Selectivity of the beta-adrenergic receptor among Gs, Gi's, and Go: assay using recombinant alpha subunits in reconstituted phospholipid vesicles.

The selective regulation of Gs (long and short forms), Gi's (1, 2, and 3), and Go by the beta-adrenergic receptor was assessed quantitatively after coreconstitution of purified receptor, purified G-protein beta gamma subunits, and individual recombinant G-protein alpha subunits that were expressed in and purified from Escherichia coli. Receptor and beta gamma subunits were incorporated into phospholipid vesicles, and the alpha subunits bound to the vesicles stoichiometrically with respect to beta gamma. Efficient regulation of alpha subunit by receptor required the presence of beta gamma. Regulation of G proteins was measured according to the stimulation of the initial rate of GTP gamma S binding, steady-state GTPase activity, and equilibrium GDP/GDP exchange. The assays yielded qualitatively similar results. GDP/GDP exchange was a first-order reaction for each subunit. The rate constant increased linearly with the concentration of agonist-liganded receptor, and the dependence of the rate constant on receptor concentration was a reproducible measurement of the efficiency with which receptor regulated each G protein. Reconstituted alpha s (long or short form) was stimulated by receptor to approximately the extent described previously for natural Gs. Both alpha i,1 and alpha i,3 were regulated with 25-33% of that efficiency. Stimulation of alpha o and alpha i,2 was weak, and stimulation of alpha o was barely detectable over its high basal exchange rate. Reduction of the receptor with dithiothreitol increased the exchange rates for all G proteins but did not alter the relative selectivity of the receptor.     

G protein beta 5 subunit interactions with alpha subunits and effectors

When the beta(5) (short form) and gamma(2) subunits of heterotrimeric G proteins were expressed with hexahistidine-tagged alpha(i) in insect cells, a heterotrimeric complex was formed that bound to a Ni-NTA-agarose affinity matrix. Binding to the Ni-NTA-agarose column was dependent on expression of hexahistidine-tagged alpha(i) and resulted in purification of beta(5)gamma(2) to near homogeneity. Subsequent anion-exchange chromatography of beta(5)gamma(2) resulted in resolution of beta(5) from gamma(2) and further purification of beta(5). The purified beta(5) eluted as a monomer from a size-exclusion column and was resistant to trypsin digestion suggesting that it was stably folded in the absence of gamma. beta(5) monomer could be assembled with partially purified hexahistidine-tagged gamma(2) in vitro to form a functional dimer that could selectively activate PLC beta2 but not PLC beta3. alpha(o)-GDP inhibited activation of PLC beta2 by beta(5)gamma(2) supporting the idea that beta(5)gamma(2) can bind to alpha(o). beta(5) monomer and beta(5)gamma(2) only supported a small degree of ADP ribosylation of alpha(i) by pertussis toxin (PTX), but beta(5) monomer was able to compete for beta(1)gamma(2) binding to alpha(i) and alpha(o) to inhibit PTX-catalyzed ADP ribosylation. These data indicate that beta(5) functionally interacts with PTX-sensitive GDP alpha subunits and that beta(5) subunits can be assembled with gamma subunits in vitro to reconstitute activity and also support the idea that there are determinants on beta subunits that are selective for even very closely related effectors.     

Regulation of signal transfer from beta 1-adrenoceptor to adenylate cyclase by beta gamma subunits in a reconstituted system

The beta gamma subunits of guanine nucleotide binding proteins from bovine brain and bovine rod outer segments have different structural and immunochemical properties. In spite of these structural differences, beta gamma subunits from these sources have been found to be fully interchangeable in terms of their interaction with alpha subunits of pertussis-toxin-sensitive G proteins. In contrast, however, there are striking differences between these beta gamma subunits with regard to their ability to deactivate fluoride-stimulated Gs. These profound differences were also observed when the interaction of the purified components of the adenylate cyclase system was studied after reconstitution into phospholipid vesicles. Addition of beta gamma purified from bovine brain to vesicles containing beta-receptor and Gs results in a biphasic effect on receptor-stimulated GTPase activity, whereas addition of transducin beta gamma was virtually without any effect. Likewise, beta gamma from bovine brain, but not transducin beta gamma, affected adenylate cyclase activity of a reconstituted system consisting of three purified components (R, Gs, C). Thus, the alpha subunit of Gs, but not the alpha subunits of pertussis-toxin-sensitive G proteins discriminate between structurally different beta gamma subunits.     

