Alfred Gilman

EXPRESSION OF G-PROTEIN ALPHA SUBUNITS IN ESCHERICHIA COLI

Publisher Summary This chapter describes a general method for expressing several G-protein α subunits in Escherichia coli (E. coli) at levels 10-100 times higher than achieved previously. G proteins are a family of guanine nucleotide-binding regulatory proteins that link a large number of cell surface receptors to regulation of several intracellular effectors. The chapter describes a method for purification of the recombinant proteins by affinity chromatography on a resin containing chelated Ni2+ after addition of an amino-terminal hexahistidine tag to the recombinant protein. Such purification is rapid and results in the isolation of highly purified protein in a single step. Furthermore, the introduction of a tobacco etch virus (TEV) polyprotein cleavage site between the hexahistidine tag and the G-protein α subunit permits the efficient removal of the tag by recombinant TEV protease.

Allosteric determinants in guanine nucleotide-binding proteins.

Members of the G protein superfamily contain nucleotide-dependent switches that dictate the specificity of their interactions with binding partners. Using a sequence-based method termed statistical coupling analysis (SCA), we have attempted to identify the allosteric core of these proteins, the network of amino acid residues that couples the domains responsible for nucleotide binding and protein–protein interactions. One-third of the 38 residues identified by SCA were mutated in the G protein Gsα, and the interactions of guanosine 5′-3-O-(thio)triphosphate- and GDP-bound mutant proteins were tested with both adenylyl cyclase (preferential binding to GTP-Gsα) and the G protein βγ subunit complex (preferential binding to GDP-Gsα). A two-state allosteric model predicts that mutation of residues that control the equilibrium between GDP- and GTP-bound conformations of the protein will cause the ratio of affinities of these species for adenylyl cyclase and Gβγ to vary in a reciprocal fashion. Observed results were consistent with this prediction. The network of residues identified by the SCA appears to comprise a core allosteric mechanism conferring nucleotide-dependent switching; the specific features of different G protein family members are built on this core.

Targeted knockdown of G protein subunits selectively prevents receptor-mediated modulation of effectors and reveals complex changes in non-targeted signaling proteins

Heterotrimeric G protein signaling specificity has been attributed to select combinations of Gα, β, and γ subunits, their interactions with other signaling proteins, and their localization in the cell. With few exceptions, the G protein subunit combinations that exist in vivo and the significance of these specific combinations are largely unknown. We have begun to approach these problems in HeLa cells by: 1) determining the concentrations of Gα and Gβ subunits; 2) examining receptor-dependent activities of two effector systems (adenylyl cyclase and phospholipase Cβ); and 3) systematically silencing each of the Gα and Gβ subunits by using small interfering RNA while quantifying resultant changes in effector function and the concentrations of other relevant proteins in the network. HeLa cells express equimolar amounts of total Gα and Gβ subunits. The most prevalent Gα proteins were one member of each Gα subfamily (Gαs, Gαi3, Gα11, and Gα13). We substantially abrogated expression of most of the Gα and Gβ proteins expressed in these cells, singly and some in combinations. As expected, agonist-dependent activation of adenylyl cyclase or phospholipase Cβ was specifically eliminated following the silencing of Gαs or Gαq/11, respectively. We also confirmed that Gβ subunits are necessary for stable accumulation of Gα proteins in vivo. Gβ subunits demonstrated little isoform specificity for receptor-dependent modulation of effector activity. We observed compensatory changes in G protein accumulation following silencing of individual genes, as well as an apparent reciprocal relationship between the expression of certain Gαq and Gαi subfamily members. These findings provide a foundation for understanding the mechanisms that regulate the adaptability and remarkable resilience of G protein signaling networks.

Sodium Acetate as a Source of Fixed Base.

Summary Studies in dogs and in man demonstrate that sodium acetate is an easily prepared and a rapidly available non-toxic source of fixed base suitable for parenteral administration when alkalinization is indicated.

