Subcellular localization of a cytochrome P-450-dependent monogenase in vesicles of the higher plant Catharanthus roseus

The intracellular location of a cytochrome P-450-dependent monoterpene hydroxylase from the higher plant, Catharanthus roseus, has been investigated. By differential and sucrose density gradient centrifugation, utilizing marker enzymes and electron microscopy, the monooxygenase was demonstrated to be associated with vesicles having a membrane thickness of 40-60 nm. The vesicles could be distinguished from endoplasmic reticulum, Golgi apparatus, mitochondria, and plasma membrane and were found in light membrane fractions containing provacuoles. Most definitive results were obtained when seedlings were ground in the presence of sand and in a medium containing sorbitol. Upon subjection of the 20,000-g pellet preparation to linear sucrose density gradient centrifugation, a threefold enrichment in hydroxylase activity was afforded in a yellow band having vesicles varying in size from 0.1 to 0.8 mum in diam and having a density of 1.09 to 1.10 g/cm3. Since the monooxygenase has been implicated in indole alkaloid biosynthesis in this plant, the data suggest the compartmentalization of at least a part of this pathway.     

Isolation and partial characterization of a lectin from a false brome grass (Brachypodium sylvaticum).

Purified rat renal brush-border membrane vesicles possess a heat-labile enzyme activity which hydrolyses NAD+. A reciprocal relationship exists between the disappearance of NAD+ and the appearance of adenosine; 2 mol of Pi are liberated from each mol of NAD+ incubated with brush-border membrane vesicles. Freezing and thawing brush-border membrane vesicles does not enhance the initial rate of NAD+ hydrolysis. Preincubation of brush-border membrane vesicles with NAD+ results in inhibition of Na+-dependent Pi-transport activity, whereas Na+-dependent glucose transport is not affected. EDTA, which prevents the release of Pi from NAD+ and which itself has no direct effect on brush-border membrane Pi transport, reverses the NAD+ inhibition of Na+-dependent Pi transport. These results suggest that it is the Pi liberated from NAD+ and not NAD+ itself that inhibits Na+-dependent Pi transport.     

Genesis of Drosophila ADH: the shaping of the enzymatic activity from a SDR ancestor

Drosophila alcohol dehydrogenase (ADH) is an NAD(H)-dependent oxidoreductase that catalyzes the oxidation of alcohols and aldehydes. Structurally and biochemically distinct from all the reported ADHs (typically, the mammalian medium-chain dehydrogenase/reductase-ethanol-metabolizing enzyme), it stands as the only small-alcohol transforming system that has originated from a short-chain dehydrogenase/reductase (SDR) ancestor. The crystal structures of the apo, binary (E.NAD(+)) and three ternary (E.NAD(+).acetone, E.NAD(+).3-pentanone and E.NAD(+).cyclohexanone) forms of Drosophila lebanonensis ADH have allowed us to infer the structural and kinetic features accounting for the generation of the ADH activity within the SDR lineage.     

Isolation of envelope membranes from bundle sheath chloroplasts of maize

Bundle sheath strands were isolated from maize (Zea mays L.) leaves treated with preparations of cellulase, hemicellulase, and pectinase. A three-phase discontinuous gradient yielded two fractions of envelope membranes from bundle sheath chloroplasts. Buoyant densities were 1.06 and 1.09 g cm⁻³. The lighter fraction contained membrane vesicles under light microscopy, but centrifugation produced a pellet that was too small and unstable for purposes of electron microscopy. The heavier fraction contained single and double membrane vesicles and was studied further. Enzymic, chemical, light microscopic, and electron microscopic examination showed less than 2% contamination by stromal contents, no contamination by microbial, microsomal, or mitochondrial membranes, and possible low levels of lamellar membrane contamination. Yields of 0.5 mg of envelope membrane protein were obtained from 56-g leaf sections. The Mg²⁺-dependent nonlatent ATPase activity, a marker enzyme for chloroplast envelope membranes, was 40 μmoles Pi released hr⁻¹ mg protein⁻¹, a value similar to that obtained with pure mesophyll chloroplast envelope membranes from other plants.     

