Reconstitution of catecholamine-stimulated adenylate cyclase activity using three purified proteins

beta-Adrenergic receptors, the GTP-binding regulatory protein that stimulates adenylate cyclase (Gs), and adenylate cyclase were each purified and reconstituted into unilamellar vesicles composed of phosphatidylethanolamine and phosphatidylserine (3:2, w/w). The molar ratio of receptor:Gs:adenylate cyclase was estimated to be about 1:10:1. Adenylate cyclase activity in the vesicles was stimulated up to 2.6-fold by beta-adrenergic agonists. Stimulation was dependent on the presence of guanine nucleotide, displayed appropriate beta-adrenergic selectivity and stereoselectivity for agonists, and was blocked appropriately by beta-adrenergic antagonists. Therefore, while additional proteins may modulate adenylate cyclase activity in native membranes, these results show that these three proteins are sufficient for the expression of hormone-stimulated adenylate cyclase.     

tRNA and the guanosinetriphosphatase activity of elongation factor Tu

Different sites of the tRNA molecule influence the activity of the elongation factor Tu (EF-Tu) center for GTP hydrolysis [Parlato, G., Pizzano, R., Picone, D., Guesnet, J., Fasano, O., & Parmeggiani, A. (1983) J. Biol. Chem. 258, 995-1000]. Continuing these studies, we have investigated some aspects of (a) the effect of different tRNA(Phe) species, including Ac-Phe-tRNA(Phe) and 3'-truncated tRNA-CCA in the presence and absence of codon-anticodon interaction, and (b) the effect of occupation of the ribosomal P-site by different tRNA(Phe) species. Surprisingly, we have found that 3'-truncated tRNA can enhance the GTPase activity in the presence of poly(U), in contrast to its inhibitory effect in the absence of codon-anticodon interaction. Moreover, Ac-Phe-tRNA(Phe) was found to have some stimulatory effect on the ribosome EF-Tu GTPase in the presence of poly(U). These results indicate that under specific conditions the 3'-terminal end and a free terminal alpha-NH2 group are not essential for the stimulation of the catalytic center of EF-Tu; therefore, the same structure of the tRNA molecule can act as a stimulator or an inhibitor of EF-Tu functions, depending on the presence of codon-anticodon interaction and on the concentration of monovalent and divalent cations. EF-Tu-GTP does not recognize a free ribosomal P-site from a P-site occupied by the different tRNA(Phe) species. When EF-Tu acts as a component of the ternary complex formed with GTP and aa-tRNA, the presence of tRNA in the P-site strongly increases the GTPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of gamma-secretase activity

gamma-Secretase is a membrane protein complex with an unusual aspartyl protease activity that catalyses the regulated intramembranous cleavage of the beta-amyloid precursor protein (APP) to release the Alzheimer's disease (AD)-associated amyloid beta-peptide (Abeta) and the APP intracellular domain (AICD). Here we show the reconstitution of gamma-secretase activity in the yeast Saccharomyces cerevisiae, which lacks endogenous gamma-secretase activity. Reconstituted gamma-secretase activity depends on the presence of four complex components including presenilin (PS), nicastrin (Nct), APH-1 (refs 3-6) and PEN-2 (refs 4, 7), is associated with endoproteolysis of PS, and produces Abeta and AICD in vitro. Thus, the biological activity of gamma-secretase is reconstituted by the co-expression of human PS, Nct, APH-1 and PEN-2 in yeast.     

Reconstitution of catecholamine-stimulated binding of guanosine 5'-O-(3-thiotriphosphate) to the stimulatory GTP-binding protein of adenylate cyclase

