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Within the past few years, the phylogenetic tree is discussed controversially regarding the position of the different bilaterian groups. There are two varying views of evolution: the classical one based on morphological structures where the annelids and arthropods are combined in the group of Articulata, and a new hypothesis based on molecular data sets, which divides the protostomian groups in Ecdysozoa (e.g. arthropods, and nematodes) and Lophotrochozoa (e.g. annelids, molluscs, and platyhelminthes). The Na+/K+-ATPase is a highly conserved protein and fulfils a very important role in physiology and maintaining the homeostasis of cells and can be found in almost all eukaryotic animals. Due to its similar molecular structure throughout the animal kingdom the Na+/K+-ATPase is an excellent marker for phylogenetic studies. Here we report the molecular cloning, sequencing and phylogenetic analysis of Na+/K+-ATPase complementary deoxyribonucleic acid (cDNA) of the medical leech Hirudo medicinalis. The cloned cDNA codes for a polypeptide of 1022 amino acids and possesses a predicted molecular mass of 113.33 kDa. Phylogenetic analysis of the complete Na+/K+-ATPase α-subunit of H. medicinalis and sequence data from other Na+/K+-ATPases supports the previously developed ‘Ecdysozoa concept’ with high posterior probabilities. A common clade comprising annelids and platyhelminthes can be defined, whereas nematodes are in a basal position at the arthropod stem line.

The involvement of a Gi- or G(o)-related G-protein as a regulator of the growth of guinea pig thoracic aorta smooth muscle (TASM) cells was studied by investigating the effects of pertussis toxin (PTX) on the growth of these cells. PTX treatment decreased the growth rate of TASM cells by 70-100%. This effect was apparent within 24 h after exposure to the toxin and persisted for at least 10 days after starting the treatment. The effect of the toxin appeared to be the result of the inactivation of a G-protein because 1) TASM cell membranes contained a 40-kilodalton substrate for the toxin in in vitro assays that was absent in membranes prepared from cells pretreated with toxin; and 2) the effect required both the enzymatic component (A-protomer) of the toxin that inactivates Gi/G(o)-related G-proteins and its B-oligomer necessary for binding and internalization of the A-protomer. The effect of the toxin was not due to an increased level of intracellular cAMP brought about by inactivation of a G-protein that normally inhibits adenylyl cyclase activity. Further, the toxin did not merely make some unknown mitogen rate limiting, because neither increasing concentrations of serum in the growth medium nor supplementation with platelet-derived growth factor could overcome its inhibition of TASM cell growth. Instead, some unknown process regulated by a PTX-sensitive G-protein appears to be required for the normal growth of these cells.

Results are presented on the evaluation of thermally fatigued E-glass composite specimens using ultrasonic techniques. The changes of the acoustic velocity and the attenuation coefficient in the ultrasonic range have been examined as a function of accumulated damage in various thermally fatigued composites. Three types of E-glass fiber reinforced composites have been used in this study having the following fiberglass weaves: (a) randomly oriented short fiber, (b) uniaxial fiber mat with continuous fiber bundles, (c) bidirectional continuous glass fiber mat. Representative specimens were subjected to thermal fatigue by thermal cycling between 25°C and 75°C. The structural integrity of these specimens was evaluated by determining the flexural strength after a number of thermal cycles via three point bending tests. It was observed that both the acoustic velocity and the flexural strength decreased whereas the ultrasonic attenuation increased with the number of thermal cycles which have been attributed to an accumulation of defects in the material and especially to debonding at the fiber-matrix interface. It has been observed that the use of ultrasonics may be very appropriate in evaluating composites in service since a good correlation has been noted between changes in the flexural strength, acoustic velocity and the attenuation coefficient in thermally fatigued composites.

The properties of a Gs alpha mutant with an Asn substituted for Ser at position 54, designated mutant 54Asn alpha s, were studied after expression in S49 alpha s-deficient (cyc-) cells. Ser-54 in alpha s is comparable to Ser-17 in Ras, which is involved in binding Mg2+ associated with bound nucleotide. 54Asn alpha s did not restore either hormone-induced cyclic AMP production in intact cyc- cells or hormone-induced adenylyl cyclase activation in membranes isolated from these cells. The defect was a failure of ligand-bound receptor to activate 54Asn alpha s, since the mutant protein retained the ability to activate adenylyl cyclase in isolated membranes in the presence of GTP or GTP gamma S. Guanine nucleotide regulation of mutant alpha s suggested that it has increased guanine nucleotide exchange rates and an increased preference for diphosphates over triphosphates. Hormone stimulation magnified the preference of 54Asn alpha s for diphosphates, which could account for its inability to be activated by receptor. The properties of this mutant are discussed in terms of similarities to and differences with the analogous RasH mutant, which has been shown to interfere with endogenous Ras function in cells.

The properties of a Gs alpha mutant with an Asn substituted for Ser at position 54, designated mutant 54Asn alpha s, were studied after expression in S49 alpha s-deficient (cyc-) cells. Ser-54 in alpha s is comparable to Ser-17 in Ras, which is involved in binding Mg2+ associated with bound nucleotide. 54Asn alpha s did not restore either hormone-induced cyclic AMP production in intact cyc- cells or hormone-induced adenylyl cyclase activation in membranes isolated from these cells. The defect was a failure of ligand-bound receptor to activate 54Asn alpha s, since the mutant protein retained the ability to activate adenylyl cyclase in isolated membranes in the presence of GTP or GTP gamma S. Guanine nucleotide regulation of mutant alpha s suggested that it has increased guanine nucleotide exchange rates and an increased preference for diphosphates over triphosphates. Hormone stimulation magnified the preference of 54Asn alpha s for diphosphates, which could account for its inability to be activated by receptor. The properties of this mutant are discussed in terms of similarities to and differences with the analogous RasH mutant, which has been shown to interfere with endogenous Ras function in cells.