Role of nonacidic endosomes in recycling of ADH-sensitive water channel structures.

Toad urinary bladder epithelial cells respond to the hormone ADH by increasing the water permeability of their luminal membrane. This action is mediated by insertion into the apical membrane of specific water channels. In the absence of ADH these channels appear to be present in tubular cytoplasmic vesicles as morphologically distinctive intramembrane structures called particle aggregates. ADH induces these vesicles to fuse with the apical membrane, transferring their aggregate-water channels into the apical membrane. When ADH stimulation is removed (ADH reversal), aggregates and fluid-phase markers from the mucosal bath appear in water-permeable vesicles in the cytoplasm. We have examined the fate of fluid-phase markers and aggregates with time after ADH reversal. Although the fluid-phase markers horseradish peroxidase and colloidal gold are initially found predominantly in tubular vesicles near the apical surface, by 30 min the markers were found in perinuclear multivesicular bodies (MVBs) of heterogeneous size and shape. These MVBs appear to be nonacidic since they fail to accumulate DAMP. Acid phosphatase (AcPase) was undetectable in these structures. After 60 min, labeled MVBs tended to be smaller, and some of these structures displayed DAMP accumulation and AcPase activity. By evaluation of uncleaned replicas it was possible to localize recycled aggregate-water channels with respect to internalized fluid-phase markers. Thirty minutes after retrieval from the apical surface in tubular vesicles, aggregates could be localized to both the central body and tubular projections of labeled MVBs. At 60 min following reversal, most MVBs had a reduced number of aggregates compared with 30 min, and compact structures could be identified that contained markers but no detectable aggregates. These observations show that aggregates and fluid-phase markers enter a nonacidic endosomal compartment with an MVB morphology following ADH reversal. At extended times following reversal, labeled MVBS having lysosomal characteristics and labeled MVBs having no detectable aggregates can be found, suggesting that aggregates are sorted or degraded prior to this stage.     

Isolation of plasma membrane vesicles, derived from transverse tubules, by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media.

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. (Na+ + K+)-stimulated, Mg2+-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca2+-stimulated, Mg2+-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell segments in an amber low-speed (800 x g) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at 100,000 x g for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of (Na+ + K+)-stimulated ATPase compared with the other fractions, but it had essentially no Ca2+-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate. Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined bu a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basememt membrane, could not be detected in this fraction.     

Membrane-bound redox proteins of the murine Friend virus-induced erythroleukemia cell

We have obtained and studied a 105,000-g pellet from T-3-Cl-2 cells, a cloned line of Friend virus-induced erythroleukemia cells. By difference spectrophotometry, the pellet was shown to contain cytochrome b5 and cytochrome P-450, hemeproteins that have been shown to participate in electron-transport reactions of endoplasmic reticulum and other membranous fractions of various tissues. The pellet also possesses NADH-cytochrome c reductase activity which is inhibited by anti-cytochrome b5 gamma-globulin, indicating the presence of cytochrome b5 reductase. This is the first demonstration of membrane-bound forms of these redox proteins in erythroid cells. Dimethyl sulfoxide-treated T-3-Cl-2 cells were also shown to possess membrane-bound cytochrome b5 and NADH-cytochrome c reductase activity. We failed to detect soluble cytochrome b5 in the 105,000-g supernatant fraction from homogenates of untreated or dimethyl sulfoxide-treated T-3-Cl-2 cells. In contrast, erythrocytes obtained from mouse blood were shown to possess soluble cytochrome b5 but no membrane-bound form of this protein. These findings are supportive of our hypothesis that soluble cytochrome b5 of erythrocytes is derived from endoplasmic reticulum or some other membrane structure of immature erythroid cells during cell maturation.     

