Reconstitution of rabbit thrombomodulin into phospholipid vesicles

The influence of phospholipid on thrombin-thrombomodulin-catalyzed activation of protein C has been studied by incorporating thrombomodulin into vesicles by dialysis from octyl glucoside-phospholipid mixtures. Thrombomodulin was incorporated into vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (30% phosphatidylserine and 70% phosphatidylcholine). Thrombomodulin is randomly oriented in vesicles of different phospholipid composition. Incorporation of thrombomodulin into phosphatidylcholine, with or without phosphatidylserine, alters the Ca2+ concentration dependence of protein C activation. Soluble thrombomodulin showed a half-maximal rate of activation at 580 microM Ca2+, whereas half-maximal rates of activation of liposome-reconstituted thrombomodulin were obtained between 500 microM Ca2+ and 2 mM Ca2+, depending on the composition (protein:phospholipid) of the liposomes. The Ca2+ dependence of protein C activation fits a simple hyperbola for the soluble activator, while the Ca2+ dependence of the membrane-associated complex is distinctly sigmoidal with a Hill coefficient greater than 2.4. In contrast, the Ca2+ dependence of gamma-carboxyglutamic acid (Gla) domainless protein C activation is unchanged by membrane reconstitution (1/2 max = 53 +/- 10 microM) and fits a simple rectangular hyperbola. Incorporation of thrombomodulin into pure phosphatidylcholine vesicles reduces the Km for protein C from 7.6 +/- 2 to 0.7 +/- 0.2 microM. Increasing phosphatidylserine to 20% decreased the Km for protein C further to 0.1 +/- 0.02 microM. Membrane incorporation has no influence on the activation of protein C from which the Gla residues are removed proteolytically (Km = 6.4 +/- 0.5 microM). The Km for protein C observed on endothelial cells is more similar to the Km observed when thrombomodulin (TM) is incorporated into pure phosphatidylcholine vesicles than into negatively charged vesicles, suggesting that the protein C-binding site on endothelial cells does not involve negatively charged phospholipids. In support of this concept, we observed that prothrombin and fragment 1, which bind to negatively charged phospholipids, do not inhibit protein C activation on endothelial cells or TM incorporated into phosphatidylcholine vesicles, but do inhibit when TM is incorporated into phosphatidylcholine:phosphatidylserine vesicles. These studies suggest that neutral phospholipids lead to exposure of a site, probably on thrombomodulin, capable of recognizing the Gla domain of protein C.     

In situ conversion of coproporphyrinogen to heme by murine mitochondria: terminal steps of the heme biosynthetic pathway.

Coproporphyrinogen oxidase (EC 1.3.3.3), protoporphyrinogen oxidase (EC 1.3.3.4), and ferrochelatase (EC 4.99.1.1) catalyze the terminal three steps of the heme biosynthetic pathway. All three are either bound to or associated with the inner mitochondrial membrane in higher eukaryotic cells. A current model proposes that these three enzymes may participate in some form of multienzyme complex with attendant substrate channeling (Grand-champ, B., Phung, N., & Nordmann, Y., 1978, Biochem. J. 176, 97-102; Ferreira, G.C., et al., 1988, J. Biol. Chem. 263, 3835-3839). In the present study we have examined this question in isolated mouse mitochondria using two experimental approaches: one that samples substrate and product levels during a timed incubation, and a second that follows dilution of radiolabeled substrate by pathway intermediates. When isolated mouse mitochondria are incubated with coproporphyrinogen alone there is an accumulation of free protoporphyrin. When Zn is added as a substrate for the terminal enzyme, ferrochelatase, along with coproporphyrinogen, there is formation of Zn protoporphyrin with little accumulation of free protoporphyrin. When EDTA is added to this incubation mixture with Zn, Zn protoporphyrin formation is eliminated and protoporphyrin is formed. We have examined the fate of radiolabeled substrates in vitro to determine if exogenously supplied pathway intermediates can compete with the endogenously produced compounds. The data demonstrate that while coproporphyrinogen is efficiently converted to heme in vitro when the pathway is operating below maximal capacity, exogenous protoporphyrinogen can compete with endogenously formed protoporphyrinogen in heme production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of the solubilized insulin receptor in phospholipid vesicles.