The stimulatory GTP-binding protein (Gs) of adenylate cyclase, purified from rabbit liver, and beta-adrenergic receptors, partially purified 1000-4000-fold from turkey erythrocyte plasma membranes, were coreconstituted into unilamellar phospholipid vesicles. The molar ratio of Gs to receptors in the vesicles varied from 3 to 10 in different preparations, as measured by guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) binding to Gs and [125I]iodocyanopindolol binding to receptors. Activation of reconstituted Gs by GTP gamma S was stimulated up to 10-fold by the addition of the beta-adrenergic agonist (-)-isoproterenol. Activation was assayed functionally by reconstitution with the catalytic unit of adenylate cyclase. Because of the relative purity of this preparation, the quasi-irreversible binding of [35S]GTP gamma S could also be measured in the vesicles and was shown to parallel the functional activation of Gs under all conditions. Most of the assayable Gs in the vesicles could interact with the receptors and undergo agonist-stimulated activation. Agonist-stimulated activation and [35S]GTP gamma S binding were complete in less than 3 min, even under suboptimal conditions, and could go to completion in less than 20 s under maximal stimulation. Agonist-stimulated binding did not require appreciable free Mg2+ (less than 0.1 mM). Activation in the absence of agonist was stimulated by free Mg2+, but maximal activation took up to 10 min in the presence of 50 mM MgCl2. Reconstitution increased the stability of Gs to thermal denaturation. The addition of beta-adrenergic agonist further stabilized Gs, presumably by the formation of a stable agonist-receptor-Gs complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characteristics of photoreceptor light-activated guanosinetriphosphatase

We describe a reconstitution of light-activated vertebrate photoreceptor GTPase and a purification of the GTP-binding protein (G protein), which is a component of the GTPase and modulates the light-activated phosphodiesterase (PDE) enzyme system. Rod outer segments (ROS) of bull frogs were treated with ethylenediaminetetraacetic acid (EDTA), and the GTPase and PDE fractions were solubilized (EDTA supernatant). When the EDTA supernatant and EDTA-treated membrane fraction (EDTA-washed membranes) were recombined, light-dependent GTPase activity appeared. In the reconstituted system, the Km for GTP as substrate was 0.5 microM; the optimum pH was 7.5-8.0. The isoelectric point of GTPase in EDTA supernatant was 4.8. G protein was purified 400-fold from ROS, and the molecular weight of G protein was determined to be 40 000 by polyacrylamide gel electrophoresis. The amount of G protein in ROS was calculated as at least 1 molecule per 400 rhodopsin molecules. By recombining (in the presence or absence of GTP) purified G protein, PDE, H fraction (an additional component of GTPase), and illuminated or unilluminated EDTA-washed membranes (as a source of rhodopsin), we showed that illuminated rhodopsin, G protein, PDE, and GTP are the minimum requirements for light-dependent PDE activity. We discuss the significance of these findings in the regulation of the light-activated GTPase and PDE activities, especially with regard to the mechanism of activation.     

Effect of oestradiol on catecholamine-stimulated lipolysis

The authors studied the effect of a single in vivo dose of oestradiol (OE) on adrenergic lipolysis in the epididymal adipose tissue of adult and juvenile male rats, and the effect of OE on plasma free fatty acids (FFA), cholesterol and beta-lipoprotein levels at various intervals after its administration. It was found that OE injected 24 h beforehand in vivo (s.c.), in doses of 100 and 200 micrograms X kg-1 body weight, significantly potentiated the lipid-mobilizing action of the catecholamines noradrenaline (NOR) and isoprenaline (ISO) in adult rats (the action of ISO was potentiated more intensively); in addition, the adipose tissue became more sensitive to the action of NOR, but not of ISO. Raising the dose of OE to 400 micrograms X kg-1 did not enhance the potentiation of the lipolytic action of the catecholamines any further; on the contrary, the lipid mobilizing effect of the catecholamines was potentiated less than after half this dose. Following the s.c. injection of an oily OE solution, the lipolytic effect was potentiated after more than 7 h; the potentiation was strongest after 12 h, but only as far as the maximum attainable degree of lipolysis was concerned. Potentiation of adrenergic lipolysis was found only in adult male rats. In male rats weighing 130-150 g the lipolytic effect of catecholamines (in mumol/g adipose tissue) was significantly greater than in adult animals and the pre-administration of OE did not potentiate adrenergic lipolysis any further. Determination of plasma FFA, cholesterol and beta-lipoprotein levels 1, 2, 4 and 6 hours after the s.c. injection of OE showed only nonsignificant changes (an increase in FFA and a decrease in cholesterol). The authors consider it important to distinguish between the effect of OE on catecholamine-stimulated lipolysis in depot adipose tissue and its effect on lipid metabolism. In their opinion, the dose-dependent effect of OE on muscular and metabolic adrenergic reactions could be one of the factors co-reversible for certain side reactions to steroid contraceptives.     