Heterotrimeric GTP-binding proteins from bovine brain were resolved by fast protein liquid chromatography chromatography using Mono Q columns. Two distinct forms of the protein Go were identified. Both forms had stochiometric amounts of alpha- and beta gamma-subunits. The a-subunits of both forms were recognized by an alpha o-specific antiserum, but not by any of the alpha i-specific antisera. The two forms showed distinct migration patterns on 9% sodium dodecyl sulfate-polyacrylamide gels containing 4-8 M urea gradients. Neither form comigrated with the recombinant alpha o1. Both the recombinant alpha o1 and the most abundant form of Go were recognized by an antiserum, H-660, against a peptide encoding amino acids 3-17 of alpha i2. H-660 has been shown previously to recognize alpha o and alpha i (Mumby, S. M., Pang, I. K., Gilman, A. G., and Sternweis, P. C. (1988) J. Biol. Chem. 263, 2020-2026). This more abundant form is called Go A most likely corresponds to the cloned alpha o1. The less abundant form, Go B, was not recognized by H-660. However, both forms of bovine brain Go were recognized by GC/2, an antiserum against the N-terminal region of alpha o1. Hence alpha oA and alpha oB may be different in their N terminus regions. Neither form of bovine brain Go was recognized by an antisera made to a peptide encoding the unique regions of the cloned alpha o2 from HIT cells (Hsu W. H., Rudolph, U., Sanford, J., Bertrand, P., Olate, J., Nelson, C., Moss, L.E., Boyd, A. E., III, Codina, J., and Birnbaumer, L. (1990) J. Biol. Chem. 265, 11220-11226). Go A and Go B have similar guanine nucleotide binding and release properties. Both release GDP within 1 min in the absence of added Mg2+. Both bind guanosine (GTP gamma S) rapidly as well. However Go A binds GTP gamma S about 2.5-fold faster than Go B, in the absence of added Mg2+ ion. Both forms of Go as well as the recombinant alpha o (alpha o1) can increase muscarinic stimulation of inositol trisphosphate-mediated Cl- current in Xenopus oocytes. These data indicate that we have identified two structurally distinct forms of Go that have different guanine nucleotide binding properties and are capable of functioning in the receptor-regulated phospholipase C pathway in Xenopus oocytes.

S49 mouse lymphoma cells contain a beta-adrenergic receptor coupled to Gs that stimulates adenylyl cyclase and a somatostatin receptor coupled to Gi that inhibits adenylyl cyclase. Membranes from these cells were used to compare the inhibitory effects of somatostatin and G protein beta gamma complex to determine under what conditions beta gamma is likely to be a mediator of somatostatin action. Somatostatin was equally effective at inhibiting basal adenylyl cyclase activity in the presence of GTP, forskolin-stimulated activity, and hormone-stimulated activity. G protein beta gamma was more effective at inhibiting basal activity than was somatostatin, and these effects were partially additive. In the presence of forskolin, the two inhibitors were equally effective and not additive. In the presence of isoproterenol, beta gamma was much less effective than somatostatin, and the two inhibitors did not have additive or synergistic effects. At very high concentrations beta gamma did inhibit isoproterenol stimulation of adenylyl cyclase, although its effects were not saturating even at 100 micrograms/ml. Under conditions where beta gamma did inhibit hormone stimulation, beta gamma was a mixed inhibitor of isoproterenol stimulation, proportionally decreasing the maximum effect of the hormone and increasing the half-maximally effective concentration. Somatostatin, on the other hand, was a simple noncompetitive inhibitor of isoproterenol stimulation. These results indicate that beta gamma and somatostatin inhibit adenylyl cyclase by different mechanisms, at least in the presence of hormones that stimulate the enzyme. It is proposed that alpha i is the primary mediator of hormone inhibition of adenylyl cyclase when Gs is activated by a hormone, but that beta gamma may have a role as a mediator of inhibition of basal activity.

The native pertussis toxin sensitive GTP-binding proteins (Gi proteins) were individually resolved, and their guanine nucleotide binding and release properties were studied. Gi2 and Gi3, the two major GTP-binding proteins of human erythrocytes, were purified to apparent homogeneity by fast protein liquid chromatography. Gi1 was purified from bovine brain. The three proteins bound 0.6-0.85 mol of guanosine 5'-O-(thio-triphosphate (GTP gamma S)/mol of protein with similar affinities (KD(app) = 50-100 nM). The rate of [35S]GTP gamma S binding to Gi2 was 5-8-fold faster than to Gi1 or Gi3 at 2 mm Mg2+. There were no observable differences in the binding characteristics between bovine brain Gi1 and human erythrocyte Gi3. At 50 mM Mg2+, all three Gi proteins exhibited fast binding, although Gi1 and Gi3 were marginally slower than Gi2. All three Gi proteins exhibited different rates of [32P]GDP release at 2 mM Mg2+. GDP release from Gi2 was severalfold faster than that from Gi1 or Gi3. GDP release rates from Gi1 and Gi3 were similar, although Gi3 was somewhat (60-80%) faster than Gi1. These data indicate that rates of GDP release and GTP binding may be independently regulated for these three proteins and that the relative proportions of Gi2/Gi1 or Gi2/Gi3 will be a crucial factor in determining the kinetics of signal transduction through Gi-coupled effectors.

The distribution and properties in brain of the alpha subunits of the major bovine brain Go isoforms, GoA, GoB and GoC, were characterized. The alpha(o)A and alpha(o)B isoforms arise from alternative splicing of RNAs from a single alpha(o) gene, whereas alpha(o)C is a deamidated form of alpha(o)A. All three Go isoforms purify from brain with different populations of betagamma dimers. This variable subunit composition of Go heterotrimers is likely a consequence of their functional differences. This study examined the biochemical properties of the alpha(o) isoforms to see if these properties explain the variable betagamma composition of their heterotrimers. The brain distribution of alpha(o)B differed substantially from that of alpha(o)A and alpha(o)C, as did its guanine nucleotide binding properties. The unique subunit composition of GoB can be explained by its expression in different brain regions. The alpha(o)A and alpha(o)C showed slight differences in guanine nucleotide binding properties but no preference for particular betagamma dimers when reassociated with a heterogeneous betagamma pool. The alpha(o)C protein occurred in a constant ratio to alpha(o)A throughout the brain, but was a much larger percent of total brain alpha(o) than previously thought, approximately 35%. These results suggest that alpha(o)A is a precursor of alpha(o)C and that the association of G(o)alpha subunits with different betagamma dimers reflects the function of an adaptive, G-protein signaling mechanism in brain.