The insulin receptor was solubilized from turkey erythrocyte membranes by extraction with 1% beta-octylglucopyranoside. Insulin binding was enhanced when the solubilized material was reconstituted in phospholipid vesicles. The affinity of the reconstituted vesicles for various insulins was similar to that of the intact membranes: porcine insulin greater than proinsulin greater than desoctapeptide insulin. A curvilinear Scatchard plot was obtained for insulin binding to the reconstituted system at 15 degrees C. A high affinity association constant of 1.4 x 10(9) M-1 was obtained from the Scatchard plot. This is a four-fold increase over the value for the turkey erythrocyte membrane, which contains more highly saturated phospholipids. This suggests that the insulin receptor may be sensitive to the lipid composition of the membranes in which it is embedded.     

Reconstitution of the Rous sarcoma virus transforming protein pp60v-src into phospholipid vesicles.

An artificial membrane system was developed to study the molecular basis for interaction of pp60v-src, the Rous sarcoma virus transforming protein, with lipid bilayers. pp60v-src was extracted from cell membranes by detergent solubilization and reincorporated into phospholipid vesicles. Reconstituted pp60v-src retained tyrosine kinase activity and was integrally associated with the liposome through a 10-kilodalton (kDa) amino-terminal domain. The same 10-kDa domain was shown to anchor pp60v-src to the plasma membrane of transformed cells. Reconstitution experiments performed with nonmyristylated pp60v-src proteins revealed that these polypeptides did not interact with phospholipid vesicles. In contrast, myristylated, soluble pp60v-src molecules (including a highly purified pp60v-src preparation) could be reconstituted into liposomes, but their interaction with the liposomal bilayer was not mediated by the 10-kDa amino-terminal domain. When membrane proteins were included during reconstitution of purified pp60v-src, binding through the 10-kDa anchor was restored. A model is presented to accommodate the different types of interactions of pp60v-src with liposomes; the model postulates the existence of an additional membrane component that anchors the pp60v-src polypeptide to the phospholipid bilayer.     

The heme biosynthetic pathway in the regenerating rat liver. The relation between enzymes of heme synthesis and growth

Enzymes of heme synthesis, porphyrins and heme content of regenerating rat livers were examined. During the first three days of regeneration the weights of livers of one-third and two-third hepatectomized rats increased 1.5-fold and 2.7-fold and the activity of porphobilinogen deaminase increased 2-fold and 4-fold and was inversely correlated with ferrochelatase activity. delta-Aminolevulinic acid synthase and delta-aminolevulinic acid dehydratase activities were reduced. Concomitantly an increase in the concentration of porphyrins and a decrease in that of heme were observed. The changes in the biosynthetic pathway of heme during rapid growth of the liver are discussed.     

Reconstitution of Saccharomyces cerevisiae phosphatidylserine synthase into phospholipid vesicles. Modulation of activity by phospholipids

Membrane-associated phosphatidylserine synthase was purified from Saccharomyces cerevisiae (Bae-Lee, M., and Carman, G. M. (1984) J. Biol. Chem. 259, 10857-10862) and reconstituted into phospholipid vesicles containing phosphatidylcholine/phosphatidylethanolamine/ phosphatidylinositol/phosphatidylserine. Reconstitution was performed by removing detergent from an octyl glucoside/phospholipid/Triton X-100/enzyme mixed micelle by Sephadex G-50 super-fine chromatography. The average diameter of the vesicles was 90 nm, and the enzyme was reconstituted asymmetrically with the active site facing outward. The enzymological properties of reconstituted phosphatidylserine synthase were determined in the absence of detergent. The enzyme was reconstituted into vesicles with phospholipid compositions approximating those of wild type and mutant strains of S. cerevisiae. Reconstituted activity was modulated by the phosphatidylinositol/phosphatidylserine ratio in the vesicles. The modulation of activity observed in the vesicles is enough to account for some of the fluctuations in the phosphatidylserine content in vivo.     

The influence of carbamazepine on the heme biosynthetic pathway

Carbamazepine, a drug which is widely used in neurological diseases, has a porphyrogenic effect in chick embryo liver cells in culture. It increased the concentration of cellular porphyrins by 80-fold and delta-aminolevulinate synthase activity by 4-fold. The increase in the accumulation of porphyrins preceded that of ALAS activity. Measurements of the activities of aminolevulinate dehydrase, porphobilinogen deaminase, and uroporphyrinogen decarboxylase showed that C inhibits UROD up to nearly 50% and PBGD activity up to 20%, but does not affect the activity of ALAD. The pattern of accumulation of porphyrins, mainly uro- and heptacarboxylporphyrin, is compatible with an inhibition of UROD. We may, therefore, conclude that the porphyrogenic effect of C in monolayers of chick embryo liver cells is the result of its inhibitory effect on the activity of UROD.     