Reconstitution of biological activity in isoagglutinins fromChlamydomonas eugametos

Gametes ofChlamydomonas eugametos produce membrane vesicles, called isoagglutinins, which are shed into the culture fluid. It is assumed that they originate from the flagellar membrane for, like flagella, they can bind to the flagellar surface of gametes of the opposite mating type (mt). The composition ofmt ^- isoagglutinin was investigated with respect to this agglutinability. When the agglutination factor present on the surface ofmt ^- isoagglutinins (PAS-1.2) was removed, together with other membrane bound glycoproteins, the membrane vesicles were rendered inactive. They could be reactivated however by incubation with the extracted glycoproteins in a time-and concentration-dependent manner. The agglutination factor proved to be necessary yet sufficient in itself for the reactivation process to occur. Experiments with CsCl density gradients showed that the agglutination factor truly bound to the vesicles during reactivation. Inactivated vesicles derived frommt ⁺ gametes could be reactivated to gainmt ^- properties. Reactivation was inhibited by prior treatment with trypsin. The results indicate that the agglutination factor inmt ^- isoagglutinins is an extrinsic membrane protein bound to an intrinsic proteinaceous receptor.Abbreviations GTC guanidine thiocyanate - mt ^+/- mating type plus or minus - PAS periodic acid Schiff     

Reconstitution of the glucose transport activity of rat adipocytes

Rat epididymal fat cell membrane proteins were extracted from adipocyte ghosts with octylglucoside and incorporated by detergent dialysis into unilamellar phosphatidylcholine vesicles approx. 200 nm in diameter. The rate of glucose transport into the vesicles under zero-trans conditions was substrate dependent, saturable and inhibited by phloretin and cytochalasin B. Their maximum specific transport activity was 35.6 mumol/min per mg protein, and their half saturation constant for glucose was 15 mM. Glucose transport into the reconstituted vesicles was inhibited by only those sugars which competitively inhibited glucose transport into intact adipocytes. A major protein component of the vesicles was a 100 kDa protein which we had previously found to react with the affinity label maltosyl isothiocyanate (Malchoff, D.M., Olansky, L., Pohl, S. and Langdon, R.G. (1981) Fed. Proc. 40, 1893). Removal of adipocyte ghost membrane extrinsic proteins with dimethylmaleic anhydride followed by extraction of the resulting membrane pellet with octylglucoside yielded a solution which contained two major proteins, of Mr 100 000 and 85 000, with very small quantities of lower Mr proteins. Vesicles into which these proteins were incorporated had average specific transport activities of 624 mumol/min per mg protein and half saturation constants of 22 mM. Our results strongly indicate that the native glucose transporter of the rat adipocyte, like that of the human erythrocyte (Shelton, R.L. and Langdon, R.G. (1983) Biochim. Biophys. Acta 733, 25-33), is a 100 kDa protein.     

Effect of Al3+ plus F- on the catecholamine-stimulated GTPase activity of purified and reconstituted Gs

The effects of Al3+ and F- on the catecholamine-stimulated GTPase cycle were studied by using reconstituted phospholipid vesicles that contained purified beta-adrenergic receptor and the stimulatory GTP-binding protein of the adenylate cyclase system, Gs. Al3+/F- activated reconstituted Gs to levels previously reported for detergent-solubilized, purified Gs, although both activation and deactivation were faster in the reconstituted preparation. Under these conditions, Al3+/F- did not inhibit by more than 15% the beta-adrenergic agonist-stimulated GTPase activity of the vesicles nor did it significantly inhibit the rates of GTP binding, GTP hydrolysis, or GDP release. When Mg2+ (50 mM) was used instead of agonist to promote GTP hydrolysis in the receptor-Gs vesicles, Al3+/F- was found to inhibit GTP gamma S binding, GDP release, and steady-state GTPase activity to unstimulated levels. These data can be interpreted as indicating that the receptor catalyzes nucleotide exchange by Gs faster or more efficiently than does Mg2+.     

