 mM) with the cholesterol oxidase immobilized beads (3.50 U) leads to ∼88% millimolar yield of cholestenone in a reaction time of 9 h at 25 °C. The immobilized enzyme retains ∼67% activity even after 12 successive batches of operation. The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone.     

Heterogeneity of glycogen synthesis upon refeeding following starvation.

1. Starvation of rats for 40 hr decreased the body weight, liver weight and blood glucose concentration. The hepatic and skeletal muscle glycogen concentrations were decreased by 95% (from 410 mumol/g tissue to 16 mumol/g tissue) and 55% (from 40 mumol/g tissue to 18.5 mumol/g tissue), respectively. 2. Fine structural analysis of glycogen purified from the liver and skeletal muscle of starved rats suggested that the glycogenolysis included a lysosomal component, in addition to the conventional phosphorolytic pathway. In support of this the hepatic acid alpha-glucosidase activity increased 1.8-fold following starvation. 3. Refeeding resulted in liver glycogen synthesis at a linear rate of 40 mumol/g tissue per hr over the first 13 hr of refeeding. The hepatic glycogen store were replenished by 8 hr of refeeding, but synthesis continued and the hepatic glycogen content peaked at 24 hr (approximately 670 mumol/g tissue). 4. Refeeding resulted in skeletal muscle glycogen synthesis at an initial rate of 40 mumol/g tissue per hr. The muscle glycogen store was replenished by 30 min of refeeding, but synthesis continued and the glycogen content peaked at 13 hr (approximately 50 mumol/g tissue). 5. Both liver and skeletal muscle glycogen synthesis were inhomogeneous with respect to molecular size; high molecular weight glycogen was initially synthesised at a faster rate than low molecular weight glycogen. These observations support suggestions that there is more than a single site of glycogen synthesis.     

Cholesterol-rich intracellular membranes: a precursor to the plasma membrane

The disposition of newly synthesized sterols in cultured human fibroblasts has been examined in this study. We began by demonstrating that cholesterol mass and exogenously added [3H]cholesterol both are markers for the plasma membrane, perhaps better than 5'-nucleotidase. Cells were incubated with radioactive acetate to label their endogenous sterols biosynthetically, treated with cholesterol oxidase to convert plasma membrane cholesterol to cholestenone, and then homogenized and spun to equilibrium on sucrose gradients. The density gradient profiles of the various organelles were monitored using these markers: plasma membrane, radioactive cholestenone; smooth endoplasmic reticulum, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase); and Golgi apparatus, galactosyltransferase. The buoyant density profiles of radioactive intracellular cholesterol and lanosterol both had a peak at 1.12 g/cm3, similar to 5'-nucleotidase and galactosyltransferase but not to HMG-CoA reductase. This result suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. Digitonin treatment shifted the profiles of both plasma membrane and intracellular cholesterol to higher densities. Pretreatment of intact cells with cholesterol oxidase abolished the digitonin shift of plasma membranes but not the intracellular cholesterol, indicating that these two membrane pools are not entirely physically associated. Because intracellular cholesterol was shifted more than any of the organelle markers, it must reside in a separate membrane. Since digitonin selectively shifts the density of membranes rich in cholesterol, we infer that newly synthesized cholesterol accumulates in such membranes prior to its delivery to the plasma membrane. Taken together, these results suggest that cholesterol may be concentrated for delivery to the plasma membrane by being synthesized from a sterol precursor such as lanosterol in a discrete but undefined intracellular membrane.     

Photosynthetic electron transport in isolated maize bundle sheath cells

Fragments of bundle sheath strands, free of mesophyll cells and showing a chlorophyll a/b ratio of 6.0 to 6.6 were prepared from Zea mays by a mechanical method. They were unable to photoreduce ferricyanide but were able to photoreduce the membrane-permeant 2,5-dimethylquinone at a rate of 250 to 420 microequivalents per hour per mg chlorophyll (μeq/hr · mg Chl) at 21 C. In the presence of the catalase inhibitor KCN, methylviologen catalyzed a Mehler reaction at a rate of 120 to 180 μeq/hr · mg Chl. This was increased to 200 to 350 μeq/hr · mg Chl when the uncoupler methylamine was added. The rate of endogenous pseudocyclic electron flow, detected as a Mehler reaction, was also considerable (100 to 150 μeq/hr · mg Chl with methylamine). Diaminodurene supported a high rate of photosystem I-mediated electron flow to methylviologen (400 to 750 μeq/hr · mg Chl).     

Membrane cholesterol oxidation inhibits ligand binding function of hippocampal serotonin(1A) receptors

We have monitored the ligand binding function of the bovine hippocampal 5-HT(1A) receptor following treatment of native membranes with cholesterol oxidase. Cholesterol oxidase is a water soluble enzyme that acts on the membrane interface to catalyze the conversion of cholesterol to cholestenone. Oxidation of membrane cholesterol significantly inhibits the specific binding of the agonist and antagonist to 5-HT(1A) receptors. Fluorescence polarization measurements of membrane probes incorporated at different locations in the membrane revealed no appreciable effect on membrane order due to the oxidation of cholesterol to cholestenone. These results therefore suggest that the ligand binding function of the 5-HT(1A) receptor is a cholesterol-dependent phenomenon that is not related to the ability of cholesterol to modulate membrane order. Importantly, these results represent the first report on the effect of a cholesterol-modifying agent on the ligand binding function of this important neurotransmitter receptor.