On the mechanism of G protein beta gamma subunit activation of the muscarinic K+ channel in guinea pig atrial cell membrane. Comparison with the ATP-sensitive K+ channel

The mechanism of G protein beta gamma subunit (G beta gamma)-induced activation of the muscarinic K+ channel (KACh) in the guinea pig atrial cell membrane was examined using the inside-out patch clamp technique. G beta gamma and GTP-gamma S-bound alpha subunits (G alpha *'s) of pertussis toxin (PT)-sensitive G proteins were purified from bovine brain. Either in the presence or absence of Mg2+, G beta gamma activated the KACh channel in a concentration-dependent fashion. 10 nM G beta gamma almost fully activated the channel in 132 of 134 patches (98.5%). The G beta gamma-induced maximal channel activity was equivalent to or sometimes larger than the GTP-gamma S-induced one. Half-maximal activation occurred at approximately 6 nM G beta gamma. Detergent (CHAPS) and boiled G beta gamma preparation could not activate the KACh channel. G beta gamma suspended by Lubrol PX instead of CHAPS also activated the channel. Even when G beta gamma was pretreated in Mg(2+)-free EDTA internal solution containing GDP analogues (24-48 h) to inactivate possibly contaminating G i alpha *'s, the G beta gamma activated the channel. Furthermore, G beta gamma preincubated with excessive GDP-bound G o alpha did not activate the channel. These results indicate that G beta gamma itself, but neither the detergent CHAPS nor contaminating G i alpha *, activates the KACh channel. Three different kinds of G i alpha * at 10 pM-10 nM could weakly activate the KACh channel. However, they were effective only in 40 of 124 patches (32.2%) and their maximal channel activation was approximately 20% of that induced by GTP-gamma S or G beta gamma. Thus, G i alpha * activation of the KACh channel may not be significant. On the other hand, G i alpha *'s effectively activated the ATP-sensitive K+ channel (KATP) in the ventricular cell membrane when the KATP channel was maintained phosphorylated by the internal solution containing 100 microM Mg.ATP. G beta gamma inhibited adenosine or mACh receptor-mediated, intracellular GTP-induced activation of the KATP channel. G i alpha *'s also activated the phosphorylated KATP channel in the atrial cell membrane, but did not affect the background KACh channel. G beta gamma subsequently applied to the same patch caused prominent KACh channel activation. The above results may indicate two distinct regulatory systems of cardiac K+ channels by PT-sensitive G proteins: G i alpha activation of the KATP channel and G beta gamma activation of the KACh channel.     

Rhodopsin/transducin interactions. II. Influence of the transducin-beta gamma subunit complex on the coupling of the transducin-alpha subunit to rhodopsin.

In these studies we have investigated the role of the beta gamma T subunit complex in promoting the rhodopsin-stimulated guanine nucleotide exchange reaction (i.e. the activation event) of the alpha T subunit. The results of these studies demonstrate that although the beta gamma T subunit complex increases the association of the alpha T subunit with lipid vesicles that lack the photoreceptor, the beta gamma T complex is not necessary for the binding of alpha T to lipid vesicles containing rhodopsin, provided sufficient amounts of rhodopsin are present. The rhodopsin-promoted GDP/guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) exchange reaction, within the rhodopsin-alpha T complex, then results in the dissociation of the alpha TGTP gamma S species from the rhodopsin-containing phospholipid vesicles. A second line of evidence for the occurrence of rhodopsin/alpha T interactions, in the absence of beta gamma T, comes from phosphorylation studies using the beta 1 isoform of protein kinase C. The phosphorylation of the alpha T subunit by protein kinase C is inhibited by beta gamma T, both in the absence and in the presence of rhodopsin, but is enhanced by rhodopsin in the absence of beta gamma T. These rhodopsin-alpha T complexes also appear to be capable of undergoing a rhodopsin-stimulated guanine nucleotide exchange event. When the guanine nucleotide exchange is allowed to occur prior to the addition of protein kinase C, the phosphorylation of the alpha T subunit is inhibited. Although beta gamma T is not absolutely required for the rhodopsin/alpha T interaction, it appears to increase the apparent affinity of the alpha T subunit for rhodopsin, both when rhodopsin was inserted into phosphatidylcholine vesicles and when soluble lipid-free preparations of rhodopsin were used. This results in a significant kinetic advantage for the rhodopsin-stimulated guanine nucleotide exchange event, such that the addition of beta gamma T causes a 10-fold promotion of the rhodopsin-stimulation [35S]GTP gamma S binding to alpha T after 1 min but provides less than a 20% promotion of the rhodopsin-stimulated binding after 1 h. The ability of beta gamma T to increase the association of alpha T with the lipid vesicle surface does not appear to contribute significantly to the ability of rhodopsin to couple functionally to alpha T subunits, and there appears to be no requirement for beta gamma T in the alpha T activation event, once the rhodopsin-alpha T complex has formed.     

