Rapid incorporation of the solubilized dihydropyridine receptor into phospholipid vesicles

We describe the rapid incorporation of the CHAPS solubilized dihydropyridine receptor into phospholipid vesicles. A series of sucrose gradient sedimentation experiments demonstrate that the (+)-[3H]PN200-110-labeled dihydropyridine receptor is associated with lipid vesicles following detergent removal by Extracti-gel chromatography. Solubilization of the receptor results in a loss of (+)-[3H]PN200-110 binding affinity relative to that observed in native membranes; the high affinity binding of (+)-[3H]PN200-110 can be restored upon reincorporation of the receptor into phospholipid vesicles. Similarly, the incorporation of the receptor restores its stability to incubation at 37 degrees C relative to that of the detergent solubilized receptor, thereby mimicking the properties of the membrane bound form of the receptor. The dissociation rate of (+)-[3H]PN200-110 from the reconstituted receptor is shown to be allosterically regulated by verapamil and diltiazem, indicating that the binding sites for these calcium antagonists have been inserted along with the dihydropyridine receptor into phospholipid vesicles. The results presented in this report, thus demonstrate the successful reconstitution of the dihydropyridine receptor into phospholipid vesicles by a variety of criteria. The reconstitution method described here is rapid and efficient, and should now facilitate structure-function studies of this receptor and its interrelationships with other regulatory components of the voltage-sensitive calcium channel system.     

Phosphorylation of membrane-bound guanylate cyclase of sea urchin spermatozoa

When Arbacia punctulata spermatozoa are incubated in seawater containing ammonium hydroxide (pH 8.8), the sperm plasma membrane-bound guanylate cyclase is dephosphorylated, its electrophoretic mobility increases (from an apparent molecular mass of 160 to 150 kD), and its enzymatic activity decreases 3.5-fold. Transfer of these cells into ammonium-free seawater (pH 7.4) results in the rephosphorylation of the cyclase, its reconversion to 160 kD, and recovery of the enzymatic activity lost upon dephosphorylation. This is the first direct demonstration that the activity of membrane-bound guanylate cyclase can be regulated by phosphorylation. A plasma membrane preparation is described that specifically supports the in vitro phosphorylation of the guanylate cyclase. This preparation will be useful in more detailed studies on the relationship between phosphorylation state and enzymatic activity of membrane-bound guanylate cyclase.     

Trypsin solubilization of rat liver membrane-bound guanylate cyclase results in a form kinetically distinct from the cytosolic enzyme

We have previously reported that treatment of rat liver plasma membranes with various proteases led to activation and solubilization of membrane-bound guanylate cyclase. We report here that the guanylate cyclase solubilized by proteolysis differed from the cytosolic cyclase and rather was similar to the membrane-bound form of the enzyme in that it exhibited a sigmoidal MnGTP concentration dependence and was not activated by an excess Mn2+ or by nitrosocompounds. Also, whereas the cytosolic guanylate cyclase activity was completely abolished by 10 to 100 microM Cd2+, a dithiol reagent, no inhibitory effect was observed on the trypsin-solubilized enzyme. Therefore, the differences in kinetic properties between cytosolic and membrane-bound rat liver guanylate cyclase reside in structural differences between both forms of the enzyme rather than in differences in their environment.     

Incorporation of rat brain adenylate cyclase into artificial phospholipid vesicles.

Adenylate cyclase was solubilized from rat brain particulate fraction with the nonionic detergent, Nonidet P-40. Incubation of detergent-solubilized adenylate cyclase with liposomes prepared from egg yolk phosphatidylcholine results in virtually quantitative incorporation of the enzyme activity into phospholipid vesicles. Incorporation of adenylate cyclase into liposomes results in an approximately 10- to 20-fold purification relative to the solubilized preparation giving a final specific activity of about 50 nmol of cAMP min-1 mg-1. The detergent-solubilized adenylate cyclase migrates as a broad band between 14 and 33% sucrose on density gradient centrifugation, separated from the endogenous phospholipid. Following overnight incubation of the solubilized enzyme with exogenous phospholipid, all enzyme activity is found in a narrow band between 7 and 9% sucrose, co-migrating with the phospholipid. The adenylate cyclase could not be released from the liposomes by extraction with high ionic strength, low ionic strength-EDTA, or sonication. Treatment of liposomal adenylates cyclase with soluble proteases or immobilized trypsin destroys enzyme activity. Thus, it is likely that a functionally important part of the enzyme molecule is exposed on the outer surface of the liposome. Optimal conditions for the incorporation of adenylate cyclase into liposomes, and some effects of manipulating the phospholipid composition on enzyme activity are reported.     