Excess salt loads in most non-mammalian vertebrates are dealt with by a variety of extra-renal salt-secreting structures collectively described as salt glands. The best studied of these are the supra-orbital nasal salt glands of birds. Two distinct types of response to osmoregulatory disturbances are shown by this structure: a progressive adaptive response on initial exposure to a salt load that results in the induction and enhancement of the secretory performance or capabilities of the gland; and the rapid activation of existing osmoregulatory mechanisms in the adapted gland in response to immediate osmoregulatory imbalance. Not only is the time-frame of these two types of response very different, but the responses usually involve fundamentally different processes: e.g., the growth and differentiation of osmoregulatory structures and their components in the former case, compared with the rapid activation of ion channels, pumps etc. in the latter. Despite marked differences in the nature and time-frame of these responses, they both are apparently triggered by neuronally released acetylcholine, which acts at muscarinic receptors on the secretory cells to induce an inositol phosphate-dependent increase in cytosolic-free calcium concentrations ([Ca2+]i). Therefore, the question arises as to how the cells produce the appropriate distinct response using a single common signal (i.e., an increase in [Ca2+]i). Examination of the features of this signaling pathway in the two conditions described, reveals that they each are uniquely tuned to generate a response with the characteristics appropriate for the cells' requirements. This tuning of the signal involves often rather subtle changes in the overall signaling pathway that are part of the adaptive differentiation process.

Go is the major G protein in bovine brain, with at least three isoforms, GoA, GoB, and GoC. Whereas alphaoA and alphaoB arise from a single Goalpha gene as alternatively spliced mRNAs, alphaoA and alphaoC are thought to differ by covalent modification. To test the hypothesis that alphaoA and alphaoC have different N-terminal lipid modifications, proteolytic fragments of alphao isoforms were immunoprecipitated with an N terminus-specific antibody and analyzed by matrix-assisted laser desorption ionization mass spectrometry. The major masses observed in immunoprecipitates were the same for all three alphao isoforms and corresponded to the predicted mass of a myristoylated N-terminal fragment. Structural differences between alphaoA and alphaoC were also compared before and after limited tryptic proteolysis using SDS-polyacrylamide gel electrophoresis containing 6 M urea. Based upon the alphao subunit fragments produced under activating and nonactivating conditions, differences between alphaoA and alphaoC were localized to a C-terminal fragment of the protein. This region, involved in receptor and effector interactions, implies divergent signaling roles for these two alphao proteins. Finally, the structural difference between alphaoA and alphaoC is associated with a difference of at most 2 daltons based upon measurements by electrospay ionization mass spectrometry.

We report the direct interaction of Gbetagamma with the third intracellular (i3) loop of the M2- and M3-muscarinic receptors (MR) and the importance of this interaction relative to effective phosphorylation of the receptor subdomain. The i3 loop of the M2- and the M3-MR were expressed in bacteria and purified as glutathione S-transferase fusion proteins for utilization as an affinity matrix and to generate substrate for receptor subdomain phosphorylation. In its inactive heterotrimeric state stabilized by GDP, brain G-protein did not associate with the i3 peptide affinity matrix. However, stimulation of subunit dissociation by GTPgammaS/Mg2+ resulted in the retention of Gbetagamma, but not the Galpha subunit, by the M2- and M3-MR i3 peptide resin. Purified Gbetagamma bound to the M3-MR i3 peptide with an apparent affinity similar to that observed for the Gbetagamma binding domain of the receptor kinase GRK2 and Bruton tyrosine kinase, whereas transducin betagamma was not recognized by the M3-MR i3 peptide. Effective phosphorylation of the M3-MR peptide by GRK2 required both Gbetagamma and lipid as is the case for the intact receptor. Incubation of purified GRK2 with the i3 peptide in the presence of Gbetagamma resulted in the formation of a functional ternary complex in which Gbetagamma served as an adapter protein. Such a complex provides a mechanism for specific spatial translocation of GRK2 within the cell positioning the enzyme on its substrate, the activated receptor. The apparent ability of Gbetagamma to act as a docking protein may also serve to provide an interface for this class of membrane-bound receptors to an expanded array of signaling pathways.

1. Bradykinin has multiple effects on differentiated NG108-15 neuroblastoma x glioma cells: it increases Ins(1,4,5)P3 production and intracellular Ca2+ concentration [Ca2+]i evokes a Ca2+ activated K+ current (IK(Ca)) and inhibits M current (IM). We studied the effect of the aminosteroid U73122 and the antibiotic neomycin, both putative blockers of phospholipase C (PLC), on these four bradykinin effects. 2. Preincubation with 1 or 5 microM U73122 for 15 min partly suppressed Ins(1,4,5)P3 generation and the increase in [Ca2+]i induced by 1 microM bradykinin. U73122 10 microM caused total and irreversible inhibition. The inactive analogue U73343 was without effect. 3. Resting levels of Ins(1,4,5)P3 were not affected. However, resting [Ca2+]i was increased by 10 microM U73122, but not by U73343. Individual cells responded to 10 microM U73122 with a small increase in [Ca2+]i, followed in some cells by a large further rise. 4. Pretreatment of whole-cell clamped cells with 1 microM U73122 for 30 min reduced the bradykinin-induced IK(Ca) to a fifth of its normal size. To suppress it totally, a 7-12 min pretreatment with 5 microM U73122 was required. Again, U73343 was without effect. 5. U73122 and U73343 at concentrations of 5-10 microM irreversibly decreased the holding current (Ih) which at a holding potential of -30 or -20 mV mainly flows through open M channels. The decrease was often preceded by a transient increase. 6. M current (IM) measured with 1 s pulses, was also decreased by 5-10 microM U73122 and U73343, but short applications of U73122 could cause a small increase. The bradykinin-induced inhibition of IM was not affected by U73122. 7. Preincubation with 1 or 3 mM neomycin for 15 min did not affect Ins(1,4,5)P3 generation and the increase in [Ca2+]i induced by bradykinin. Pretreatment with 3 mM neomycin for about 20 min diminished the bradykinin-induced IK(Ca) to a fifth of its normal size. 8. The four main conclusions drawn from the results are: (a) U73122 suppresses bradykinin-induced PLC activation and IK(Ca), but not IM inhibition. (b) This indicates that the transient outward current IK(Ca), but not the decrease of IM in response to bradykinin, is mediated by PLC. (c) U73122 itself inhibits IM and mobilizes Ca2+ from intracellular stores. (d) Externally applied neomycin is not an effective inhibitor of PLC-mediated signalling pathways in NG108-15 cells.