Functional differences in the beta gamma complexes of transducin and the inhibitory guanine nucleotide regulatory protein

We have examined the mechanism of inhibition of adenylate cyclase using the purified alpha and beta gamma subunits of bovine brain inhibitory guanine nucleotide regulatory protein (Ni) (i.e., alpha i and beta gamma N) and bovine retinal transducin (alpha T and beta gamma T) in reconstituted phospholipid vesicle systems. The addition of beta gamma N or beta gamma T to lipid vesicles containing the pure stimulatory guanine nucleotide regulatory protein (Ns) from human erythrocytes as well as a resolved preparation of the catalytic moiety (C) of bovine caudate adenylate cyclase results in significant inhibition of guanine nucleotide stimulated cyclase activity (80-90%). The inhibition by these beta gamma subunit complexes appears to fully account for the inhibitory effects observed with holo-Ni or holotransducin. A variety of structure-function comparisons of the beta gamma N and beta gamma T complexes were performed in order to further probe the molecular mechanisms involved in the inhibitory pathway. Whereas the beta subunits of beta gamma N and beta gamma T appear to be very similar, if not identical, on the basis of comparisons of their gel electrophoretic mobility and immunological cross-reactivity, clear differences exist in the apparent structures of gamma N and gamma T. Interestingly, functional differences are observed in the effectiveness of these two beta gamma complexes to inhibit adenylate cyclase activity. Specifically, while both beta gamma N and beta gamma T are capable of effecting significant levels of inhibition of the guanine nucleotide stimulated activities, the beta gamma N complex is consistently more potent than beta gamma T in inhibiting these activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Major pertussis-toxin-sensitive GTP-binding protein of bovine lung. Purification, characterization and production of specific antibodies

Two GTP-binding proteins which can be ADP-ribosylated by islet-activating protein, pertussis toxin, were purified from the cholate extract of bovine lung membranes. Both proteins had the same heterotrimeric structure (alpha beta gamma), but the alpha subunits were dissociated from the beta gamma when they were purified in the presence of AlCl3, MgCl2 and NaF. The molecular mass of the alpha subunit of the major protein (designated GLu, with beta gamma) was 40 kDa and that of the minor one was 41 kDa. The results of peptide mapping analysis of alpha subunits with a limited proteolysis indicated that GLu alpha was entirely different from the alpha of brain Gi or Go, while the 41-kDa polypeptide was identical with the alpha of bovine brain Gi. The kinetics of guanosine 5'-[3-O-thio]triphosphate (GTP[gamma S]) binding to GLu was similar to that to lung Gi but quite different from that to brain Go. On the other hand, incubation of GLu alpha at 30 degrees C caused a rapid decrease of GTP[gamma S] binding, the inactivation curve being similar to that of Go alpha but different from that of Gi alpha. The alpha subunits of lung Gi and GLu did not react with the antibodies against the alpha subunit of bovine brain Go. The antibodies were raised in rabbits against GLu alpha and were purified with a GLu alpha-Sepharose column. The purified antibodies reacted not only with GLu alpha but also with the 41-kDa protein and purified brain Gi alpha. However, the antibodies adsorbed with brain Gi alpha reacted only with GLu alpha, indicating antisera raised with GLu alpha contained antibodies that recognize both Gi alpha and GLu alpha, and those specific to GLu alpha. These results further indicate that GLu is different from Gi or Go. Anti-GLu alpha antibodies reacted with the 40-kDa proteins in the membranes of bovine brain and human leukemic (HL-60) cells. The beta gamma subunits were also purified from bovine lung. The beta subunit was the doublet of 36-kDa and 35-kDa polypeptides. The lung beta gamma could elicit the ADP-ribosylation of GLu alpha by islet-activating protein, increase the GTP[gamma S] binding to GLu and protect the thermal denaturation of GLu alpha. The antibodies raised against brain beta gamma cross-reacted with lung beta but not with lung gamma.     