Incorporation of the purified human placental insulin receptor into phospholipid vesicles

Purified human placental insulin receptors were incorporated into small unilamellar phospholipid vesicles by the addition of n-octyl beta-glucopyranoside solubilized phospholipids, followed by removal of the detergent on a Sephadex G-50 gel filtration column and extensive dialysis. The vesicles have an average diameter of 142 +/- 24 nm by Sephacryl S-1000 gel filtration chromatography and 119 +/- 20 nm by transmission electron microscopy. These vesicles are impermeant to small molecules as indicated by their ability to retain [gamma-32P]ATP, which could be released by the addition of 0.05% Triton X-100. Detergent permeabilization or freeze-thawing of the insulin receptor containing vesicles in the presence of 125I-insulin indicated that approximately 75% of the insulin binding sites were oriented right side out (extravesicularly). Sucrose gradient centrifugation of insulin receptors incorporated at various protein to phospholipid mole ratios demonstrated that the insulin receptors were inserted into the phospholipid bilayer structure in a concentration-dependent manner. Addition of [gamma-32P]ATP to the insulin receptor containing vesicles was relatively ineffective in promoting the autophosphorylation of the beta subunit in the absence or presence of insulin. Permeabilization of the vesicles with low detergent concentrations, however, stimulated the beta-subunit autophosphorylation approximately 2-fold in the absence and 10-fold in the presence of insulin. Insulin-stimulated beta-subunit autophosphorylation was also observed under conditions such that 94% of those vesicles containing insulin receptors had a single receptor per vesicle, suggesting that the initial beta-subunit autophosphorylating activity is intramolecular. Phospho amino acid analysis of the vesicle-incorporated insulin receptors demonstrated that the basal and insulin-stimulated beta-subunit autophosphorylation occurs exclusively on tyrosine residues. It is concluded that when purified insulin receptors are incorporated into a phospholipid bilayer, they insert into the vesicles primarily in the same orientation as occurs in the plasma membrane of intact cells and retain insulin binding as well as insulin-stimulated beta-subunit autophosphorylating activities.     

Highly purified particulate guanylate cyclase from rat lung: characterization and comparison with soluble guanylate cyclase

Guanylate cyclase was purified 1000-fold from washed rat lung particulate fractions to a final specific activity of 500 nmoles cyclic GMP produced/min/mg protein by a combination of detergent extraction and chromatography on concanavalin A-Sepharose, GTP-agarose, and blue agarose. Particulate guanylate cyclase has a molecular weight of 200 000 daltons, a Stokes radius of 48 A and a sedimentation coefficient of 9.4 while the soluble form has a molecular weight of 150 000 daltons, a Stokes radius of 44 A, and a sedimentation coefficient of 7.0. Whereas the particulate enzyme is a glycoprotein with a specific affinity for concanavalin A and wheat germ agglutinin, the soluble form of guanylate cyclase did not bind to these lectins. Purified particulate guanylate cyclase did not cross-react with a number of monoclonal antibodies generated to the soluble enzyme. While both forms of the enzyme could be regulated by the formation of mixed disulfides, the particulate enzyme was relatively insensitive to inhibition by cystine. With GTP as substrate both forms of the enzyme demonstrated typical kinetics, and with GTP analogues negative cooperativity was observed with both enzyme forms. These data support the suggestion that the two forms of guanylate cyclase possess similar catalytic sites, although their remaining structure is divergent, resulting in differences in subcellular distribution, physical characteristics, and antigenicity.     

Isolation and incorporation of rabbit liver epoxide hydrase into phospholipid vesicles.