The region encoded by amino acids 956-982 of adenylyl cyclase 2 is important for Gbetagamma stimulation. Interactions of a peptide encoding the 956-982 region of adenylyl cyclase 2 (QEHAQEPERQYMHIGTMVEFAYALVGK (QEHA peptide)) with Gbetagamma subunits were studied. QEHA peptide was covalently attached to beta subunit of free Gbetagamma by the cross-linker N-succinimidyl(4-iodoacetyl)aminobenzoate. Cross-linking was proportional to the amount of QEHA peptide added; other control peptides cross-linked minimally. When Go was used, very little cross-linking was observed with GDP and EDTA, but upon activation by guanosine 5'-3-O-(thio)triphosphate and Mg2+, specific cross-linking of the QEHA peptide to Gbeta was observed. We conclude that beta subunits of G proteins contain effector interaction domains that are occluded by Galpha subunits in the heterotrimer. Molecular modeling studies used to dock the QEHA peptide on to Gbeta indicate that amino acids 75-165 of Gbeta may be involved in effector interactions.

Among subfamilies of G-protein-coupled receptors, agonists initiate several cell signaling events depending on the receptor subtype (R) and the type of G-protein (G) or effector molecule (E) expressed in a particular cell. Determinants of signaling specificity/efficiency may operate at the R-G interface, where events are influenced by cell architecture or accessory proteins found in the receptor's microenvironment. This issue was addressed by characterizing signal transfer from R to G following stable expression of the alpha 2A/D adrenergic receptor in two different membrane environments (NIH-3T3 fibroblasts and the pheochromocytoma cell line, PC-12). Receptor coupling to endogenous G-proteins in both cell types was eliminated by pertussis toxin pretreatment and R-G signal transfer restored by reconstitution of cell membranes with purified brain G-protein. Thus, the receptor has access to the same population of G-proteins in the two different environments. In this signal restoration assay, agonist-induced activation of G was 3-9-fold greater in PC-12 as compared with NIH-3T3 alpha 2-adrenergic receptor transfectants. The cell-specific differences in signal transfer were observed over a range of receptor densities or G-protein concentration. The augmented signal transfer in PC-12 versus NIH-3T3 transfectants occurred despite a 2-3-fold lower level of receptors existing in the R-G-coupled state (high affinity, guanyl-5'-yl imidodiphosphate-sensitive agonist binding), suggesting the existence of other membrane factors that influence the nucleotide binding behavior of G-protein in the two cell types. Detergent extraction of PC-12 but not NIH-3T3 membranes yielded a heat-sensitive, macromolecular entity that increased 35S-labeled guanosine 5'-O-(thiotriphosphate) binding to brain G-protein in a concentration-dependent manner. These data indicate that the transfer of signal from R to G is regulated by a cell type-specific, membrane-associated protein that enhances the agonist-induced activation of G.

The effects of acute exposure to 25 mM ethanol on high voltage-activated, L-type Ca2+ channels in undifferentiated and nerve growth factor-treated pheochromocytoma (PC-12) cells were examined using conventional, whole-cell, patch-clamp techniques. Acute exposure to 25 mM ethanol inhibited macroscopic L-type Ca2+ currents in undifferentiated PC-12 cells significantly more than in nerve growth factor-treated PC-12 cells. Intracellular infusion with guanosine-5'-O-(2-thio)diphosphate or pretreatment with pertussis toxin reduced ethanol inhibition in undifferentiated cells without altering inhibition in nerve growth factor-treated cells, suggesting the involvement of a G protein in ethanol inhibition of Ca2+ channels in undifferentiated cells. Intracellular infusion with an affinity-purified antibody that recognizes the carboxyl termini of alpha i1 and alpha i2 significantly reduced ethanol inhibition in undifferentiated cells, in contrast to the effects of antibodies that recognize the carboxyl termini of alpha oA and alpha oB. None of these antibodies reduced ethanol inhibition in nerve growth factor-treated cells. These results indicate that Gi1 alpha or Gi2 alpha mediates ethanol inhibition of L-type Ca2+ channel currents in undifferentiated but not in nerve growth factor-treated PC-12 cells.

The diversity of the gamma subunits in bovine brain G protein preparations was investigated using matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Analysis of these G protein mixtures revealed at least four gamma subunit masses by the following four criteria. 1) The measured masses were in the same mass range as the predicted molecular weights of gamma isoforms. 2) The masses were reproducible between the same or different preparations of G proteins. 3) The masses were independent of the matrix used for MALDI analysis. 4) The masses comigrated with the gamma subunit, as part of the heterotrimer, the beta gamma dimer, or the separated gamma subunit. These measured masses were compared with those calculated from cDNA sequences of known bovine brain gamma isoforms with the addition of plausible post-translational modifications. The mass of each spectral peak was consistent with the calculated mass for only one of four known bovine brain gamma subunit isoforms, but the data suggest modifications of the gamma subunits in addition to those already known or suspected at their carboxyl termini. Besides these four major masses, several additional, less resolved spectral peaks were observed whose measured masses did not correlate with any known gamma subunit or plausible modification. MALDI mass spectrometry promises to be a powerful technique for the analysis of the diversity of the gamma subunit in G proteins and for the characterization of their post-translational modifications.

Macrodystrophia lipomatosa is a rare form of gigantism involving an extremity consisting of predominately fibroadipose tissue. Radiographically, it appears as hypertrophy of soft tissues and bone. CT and MR scanning delineate the tissues involved and are virtually diagnostic, demonstrating the predominantly adipose component as well as the overgrowth of the bones, nerves, and vessels also involved.