G protein beta gamma subunits from bovine brain and retina: equivalent catalytic support of ADP-ribosylation of alpha subunits by pertussis toxin but differential interactions with Gs alpha

We have examined the ability of the beta gamma subunits of guanine nucleotide binding regulatory proteins (G proteins) to support the pertussis toxin (PT) catalyzed ADP-ribosylation of G protein alpha subunits. Substoichiometric amounts of the beta gamma complex purified from either bovine brain G proteins or the bovine retinal G protein, Gt, are sufficient to support the ADP-ribosylation of the alpha subunits of Gi (the G protein that mediates inhibition of adenylyl cyclase) and Go (a G protein of unknown function) by PT. This observation indicates that ADP-ribosylated G protein oligomers can dissociate into their respective alpha and beta gamma subunits in the absence of activating regulatory ligands, i.e., nonhydrolyzable GTP analogues or fluoride. Additionally, the catalytic support of ADP-ribosylation by bovine brain beta gamma does not require Mg2+. Although the beta gamma subunit complexes purified from bovine brain G proteins and the beta gamma complex of Gt support equally the ADP-ribosylation of alpha subunits by PT, there is a marked difference in their abilities to interact with Gs alpha. The enhancement of deactivation of fluoride-activated Gs alpha requires 25-fold more beta gamma from Gt than from brain G proteins to produce a similar response. This difference in potency of beta gamma complexes from the two sources was also observed in the ability of beta gamma to produce an increase in the activity of recombinant Gs alpha produced in Escherichia coli.     

Two guanine nucleotide-binding proteins in rat brain serving as the specific substrate of islet-activating protein, pertussis toxin. Interaction of the alpha-subunits with beta gamma-subunits in development of their biological activities

Two proteins serving as substrates for ADP-ribosylation catalyzed by islet-activating protein (IAP), pertussis toxin, and binding guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) with high affinities were purified from the cholate extract of rat brain membranes. The purified proteins had the same heterotrimeric structure (alpha beta gamma) as the IAP substrates previously purified from rabbit liver and bovine brain and differed from each other in alpha only; the molecular weight of alpha was 41,000 (alpha 41 beta gamma) and 39,000 (alpha 39 beta gamma). Both were further resolved into alpha (alpha 41 or alpha 39) and beta gamma which were also purified to homogeneity to compare the activities of alpha-monomers with the original trimers. The maintenance of the rigid trimeric structure by combining alpha 41 or alpha 39 with beta gamma in the absence of Mg2+ was essential for the alpha-subunit to be ADP-ribosylated by IAP. The alpha-subunit was very stable but displayed the only partial GTP gamma S-binding activity under these conditions. Isolated alpha-monomers exhibited high GTPase activities when assayed in the presence of submicromolar Mg2+ but were very unstable at 30 degrees C and not ADP-ribosylated by IAP. The most favorable conditions for the GTP gamma S binding to alpha-subunits were achieved by combining alpha 41 or alpha 39 with beta gamma in the presence of millimolar Mg2+, probably due to the increase in stability and unmasking of the GTP-binding sites. There was no qualitative difference in these properties between alpha 41 beta gamma (alpha 41) and alpha 39 beta gamma (alpha 39). But alpha 39 beta gamma (or alpha 39) was usually more active than alpha 41 beta gamma (or alpha 41), at least partly due to its higher affinity for Mg2+ and lower affinity for beta gamma. Relation of these differences in activity between alpha 41 beta gamma and alpha 39 beta gamma to their physiological roles in signal transduction is discussed.     