Methods are described for the incorporation into phospholipid vesicles of epoxide hydrase isolated from liver microsomes of phenobarbital-treated rabbits. Chromatography on a Sephadex G-50 column of epoxide hydrase and egg yolk phosphatidylcholine treated with sodium cholate yielded homogeneous vesicles with a diameter of about 25 nm and containing 80 to 85% of the protein applied. At high substrate concentrations, the vesicles catalyzed the hydration of benzo(a)pyrene-4,5-oxide and styrene-7,8-epoxide at a rate similar to that obtained with the enzyme in a soluble form. However, the kinetics of styrene glycol formation catalyzed by the vesicular or microsomal preparations were complex. Convex Lineweaver-Burk plots and concave Hill plots were obtained, whereas normal Michaelis-Menten kinetics characterized the hydration catalyzed by the enzyme in a soluble form. The results could be explained if reconstitution of the enzyme into the vesicles gives rise to low affinity high capacity sites for the substrate on the enzyme, or alternatively facilitates the interaction of the substrate with such sites already present. It is suggested that reconstituted liposomes containing both the liver microsomal hydroxylase system and epoxide hydrase may prove to be a good model system for evaluating substrate specificity and factors of importance in the formation of toxic and carcinogenic metabolites by these enzymes.     

Asymmetric incorporation of Na+, K+-ATPase into phospholipid vesicles

Purified lamb kidney Na⁺, K⁺-ATPase, consisting solely of the Mτ = 95,000 catalytic subunit and the M_τ～- 44,000 glycoprotein, was solubilized with Triton X-100 and incorporated into unilamellar phospholipid vesicles. Freeze-fracture electron microscopy of the vesicles showed intramembranous particles of approximately 90–100 Å in diameter, which are similar to those seen in the native Na⁺,K⁺-ATPase fraction. Digestion of the reconstituted proteins with neuraminidase indicated that the glycoprotein moiety of the Na⁺,K⁺-ATPase was asymmetrically oriented in the reconstituted vesicles, with greater than 85% of the total sialic acid directed toward the outside of the vesicles. In contrast, in the native Na⁺,K⁺-ATPase fraction, the glycoprotein was symmetrically distributed. Purified glycoprotein was also asymmetrically incorporated into phospholipid vesicles using Triton X-100 and without detergents as described by R. I. MacDonald and R. L. MacDonald (1975, J. Biol. Chem.250, 9206–9214). The glycoprotein-containing vesicles were 500–1000 Å in diameter, unilamellar, and, in contrast to the vesicles containing the Na⁺,K⁺-ATPase, did not contain the 90- to 100-Å intramembranous particles. These results indicate that the intramembranous particles observed in the native Na⁺,K⁺-ATPase and in the reconstituted Na⁺,K⁺-ATPase are not due to the glycoprotein alone, but represent either the catalytic subunit, or the catalytic plus the glycoprotein subunit.     

Isolation and incorporation into lipid vesicles of a concanavalin A receptor from human erythrocytes

Affinity chromatography has been used to isolate a concanavalin A receptor portion of Band 3 from humen erythrocytes in the presence of the readily-dialysable detergent, dodecyltrimethylammonium bromide. Addition of phospholipids to the isolated fraction and removal of detergent by dialysis leads to formation of vesicles containing the receptor. Intramembranous particles similar in size and shape to those seen in intact erythrocytes are a characteristic of the reconstituted preparations. Vesicles containing receptor bind concanavalin A with high affinity.     

Atrial natriuretic factor activates membrane-bound guanylate cyclase of chief cells

The present study examined the effect of atrial natriuretic factor (ANF) on cGMP generation by dispersed chief cells from guinea pig stomach. ANF caused a rapid dose-dependent increase in cGMP, a 7-fold increase in cGMP caused by 1 microM ANF, with or without 3-isobutyl-1-methylxanthine present. Methylene blue reduced cGMP in response to nitroprusside but not ANF. Guanylate cyclase activity of a chief cell membrane fraction doubled in response to ANF, but was not affected by nitroprusside. ANF had no effect on guanylate cyclase activity of the soluble fraction of lysed chief cells. Dose-response curves for whole cell cGMP production and membrane guanylate cyclase activity in response to ANF were closely related. These data indicate that ANF increases chief cell cGMP production by activating particulate guanylate cyclase, providing functional evidence that chief cells possess surface membrane receptors for ANF.     

