Structure and function of tyrosine kinase receptors

Over the past ten years, several growth factor receptors have been shown to be ligand-regulated tyrosine kinases. Tyrosine kinase activity is essential for signal transmission, suggesting that phosphorylation cascades may play an important role. Considerable effort has gone into understanding the structure and function of tyrosine kinase receptors in order to define their mechanisms of signal transmission. However, the protein substrates of the receptor kinases have proven to be difficult to isolate and clone. This review focuses on the receptors for insulin, epidermal growth factor, and platelet-derived growth factor. They are all tyrosine kinases, but emerging evidence suggests that they utilize multiple separate signal transduction pathways. Work carried out during the next several years should yield considerable insight into the complexity of the components which interact with these tyrosine kinase receptors to regulate cellular growth and metabolism.     

Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport.

Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically.     

Measurements of transmembrane pH differences of low extents in bacterial chromatophores

In chromatophores from photosynthetic bacteria the interaction of the fluorescent monoamine, 9-amino, 6-chloro, 2-methoxyacridine (ACMA), with the membrane is evaluated and described by an S-shaped adsorption isotherm. This phenomenon is hysteretic, as indicated by the difference between the adsorption and desorption branches of the binding isotherm. Maximal saturation of adsorption is reached at one ACMA per one to four lipid molecules, indicating that the probe binds in its neutral form. Adsorption of the probe on the membrane causes a large quenching of its fluorescence, which is explaind as being due to hypochromic effects following stacking and aggregation in a medium of low dielectric constant. A further quenching of fluorescence is brought about by imposing artificially induced transmembrane pH's. This latter phenomenon titrates in at increasing pH values and approaches saturation when pH is 2. The dependence of pH on the observed quenching of fluorescence is predicted by considering a model based on the equilibrium distribution of the amine between two phases at different pH's, in which adsorption of the probe on the membrane is used to evaluate its free concentration in the inner and outer compartments of the chromatophore vesicle. It is proposed that the equation thus obtained should be used to measure pH from the quenching of ACMA fluorescence.Abbreviations pH transmembrane pH difference between the inner and outer compartments - Q quenching of fluorescence - BChl Bacteriochlorophyll - ACMA 9-amino-6-chloro-2-methoxyacridine - 9AA 9-aminoacridine - Tricine N-tris-(hydroxymethyl)methylglycine - MES 2-morpholinoethanesulfonic acid - FCCP carbonylcyanide-p-trifluoro-methoxy-phenylhydrazone - CCCP carbonylcyanide-m-chloro-phenylhydrazone     

Role of protein phosphorylation and inositol phospholipid turnover in rat parotid gland proliferation

The involvement of protein phosphorylation in isoproterenol (ISO)-mediated proliferation in the rat parotid gland was investigated by labeling the cells with [³²P] orthophosphate. An increased (4–6 fold) incorporation of the radiolabel was noted in the total parotid gland homogenates of ISO-treated animals when compared to controls. Plasma membrane, nuclear membrane and cytoplasm were isolated, the proteins separated by SDS/PAGE and the phosphoproteins detected by autoradiography. Two phosphoproteins with apparent M_r of 45 and 170 kDa were identified in the cytoplasm while the 170 kDa phosphoprotein also appeared as part of plasma membrane. Transfer of these proteins to nitrocellulose followed by Western blot detection with an antiphosphotyrosine monoclonal antibody showed reactivity with the 170 kDa region of the plasma membrane and cytoplasm. Separate in vitro studies involving incubations of rat parotid slices with 0.2 mM ISO and [³H] myo-inositol for 1 min induced inositol phosphate hydrolysis resulting in a significant increase in inositol-bis and -tris phosphate production. Inositol phosphate production can be blocked by pre-incubation with a mixed -adrenergic receptor antagonist but not with physiological concentrations of - or ₁-specific adrenergic receptor antagonists, indicating the ISO effects are mediated through the ₂-adrenergic receptors. The inclusion of calmodulin antagonists along with ISO prevented the expression of cell-surface galactosyltransferase and retarded gland hypertrophy and hyperplasia. These results suggest that ISO treatment leads to the phosphorylation of target proteins which may be involved in signal transduction pathways leading to cell proliferation.Abbreviations InsP₁, InsP₂, InSP₃ inositol mono-, bis-, and tris-phosphates - UDP Uridine diphosphate - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate - TFP Trifluoperazine - P-tyr phosphotyrosine - Gal Tase galactosyltransferase     