Development of an Assay for Phospholipase C Using Column-Reconstituted, Extruded Phospholipid Vesicles

The reconstitution of heterotrimeric G proteins into phospholipid vesicles has been widely used for the measurement of PLC-beta activity in vitro. We have developed an improved and sensitive method for the assay of PLC-beta activity. This approach involves reconstitution of purified betagamma dimers into extruded phospholipid vesicles containing phosphatidylinositol 4, 5-bisphosphate and using a gel-filtration technique to separate the reconstituted vesicles from monodispersed betagamma dimers and the detergent used to solubilize G proteins. The method provides physical information about the partitioning of betagamma dimers into phospholipid vesicles and was used to examine the effect of different prenyl groups on the gamma subunits in the activation of PLC-beta. The beta1gamma1 dimer (containing the farnesyl group) and the beta1gamma2 dimer (containing the geranylgeranyl group) were purified from baculovirus-infected Sf9 insect cells and were found to partition equally into phospholipid vesicles. The beta1gamma2 dimer is more potent and effective in stimulating PLC-beta activity than the beta1gamma1 dimer. The EC50 values of betagamma dimers for the activation of PLC-beta determined with this method were lower than those determined by previous methodology, showing that betagamma subunits have a subnanomolar affinity for PLC-beta.     

Studies on the interaction of alpha subunits of GTP-binding proteins with beta gamma dimers.

The interaction of several preparations of purified beta gamma dimers with two types of guanosine-nucleotide-binding-regulatory-(G)-protein alpha subunits, a recombinant bv alpha i3, made in Sf9 Spodoptera frugiperda cells by the baculovirus (bv) expression system, and alpha s, either purified from human erythrocyte Gs-type GTP-binding protein, and activated by NaF/AlCl3, or unpurified as found in a natural membrane, were studied. The beta gamma dimers used were from bovine rod outer segments (ROS), bovine brain, human erythrocytes (hRBC) and human placenta and contained distinct ratios of beta subunits that, upon electrophoresis, migrated as two bands with approximate M(r) of 35,000 and 36,000, as well as distinct complements of at least two gamma subunits each. When tested for their ability to recombine at submaximal concentrations with bv alpha i3, ROS, brain, hRBC and placental beta gamma dimers exhibited apparent affinities that were the same within a factor of two. When bovine brain, placental and ROS beta gamma dimers were tested for their ability to promote deactivation of Gs, brain and placental beta gamma dimers were equipotent and at least 10-fold more potent than that of ROS beta gamma dimers; likewise, brain beta gamma and placental dimers were equipotent in inhibiting GTP-activated and GTP-plus-isoproterenol-activated adenylyl cyclase, while ROS beta gamma dimers were less potent when assayed at the same concentration. The possibility that different alpha subunits may distinguish subsets of beta gamma dimers from a single cell was investigated by analyzing the beta gamma composition of three G proteins, Gs, Gi2 and Gi3, purified to near homogeneity from a single cell type, the human erythrocyte. No evidence for an alpha-subunit-specific difference in beta gamma composition was found. These findings suggests that, in most cells, alpha subunits interact indistinctly with a common pool of beta gamma dimers. However, since at least one beta gamma preparation (ROS) showed unique behavior, it is clear that there may be mechanisms by which some combinations of beta gamma dimers may exhibit selectivity for the alpha subunits they interact with.     

Functional equivalence of monomeric and dimeric forms of purified acetylcholine receptors from Torpedo californica in reconstituted lipid vesicles

Acetylcholine receptors from Torpedo californica electric organ were solubilized and purified under conditions which prevent inactivation of the agonist-regulated cation channels. The dimer form of the receptors was preserved during purification. Treatment with reducing agents converted dimers into monomers. Receptor monomers and dimers were separately reconstituted into soybean lipid vesicles by the cholate dialysis technique. Reconstituted monomers and dimers were functionally equivalent with respect to their carbamylcholine-induced dose-dependent uptake of 22Na+, the total flux of 22Na+ per receptor during the permeability response, and the occurrence of desensitization. Evidence against non-covalent association of monomers to produce dimeric functional units was obtained using glutaraldehyde as a crosslinking agent. These results show that both the acetylcholine-binding sites and the agonist-regulated cation-specific channel are contained within the alpha 2 beta gamma delta subunit structure of the acetylcholine receptor monomer.     