Reconstitution of bacteriorhodopsin into cyclic lipid vesicles

Bacteriorhodopsin (bR), a membrane protein that can generate a light-driven proton pump, was successfully reconstituted into vesicles composed of an artificial cyclic lipid that mimics archaeal membrane lipids. Unlike reconstituted bR in 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles, the net topology and structure of bR molecules in cyclic lipid vesicles are identical to those in the native purple membrane of Halobacterium salinarum.     

Reconstitution of monomeric cytochrome c oxidase into phospholipid vesicles yields functionally interacting cytochrome aa3 units

When the carbon monoxide complex of fully reduced cytochrome c oxidase, reconstituted into liposomes, is mixed with oxygen-containing buffer, complex kinetic progress curves are observed. This pattern is seen irrespective of whether the oxidase used in reconstitution is the dimeric or monomeric (subunit III-depleted) enzyme. These findings are interpreted in the light of similar experiments on the detergent-solubilized enzyme reported by Gibson and Greenwood (Gibson, Q.H., and Greenwood, C. (1963) Biochem. J. 86, 541-554) and confirmed by ourselves. We conclude that reconstitution of monomeric (subunit III-less) enzyme yields, preferentially, vesicles containing more than one functional unit, possibly associated as dimers. This result is of significance to our understanding of the relationships between aggregation state and proton pumping capacity of cytochrome oxidase.     

Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana

The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g(-1) FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.     

Localization of two enzymes of the tetrahydrobiopterin biosynthetic pathway in embryonic chick retina.

Tetrahydrobiopterin (BH4) is an essential co-factor for the biosynthesis of catecholamine-type neurotransmitters and of nitric oxide (NO). The expression of the enzymes catalyzing the first two steps of the BH4 biosynthetic pathway was studied in the developing chicken retina by in situ hybridization and immunocytochemistry. GTP-cyclohydrolase-I (GTP-CH-I) and 6-pyruvoyl-tetrahydropterin synthase (PTPS) were already expressed in the undifferentiated and proliferating retina of E7. At stage E11 both enzymes were expressed in photoreceptors, amacrine cells, displaced amacrine cells, and ganglion cells, and in the plexiform layers in which synaptic connections take place. At stage E18 the labeling was comparable to E11 but appeared to be more concentrated in photoreceptors and ganglion cells.     

Enzymatic detection of phospholipid biosynthetic enzymes after electroblotting.

The membrane-associated enzymes phosphatidylinositol synthase (CDPdiacylglycerol:myo-inositol 3-phosphatidyltransferase; EC 2.7.8.11) and phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase; EC 2.7.8.8) from Saccharomyces cerevisiae were detected enzymatically after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting. Enzyme activities were measured on nitrocellulose blots by using pure enzyme preparations as well as Triton X-100-solubilized membranes. Phosphatidylinositol synthase activity migrated to Mr 34,000, and phosphatidylserine synthase activity migrated to Mr 23,000.     

Effect of phospholipid vesicles on the activity of terminal transferase.

The terminal transferase activity is modified in the presence of lipid vesicles. A deep inhibitory effect takes place with phosphatidylserine and phosphatidylinositol, while some stimulation is present with sphingomyelin and almost no effect has been detected with phosphatidylethanolamine vesicles. These effects seem to be related to the charge properties of the lipid membranes.A possible involvement of phospholipids in the mechanism of action of the terminal transferase is suggested.     

Reconstitution of the peptidoglycan cytoplasmic precursor biosynthetic pathway in cell-free system and rapid screening of antisense oligonucleotides for Mur enzymes

Bacterial peptidoglycan is the cell wall component responsible for various biological activities. Its cytoplasmic precursor UDP-N-acetylmuramyl pentapeptide is biosynthesized by the first six enzymes of peptidoglycan synthetic pathways (Mur enzymes), which are all proved to be important targets for antibiotic screening. In our present work, the genes encoding Mur enzymes from Escherichia coli were co-expressed in the cell-free protein synthesis (CFPS) system, and the activities of Mur enzymes derived from CFPS system were validated by the synthesis of the final product UDP-N-acetylmuramyl pentapeptide. Then this in vitro reconstituted Mur biosynthetic pathway was used to screen a panel of specific antisense oligonucleotides for MurA and MurB. The selected oligonucleotides were proved to eliminate the expression of Mur enzymes, and thus inhibit the Mur biosynthetic pathway. The present work not only developed a rapid method to reconstruct and regulate a biosynthetic pathway in vitro, but also may provide insight into the development of novel antibiotics targeting on peptidoglycan biosynthetic pathway.     