Solubilization of proteins from bovine brain coated vesicles by protein perturbants and Triton X-100

To identify integral and peripheral membrane proteins, highly purified coated vesicles from bovine brain were exposed to solutions of various pH, ionic strength, and concentrations of the nonionic detergent Triton X-100. At pH 10.0 or above most major proteins were liberated, but four minor polypeptides sedimented with the vesicles. From quantitative analysis of phospholipids in the pellet and extract, we determined that at a pH of up to 12 all phospholipids could be recovered in the pellet. Electron microscopic examination of coated vesicles at pH 12.0 showed all vesicles devoid of coat structures. Treatment with high ionic strength solutions (0-1.0 M KCl) at pH 6.5-8.5 also liberated all major proteins, except tubulin, which remained sedimentable. The addition of Triton X-100 to coated vesicles or to stripped vesicles from which 90% of the clathrin had been removed resulted in the release of four distinct polypeptides of approximate Mr 38,000, 29,000, 24,000 and 10,000. The 38,000-D polypeptide (pK approximately 5.0), which represents approximately 50% of the protein liberated by Triton X-100, appears to be a glycoprotein on the basis of its reaction with periodic acid-Schiff reagent. Extraction of 90% of the clathrin followed by extraction of 90% of the phospholipids with Triton X-100 produced a protein residue that remained sedimentable and consisted of structures that appeared to be shrunken stripped vesicles. Together our data indicate that most of the major polypeptides of brain coated vesicles behave as peripheral membrane proteins and at least four polypeptides behave as integral membrane proteins. By use of a monoclonal antibody, we have identified one of these polypeptides (38,000 mol wt) as a marker for a subpopulation of calf brain coated vesicles.     

Crystal structure of the vertebrate NADP(H)-dependent alcohol dehydrogenase (ADH8)

The amphibian enzyme ADH8, previously named class IV-like, is the only known vertebrate alcohol dehydrogenase (ADH) with specificity towards NADP(H). The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP(+) have been now determined and refined to resolutions of 2.2A and 1.8A, respectively. The coenzyme and substrate specificity of ADH8, that has 50-65% sequence identity with vertebrate NAD(H)-dependent ADHs, suggest a role in aldehyde reduction probably as a retinal reductase. The large volume of the substrate-binding pocket can explain both the high catalytic efficiency of ADH8 with retinoids and the high K(m) value for ethanol. Preference of NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225 and the recruitment of conserved Lys228, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds to ADH8 in an extended conformation that superimposes well with the NAD(H) molecules found in NAD(H)-dependent ADH complexes. No additional reshaping of the dinucleotide-binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. The structural features support the classification of ADH8 as an independent ADH class.     

Solubilization of the ileal receptor for intrinsic factor--vitamin B-12 complex by digestion with papain.

Brush-border-membrane vesicles isolated from hamster ileum were incubated with either papain or Pronase P and subsequently centrifuged to obtain soluble (supernatant) and insoluble (pellet) fractions. Papain (4 units/ml) solubilized 95--100% of the sucrase and leucine naphthylamide-hydrolysing activities but only 30% of the alkaline phosphatase. Digestion with papain also resulted in the solubilization of more than 75% of the ileal receptor for intrinsic factor-vitamin B-12 complex with a corresponding decrease in receptor activity in the pellet. Essentially 100% of the receptor activity was recovered. In contrast, digestion with Pronase P resulted in a decrease in total receptor activity. Papain-solubilized receptor was not sedimented by centrifugation at 105 000 g for 90 min and was eluted in the included volume of Sepharose 6B. Like the binding to more intact preparations, binding of intrinsic factor-vitamin B-12 complex to papain-solubilized receptor was rapid, reaching 50% of maximum in 8 min, and required Ca2+. Although Mg2+ could not completely substitute for Ca2+, Mg2+ did stimulate Ca2+-dependent binding at low Ca2+ concentrations. These results demonstrate that the ileal receptor for intrinsic factor-vitamin B-12 complex can be solubilized with papain, and suggest that papain solubilization may be a useful first step in the isolation and purification of this receptor.     

Alcohol dehydrogenase (ADH) in yeasts. II. NAD+-and NADP+-dependent alcohol dehydrogenases in Saccharomycopsis lipolytica.