Transmembrane diffusion of hydrophobic antimicrobial agents and cell surface hydrophobicity in Bacteroides fragilis

The transmembrane diffusion of hydrophobic antimicrobial agents, e.g. lincomycin and clindamycin, was examined in Bacteroides fragilis which is sensitive to these agents. The results showed that these agents penetrate efficiently through the outer membrane. Cell surface hydrophobicity measured by the partition assay between water and p-xylene revealed that the cell surface of B. fragilis is more hydrophobic than that of Salmonella typhimurium or Pseudomonas aeruginosa. Furthermore, treatment with low concentrations of surfactant caused cell lysis. These results suggest that the cell surface hydrophobicity in B. fragilis plays an important role in the efficient transmembrane penetration of hydrophobic compounds. This efficiency explains the susceptibility of B. fragilis to hydrophobic antimicrobial agents.     

Cyclic AMP-Dependent Protein Phosphorylation in Chemosensory Neurons: Identification of Cyclic Nucleotide-Regulated Phosphoproteins in Olfactory Cilia

Chemosensory dendritic membranes (olfactory cilia) contain protein kinase activity that is stimulated by cyclic AMP and more efficiently by the nonhydrolyzable GTP analog guanosine-5'-O-(3-thio)triphosphate (GTP gamma S). In control nonsensory (respiratory) cilia, the cyclic AMP-dependent protein kinase is practically GTP gamma S-insensitive. GTP gamma S activation of the olfactory enzyme appears to be mediated by a stimulatory GTP-binding protein (G-protein) and adenylate cyclase previously shown to be enriched in the sensory membranes. Protein kinase C activity cannot be detected in the chemosensory cilia preparation under the conditions tested. Incubation of olfactory cilia with [gamma-32P]ATP leads to the incorporation of [32P]phosphate into many polypeptides, four of which undergo covalent modification in a cyclic nucleotide-dependent manner. The phosphorylation of one polypeptide, pp24, is strongly and specifically enhanced by cyclic AMP at concentrations lower than 1 microM. This phosphoprotein is not present in respiratory cilia, but is seen also in membranes prepared from olfactory neuroepithelium after cilia removal. Cyclic AMP-dependent protein kinase and phosphoprotein pp24 may be candidate components of the molecular machinery that transduces odor signals.     

Properties and Partial Purification of Choline Acetyltransferase from the Nematode Caenorhabditis elegans

We have stabilized and studied choline acetyltransferase from the nematode Caenorhabditis elegans. The enzyme is soluble, and two discrete forms were resolved by gel filtration. The larger of these two forms (MW approximately 154,000) was somewhat unstable and in the presence of 0.5 M NaI was converted to a form indistinguishable from the "native" small form (MW approximately 71,000). We have purified the small form of the enzyme greater than 3,300-fold by a combination of gel filtration, ion-exchange chromatography, and nucleotide affinity chromatography. The purified preparation has a measured specific activity of 3.74 mumol/min/mg protein, and is free of acetylcholinesterase and acetyl-CoA hydrolase activities. The Vmax of the purified enzyme is stimulated by NaCl, with half-maximal stimulation at 80 mM NaCl. The Km for each substrate is also affected by salt, but in different manners from each other and the Vmax; the kinetic parameter Vmax/Km thus changes significantly as a function of the salt concentration.     