Influence of differential stability of G protein βγ dimers containing the γ11 subunit on functional activity at the M1 muscarinic receptor, A1 adenosine receptor, and phospholipase C-β

Ggamma11 is an unusual guanine nucleotide-binding regulatory protein (G protein) subunit. To study the effect of different Gbeta-binding partners on gamma11 function, four recombinant betagamma dimers, beta1gamma2, beta4gamma2, beta1gamma11, and beta4gamma11, were characterized in a receptor reconstitution assay with the G(q)-linked M1 muscarinic and the G(i1)-linked A1 adenosine receptors. The beta4gamma11 dimer was up to 30-fold less efficient than beta4gamma2 at promoting agonist-dependent binding of [35S]GTPgammaS to either alpha(q) or alpha(i1). Using a competition assay to measure relative affinities of purified betagamma dimers for alpha, the beta4gamma11 dimer had a 15-fold lower affinity for G(i1) alpha than beta4gamma2. Chromatographic characterization of the beta4gamma11 dimer revealed that the betagamma is stable in a heterotrimeric complex with G(i1) alpha; however, upon activation of alpha with MgCl2 and GTPgammaS under nondenaturing conditions, the beta4 and gamma11 subunits dissociate. Activation of purified G(i1) alpha:beta4gamma11 with Mg+2/GTPgammaS following reconstitution into lipid vesicles and incubation with phospholipase C (PLC)-beta resulted in stimulation of PLC-beta activity; however, when this activation preceded reconstitution into vesicles, PLC-beta activity was markedly diminished. In a membrane coupling assay designed to measure the ability of G protein to promote a high-affinity agonist-binding conformation of the A1 adenosine receptor, beta4gamma11 was as effective as beta4gamma2 when coexpressed with G(i1) alpha and receptor. However, G(i1) alpha:beta4gamma11-induced high-affinity binding was up to 20-fold more sensitive to GTPgammaS than G(i1) alpha:beta4gamma2-induced high-affinity binding. These results suggest that the stability of the beta4gamma11 dimer can modulate G protein activity at the receptor and effector.     

Identification and characterization of the 35-kDa beta subunit of guanine-nucleotide-binding proteins by an antiserum raised against transducin

Antisera were raised against the retinal guanine-nucleotide-binding protein (N-protein), transducin, purified from bovine rod outer segments. Sera obtained after repeated injections of antigen recognized all transducin subunits (alpha, beta and gamma). One antiserum, tested for cross-reactivity with non-retinal N-proteins, was found to cross-react with the beta subunits of the ubiquitously occurring N-proteins, Ns and Ni, but not with their respective alpha and gamma subunits. The antiserum also cross-reacted with the beta subunit of the recently identified N-protein, No, which has been found in high abundance in the central nervous system. These data support the similarity of the beta subunits of the N-proteins identified so far. Purification of N-proteins from porcine cerebral cortex without the use of activating ligands yielded fractions containing the isolated alpha subunit of No, free beta gamma complex, Ni, No and fractions containing both N-proteins in various proportions. The purity of the preparations was at least 80% as judged by Coomassie-blue-stained SDS gels. No pure Ns was obtained. Use of the transducin antibody during the course of the purification revealed that the beta subunits coeluted from a gel filtration column largely with the alpha subunits of Ni and No but were hardly detectable in fractions that were able to reconstitute Ns activity into membranes of an Ns-deficient cell line (S49 cyc- lymphoma cells). This indicates that in the central nervous system the concentrations of Ni and No are of magnitudes higher than that of Ns. Two-dimensional gel electrophoresis of N-proteins, purified from porcine cerebral cortex, resulted in the resolution of two major peptides in the 35-kDa region, which differed in their pI values and were identified as beta subunits by the use of the antiserum. Identical results were achieved using crude cholate extracts from membranes of the same tissue instead of purified proteins. The occurrence of different beta subunits may be explained by posttranslational N-protein modification.     

Role of the subunits of the energy-transducing adenosine triphosphatase from Micrococcus lysodeikticus membranes studied by proteolytic digestion and immunological approaches.