Reconstitution of the Turkey erythrocyte adenylate cyclase sensitivity to l-epinephrine upon re-insertion of the Lubrol solubilized components into phospholipid vesicles

Turkey erythrocyte adenylate cyclase was activated by GppNHp and l-epinephrine to its stable, highly active form. In this form the enzyme could be solubilized by Lubrol-PX and subsequently re-inserted into phospholipid vesicles concomitantly with the removal of up to 99.3% of the Lubrol. The ability of GTP and l-epinephrine to reverse the GppNHp/epinephrine activated state was taken as a measure for the reappearance of hormone sensitivity in the reconstituted vesicles. An incomplete but significant reappearance of hormone sensitivity in the reconstituted adenylate cyclase was achieved. This hormone sensitivity was found to be stereospecific for (?)epinephrine. The ¹²⁵I-cyanopindolol binding properties of the reconstituted β-receptor depend on the nature of the detergent and the phospholipids used in the reconstitution.     

Reconstitution in phospholipid vesicles of calcium-activated potassium channel from outer renal medulla

Summary A barium-sensitive Ca-activated K⁺ channel in the luminal membrane of the tubule cells in thick ascending limb of Henle's loop is required for maintenance of the lumen positive transepithelial potential and may be important for regulation of NaCl reabsorption. In this paper we examine if the K⁺ channel can be solubilized and reconstituted into phospholipid vesicles with preservation of its native properties. The K⁺ channel in luminal plasma membrane vesicles can be quantitatively solubilized in CHAPS at a detergent/protein ratio of 3. For reconstitution, detergent is removed by passage over a column of Sephadex G 50 (coarse). K⁺-channel activity is assayed by measurement of⁸⁶Rb⁺ uptake against a large opposing K⁺ gradient. The reconstituted K⁺ channel is activated by Ca²⁺ in the physiological range of concentration (K_1/22×10^–7 m at pH 7.2) as found for the K⁺ channel in native plasma membrane vesicles and shows the same sensitivity to inhibitors (Ba²⁺, trifluoperazine, calmidazolium, quinidine) and to protons. Reconstitution of the K⁺ channel into phospholipid vesicles with full preservation of its native properties is an essential step towards isolation and purification of the K⁺-channel protein.Titration with Ca²⁺ shows that most of the active K⁺ channels in reconstituted vesicles have their cytoplasmic aspect facing outward in contrast to the orientation in plasma membrane vesicles, which requires also addition of Ca²⁺ ionophore in order to observe Ca²⁺ stimulation. The reconstituted K⁺ channel is highly sensitive to tryptic digestion. Brief digestion leads to activation of the K⁺ channel in absence of Ca²⁺, to the level of activity seen with saturating concentrations of Ca²⁺. This tryptic split is located in a cytoplasmic aspect of the K⁺ channel that appears to be involved in opening and closing the K⁺ channel in response to Ca²⁺ binding.     

Reconstitution of cytochrome c oxidase in phospholipid vesicles containing polyvinylic polymers.

Cytochrome c oxidase was reconstituted in phospholipid vesicles in the presence of highly hydrophobic poly(vinyl alkanoate) polymers. Electron-microscopy observations demonstrated that polymer interaction with the lipid phase induces vesicles to adopt smaller diameters than those typical of standard proteoliposomes. Functional characterization of these polymer-proteoliposome structures indicates that the reconstitution of the enzyme proceeds efficiently without causing either scrambling of the protein orientation in the membrane or loss of respiratory control. A clear dependence of respiratory control ratio on vesicle size was also demonstrated, which is in agreement with a previous model proposed for control of activity of cytochrome c oxidase vesicles [Brunori, Sarti, Colosimo, Antonini, Malatesta, Jones & Wilson (1985) EMBO J. 4, 2365-2368].     