All eukaryotic cells use heterotrimeric GTP-binding proteins, G-proteins, in their responses to extracellular signals. Our current understanding of the transduction processes mediated by these proteins began with the discovery that GTP regulates glucagon binding to its receptor (Rodbell et al. 1971b), and that the nucleotide is required for hormone stimulation of adenylyl cyclase (Rodbell et al. 1971a). Those observations led to the discovery of the family of G-proteins and their widespread involvement in many different signal transduction pathways (Birnbaumer 1990; Gilman 1984). The responses of cells to individual signals using G-proteins are probably complex and seem to depend upon a large number of factors related to the diversity of G-proteins within the cell and their mechanism(s) of activation. The identification of these mechanisms has often involved studies of receptor regulation of adenylyl cyclase activity.

Heterotrimeric GTP-binding proteins from bovine brain were resolved by fast protein liquid chromatography chromatography using Mono Q columns. Two distinct forms of the protein Go were identified. Both forms had stochiometric amounts of alpha- and beta gamma-subunits. The a-subunits of both forms were recognized by an alpha o-specific antiserum, but not by any of the alpha i-specific antisera. The two forms showed distinct migration patterns on 9% sodium dodecyl sulfate-polyacrylamide gels containing 4-8 M urea gradients. Neither form comigrated with the recombinant alpha o1. Both the recombinant alpha o1 and the most abundant form of Go were recognized by an antiserum, H-660, against a peptide encoding amino acids 3-17 of alpha i2. H-660 has been shown previously to recognize alpha o and alpha i (Mumby, S. M., Pang, I. K., Gilman, A. G., and Sternweis, P. C. (1988) J. Biol. Chem. 263, 2020-2026). This more abundant form is called Go A most likely corresponds to the cloned alpha o1. The less abundant form, Go B, was not recognized by H-660. However, both forms of bovine brain Go were recognized by GC/2, an antiserum against the N-terminal region of alpha o1. Hence alpha oA and alpha oB may be different in their N terminus regions. Neither form of bovine brain Go was recognized by an antisera made to a peptide encoding the unique regions of the cloned alpha o2 from HIT cells (Hsu W. H., Rudolph, U., Sanford, J., Bertrand, P., Olate, J., Nelson, C., Moss, L.E., Boyd, A. E., III, Codina, J., and Birnbaumer, L. (1990) J. Biol. Chem. 265, 11220-11226). Go A and Go B have similar guanine nucleotide binding and release properties. Both release GDP within 1 min in the absence of added Mg2+. Both bind guanosine (GTP gamma S) rapidly as well. However Go A binds GTP gamma S about 2.5-fold faster than Go B, in the absence of added Mg2+ ion. Both forms of Go as well as the recombinant alpha o (alpha o1) can increase muscarinic stimulation of inositol trisphosphate-mediated Cl- current in Xenopus oocytes. These data indicate that we have identified two structurally distinct forms of Go that have different guanine nucleotide binding properties and are capable of functioning in the receptor-regulated phospholipase C pathway in Xenopus oocytes.

S49 mouse lymphoma cells contain a beta-adrenergic receptor coupled to Gs that stimulates adenylyl cyclase and a somatostatin receptor coupled to Gi that inhibits adenylyl cyclase. Membranes from these cells were used to compare the inhibitory effects of somatostatin and G protein beta gamma complex to determine under what conditions beta gamma is likely to be a mediator of somatostatin action. Somatostatin was equally effective at inhibiting basal adenylyl cyclase activity in the presence of GTP, forskolin-stimulated activity, and hormone-stimulated activity. G protein beta gamma was more effective at inhibiting basal activity than was somatostatin, and these effects were partially additive. In the presence of forskolin, the two inhibitors were equally effective and not additive. In the presence of isoproterenol, beta gamma was much less effective than somatostatin, and the two inhibitors did not have additive or synergistic effects. At very high concentrations beta gamma did inhibit isoproterenol stimulation of adenylyl cyclase, although its effects were not saturating even at 100 micrograms/ml. Under conditions where beta gamma did inhibit hormone stimulation, beta gamma was a mixed inhibitor of isoproterenol stimulation, proportionally decreasing the maximum effect of the hormone and increasing the half-maximally effective concentration. Somatostatin, on the other hand, was a simple noncompetitive inhibitor of isoproterenol stimulation. These results indicate that beta gamma and somatostatin inhibit adenylyl cyclase by different mechanisms, at least in the presence of hormones that stimulate the enzyme. It is proposed that alpha i is the primary mediator of hormone inhibition of adenylyl cyclase when Gs is activated by a hormone, but that beta gamma may have a role as a mediator of inhibition of basal activity.

The native pertussis toxin sensitive GTP-binding proteins (Gi proteins) were individually resolved, and their guanine nucleotide binding and release properties were studied. Gi2 and Gi3, the two major GTP-binding proteins of human erythrocytes, were purified to apparent homogeneity by fast protein liquid chromatography. Gi1 was purified from bovine brain. The three proteins bound 0.6-0.85 mol of guanosine 5'-O-(thio-triphosphate (GTP gamma S)/mol of protein with similar affinities (KD(app) = 50-100 nM). The rate of [35S]GTP gamma S binding to Gi2 was 5-8-fold faster than to Gi1 or Gi3 at 2 mm Mg2+. There were no observable differences in the binding characteristics between bovine brain Gi1 and human erythrocyte Gi3. At 50 mM Mg2+, all three Gi proteins exhibited fast binding, although Gi1 and Gi3 were marginally slower than Gi2. All three Gi proteins exhibited different rates of [32P]GDP release at 2 mM Mg2+. GDP release from Gi2 was severalfold faster than that from Gi1 or Gi3. GDP release rates from Gi1 and Gi3 were similar, although Gi3 was somewhat (60-80%) faster than Gi1. These data indicate that rates of GDP release and GTP binding may be independently regulated for these three proteins and that the relative proportions of Gi2/Gi1 or Gi2/Gi3 will be a crucial factor in determining the kinetics of signal transduction through Gi-coupled effectors.