Studies on 9-amino-6-chloro-2-methoxy acridine fluorescence quenching and enhancement in relation to energy transduction in spheroplasts and intact cells of the cyanobacterium Plectonema boryanum

The fluorescent probe 9-amino-6-chloro-2-methoxy acridine was used to study the energy transduction in the thylakoid and cell membranes of the cyanobacterium Plectonema boryanum. Apart from light-driven electron transfer, the dark endogenous respiration also leads to energization resulting in an ACMA fluorescence response, that is sensitive to the electron flow inhibitor 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, to the energy transfer inhibitors dicyclohexylcarbodiimide and venturicidine and to the uncoupler 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide.In spheroplasts, in which the cell membranes have lost their capacity to maintain a proton gradient, the respiration-and light-induced ACMA fluorescence changes (quenching) are similar to those in chloroplasts. In intact cells a combination of reversible quenching and enhancement of ACMA fluorescence was found. This dualistic behaviour is supposedly caused by an opposite orientation of the thylakoid and cell membranes. ACMA quenching at the level of the thylakoids was obtained either by respiratory or photosynthetic electron transfer and gave similar responses to those obtained in the spheroplasts. The slower ACMA fluorescence enhancement, only observed in cells with intact cell membranes, also evoked by both respiration and light-induced energization is sensitive to the compounds mentioned above and in addition to KCN.Our results support the view [8] that dark oxidation of substrates by O₂ proceeds via the thylakoid membrane and terminates at a CN^- sensitive oxidase located in the cell membrane which requires the involvement of a mobile cytoplasmic redox mediator.Abbreviations ACMA 9-amino-6-chloro-2-methoxy acridine - chl a chlorophyll a - DBMIB 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD dicyclohexylcarbodiimide - DNP dinitrophenol - DNP-INT dinitrophenyl ether of 2-iodo-4-nitrothymol - FCCP carbonylcyanide-p-trifluoro-methoxy phenylhydrazone - S-13 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide - tricine N-2 (2-Hydroxy-1, 1-bis (hydroxymethyl) ethyl)-glycine - Tris Tris (hydroxymethyl) amino methane     

Ethylene biosynthesis in isolated vacuoles of Vicia faba L. — requirement for membrane integrity

The ability of vacuoles prepared from V. faba leaves to convert 1-aminocyclopropane-1-carboxylic acid to C₂H₄ was destroyed when vacuoles were lysed by passage through a hypodermic needle, freezing and thawing, osmotic shock, treatment with ethanol or with a detergent. Ethylene synthesis in the vacuolar fraction was also inhibited by the uncouplers carbonyl cyanide m-chlorophenyl hydrazone and dinitrophenol and by the ionophores valinomycin, nigericin, and A23187. Ethylene formation increased with increasing pH of the incubation medium over the pH range of 5.0–7.5. These observations support the hypothesis that C₂H₄ biosynthesis in vacuolar preparations is dependent on membrane integrity, possibly because of the requirement for a transmembrane ion gradient.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CCCP carbonyl cyanide m-chlorophenyl hydrazone     

Conformation of antibiotic X-537A (lasalocid-A) and its calcium complex in acetonitrile solvent using circular dichroism spectroscopy

The calcium binding characteristics of antibiotic X-537A (lasalocid-A) in a lipophilic solvent, acetonitrile (CH3CN), have been studied using circular dichroism (CD) spectroscopy. The analysis of the data indicated that in this medium polar solvent, X-537A forms predominantly the charged complexes of stoichiometries 2:1 and 1:1, the relative amounts of the two being dependent on [Ca2+]. The conformations of the complexes, arrived at on the basis of the data, seem to indicate a rigid part encompassing Ca2+, liganded to 3 oxygens of the molecule, viz., the carbonyl, the substituted tetrahydrofuran ring and the substituted pyran ring oxygens (apart from, possibly, the liganding provided by nitrogen atoms of the solvent molecules), and a flexible part consisting of the salicylic acid group of the molecule.