Molecular probes for muscarinic receptors: derivatives of the M1-antagonist telenzepine.

Functionalized congeners of the M1-selective muscarinic antagonist telenzepine (4,9-dihydro-3-methyl-4-[(4-methyl-1-piperazinyl)acetyl]-10H- thieno[3,4-b][1,5]benzodiazepin-10-one) were developed and found to bind to the receptor with affinities (Ki values) in approximately the nanomolar range. The derivatives contain a 10-aminodecyl group, which provides a nucleophilic functionality for further derivatization. The attachment of a spacer chain to the distal piperazinyl nitrogen was based on previous findings of enhanced affinity at muscarinic receptors in an analogous series of alkylamino derivatives of pirenzepine [J. Med. Chem. (1991) 34, 2133-2145]. The telenzepine derivatives contain prosthetic groups for radioiodination, protein cross-linking, photoaffinity labeling, and fluorescent labeling and biotin for avidin complexation. The affinity for muscarinic receptors in rat forebrain (mainly m1 subtype) was determined in competitive binding assays vs [3H]-N-methylscopolamine. A (p-aminophenyl)-acetyl derivative for photoaffinity labeling had a Ki value of 0.29 nM at forebrain muscarinic receptors (16-fold higher affinity than telenzepine). A biotin conjugate displayed a Ki value of 0.60 nM at m2-receptors and a 5-fold selectivity versus forebrain. The high affinity of these derivatives makes them suitable for the characterization of muscarinic receptors in pharmacological and spectroscopic studies, for peptide mapping, and for histochemical studies.     

Lateral diffusion of membrane-spanning and glycosylphosphatidylinositol-linked proteins: toward establishing rules governing the lateral mobility of membrane proteins.

In the plasma membrane of animal cells, many membrane-spanning proteins exhibit lower lateral mobilities than glycosylphosphatidylinositol (GPI)-linked proteins. To determine if the GPI linkage was a major determinant of the high lateral mobility of these proteins, we measured the lateral diffusion of chimeric membrane proteins composed of normally transmembrane proteins that were converted to GPI-linked proteins, or GPI-linked proteins that were converted to membrane-spanning proteins. These studies indicate that GPI linkage contributes only marginally (approximately twofold) to the higher mobility of several GPI-linked proteins. The major determinant of the high mobility of these proteins resides instead in the extracellular domain. We propose that lack of interaction of the extracellular domain of this protein class with other cell surface components allows diffusion that is constrained only by the diffusion of the membrane anchor. In contrast, cell surface interactions of the ectodomain of membrane-spanning proteins exemplified by the vesicular stomatitis virus G glycoprotein reduces their lateral diffusion coefficients by nearly 10-fold with respect to many GPI-linked proteins.     

Crystallization of beta-galactosidase from Escherichia coli.

Two crystal forms of beta-galactosidase have been obtained from Escherichia coli. One crystal form is hexagonal space group P6222 or enantiomorph, with cell dimensions a = b = 154 A, c = 750 A. The second form is monoclinic, space group P21, with cell dimensions a = 107.9 A, b = 207.5 A, c = 509.9 A, beta = 94.7 degrees. The monoclinic form seems better suited to detailed structural analysis. The crystals are radiation-sensitive, but by using synchrotron radiation in conjunction with a long (400 mm) crystal-to-film distance it was possible to resolve the individual reflections. On the basis of crystal density measurements, there are four tetramers each of molecular weight 465,000 per asymmetric unit. The Patterson function strongly suggests that two of the tetramers are related to the other two by translation. The data are consistent with the tetramers having 222 point symmetry, but this is not proven.     

Structure and dynamics of α-chymotrypsin-N-trifluoroacetyl-4-fluorophenylalanine complexes

Summary Fluorine NMR lineshape, relaxation and Overhauser effect data collected at 282 and 470 MHz have been used to obtain information about the nature of complexes formed betweenN-trifluoroacetyl-4-fluorophenylalanine and the enzyme chymotrypsin. Systems involving both enantiomers have been examined as well as derivatives of these in which the aromatic ring hydrogens have been replaced by deuterium. The enzyme-induced fluorine chemical shift effects and the dynamics of molecular motions of the fluorophenyl ring at the respective binding sites appear to be similar in both complexes and, where comparable, the results are in agreement with data obtained at lower frequencies that have been reported by other workers. The dynamics of the fluoroaromatic ring in these complexes are significantly different from those observed in a closely related acylated enzyme.     

Characterization of the locomotor depression produced by an A2-selective adenosine agonist

Adenosine analogs, such as N6-cyclohexyladenosine (CHA) that are selective for A1-adenosine receptors, and analogs, such as 5'-N-ethylcarboxamidoadenosine (NECA) that are active at both A1 and A2 receptors, cause a profound depression of locomotor activity in mice via a central mechanism. The depression is effectively reversed by non-selective adenosine antagonists such as theophylline. We report that 2-([2-aminoethylamino) carbonylethylphenylethylamino]-5'-N-ethylcarboxamidoadenosine (APEC), an amine derivative of the A2-selective agonist, CGS21680, is a potent locomotor depressant in mice. The in vivo pharmacology is consistent with A2-selectivity at a central site of action. Two parameters indicative of locomotor activity, horizontal activity and total distance travelled, were measured using a computerized activity monitor. From dose-response curves it was found that APEC (ED50 16 micrograms/kg) is more potent than CHA (ED50 60 micrograms/kg) and less potent than NECA (ED50 2 micrograms/kg). The locomotor depression by APEC was reversible by theophylline, but not by the A1-selective antagonists 8-cyclopentyltheophylline (CPT) and 8-cyclopentyl-1, 3-dipropyl-2-thioxanthine, nor by the peripheral antagonists 8-p-sulfophenyltheophylline (8-PST) and 1,3-dipropyl-8-p-sulfophenylxanthine. The locomotor activity depression elicited by NECA and CHA was reversed by A1-selective antagonists. These results suggest that the effects of APEC are due to stimulation of A2 adenosine receptors in the brain.     