Thyroid iodide was separated from organic iodine by gel electrophoresis of thyroid lobes 72 h after their labeling with radioiodine. Iodide thus measured accounted for 0.233% of total thyroid iodine, on the average. The lower and upper limits of the contribution of iodide due to a method artifact were established, respectively, by a second electrophoresis of thyroid lobes from which iodide had already been removed and by discharging transported iodide with perchlorate and blocking formation of iodide from organic precursors in vivo with 100 μmol mononitrotyrosine (MNT) and 20 mg propylthiouracil (PTU). They were thus found to be 0.042% and 0.064% of total thyroid iodine. In experiments in which iodide (corrected for 0.05% of the label taken to represent artifactitious iodide) averaged 0.131% of the total thyroid iodine, MNT lowered its level by 50%, perchlorate by 23%, and PTU by 15%. TSH (maximally effective dose, 1 IU) elevated thyroid iodide concentration within 1 h after its injection. Prior or concur...

Modification of bovine brain G proteins by an N-hydroxysuccinimide ester of biotin has been studied. In the presence of GDP, but in the absence of Mg2+, neither guanine nucleotide binding nor GTPase activity of the protein was altered by modification using less than 1.25 mM biotin derivative with 1 mg/ml G protein. Under these conditions the alpha subunit was modified more extensively than the beta and gamma subunits. However, biotinyl-alpha was less readily bound to streptavidin-agarose than was the less modified beta subunit. Biotinyl-beta gamma was isolated from the modified, intact G protein and further characterized to determine if biotinylation alters its functional properties. Isolated biotinyl-beta gamma and unmodified beta gamma were equivalent based upon: 1) inhibition of the S49 cell membrane adenylyl cyclase, 2) changes in hydrodynamic parameters after being recombined with isolated alpha and treated with guanine nucleotides or complexes of fluoride and aluminum, and 3) competition for isolated alpha binding to biotinyl-beta gamma immobilized previously on streptavidin-agarose. Biotinyl-beta gamma prebound to streptavidin-agarose was 70-100% functional, based upon binding of isolated alpha subunits. Estimates of the affinity of alpha binding to biotinyl-beta gamma indicate that bovine brain alpha 41 has a 10-15-fold higher affinity for beta gamma than does alpha 39. Nonhydrolyzable guanine nucleotides and complexes of fluoride and aluminum decreased binding of either alpha 39 or alpha 41 to biotinyl-beta gamma, and these effects were dependent upon the amount of Mg2+ present. GTP decreased binding of alpha 39, but not alpha 41, to biotinyl-beta gamma. These results indicate that GTP can affect G protein subunit interactions and that its effects do not necessarily require an intact membrane environment or the participation of activating receptors or other membrane-associated proteins. They further indicate that biotinylation of beta gamma does not alter its functional properties and that it can be used for studying G protein subunit interactions.

Thyroid radioiodide to serum radioiodide concentration (*T/*S) ratios 1 h after 131I administration were 3 times higher in rats pretreated with propylthiouracil (PTU) than in rats with binding thyroids. Although indications of a preponderance of transported iodide in the thyroid early after 131I injection were found, a reduction of the 1-h *T/*S ratio in TSH-treated rats by the inhibitor of iodotyrosine dehalogenation, mononitrotyrosine, points to an early contribution of intrathyroidally generated (internal) iodide to total thyroid iodide. In rat thyroids labeled 72 h earlier, there was no rise in electrophoretically separated radioiodide 2 h after the administration of PTU alone, but the elevation of thyroid iodide due to TSH was augmented by concurrently given PTU. On the assumption that PTU affects transported components of thyroid iodide identically whether they are labeled 1 or 72 h earlier, we conclude that PTU must severely depress the generation of internal iodide. This effect is no longer evident if TSH is given simultaneously. Further, our results are consistent with the hypothesis that internal iodide passes through a stage in which it is not available for organic binding before it mixes with transported iodide. TSH appears to facilitate the transfer of internal iodide into the pool which it shares with external iodide.

This study was undertaken to determine if the categorization of Neisseria gonorrhoeae strains into disseminated gonococcal infection (DGI) and non-DGI types was also paralleled by some common characteristic in their penicillin-binding proteins (PBPs). Fluorography of sodium dodecyl sulfate-containing polyacrylamide gels, on which the [14C]penicillin-labeled PBPs had been separated, was used to visualize the PBPs. No common characteristic PBP for genital or DGI strains was observed. Apart from PBPs 1 and 4, which were observed in all cases, there was an apparently random distribution of other PBPs in both types of N. gonorrhoeae. It was concluded from these data that the penicillin susceptibility associated with most DGI strains of N. gonorrhoeae cannot be correlated with any specific changes in their PBP patterns.

A new femur distractor has been developed, lengthening the leg. This instrument is electronically controllable and can be implanted totally. It is possible to regulate transcutaneously by radio control the operation "forward motion", "stop" and "backward motion". After testing this instrument in sheep, the lengthening of human femur by this system can be introduced.

Zur operativen Beinverlangerung wurde ein neues Femur-Distraktionsgerat entwickelt. Das Gerat besteht aus einem elektronisch programmierbaren Distraktionsaggregat, einschlieslich Kraftquelle und einer zweiteiligen Fuhrungsschiene. Das Gesamtsystem ist voll versenkbar und schafft Ubungsstabilitat wahrend der Distraktionsphase. Die Funktionen „Vorlauf — Stop — Rucklauf“ konnen transkutan induktiv gesteuert werden. Ein fur das Tierexperiment speziell entwickelter Prototyp des Gerates wurde an Schafen erfolgreich erprobt. Die Langendifferenzen der unteren Extremitaten stellen eine schwere Storung der kinetischen Gliederkette dar. Die Storung der Statik fuhrt zur Fehlbelastung der Gelenke und damit zur konsekutiven Arthrose. Der durch die Beinlangendifferenz verursachte Schaden ubersteigt somit weitaus den Grad der „kosmetischen Storung“.

cyc- S49 cell membranes contain an adenylyl cyclase activity which is stimulated by forskolin and inhibited by guanine nucleotides and NaF. These inhibitory effects are mediated by an inhibitory guanine nucleotide-binding regulatory component (Ni) affecting the adenylyl cyclase catalytic unit (Hildebrandt, J. D., Sekura, R. D., Codina, J., Iyengar, R., Manclark, C. R., and Birnbaumer, L. (1983) Nature (Lond.) 302, 706-709). Since cyc- S49 cells do not contain a stimulatory guanine nucleotide-binding regulatory component (Ns), these membranes were used to study the requirements and kinetics of activation of Ni in the absence of Ns. Activation of Ni by guanyl-5'-yl imidodiphosphate was time-dependent (i.e. hysteretic) and pseudo-irreversible. Although GTP and guanosine 5'-(beta-thio)diphosphate could prevent the inhibition caused by guanyl-5'-yl imidodiphosphate if added simultaneously with it, they could not reverse the inhibited state induced by previous exposure to guanyl-5'-yl imidodiphosphate. Activation of Ni had an absolute requirement for Mg2+. Unlike the activation of Ns, however, which requires millimolar concentrations of Mg2+ in the absence of hormonal stimulation, activation of Ni requires only micromolar concentrations of the divalent cation. These results support the contention that hormones which activate Ni or Ns do so by altering different parameters of a similar activation mechanism.