Reconstitution of the fusogenic activity of vesicular stomatitis virus.

Enveloped virus glycoproteins exhibit membrane fusion activity. We have analysed whether the G protein of vesicular stomatitis virus, reconstituted into liposomes, is able to fuse nucleated cells in a pH-dependent fashion. Proteoliposomes produced by octylglucoside dialysis did not exhibit cell fusion activity of the G protein. However, by making use of n-dodecyl octaethylene monoether (C12E8) as the solubilizing agent and by removal of the detergent in two steps, we were able to produce fusogenic G protein liposomes. These G protein liposomes fuse to the BHK-21 cell surface at pH 5.7-6.0 with an efficiency of fusion comparable with that of the parent virus. Physical and chemical analysis revealed that the fusogenic liposomes exhibited a protein to lipid weight ratio of 0.67 and showed an average diameter of 130 nm.     

The interaction of dietary fatty acid and cholesterol on catecholamine-stimulated adenylate cyclase activity in the rat heart

Diets supplemented with high levels of saturated or unsaturated fatty acids supplied by addition of sheep kidney fat or sunflower seed oil, respectively, were fed to rats with or without dietary cholesterol. The effects of these diets on cardiac membrane lipid composition, catecholamine-stimulated adenylate cyclase and beta-adrenergic receptor activity associated with cardiac membranes, were determined. The fatty acid-supplemented diets, either with or without cholesterol, resulted in alterations in the proportion of the (n-6) to (n-3) series of unsaturated fatty acids, with the sunflower seed oil increasing and the sheep kidney fat decreasing this ratio, but did not by themselves significantly alter the ratio of saturated to unsaturated fatty acids. However, cholesterol supplementation resulted in a decrease in the proportion of saturated and polyunsaturated fatty acids and a dramatic increase in oleic acid in cardiac membrane phospholipids irrespective of the nature of the dietary fatty acid supplement. The cholesterol/phospholipid ratio of cardiac membrane lipids was also markedly increased with dietary cholesterol supplementation. Although relatively unaffected by the nature of the dietary fatty acid supplement, catecholamine-stimulated adenylate cyclase activity was significantly increased with dietary cholesterol supplementation and was positively correlated with the value of the membrane cholesterol/phospholipid ratio. Although the dissociation constant for the beta-adrenergic receptor, determined by [125I](-)-iodocyanopindolol binding, was unaffected by the nature of the dietary lipid supplement, the number of beta-adrenergic receptors was dramatically reduced by dietary cholesterol and negatively correlated with the value of the membrane cholesterol/phospholipid ratio. These results indicate that the activity of the membrane-associated beta-adrenergic/adenylate cyclase system of the heart can be influenced by dietary lipids particularly those altering the membrane cholesterol/phospholipid ratio and presumably membrane physico-chemical properties. In the face of these dietary-induced changes, a degree of homeostasis was apparent both with regard to membrane fatty acid composition in response to an altered membrane cholesterol/phospholipid ratio, and to down regulation of the beta-adrenergic receptor in response to enhanced catecholamine-stimulated adenylate cyclase activity.     

Reconstitution of protein translocation activity from partially solubilized microsomal vesicles

We have used a reconstitution assay to demonstrate that protein translocation activity can be recovered after microsomal vesicles derived from the rough endoplasmic reticulum have been partially solubilized with n-octyl-beta-glucopyranoside. Two independent approaches were used to establish conditions for partially solubilizing microsomal membranes. When the lipid bilayer was disrupted by detergents to the extent that the integrity of the lipid bilayer had been perturbed, membranes were inactive for translocation. However, detergent-treated membranes could be reconstituted in good yield into a translocation competent form once the detergent was removed.     