Effect of GTP analogues on purified soluble guanylate cyclase

In our studies with purified soluble guanylate cyclase from rat lung, we have tested a number of guanosine 5'-triphosphate (GTP) analogues as substrates and inhibitors, 5'-Guanylylimidodiphosphate (GMP-P(NH)P), guanylyl (beta, gamma-methylene) diphosphate (GMP-P(CH2)P), and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) were found to be substrates for guanylate cyclase. GTP gamma S supported cyclic GMP formation at 20 or 75% of the rate seen with Mn2+-GTP and Mg2+-GTP, respectively. GMP-P(NH)P and GMP P(CH2)P supported cyclic GMP formation at 10-20% of the GTP rate with either cation cofactor. These analogues were found to have multiple Km values; one Km value was similar to GTP (150 microM with Mg2+, 20-70 microM with Mn2+), but an additional high affinity catalytic site (3 microM) was also observed. Guanosine tetraphosphate (Ki = 10 microM), adenosine triphosphate (Ki = 9 microM) and the 2'3'-dialdehyde derivative of GTP (dial GTP) (Ki = 1 microM) were not good substrates for the enzyme; however, they were potent competitive inhibitors. These GTP analogues will be useful tools for the study of GTP binding sites on guanylate cyclase and they may also help elucidate the effects of free radicals and other agents on guanylate cyclase regulation.     

The insertion of D-beta-hydroxybutyrate apodehydrogenase into phospholipid monolayers and phospholipid vesicles

The strong interaction of D-beta-hydroxybutyrate dehydrogenase with phospholipid monomolecular films is demonstrated by the surface pressure increase of a film compressed up to 33 mN/m. Although the D-beta-hydroxybutyrate apodehydrogenase was able to penetrate many phospholipid monolayers, it interacted preferentially with negatively charged monolayers such as those made from diphosphatidylglycerol. The weakest interaction was found with phosphatidylcholine, which is the reactivating phospholipid for the enzyme. These interactions were dependent on the phospholipid chain length, ionic strength, and pH. At basic pH the apoenzyme lost its specificity for negatively charged phospholipids, suggesting the deprotonation of a cationic amino acid residue of the enzyme polypeptide chain. The charge effects are in agreement with results obtained using phospholipid vesicles. Beside the electrostatic interactions, the influence of phospholipid chain length and the ionic strength indicate that D-beta-hydroxybutyrate apodehydrogenase penetrates into the hydrophobic part of the lipid interface.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37°C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20°C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20°C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freezethaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Purification and properties of the phosphorylated form of guanylate cyclase

Guanylate cyclase is dephosphorylated in response to the interaction of egg peptides with a spermatozoan surface receptor (Suzuki, N., Shimomura, H., Radany, E. W., Ramarao, C. S., Ward, G. E., Bentley, J. K., and Garbers, D. L. (1984) J. Biol. Chem. 259, 14874-14879). Here, the phosphorylated form of guanylate cyclase was purified to apparent homogeneity from detergent-solubilized spermatozoan membranes by the use of GTP-agarose, DEAE-Sephacel, and concanavalin A-Sepharose chromatography. To prevent dephosphorylation of the enzyme during purification, glycerol (35%) was required in all buffers. Following purification, a single protein-staining band of Mr 160,000 was obtained on sodium dodecyl sulfate-polyacrylamide gels. The final specific activity of the purified enzyme was 83 mumol of cyclic GMP formed/min/mg of protein at 30 degrees C, an activity 5-fold higher than that observed with the purified, dephosphorylated form of guanylate cyclase. A preparation containing protein phosphatase from spermatozoa, or highly purified alkaline phosphatase (from Escherichia coli), catalyzed the dephosphorylation of the enzyme; this resulted in a subsequent decrease in guanylate cyclase activity and a shift in the Mr from 160,000 to 150,000. The phosphate content of the high Mr form of the enzyme was 14.6 mol/mol protein whereas the phosphate content of the low Mr form was 1.6 mol/mol protein. All phosphate was localized on serine residues. The Mr 160,000 form of guanylate cyclase demonstrated positive cooperative kinetics with respect to MnGTP while the Mr 150,000 form displayed linear, Michaelis-Menten type kinetics. The phosphorylation state of the membrane form of guanylate cyclase, therefore, appears to dictate not only the absolute activity of the enzyme but also the degree of cooperative interaction between catalytic or GTP-binding sites.     