In Sm. lipolytica one NAD+-dependent and three NADP+-dependent alcohol dehydrogenases are detectable by polyacrylamide gelelectrophoresis. The NAD+-dependent ADH (ADH I), with a molecular weight of 240,000 daltons, reacts more intensively with long-chain alcohols (octanol) than with short-chain alcohols (methanol, ethanol). The ADH I is not or only minimally subject to glucose repression. Besides the ADH I band no additional inducible NAD+-dependent ADH band is gel-electrophoretically detectable during growth of yeast cells in medium containing ethanol or paraffin. The ADH I band is very probably formed by two ADH enzymes with the same electrophoretic mobility. The NADP+-dependent alcohol dehydrogenases (ADH II--IV) react with methanol, ethanol and octanol with different intensity. In polyacrylamide gradients two bands of NADP+-dependent ADH are detectable: one with a molecular weight of 70,000 daltons and the other with 120,000 daltons. The occurrence of the three NADP+-dependent alcohol dehydrogenases is regulated by the carbon source of the medium. Sm. lipolytica shows a high tolerance against allylalcohol. Resistant mutants can be isolated only at concentrations of 1 M allylalcohol in the medium. All isolates of allylalcohol-resistant mutants show identical growth in medium containing ethanol as the wild type strain.     

NAD (P)H-Duroquinone Reductase in the Plant Plasma Membrane

The intracellular distribution of NADPH- and NADH-dependentduroquinone reductase (NAD (P)H-DQR) from etiolated zucchinihypocotyls (Cucurbita pepo L.) was investigated. About 80% ofthis enzyme is in the supernatant fraction and is probably cytosolic.Particulate NAD (P)H-DQR was largely (42%) found in associationwith the plasma membrane and was strongly stimulated by TX100.Another 33% of NAD (P)H-DQR was associated with mitochondria,and minor fractions with the endoplasmic reticulum (8%) andother particles. All these fractions were little or not stimulatedby TX100. The distribution of detergent-activated NAD (P)H-DQRis thus distinct from microsomal NADH- and NADPH-CCR. The plasma membrane was purified from microsomal fractions bymetrizamide plus sucrose density gradient centrifugation orby PEG/dextran phase partitioning. Both types of particle preparationspeaked at a density (d) of 1.165 g cm^–3 in sucrose gradientsand contained substantial TX100-sensitive NADH-DQR, TX100-stimulatedNAD (P)H-DQR, together with traces of NADH-CCR and trapped ‘soluble’enzyme (MDH, NADP-malic enzyme) activities. In isopycnic gradientsof unfractionated microsomes, however, trapped enzymes peakedat d 1.155 whereas NAD (P)H-DQR peaked at d 1.165 and GSII atd 1.170, probably revealing plasma membrane heterogeneity. Furtherevidence of heterogeneity was provided by fractionation of plasmamembrane vesicles on dextran step-gradients. Most of the trapped MDH was released to the supernatant by sonicationor treatment with 0.0125% TX100. Under these conditions mostof the NAD (P)H-DQR sedimented with the membranes. It is concludedthat NAD (P)H-DQR is bound to the inside of plasma membranevesicles, but a fraction (7 to 31%) may be ‘soluble’and sequestered within the vesicle lumen. Part of the detergent-sensitiveNADH-DQR may be externally bound and accessible to non-permeatingsubstrates. Key words: Cucurbita, NAD (P)H-quinone reductase, plasma membrane     

The intracellular location of adenylyl cyclase in the cellular slime molds Dictyostelium discoideum and Polysphondylium pallidum

Adenylyl cyclase activity was low or not detectable on intact cells and in isolated plasma membranes, phagocytic vacuoles and nuclei of the two slime mold species examined. The entire activity of homogenates was sedimentable and concentrated in a light membrane fraction. When this fraction was centrifugated through sucrose density gradients the adenylyl cyclase activity sedimented differently from all other enzymes measured. The gradient fractions with the highest specific activity of adenylyl cyclase consisted mainly of small vesicles. No changes in adenylyl cyclase distribution were associated with development. The possibility that cellular slime mold adenylyl cyclase activity is associated with vesicles in vivo, as already suggested by Maeda & Gerisch [10], is discussed.     