Isolation and characterization of human glucose-6-phosphate isomerase isoforms containing two different size subunits

Previously undetected isoforms of human glucose-6-phosphate isomerase (GPI) have been isolated utilizing substrate-induced elution of the enzyme from spherical cross-linked phosphocellulose as an affinity ligand and subjected to a series of physical and chemical studies. The two major isoforms (1, 48%, pI 9.13; 2, 36%, pI 9.00) are homodimers of subunits of 63.2 kDa (Type-A) and are charge isomers, probably representing deamidation of specific Asn-Gly sequences as in other species. Isoform 3 (13%, pI 8.84) is a heterodimer composed of the Type-A subunit and a previously unreported larger subunit of 69.8 kDa (Type-B). Isoform 4 (3%, pI 8.62) is a BB-homodimer. Structural differences in the two types of subunits are also apparent from CNBr fragmentation patterns. Carbohydrate analyses show that, even though potential N- and O-linked glycosylation sites exist, the isoforms are not due to glycosylation. Recently recognized sequence similarities between GPI and the neurotropic lymphokine, neuroleukin (NLK) suggest that GPI and NLK are either derived from the same gene or represent modifications of the same protein. The possibility of NLK-GPI dimers exists, but the new isoforms identified in this study do not appear to represent hybrids of GPI subunits with mature NLK.     

Stimulation and priming of protein kinase C translocation by a Ca2+ transient-independent mechanism. Studies in human neutrophils challenged with platelet-activating factor and other receptor agonists

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 stimulate human polymorphonuclear neutrophils (PMN) to translocate protein kinase C from the cytosol to plasmalemma as judged by their abilities to increase PMN binding of and receptor numbers for [3H]phorbol dibutyrate [( 3H]PDB) (O'Flaherty, J.T., Jacobson, D.P., Redman, J.F., and Rossi, A.G. (1990) J. Biol. Chem. 265, 9146-9152). Platelet-activating factor (PAF) had these same effects. Moreover, two potent PAF analogs (but not an inactive analog) increased [3H]PDB binding; a PAF antagonist blocked responses to PAF without altering those to fMLP; and PMN treated with PAF became desensitized to PAF while retaining sensitivity to fMLP. Indeed, PMN incubated with 1-100 nM PAF for 5-40 min had markedly enhanced [3H]PDB binding responses to fMLP. PAF thus acted through its receptors to stimulate and prime protein kinase C translocation. Its effects, however, did not necessarily proceed by a standard mechanism: Ca2(+)-depleted PMN failed to raise Fura-2-monitored cytosolic Ca2+ concentrations [( Ca2+]i), yet increased [3H]PDB binding and receptor numbers almost normally after PAF challenge. PAF also primed Ca2(+)-depleted PMN to fMLP. Nevertheless, [3H]PDB binding responses to PAF were blocked in PMN loaded with Ca2+ chelators, viz. Quin 2, Fura-2, or 5,5'-dimethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Exogenous Ca2+ reversed Quin 2 inhibition, and a weak chelator 4,4'-difluoro-BAPTA, lacked inhibitory actions. The chelators similarly influenced fMLP and leukotriene B4. Thus, PMN can by-pass [Ca2+]i to translocate protein kinase C. They may achieve this using a regulatable pool of Ca2+ that evades conventional [Ca2+]i monitors or a signal that needs cell Ca2+ to form and/or act. This signal may mediate function in Ca2(+)-depleted cells, the actions of [Ca2+]i-independent stimuli, cell priming, and protein kinase C movements that otherwise seem [Ca2+]i-induced.     

A hybrid Azotobacter vinelandii-Clostridium pasteurianum nitrogenase iron protein that has in vivo and in vitro catalytic activity

Site-directed mutagenesis and gene replacement procedures were used to construct a mutant strain of Azotobacter vinelandii which expresses a hybrid nitrogenase Fe protein. This hybrid Fe protein has its carboxyl-terminal 18 residues replaced with the 5 analogous residues from the Clostridium pasteurianum Fe protein sequence. The hybrid Fe protein is 13 amino acids smaller than the wild-type A. vinelandii Fe protein and has a net loss of 4 negatively charged residues, resulting in a change in size and charge. The strain which produces the hybrid Fe protein remained capable of diazotrophic growth, albeit at a reduced rate. Also, the purified hybrid Fe protein exhibited a maximum activity about one-half that of native Fe protein. These results demonstrate that the tight, inactive complex which is formed when A. vinelandii MoFe protein and C. pasteurianum Fe protein are mixed in heterologous reconstitution experiments cannot be accounted for only by differences in the A. vinelandii and C. pasteurianum Fe protein primary sequences located at their respective carboxyl termini.