CLINICAL USES OF 2,3-DIMERCAPTOPROPANOL (BAL). X. THE TREATMENT OF ACUTE SYSTEMIC MERCURY POISONING IN EXPERIMENTAL ANIMALS WITH BAL, THIOSORBITOL AND BAL GLUCOSIDE

Although chemical agents were not employed as offensive weapons during the past war, nevertheless it was imperative to pursue vigorously investigations on the treatment of chemical warfare casualties. Inasmuch as a number of chemical warfare agents contain arsenic, attention was focused on the synthesis of compounds capable of antagonizing the local and systemic effects of ar-senical vesicants. These investigations, initiated by the British, led to the synthesis of 2,3-dimer-captopropanol, which is now more familiarly known as BAL (British Anti-Lewisite). In addition to BAL, numerous other SH-containing compounds, including monothiols and dithiols of polyhydric alcohols, were synthesized by the American and British investigators. Among these were thiosorbitol, prepared at the du Pont de Nemours and Company laboratories, and the glu-coside of BAL, first synthesized by Danielli and co-workers (1). The historical background which led to the synthesis of BAL and its derivatives, and the basic contributions which have resulted-from studies on the effect of mercaptans on the toxic action of arsenic and other heavy metals, have been summarized by Peters (2) and Waters and Stock (3). Pertinent to the background of the present study were the observations supporting the general hypothesis that heavy metals are toxic to biological systems because of their reaction with SH groups of the protein moiety of cellular enzymes to form mercaptides. That mercury shares in this action has been demonstrated by Barron and co-workers (3). Moreover, BAL is capable of reactivating enzyme systems poisoned by mercury, a fact which both affords support to the theory of the mechanism of inactivation, and gives promise for the therapeutic efficacy of mercaptans in the treatment of mercury poisoning. The present report is concerned with the efficacy of BAL, BAL glucoside and thiosorbitol in the treatment of experimental , acute, systemic mercury poisoning in rabbits and dogs. The reactions between mercaptans and Hg++ in uitro The reactions between mercaptans and cationic mercury which occur in vitro are presumably indicative of the expected interaction of the two agents in vivo. A brief study of the chemical reactions between the various mercaptans and mercury was, therefore, undertaken. Reactions zvith BAL. When solutions of HgCl, (0.1 M) were added to non-buffered, aqueous solutions of BAL (0.05 M) a copious, flocculent white precipitate formed. Titration with phenolphthalein as an indicator revealed the formation of 2 equivalents of H+ for each mol of HgCl, added. The white precipitate was insoluble in alkali, but could be …

Assay of RGS protein activity in vitro using purified components

Single-turnover and steady-state GTPase assays are an effective means to identify and characterize interactions between RGS and G alpha proteins in vitro. The advantage of the single turnover GTPase assay is that it permits simple and rapid assessment of RGS protein activity toward a putative G alpha-GTP substrate. Moreover, once an interaction between an RGS protein and a G alpha-GTP subunit has been identified, the single-turnover assay can be used to determine Michaelis-Menten constants and/or KI values for other competing G alpha substrates. A disadvantage of the single-turnover assay is that a negative result does not preclude the possibility of an interaction between given RGS and G alpha proteins in vivo. Inappropriate reaction conditions or the presence (or absence) of appropriate posttranslational modifications may result in small or undetectable increases in RGS protein-dependent GTPase activity. In these cases it may be tempting to examine RGS protein activity using steady-state GTPase assays in phospholipid vesicles reconstituted with receptors and heterotrimetric G proteins. The advantage to monitoring steady-state GTPase activity in reconstituted proteoliposomes is that ligand-dependent activation of the receptor facilitates GDP dissociation, such that effects of RGS proteins can be observed; multiple cycles of GTP binding and hydrolysis then amplify the GTPase signal. Additionally, the presence of the phospholipid membrane can increase the local RGS protein concentration approximately 10(4)-fold, permitting observation of interactions that are weak in solution. The primary disadvantage of the reconstituted system is the requirement for receptor purification, a technically demanding undertaking in comparison to the purification of G alpha, G beta gamma, and most RGS proteins.