The membrane form of guanylate cyclase. Homology with a subunit of the cytoplasmic form of the enzyme

A cDNA clone for the membrane form of guanylate cyclase has been isolated from the testis of the sea urchin Strongylocentrotus purpuratus. An open reading frame predicts a protein of 1125 amino acids including an apparent signal peptide of 21 residues; a single transmembrane domain of 25 amino acids divided the mature protein into an amino-terminal, extracellular domain of 485 amino acids and a carboxyl domain of 594 intracellular amino acids. Three potential Asn-linked glycosylation sites were present in the proposed extracellular domain. The deduced protein sequence was homologous to the protein kinase family and contained limited but significant regions of identity with a low molecular weight atrial natriuretic peptide receptor. The carboxyl region (202 amino acids) was 42% identical with a subunit of the cytoplasmic form of guanylate cyclase recently cloned from bovine lung (Koesling, D., Herz, J., Gausepohl, H., Niroomand, F., Hinsch, K.-D., Mulsch, A., Bohme, E., Schultz, G., and Frank, R. (1988) FEBS Lett. 239, 29-34). Therefore, the membrane form of guanylate cyclase is a member of an apparently large family of proteins that includes the low molecular weight atrial natriuretic peptide receptor, the soluble form of guanylate cyclase and protein kinases.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles.

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37 degrees C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20 degrees C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20 degrees C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freeze-thaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Electron microscopy of phospholipid vesicles reconstituted with purified renal Na,K-ATPase

Purified Na,K-ATPase after reconstitution into phospholipid vesicles catalyzed an active coupled transport with a ratio close to 3Na/2K. A uniform population of closed vesicles with average diameters close to 900 A are observed after freeze-fracture and thin sectioning. After freeze-fracture intramembranous particles with diameters of 80-100 A are observed. The data suggest that these particles correspond to Na,K-ATPase molecules.     

Production and properties of antibody to soluble guanylate cyclase purified from bovine brain

Guanylate cyclase was purified 12,700-fold from bovine brain supernatant, and the purified enzyme exhibited essentially a single protein band on polyacrylamide gel electrophoresis. Repeated injection of the purified enzyme into rabbits produced an antibody to guanylate cyclase. The immunoglobulin G fraction from the immunized rabbit gave only one precipitin line against the purified guanylate cyclase and the crude supernatant of bovine brain on double immunodiffusion and immunoelectrophoreis. The antibody completely inhibited the soluble guanylate cyclase activity from bovine brain, various tissues of rat and mouse and neuroblastoma N1E 115 cells, whereas the Triton-dispersed particulate guanylate cyclase from these tissues was not inhibited by the antibody.     

Properties of purified soluble guanylate cyclase activated by nitric oxide and sodium nitroprusside

Highly purified rat lung soluble guanylate cyclase was activated with nitric oxide or sodium nitroprusside and the degree of activation varied with incubation conditions. With Mg2+ as the action cofactor, about 2- to 8-fold activation was observed with nitric oxide or sodium nitroprusside alone. Markedly enhanced activation (20-40 fold) was observed when 1 muM hemin added to the enzyme prior to exposure to the activating agent. The activation with hemin and sodium nitroprusside was prevented in a dose-dependent manner by sodium cyanide. The level activation was also increased by the addition of 1 mM dithiothreitol, but unlike hemin which had no effect on basal enzyme activity, dithiothreitol led to a considerable increase in basal activity. Activated guanylate cyclase decayed to basal activity within one hour at 2 degrees C and the enzyme could be reactivated upon re-exposure to nitroprusside or nitric oxide. Under basal conditions, Michaelis-Menten kinetics were observed, with a Km for GTP of 140 muM with Mg2+ cofactor. Following activation with nitroprusside or nitric oxide, curvilinear Eadie-Hofstee transformations of kinetic data were observed, with Km's of 22 MuM and 100 MuM for Mg-GTP. When optimal activation (15-40 fold) was induced by the addition of hemin and nitroprusside, multiple Km's were also seen with Mg-GTP and the high affinity form was predominant (22 MuM). Similar curvilinear Eadie-Hofstee transformations were observed with Mn2+ as the cation cofactor. These data suggest that multiple GTP catalytic sites are present in activated guanylate cyclase, or alternatively, multiple populations of enzyme exist.