Principal outer membrane protein (protein I) of Neisseria gonorrhoeae was prepared nearly free of lipopolysaccharide (LPS) and substantially purified from other membrane proteins by chromatography of partially purified gonococcal outer membranes over Sepharose 6B in the presence of deoxycholate at pH 9.0. This protein I of nine separate antigenic types was coated to polystyrene tubes and used in the enzyme-linked immunosorbent assay (ELISA) to measure antibody to protein I or in inhibition tests to quantitate protein I antigen. No significant inhibition of the ELISA test was produced by purified LPS from the strain used to prepare each of the protein I types or by whole gonococci bearing the same LPS but different protein I antigens as the strain used to produce a given protein I antigen. Of 125 strains of gonococci used as whole organisms to inhibit the protein I ELISA, 124 (99%) typed with one or more of the nine protein I types, and 35% of these typed with a single protein I serotype. Sixty-one of 65 (94%) strains from Seattle and Atlanta patients with disseminated gonococcal infection contained protein I serotype 1, and 16 of 24 (64%) strains from Seattle patients with salpingitis bore one or both of protein I serotypes 1 and 2.

Cyc- S49 mouse lymphoma cell membranes contain an adenylyl cyclase system which is deficient in the regulatory properties characteristic of a stimulatory guanine nucleotide-binding regulatory component (Ns), such as enhancement of activity in the presence of GTP, GTP analogues, and NaF. Detailed kinetic analysis of cycl- adenylyl cyclase showed, however, that it is not unresponsive to these agents, for their addition to assays elicited inhibitory effects. Inhibition of cyc- activity was seen in 11 different membrane preparations obtained from two different strains and was observable both in the absence and presence of the strongly stimulatory diterpene forskolin. The GTP analogues GTP gamma S and guanyl-5'-yl imidodiphosphate caused a maximum of 60% inhibition with IC50 values of 2 and 12 nM, respectively. GTP itself was less potent than its analogues, with an IC50 of 100 mM, and elicited less inhibition as well (a maximum of 25%). Cholera toxin treatment of cyc- S49 cell membranes, under conditions which appeared to maximally activate the adenylyl cyclase system of wild type S49 cell membranes, had no effect on inhibition of catalytic activity. Our results indicate the presence in cyc- S49 cell membranes of a guanine nucleotide-binding component which is inhibitory to adenylyl cyclase activity (Ni). This suggests (a) that these membranes cannot be considered as completely deficient of adenylyl cyclase guanine nucleotide-binding regulatory components, and (b) that these membranes offer a unique opportunity to study Ni-mediated effects of guanine nucleotides and fluoride in the functional absence of Ns-mediated effects of these agents.

Thyroid iodide labeled 72 h earlier with 131I was separated from organic iodine by polyacrylamide gel electrophoresis. The concentration of thyroid radioiodide was not significantly diminished 2 h after the administration of perchlorate (100 mg NaClO4) alone, but perchlorate reduced the rise in glandular radioiodide caused by simultaneously given TSH (1 IU). When propylthiouracil (PTU; 20 mg) was given along with it, perchlorate decreased thyroid radioiodide even in rats not treated with TSH. In rats given perchlorate, PTU caused a much slighter augmentation of the TSH-induced increase in the thyroid radioiodide concentration than in the absence of perchlorate. These results are interpreted as follows. Perchlorate-discharged iodide in rats not given TSH is largely transported iodide. Perchlorate can discharge intrathyroidally generated (internal) iodide too, but this is unequivocally reflected by a decrease in the thyroid iodide concentration only when the production of internal iodide is enhanced by TSH. Perchlorate in its own right interferes with organic binding of internal iodide, thereby partially preempting the effect whereby PTU causes accumulation of internal iodide in TSH-treated rats. We suggest that internal thyroid iodide is transported from its site of generation, the follicular cell, to its site of organic binding, the follicular lumen, by perchlorate-sensitive transport. It is also possible that internal iodide escaping from a follicle is conserved by perchlorate-inhibitable reuptake into follicular cells closer to the venous end of the capillary bed.

Modification of bovine brain G proteins by an N-hydroxysuccinimide ester of biotin has been studied. In the presence of GDP, but in the absence of Mg2+, neither guanine nucleotide binding nor GTPase activity of the protein was altered by modification using less than 1.25 mM biotin derivative with 1 mg/ml G protein. Under these conditions the alpha subunit was modified more extensively than the beta and gamma subunits. However, biotinyl-alpha was less readily bound to streptavidin-agarose than was the less modified beta subunit. Biotinyl-beta gamma was isolated from the modified, intact G protein and further characterized to determine if biotinylation alters its functional properties. Isolated biotinyl-beta gamma and unmodified beta gamma were equivalent based upon: 1) inhibition of the S49 cell membrane adenylyl cyclase, 2) changes in hydrodynamic parameters after being recombined with isolated alpha and treated with guanine nucleotides or complexes of fluoride and aluminum, and 3) competition for isolated alpha binding to biotinyl-beta gamma immobilized previously on streptavidin-agarose. Biotinyl-beta gamma prebound to streptavidin-agarose was 70-100% functional, based upon binding of isolated alpha subunits. Estimates of the affinity of alpha binding to biotinyl-beta gamma indicate that bovine brain alpha 41 has a 10-15-fold higher affinity for beta gamma than does alpha 39. Nonhydrolyzable guanine nucleotides and complexes of fluoride and aluminum decreased binding of either alpha 39 or alpha 41 to biotinyl-beta gamma, and these effects were dependent upon the amount of Mg2+ present. GTP decreased binding of alpha 39, but not alpha 41, to biotinyl-beta gamma. These results indicate that GTP can affect G protein subunit interactions and that its effects do not necessarily require an intact membrane environment or the participation of activating receptors or other membrane-associated proteins. They further indicate that biotinylation of beta gamma does not alter its functional properties and that it can be used for studying G protein subunit interactions.