Amphibian intestinal brush border membranes-I. Isolation from Rana catesbeiana tadpole.

1. Intestinal brush border membrane vesicles have been isolated form Rana catesbeiana tadpole. 2. Electron microscopy of brush border membrane vesicles demonstrates a fairly homogenous preparation of vesicles, some of them still containing electron dense material. 3. The dense vesicles probably comprise both microvillus core and membrane. 4. Negative staining of vesicles reveals the presence of knob-like structures (particles) covering the outer surface of the membrane. 5. The membranous fraction is characterized by a high specific activity of alkaline phosphatase, trehalase, glucoamylase, maltase and gamma-glutamyltranspeptidase.     

Plasma membranes from Candida tropicalis grown on glucose or hexadecane. I. Isolation, identification and purification.

Plasma membranes from Candida tropicalis grown on glucose or hexadecane were isolated using a method based on the difference in surface charge of mitochondria and plasma membranes. After mechanical disruption of the cells, a fraction consisting of mitochondrial and plasma membrane vesicles was obtained by differential centrifugation. Subsequently the mitochondria were separated from the plasma membrane vesicles by aggregation of the mitochondria at a pH corresponding to their isoelectric point. Additional purification of the isolated plasma membrane vesicles was achieved by osmolysis. Surface charge densities of mitochondria and plasma membranes were determined and showed substrate-dependent differences. The isolated plasma membranes were morphologically characterized by electron microscopy and, as a marker enzyme, the activity of Mg2+-dependent ATPase was determine. By checking for three mitochondrial marker enzymes the plasma membrane fractions were estimated to be 94% pure with regard to mitochondrial contamination.     

Localization of phosphatases in Trypanosoma rhodesiense

Phosphatase activity in Trypanosoma rhodesiense has been examined histochemically by light and electron microscopy and by enzymatic assay in homogenate fractions. Using a method with lead as capture ion, acid phosphatase was found in lysosome-like vesicles and in the flagellar pocket. No alkaline adenosine triphosphatase (ATPase) was detectable by this method. Direct assay of p-nitrophenylphosphatase activity in homogenate fractions showed that acid phosphatase activity was strongly membrane-bound, but that activity at pH 9 was minimal in both soluble and particulate fractions. "Endogenous" ATPase activity was localized specifically and reproducibly in the mitochondrial membranes and under the plasma membrane of he flagellum. This nonenzymic reaction product could not be eradicated by glycerol extraction or glucose depletion. Unlike the membrane staining, which was manifest only after lead treatment, heat-resistant electron-dense material was found in the matrix of lysosomal vesicles in trypanosomes fixed in glutaraldehyde only and not subjected to further treatment with heavy metal reagents. X-ray emission analysis showed the presence of calcium and phosphorus, indicating that the matrix might have a phosphate storage function.     

An alcohol dehydrogenase ribozyme

We report an RNA molecule that exhibits activity analogous to that of alcohol dehydrogenase (ADH). Directed in vitro evolution was used to enrich nicotinamide adenine dinucleotide (NAD+)-dependent redox-active RNAs from a combinatorial pool. The most active ribozyme in the population forms a compact pseudoknotted structure and oxidizes an alcohol seven orders of magnitude faster than the estimated spontaneous rate. Moreover, this ADH RNA was coupled with a redox relay between NADH and flavin adenine dinucleotide to give a NAD+-regeneration system. Our demonstration of the redox ability of RNA adds support to an RNA-based metabolic system in ancient life.     

Role of aspartic acid 38 in the cofactor specificity of Drosophila alcohol dehydrogenase.