Reconstitution of quinone reduction and characterization of Escherichia coli fumarate reductase activity

Resolution of the fumarate reductase complex (ABCD) of Escherichia coli into reconstitutively active enzyme (AB) and a detergent preparation containing peptides C and D resulted in loss of quinone reductase activity, but the phenazine methosulfate or fumarate reductase activity of the enzyme was unaffected. An essential role for peptides C and D in quinone reduction was confirmed by restoration of this activity on recombination of the respective preparations. Neither peptide C nor peptide D by itself proved capable of permitting quinone reduction and membrane binding by the enzyme when E. coli cells were transformed with plasmids coding for the enzyme and the particular peptides. Transformation of a plasmid coding for all subunits resulted in a 30-fold increase in membrane-bound complex, which exhibited, however, turnover numbers for succinate oxidation and fumarate reduction that were intermediate between the high values characteristic of chromosomally produced complex and the relatively low values found for the isolated complex. It is also shown that preparations of the isolated complex and membrane-bound form of the enzyme, as obtained from anaerobically grown cells, are in the deactivated state owing to the presence of tightly bound oxalacetate and thus must be activated prior to assay.     

Reconstitution of beta-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase

CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards beta-carotene. Activity of CYP175A1 was reconstituted in vitro using artificial electron transport proteins. First results were obtained in the mixture with a crude Escherichia coli cell extract at 37 degrees C. In this system, beta-carotene was hydroxylated to beta-cryptoxanthin. The result indicated the presence of electron transport enzymes among the E. coli proteins, which are suitable for CYP175A1. However, upon in vitro reconstitution of CYP175A1 activity with purified recombinant flavodoxin and flavodoxin reductase from E. coli, only very low beta-cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated beta-carotene at 3- and also 3'-positions, resulting in beta-cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol beta-cryptoxanthin produced per nmol P450 per min.     

Reconstitution of human atlastin fusion activity reveals autoinhibition by the C terminus

ER network formation depends on membrane fusion by the atlastin (ATL) GTPase. In humans, three paralogs are differentially expressed with divergent N- and C-terminal extensions, but their respective roles remain unknown. This is partly because, unlike Drosophila ATL, the fusion activity of human ATLs has not been reconstituted. Here, we report successful reconstitution of fusion activity by the human ATLs. Unexpectedly, the major splice isoforms of ATL1 and ATL2 are each autoinhibited, albeit to differing degrees. For the more strongly inhibited ATL2, autoinhibition mapped to a C-terminal α-helix is predicted to be continuous with an amphipathic helix required for fusion. Charge reversal of residues in the inhibitory domain strongly activated its fusion activity, and overexpression of this disinhibited version caused ER collapse. Neurons express an ATL2 splice isoform whose sequence differs in the inhibitory domain, and this form showed full fusion activity. These findings reveal autoinhibition and alternate splicing as regulators of atlastin-mediated ER fusion.     

Reconstitution of human azurocidin catalytic triad and proteolytic activity by site-directed mutagenesis

Azurocidin belongs to the serprocidin family, but it is devoid of proteolytic activity due to a substitution of His and Ser residues in the catalytic triad. The aim of this study was to reconstitute the active site of azurocidin by site-directed mutagenesis, analyze its processing and restored proteolytic activity. Azurocidin expressed in Sf9 insect cells possessing the reconstituted His41-Asp89-Ser175 triad exhibited significant proteolytic activity toward casein with a pH optimum of approximately 8-9, but a reconstitution of only one active site amino acid did not result in proteolytically active protein. Enzymatically active recombinant azurocidin caused cleavage of the C-terminal fusion tag with the primary cleavage site after lysine at Lys-Leu and after alanine at Ala-Ala, and the secondary cleavage site after arginine at Arg-Gln, as well as with low efficiency caused cleavage of insulin chain B after leucine at Leu-Tyr and Leu-Cys, and after alanine at Ala-Leu. We demonstrate that cleavage of the azurocidin C-terminal tripeptide is not necessary for its enzymatic activity. The first isoleucine present in mature azurocidin can be replaced by similar amino acids, such as leucine or valine, but its substitution by histidine or arginine decreases proteolytic activity.