Plastid DNA of the light green Oenothera plastome mutant sigma, from plastome I, which is deficient in ribulose bisphosphate carboxylase, has been compared with wild-type chloroplast DNA from plastome I and the related plastome IV. For this, double digestions with the restriction endonucleases Sal I, Pst I and Kpn I were used. Chloroplast DNA from plastomes I and IV differs in the sizes of several fragments, with the changes being from under 0.1 to about 0.6 Md in size. In the cleavage patterns of the mutant DNA compared to the wild-type DNA from plastome I, the only differences observed are two possible deletions of less than 0.1 Md from a fragment known to partly cover the genes for the ribosomal RNAs and from a fragment located in the small single-copy region of the molecule. It is concluded that the ribulose bisphosphate carboxylase deficiency in this mutant is not caused by a major deletion in the plastid DNA.

The autotrophic eukaryotic cell is a highly complex system; its biogenesis demands a subtle interplay of three genetic compartments: nucleus/cytosol, plastids, and mitochondria. The genetic material of plastids, the plastome (Renner 1934), possesses unique features which allow us to probe the cooperation between intracellular genetic compartments as well as gain insight into the evolution of this system. The primary attributes of plastid DNA (ptDNA) are that it is moderately complex, structurally defined, and does not appear to encode only ancillary functions. In vascular plants the plastid genetic information is deposited in a single, highly reiterated circular DNA molecule of about 150 kilobase pairs (kbp), a size that corresponds roughly to a coding potential of 200 polypeptides of average molecular weight. Although the sizes of plastid chromosomes of the vascular plants studied to date are remarkably uniform (for review see Herrmann and Possingham 1980), they can vary from at least 120 kbp in the liverwort Sphaerocarpos (Herrmann et al. 1980) or the Xanthophycean alga Vaucheria (Kowallik and Hennig unpublished) to 200 kbp in Chlamydomonas reinhardii (Behn and Herrmann 1977).

The effect of GTP analogs and Mg on the structure of Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclase, were studied in a comparative manner. Both N proteins, which are alpha beta gamma heterotrimers that differ in their alpha subunits, when exposed to GTP analogs underwent a Mg-dependent conformational change that was not dependent on subunit dissociation. This was seen both as change in sedimentation behavior at 4 degrees C from 4 S to about 3 S and by a property of the new conformation to retain guanine nucleotide tightly bound to it. Warming to 32 degrees C promoted subunit dissociation, each protein giving a mixture of 2 S alpha G and 2 S beta gamma complexes. For both Ns and Ni, these reactions were reversible: 2 S complexes of Ns and Ni associated to 3 S forms on cooling to 4 degrees C, provided the Mg concentration was at or below 10 mM and detergent concentration was below 1%, and the 3 S complexes of these proteins reverted to 4 S forms and released the nucleotide in the cold on chelation of free Mg with EDTA. Reconstitution assays with Ns-deficient membranes from cyc- S49 lymphoma cells revealed that the 3 S form of Ns is a "pre-active" form of the protein. The scheme below summarizes these findings, where G represents a guanine nucleotide. (Formula: see text) Ns and Ni differ in that more Mg is necessary to promote the 4 S to 3 S conversion of Ns than of Ni, and in that both the 2 S to 3 S to 4 S conversions proceeded more readily with Ni than with of Ns. Mg could not be shown to promote subunit dissociation. The above scheme is suggested as a plausible description of the reaction sequence leading from an unactivated to an activated N protein.

The adenylate cyclase (AC) system is responsible for the synthesis of the second messenger cAMP which mediates the effects of many hormones and neurotransmitters on their target tissues. This system is located within the plasma membrane of target cells and is responsible for the vectorial transduction of receptor occupancy by hormones into an altered rate of cyclic AMP synthesis. A complete AC system (Figure 1) responds to both stimulatory and inhibitory hormonal regulation (Cooper, 1982). In addition to receptors (R) and the catalytic unit (C), which actually synthesizes cAMP, an AC system is composed of two N (or G) coupling proteins that upon activation regulate the activity of C (Figure 2).

Treatment of MDCK cells with glucagon results in decreases in glucagon, NaF and prostaglandin E1-stimulated adenylyl cyclase activities, indicating the occurrence of a heterologous desensitization process. The extent of desensitization was time and glucagon concentration dependent. Maximal desensitization (30-50% decrease in stimulation by various effectors) was obtained by 4 h at 100 nM glucagon. Glucagon also induced homologous desensitization since after treatment, the Kact of glucagon was specifically increased. Treatment of cells with 10 microM 8-bromoadenosine 3':5'-monophosphate or 10 microM forskolin resulted in decreased hormonal (glucagon and prostaglandin E1) stimulation without any decrease in the stimulation by nonhormonal effectors (NaF, forskolin, and guanyl-5'-yl imidodiphosphate). The stimulatory regulator (Ns) of the adenylyl cyclase system was analyzed after desensitization with glucagon and no measurable changes in the apparent levels of the alpha s subunits of Ns or the activity of Ns as assessed by reconstitution of the cyc- S49 cell membrane adenylyl cyclase were detected. Levels of the alpha i subunit of the inhibitory regulator (Ni) were monitored by labeling with [32P]NAD and pertussis toxin. Membranes of glucagon-treated cells showed a 2-fold increase in the amount of alpha i labeled. Addition of pure Ns to glucagon-treated MDCK cell membranes restored full stimulation by NaF but did not restore stimulation by prostaglandin E1 or glucagon. It is concluded that glucagon induces heterologous and homologous desensitization of the MDCK cell adenylyl cyclase. The locus of the heterologous desensitization is at the level of the regulatory components. Decreased stimulation is thought to occur due to either an increase in the levels of Ni or due to altered interactions between the subunits of Ni.