Drosophila alcohol dehydrogenase (ADH), an NAD(+)-dependent dehydrogenase, shares little sequence similarity with horse liver ADH. However, these two enzymes do have substantial similarity in their secondary structure at the NAD(+)-binding domain [Benyajati, C., Place, A. P., Powers, D. A. & Sofer, W. (1981) Proc. Natl Acad. Sci. USA 78, 2717-2721]. Asp38, a conserved residue between Drosophila and horse liver ADH, appears to interact with the hydroxyl groups of the ribose moiety in the AMP portion of NAD+. A secondary-structure comparison between the nucleotide-binding domain of NAD(+)-dependent enzymes and that of NADP(+)-dependent enzymes also suggests that Asp38 could play an important role in cofactor specificity. Mutating Asp38 of Drosophila ADH into Asn38 decreases Km(app)NADP 62-fold and increases kcat/Km(app)NADP 590-fold at pH 9.8, when compared with wild-type ADH. These results suggest that Asp38 is in the NAD(+)-binding domain and its substituent, Asn38, allows Drosophila ADH to use both NAD+ and NADP+ as its cofactor. The observations from the experiments of thermal denaturation and kinetic measurement with pH also confirm that the repulsion between the negative charges of Asp38 and 2'-phosphate of NADP+ is the major energy barrier for NADP+ to serve as a cofactor for Drosophila ADH.     

Studies on the heterogeneity of sarcoplasmic reticulum vesicles.

Isolated sarcoplasmic reticulum vesicles from rabbit white muscle were separated into a light (15--20% of total microsomes) and a heavy (80--85%) fraction by density gradient centifugation. The ultrastructure, chemical composition, enzymic activities and localization of membrane components in the vesicles of both fractions were investigated. From the following results it was concluded that both fractions are derived from the membranes of the sarcoplasmic reticulum system of the muscle: (i) The protein pattern of both fractions is essentially the same, except for different ratios of acidic, Ca2+-binding proteins. (ii) The 105000 dalton protein of the light fraction cross-reacts immunologically with the Ca2+-dependent ATPase of the heavy fraction. (iii) Ca2+-dependent ATPase, although of different specific activity, is found in both fractions. After rendering the vesicles leaky, specific activities in both fractions reach the same value. The light fraction was found to consist of "inside-out" vesicles by the following criteria: (i) No Ca2+ accumulation can be measured and the Ca2+-dependent ATPase activity is low and variable. (ii) The rate of trypsin digestion is lower and, compared to the heavy microsomes, a different ratio of degradation products is obtained. (iii) The sarcoplasmic reticulum membrane has a highly asymmetrical lipid distribution. This distribution of aminophospholipids is opposite to that in vesicles of heavy fraction. The light sarcoplasmic reticulum fraction has a higher phospholipid to protein ratio than the heavy one. This is consistent with the possibility that the two fractions derive from different parts of the sarcoplasmic reticulum system.     

Catecholamine-rich cells and varicosities in bovine splenic nerve, vesicle contents and evidence for exocytosis.

The bovine splenic nerve trunk contains mast cells, ganglion cells, small intensely fluorescent (SIF) cells, and varicosities which exhibit a brilliant fluorescence characteristic for noradrenaline (NA) and dopamine (DA) after formaldehyde exposure. All these catecholamine-rich structures could contribute particles to isolated nerve vesicle fractions. Mast cells are recognized ultrastructurally by their large (300-800 nm) dense granules. SIF cells may be represented by cells and processes containing dense cored vesicles (120-140 nm) which are larger than the typical vesicles in axons and terminals. Terminal-like areas with typical large dense cored vesicles (LDV, 75 nm) and small dense cored vesicles (SDV, 45-55 nm) probably correspond to the fluorescent varicosities. The LDV constitute about 40% of all vesicles in terminal-like areas and terminals. Their staining properties indicate the presence of protein, phospholipids, and ATP. Tyramine depletes NA without loss of matrix density. The LDV can fuse with the terminal membrane, and released material outside omega profiles is interpreted to depict exocytosis. Large and small vesicles are easily distinguished from the very large mast cell granules and the moderately dense Schwann cell vesicles. Neither appear to contaminate the LDV fractions but the latter may contain a small population of SIF cell vesicles. Golgi vesicles from the Schwann cells mainly occur in the lighter zones of the gradient.