Charge transfer photochemistry of quadruply bonded ditungsten halophosphine complexes

The quadruple metal-metal bonded complexes, W₂Cl₄(PR₃)₄ (PR₃ = PMe₃, PMe₂Ph, PBu₃), photoreact in dichloromethane with near-UV excitation (λ>375 nm) to yield a mixed valence W₂(II,III) photoproduct. Electronic absorption and EPR spectra of photolyzed solutions are identical to those obtained from the thermal oxidation of W₂Cl₄(PR₃)₄ by PhICI₂, which is known to yield W₂Cl₅(PR₃)₃. Subsequent reaction of the photolyzed solution yields the oxidized, confacial biotahedral W₂(III,III) halophosphine. Analysis of the organic photoproduct reveals that the halocarbon solvent is reduced by one electron to yield the chloromethyl radical. When the radical is produced in low yields, hydrogen abstraction from solvent appears to be sufficiently efficient to compete with dimerization and only chloromethane is observed; however, at higher concentrations, the chloromethyl radicals couple to produce dichloroethane. Photoreaction is observed only with near-UV excitation of the LMCT absorption manifold of W₂Cl₄(PR₃)₄. At lower energy wavelengths, transient absorption spectroscopy shows the production of the ¹δδ^* excited state, which decays to ground state over times commensurate with the decay of ¹δδ^* luminescence. In hydrocarbon solutions, no transient intermediate or photochemistry is observed, indicating that the LMCT excited state, although capable of reducing a C---X bond, cannot activate the stronger C---H bonds of hydrocarbons. The photochemistry and transient absorption spectroscopy results of the W₂Cl₄(PR₃)₄ complexes are compared to our previous studies of the homologs.     

The application of molecular markers to the study and conservation of fish populations, with special reference to Salmo

The main molecular techniques which can be used to generate genetic markers, and the applications of these markers to studies of fish populations are outlined. Published and ongoing studies, in the authors' laboratories, on brown trout and Atlantic salmon are used to compare the resolution and applicability of allozyme, mitochondrial DNA and minisatellite (variable number of tandem repeats) markers for studies on population structuring, genetic variation within populations, and the impact of the accidental and deliberate introduction of non-native salmonids on the genetic make-up of natural populations.     

Mutant Escherichia coli penicillin acylase with enhanced stability at alkaline pH

Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins into 6-aminopenicillanic acid, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding for Escherichia coli penicillin acylase was subjected to oligonucleotide-directed random mutagenesis at regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned in E. coli, was screened by a filter paper assay of the colonies for the presence of penicillin acylase activity with enhanced stability at alkaline pH. Characterization of one of the selected clones revealed the presence of a mutation, Trp431-Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half-life of the mutant enzyme when stored at pH 8.5 as compared with the wild-type enzyme, with a comparable specific activity at several pH values. In general, the mutant displayed increased stability toward the basic side in the pH-stability profile. (c) 1995 John Wiley & Sons, Inc.     

Mammalian expression of single chain variable region fragments dimerized by Fc regions

To promote application of a single chain variable region fragment (sFv) in immunoglobulins, a sFv gene was connected to an IgG1 Fc gene, designated as a sFvc gene, and used for transfection of Sp2/0. As a result, the sFvc protein was found to be secreted in a dimeric form. It is thus felt that the sFvc protein, which mimicks the shape of a naturally occurring antibody, can be simple and useful to reproduce divalency and Fc-associated effecter functions as seen in a natural antibody.Abbreviations Abbreviations sFv single chain variable region fragment - Fc constant region of immunoglobulin - sFvc single chain variable region fragment with an Fc region     

Charge clusters and the orientation of membrane proteins

Summary Although hydrophobic forces probably dominate in determining whether or not a protein will insert into a membrane, recent studies in our laboratory suggest that electrostatic forces may influence the final orientation of the inserted protein. A negatively charged hepatic receptor protein was found to respond totrans-positive membrane potentials as though electrophoresing into the bilayer. In the presence of ligand, the protein appeared to cross the membrane and expose binding sites on the opposite side. Similarly, a positively charged portion of the peptide melittin crosses a lipid membrane reversibly in response to atrans-negative potential. These findings, and others by Date and co-workers, have led us to postulate that transmembrane proteins would have hydrophobic transmembrane segments bracketed by positively charged residues on the cytoplasmic side and negatively charged residues on the extra-cytoplasmic side. In the thermodynamic sense, these asymmetrically placed charge clusters would create a compelling preference for correct orientation of the protein, given the inside-negative potential of most or all cells. This prediction is borne out by examination of the few transmembrane proteins (glycophorin, M13 coat protein, H-2K^b, HLA-A2, HLA-B7, and mouse Ig heavy chain) for which we have sufficient information on both sequence and orientation.In addition to the usual diffusion and pump potentials measurable with electrodes, the microscopic membrane potential reflects surface charge effects. Asymmetries in surface charge arising from either ionic or lipid asymmetries would be expected to enhance the bias for correct protein orientation, at least with respect to plasma membranes. We introduce a generalized form of Stern equation to assess surface charge and binding effects quantitatively. In the kinetic sense, dipole potentials within the membrane would tend to prevent positively charged residues from crossing the membrane to leave the cytoplasm. These considerations are consistent with the observed protein orientations. Finally, the electrostatic and hydrophobic factors noted here are combined in two hypothetical models of translocation, the first involving initial interaction of the presumptive transmembrane segment with the membrane; the second assuming initial interaction of a leader sequence.     

Ionic charge on phospholipids and their interaction with the mitochondrial adenosine triphosphatase

The activity of the lipid-depleted, oligomycin-sensitive mitochondrial ATPase has been measured in the presence of liposomes prepared from mixtures of phosphatidylglycerol and phosphatidylglycerol lysine. Enzyme activity increased linearly with an increase in the negative charge of liposomes prepared from the phosphatidylglycerol-phosphatidylglycerol lysine mixtures. The electrophoretic mobility and activating capacity of liposomes of several other phospholipids were determined. A linear relationship between electrophoretic mobility of the liposomes and oligomycin-sensitive activity was again apparent. These observations demonstrate that the activity of the ATPase is directly proportional to the ionic charge on phospholipid activators if the acyl chain composition of the phosphoglycerides is relatively constant.     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

Lateral enzyme topology in the rough endoplasmic reticulum of rat liver

Summary Rough microsomes were subfractionated on the basis of different properties in order to investigate the nature and extent of the enzyme heterogeneity of these vesicles. A discontinuous gradient, containing monovalent cations allowed the separation of a ribosome-poor membrane fraction which was enriched in electron transport enzymes and relatively poor in phosphatases. Zonal centrifugation on a stabilizing gradient separated 3 fractions characterized by enrichment of electron transport enzymes, glucose-6-phosphatase and adenosinetriphosphatase, respectively. An essentially similar pattern was seen when ribosomes were removed with EDTA and the denuded vesicles subfractionated on a sucrose gradient. Rough microsomes from phenobarbitaltreated rats exhibited the same pattern both qualitatively and quantitatively. It appears that electron transport enzymes and two types of phosphatases are heterogeneously distributed among rough microsomal vesicles.This work has been supported by grants from the Swedish Medical Research Council. The authors wish to thank Mrs. Ulla-Britta Torndal for her valuable technical assistance