Enzymes of the heme biosynthetic pathway in the nonphotosynthetic alga Polytomella sp

Heme biosynthesis involves a number of enzymatic steps which in eukaryotes take place in different cell compartments. Enzyme compartmentalization differs between photosynthetic and nonphotosynthetic eukaryotes. Here we investigated the structures and subcellular localizations of three enzymes involved in the heme pathway in Polytomella sp., a colorless alga evolutionarily related to the green alga Chlamydomonas reinhardtii. Functional complementation of Escherichia coli mutant strains was used to isolate cDNAs encoding three heme biosynthetic enzymes, glutamate-1-semialdehyde aminotransferase, protoporphyrinogen IX oxidase, and ferrochelatase. All three proteins show highest similarity to their counterparts in photosynthetic organisms, including C. reinhardtii. All three proteins have N-terminal extensions suggestive of intracellular targeting, and immunoblot studies indicate their enrichment in a dense cell fraction that is enriched in amyloplasts. These results suggest that even though the plastids of Polytomella sp. are not photosynthetically active, they are the major site of heme biosynthesis. The presence of a gene for glutamate-1-semialdehyde aminotransferase suggests that Polytomella sp. uses the five-carbon pathway for synthesis of the heme precursor 5-aminolevulinic acid.     

Preparation of inside-out vesicles from erythrocyte membranes inactivates the pathway for oleic acid incorporation into phospholipid

The pathway for membrane phospholipid fatty acid turnover in situ may be important in the regulation of the composition and turnover of the lipid microenvironment of membrane proteins. This pathway has been characterized further by studying the activation and incorporation of [9,10(n)-3H]oleic acid and transesterification of [1-14C]oleoyl-CoA into membrane phospholipids by isolated erythrocyte membrane ghosts and inside-out vesicles derived from these ghosts. Erythrocyte ghosts and sealed vesicles of defined orientation prepared from them have been widely employed in studies of the function of membrane proteins, particularly those which mediate the transport of ions and sugars. Preparation of inside-out vesicles from ghosts by exposure to alkaline hypotonic conditions results in elution of some membrane proteins but no loss of membrane phospholipid. Compared to ghosts, the ability of inside-out vesicles to activate and incorporate [9,10(n)-3H]oleic acid into phospholipid is diminished by over 90% and the ability of inside-out vesicles to transesterify [1-14C]oleoyl-CoA to phospholipid is diminished by over 50%. These findings indicate that exposure of erythrocyte membranes to the alkaline hypotonic conditions required for inside-out vesicle preparation results in loss or inactivation of both acyl-CoA ligase and acyl-CoA-lysophospholipid acyltransferase activities. This lability of the enzymes for in situ phospholipid fatty acid turnover should be considered in the design and interpretation of studies concerned with elucidation of the relationship between phospholipid fatty acid turnover and the regulation of membrane protein function in this membrane preparation.     

Reconstitution of (Na+ + K+)-ATPase into phospholipid vesicles with full recovery of its specific activity

(Na+ + K+)-ATPase from rectal glands of the spiny dogfish has been reconstituted into phospholipid vesicles. The nonionic detergent octaethyleneglycoldodecyl monoether ( C12E8 ) is used to dissolve both the enzyme and the lipids and reconstitution is accomplished by subsequent removal of the detergent by adsorption to polystyrene beads. About 60% of the enzyme incorporates in the right-side-out orientation (r/o). The fraction of molecules in the inside-out orientation (i/o) increases from about 10% to about 30% with a parallel decrease in the fraction of 'non-oriented' (n-o) molecules (both sides exposed) when the protein/lipid ratio decreases from 1:10 to 1:75. The orientation of enzyme molecules detected from vanadate binding is the same as measured from activity, i.e., the turnover of the enzyme molecule in the different orientations is the same. The recovery of the specific activity of the incorporated enzyme increases with an increase in the protein/lipid ratio and is 100% with a protein/lipid ratio of about 1:20 or higher. Full recovery is only obtained provided a proper lipid composition is chosen which includes both negatively charged phospholipids, preferably phosphatidylinositol, and cholesterol. The ATP-dependent, K+-stimulated Na+-influx is found to be about 35 mumol Na+ per mg (i/o)-protein per min at 22 degrees C in 1:10 protein/lipid liposomes. The specific activity corresponds to 3 Na+ transported per ATP molecule hydrolyzed.