An energy-transducing adenosine triphosphatase (ATPase, EC 3.6.1.3) that contains an extra polypeptide (delta) as well as three intrinsic subunits (alpha, beta, gamma) was purified from Micrococcus lysodeikticus membranes. The apparent subunit stoichiometry of this soluble ATPase complex is alpha 3 beta 3 gamma delta. The functional role of the subunits was studied by correlating subunit sensitivity to trypsin and effect of antibodies raised against holo-ATPase and its alpha, beta and gamma subunits with changes in ATPase activity and ATPase rebinding to membranes. A form of the ATPase with the subunit proportions 1.67(alpha):3.00(beta:0.17(gamma) was isolated after trypsin treatment of purified ATPase. This form has more than twice the specific activity of native enzyme. Other forms with less relative proportion of alpha subunits and absence of gamma subunit are not active. Of the antisera to subunits, only anti-(beta-subunit) serum shows a slight inhibitory effect on ATPase activity, but its combination with either anti-(alpha-subunit) or anti-(gamma-subunit) serum increases the effect. The results suggest that beta subunit is required for full ATPase activity, although a minor proportion of alpha and perhaps gamma subunit(s) is also required, probably to impart an active conformation to the protein. The additional polypeptide not hitherto described in Micrococcus lysodeikticus ATPase had a molecular weight of 20 000 and was found to be involved in ATPase binding to membranes. This 20 000-dalton component can be equated with the delta subunit of other energy-transducing ATPases and its association with the (alpha, beta, gamma) M. lysodeikticus ATPase complex appears to be dependent on bivalent cations. The present results do not preclude the possibility that the gamma subunit also plays a role in ATPase binding, in which, however, the major subunits do not seem to play a role.     

F1-ATPase from Micrococcus sp. ATCC 398. Purification by ion-exchange chromatography and further characterization. (Auto)proteolysis and dissociative effects.

The preparation of highly purified F1-ATPase from Micrococcus sp. ATCC 398 by application of DEAE-Sepharose CL-6B chromatography as final step is described. This enzyme consists of five subunits of different molecular weight: alpha (65000), beta (55000),gamma (35000), delta (20000), and epsilon (17000). Disc electrophoresis on 5% polyacrylamide gels removes the epsilon-polypeptide yielding an active ATPase complex with four different subunits: alpha, beta, gamma, delta. Additionally, by variation of the ionic strength delta can (partly) removed allowing the isolation by disc electrophoresis of an active ATPase complex which consists only of three different subunits alpha, beta, and gamma. If the DEAE-Sepharose chromatography is carried out in the absence of diisopropyl phosphofluoridate (auto)proteolysis yields both an active ATPase with the subunits alpha+ (mol. wt 61000), beta, gamma, and delta and an inactive protein complex with the subunits alpha+, beta, gamma, delta, and two additional polypeptides a (mol. wt 38000) and b (mol. wt 23000). The latter two polypeptides are supposedly fragments of alpha+-chains which have become partially cleaved by (auto)proteolysis.     

Functional properties of rat brain sodium channels lacking the beta 1 or beta 2 subunit

The sodium channel purified from rat brain is a heterotrimeric complex of alpha (Mr 260,000), beta 1 (Mr 36,000), and beta 2 (Mr 33,000) subunits. alpha and beta 2 are attached by disulfide bonds. Removal of beta 1 subunits by incubation in 1.0 M MgCl2 followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 2 which did not bind [3H]saxitoxin, mediate veratridine-activated 22Na+ influx, or bind the 125I-labeled alpha-scorpion toxin from Leiurus quinquestriatus (LqTx). In contrast, removal of beta 2 subunits by reduction of disulfide bonds with 1.5 mM dithiothreitol followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 1 that retained full sodium channel function. Alpha beta 1 bound [3H]saxitoxin with a KD of 4.1 nM at 36 degrees C. It mediated veratridine-activated 22Na+ influx at a comparable initial rate as intact sodium channels with a K0.5 for veratridine of 46 microM. Tetracaine and tetrodotoxin blocked 22Na+ influx. Like intact sodium channels, alpha beta 1 bound 125I-LqTx in a voltage-dependent manner with a KD of approximately 6 nM at a membrane potential of -60 mV and was specifically covalently labeled by azidonitrobenzoyl 125I-LqTx. When incorporated into planar phospholipid bilayers, alpha beta 1 formed batrachotoxin-activated sodium channels of 24 pS whose voltage-dependent activation was characterized by V50 = -110 mV and an apparent gating charge of 3.3 +/- 0.3. These results indicate that beta 2 subunits are not required for the function of purified and reconstituted sodium channels while a complex of alpha and beta 1 subunits is both necessary and sufficient for channel function in the purified state.     