[16] Purification of recombinant Gqα, G11α, and G16α from Sf9 cells

Publisher Summary This chapter discusses purification of recombinant G q α, G 11 α, and G 16 α from Sf9 cells. The G q α subfamily consists of five members: G q α, G 11 α, G 14 α, G 15 α, and G 16 α. The G 15 α and G 16 α are similar to one another (85% identity) and may represent the mouse and human homologs of the same gene. All members of the Gq class lack the cysteine residue near the carboxyl terminus that is covalently modified (ADP-ribosylated) by pertussis toxin in members of the G i class of α subunits. Thus, G q family members are thought to couple cell surface receptors to signaling pathways that are insensitive to this toxin. A wide variety of hormones, neurotransmitters, and growth factors activate phospholipase C (PLC) which, in turn, generates two second messengers, inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] and diacylglycerol. Members of the Gq subfamily have been shown to link cell surface receptors to stimulation of the three known β isoforms of PLC.

Purification and functional analysis of Ric-8A: A guanine nucleotide exchange factor for G-protein α subunits

Abstract Ric-8A (synembryn) has been shown to accelerate the in vitro guanine nucleotide exchange activities of most G-protein α subunits (with the exception of Gαs). Methods are presented in this article for the purification of Ric-8A and functional analysis of the effects Ric-8A has on G-protein α subunit guanine nucleotide-binding activities. The use of Ric-8A to prepare GTPγS-Gα and nucleotide-free Gα (in complex with Ric-8A) is described.

Ion Transport in the Gut

The in vitro preparation of the small intestine of the rat is well suited for studies of ion transport. Data from this preparation indicate that the passage of water and electrolytes across the intestinal epithelium depends on active transport. Glycolysis seems to be involved as a source of energy for the transport of ions from the mucosal to the serosal aspect of the intestinal wall.

The mechanism of diuretic action of the carbonic anhydrase inhibitors.

A discussion of the mechanism of action of carbonic anhydrase (CA) inhibitors is somewhat simplified by the information implicit in the title of this paper. We are dealing with a group of drugs highly specific in action. Moreover, the action of these compounds is restricted to the inhibition of a single enzyme, carbonic anhydrase. There are only a few pharmacodynamic agents whose mechanism of action has been so sharply defined. As a result, compounds such as acetazolamide have not only proved useful in pharmacotherapy, but have also been of great heuristic value in the study of the formation of urine, gastric juice, pancreatic juice, cerebrospinal fluid, and the ocular fluids. The carbonic anhydrase inhibitors are exemplary drugs in another way. They were developed, not as a result of blind screening, but rather as the result of a planned approach. The chemical agents were deliberately fashioned to inhibit an enzyme that was presumed to have important functions in the sequence of events involved in H+ transport. Their potent pharmacodynamic actions attest to the validity of the presumption. No discussion of the carbonic anhydrase inhibitors can be initiated without tribute to the pioneer investigations of F. J. W. Roughton and H. W. Davenport and their associates, which defined the function and distribution of the enzyme. However, in the brief time available, consideration must be limited to the effects of carbonic anhydrase inhibitors on renal tubular function and their role as diuretic drugs. Soon after the introduction of sulfanilamide as a chemotherapeutic agent it was appreciated that the drug produced abnormalities in acid-base metabolism; namely, a metabolic acidosis that was initiated by a rise in urine pH and an increase in bicarbonate Moreover, it was demonstrated that sulfanilamide was a potent inhibitor of carbonic anhydraseP Soon thereafter, Davenport and Wilhelmi4 reported that this enzyme was present in renal tissue in a high concentration. It was therefore assumed that carbonic anhydrase was essential for the renal tubular reabsorption of bicarbonate ion, and that inhibition of the enzyme depressed the transport of bicarbonate ion from tubular urine to tubular cell. This was a t a time when Arthur R. Cushny’s theory of renal function dominated thought on the subject. It will be recalled that Cushny was of the opinion that all the eventual constituents of the urine were present in the glomerular filtrate. Acidification of the urine was believed to result from the selective reabsorption of the basic components of the buffer systems present in the glomerular filtrate. Thus, inhibition of bicarbonate reabsorption was believed to be an adequate explanation of the effects of sulfanilamide on acid-base metabolism and renal function.