Analysis and purification of acyl coenzyme A thioesters by reversed-phase ion-pair liquid chromatography.

A rapid method is described for the analysis of mixtures of short-chain acyl coenzyme A thioesters by reversed-phase ion-pair chromatography on LiChrosorb RP-8 and μBondapak C₁₈ columns. The technique is applicable to separation of CoASH, acetyl-CoA propionyl-CoA, and 3-hydroxy-3-methylglutaryl-CoA, as well as dicarboxylic acids and several nucleotides commonly used as cofactors for biosynthetic reactions. The method was utilized on a preparative scale for purification of 3-hydroxy-3-ethylglutaryl-CoA from CoASH and 3-hydroxy-3-ethylglutaric acid. The counterion employed was tetrabutylammonium (phosphate), pH 5.5, in various methanol:water mixtures. Elution profiles and retention values of compounds were influenced by the concentration of counterion and mass of injected sample. Tetrabutylammonium ions could be removed from effluent by ionexchange chromatography on Amberlite IR-120 resin.     

Effect of high pH on the spectral and catalytic properties of beef heart cytochrome oxidase

Incubation of cytochrome oxidase at high pH induces changes in several spectral properties. The optical Soret maximum shifts to longer wavelength, and there is an apparent loss in intensity of the 655-nm band, effects that are normally assigned either to a spin-state transition in cytochrome a3 or to a reduction of heme a. However, magnetic circular dichroism spectra show that cytochrome a3 remains high spin and that both cytochrome a and cytochrome a3 are oxidized. At the same time, there is the appearance of a low-spin signal indicative of hydroxide-imidazole coordination which we assign as arising from a structural transition at cytochrome a, rather than at cytochrome a3, as has been proposed previously. With longer incubation times, a new copper signal appears with electron paramagnetic resonance parameters markedly different from those obtained from copper centers which have undergone denaturation. Spin quantitation establishes that this new resonance does not arise from CuA and suggests that high pH breaks the magnetic coupling present at the cytochrome a3-CuB center. A significant proportion of cytochrome a3 may be converted to a low-spin thiolate during this process.     

Cytoplasmic network arrays demonstrated by immunolocalization using antibodies to a high molecular weight protein present in cytoskeletal preparations from cultured cells

Antisera were raised in rabbits against the two major components of intermediate filament preparations from glia-derived C6 cells, polypeptides of M_r around 300 000 and 58 000 (vimentin). These, and a third antiserum raised against microtubule proteins from hog brain, were shown to be specific for their respective immunogens. The assay employed involved the separation of components of crude cell extracts or filament preparations by SDS-polyacrylamide gel electrophoresis, and their subsequent transfer to and immobilization on nitrocellulose sheets. Cross-reacting counterparts of the immunogens were found in various cell lines, including C6, BALB/c 3T3, SV101, CHO, HeLa and PtK2 cells. In indirect immunofluorescence studies, antibodies to the high-M_r polypeptide component stained dense cytoplasmic network arrays of seemingly short, irregularly oriented fibres and lines of dots, in fibroblasts and in HeLa cells, but not in PtK2 cells. In well spread cells these networks were clearly distinguishable in morphology from the fibres decorated by antibodies to either microtubule protein or vimentin. The network arrays were resistant towards treatments with Triton X-100 and colcemid. By double immunofluorescence microscopy of single cells, using an additional antibody preparation to vimentin raised in guinea pigs, it was shown that after prolonged colcemid treatment of BALB/c 3T3 cells both; vimentin filaments and the structures stained by antibodies to the high-M_r component, accumulated in corresponding areas of the cytoplasm. The possibilities are discussed that this novel network-like structure is of the intermediate filament type and that it might function as a cross-linker of cytoplasmic—in particular cytoskeletal—elements. To signify its fluorescent localization and its possible linking role it is proposed to call the high-M_r component of intermediate filament preparations from cultured cells ‘plectin’.     

Preliminary crystallographic studies on bovine lactoferrin

The purification of bovine lactoferrin, its crystallization at low ionic strength, and preliminary X-ray crystallographic data are reported. The crystals, which grow from a two-phase system, are radiation-stable and suitable for a medium-resolution X-ray analysis. They are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 138.4 A, b = 87.1 A, c = 73.6 A, and one protein molecule in the asymmetric unit.     

Characterization of PTPRG in Knockdown and Phosphatase-Inactive Mutant Mice and Substrate Trapping Analysis of PTPRG in Mammalian Cells

Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98–99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, “substrate trapping” mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.     

Distribution of feeding and drinking by groups of captive house mice

Locations of feeding and drinking by social groups of commensal house mice (Mus domesticus), were monitored daily for 30 to 90 minutes for 1 to 3 months. Eighteen foodcups and 18 water bottles were distributed in a 2.4 × 5.1 m population cage. Social groups concentrated their feeding at a few of the foodcups which varied among replicates but drank from most of the water bottles. A mouse feeding at the same sites that are used by others implies that mice use social cues in choosing feeding sites. This is the first documentation of socially influenced choice of feeding sites by wild-caught commensal house mice. A mouse drinking at several sites without concentrating on any sites implies social cues are less important in choosing drinking sites.     

Protease pro region required for folding is a potent inhibitor of the mature enzyme.

alpha-Lytic protease, an extracellular bacterial serine protease, is synthesized with a large pro region that is required in vivo for the proper folding of the protease domain. To allow detailed mechanistic study, we have reconstituted pro region-dependent folding in vitro. The pro region promotes folding of the protease domain in the absence of other protein factors or exogenous energy sources. Surprisingly, we find that the pro region is a high affinity inhibitor of the mature protease. The pro region also inhibits the closely related Streptomyces griseus protease B, but not the more distantly related, yet structurally similar protease, elastase. Based on these data, we suggest a mechanism in which pro region binding reduces the free energy of a late folding transition state having native-like structure.