Binding of alpha- and beta gamma-subunits of Go to beta 1-adrenoceptor in sealed unilamellar lipid vesicles.

First, we describe a preparation of sealed unilamellar lipid vesicles. When this preparation was subjected to sucrose density gradient centrifugation, two rather uniform fractions emerged, one consisting of lighter lipid-rich vesicles with average diameters ranging over 150-200 nm (fraction I), the other consisting of heavier vesicles with average diameters ranging over 30-70 nm (fraction II). When the lipid mixture containing dimyristoylglycerophosphocholine, cholesterol, dipalmitoylglycerophosphoserine and dipalmitoylglycerophosphoethanolamine at molar ratios of 54:35:10:1 was reconstituted with alpha- and beta gamma-subunits of Go-proteins purified to homogeneity from bovine brain, the lipid-rich lighter vesicle fraction I took up these subunits nearly exclusively. Whereas, when a beta 1-adrenoceptor preparation purified from turkey erythrocyte membranes was reconstituted, it was found nearly completely in the smaller heavier vesicle fraction II where it was incorporated inside-out. On co-reconstitution of either alpha o or beta gamma alone with beta 1-adrenoceptors, some of these subunits appear together with beta 1-adrenoceptors in the small vesicle fraction II, but much more alpha o was bound to the receptor in the presence of beta gamma-subunits. The observations reported are novel and surprising in several respects: firstly, they suggest that beta gamma-subunits can bind to the non-activated beta 1-receptor where they may serve as an anchor for alpha-subunits. Secondly, the binding of alpha o- and beta gamma-subunits to the beta 1-adrenoceptors enhances the basal GTPase activity of alpha o. Thirdly, since the binding domains of the beta 1-adrenoceptor for G-proteins were facing outwards in our sealed vesicle preparations, it follows that interactions of G-proteins with the beta-receptor can occur at the aqueous membrane interface as was postulated originally by M. Chabre [Trends Biochem. Sci. 12, 213-215 (1987)] for the transducin-rhodopsin interactions. Finally, the binding of Go-subunits from bovine brain to a beta 1-adrenoceptor from turkey erythrocytes was not expected, since these polypeptides are not likely to be physiological partners.     

Identification of subunits mediating clustering of GABA(A) receptors by rapsyn.

Human embryonic kidney 293 cells transfected with alpha1beta1gamma2, alpha1beta2gamma2, alpha1beta3gamma2, alpha1beta1, alpha1beta2, alpha1beta3, beta3gamma2, or beta3 subunits formed gamma-aminobutyric acidA receptors on the cell surface that could be clustered by rapsyn. In contrast, alpha1, beta1, beta2, or gamma2 subunits, or alpha1gamma2 subunit combinations could not be detected on the surface of transfected cells and could not be clustered by rapsyn. Experiments investigating the ability of rapsyn to cluster chimeras consisting of the N-terminus of the beta3 subunit and the remaining part of the alpha1, beta2 or gamma2 subunits indicated that the intracellular domains of beta1, beta2, beta3 or gamma2 subunits, but not those of alpha1 subunits are able to form sites mediating clustering by rapsyn. These results demonstrate that rapsyn has the potential to cluster the majority of GABA(A) receptor subtypes via beta or gamma2 subunits. Further experiments will have to clarify the physiological importance of this observation.     

Immunoassay for the beta gamma subunits of GTP-binding proteins and their regional distribution in bovine brain

Antibodies were raised in rabbits against the beta gamma subunits of bovine brain GTP-binding proteins, and were purified with a beta gamma-coupled Sepharose column. Purified antibodies reacted strongly with 36,000-dalton beta subunit and slightly with 35,000-dalton beta and gamma subunits, but not with other proteins in an immunoblot assay. Using these purified antibodies, a sensitive enzyme immunoassay method for the quantification of brain beta gamma was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimum detection limit of the assay was 3 fmol, or 130 pg. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of beta gamma were determined. The beta gamma were detected in all the regions, and the highest concentrations were observed in the cerebral cortex and nucleus caudatus. The concentrations of beta gamma were higher than those of alpha subunit of GTP-binding protein, Go, in all the regions.