Nonviral, host-derived proteins on lentiviral vector surfaces can have a profound effect on the vector's biology as they can both promote infection and provide resistance to complement inactivation. We have exploited this to engineer a specific posttranslational modification of a "nonenvelope," virally associated protein. The bacterial biotin ligase (BirA) and a modified human DeltaLNGFR have been introduced into HEK293T cells and their protein products directed to the lumen of the endoplasmic reticulum. The BirA then couples biotin to an acceptor peptide that has been fused to the DeltaLNGFR. This results in the covalent linkage of biotin to the extracellular domain of the DeltaLNGFR expressed on the cell surface. Lentiviral vectors from these cells are metabolically labeled with biotin in the presence of free biotin. These biotinylated lentiviral vectors have a high affinity for streptavidin paramagnetic particles and, once captured, are easily manipulated in vitro. This is illustrated by the concentration of lentiviral vectors pseudotyped with either the VSV-G or an amphotropic envelope in excess of 4500-fold. This new cell line has the potential for widespread application to envelope pseudotypes compatible with lentiviral vector production.
Prolamellar bodies (PLB) contain two photochemically active forms of the enzyme protochlorophyllide oxidoreductase POR-PChlide 640 and POR-PChlide 650 (the spectral forms of POR-Chlide complexes with absorption maxima at the indicated wavelengths). Resuspension of maize PLB in media with a pH below 6.8 leads to a rapid conversion of POR-PChlide 650 to POR-PChlide 640 and a dramatic re-organization of the PLB membrane system. In the absence of excess NADPH, the absorption maximum of the POR complex undergoes a further shift to about 635 nm. This latter shift is reversible on the re-addition of NADPH with a half-saturation value of about 0.25 mM NADPH for POR-PChlide 640 reformation. The disappearance of PORPChlide 650 and the reorganization of the PLB, however, are irreversible. Restoration of low-pH treated PLB to pH 7.5 leads to a further breakdown down of the PLB membrane and no reformation of POR-PChlide 650 . Related spectral changes are seen in PLB aged at room temperature at pH 7.5 in NADPH-free assay medium. The reformation of PORPChlide 650 in this system is readily reversible on re-addition of NADPH with a half-saturation value about 1.0 lM. Comparison of the two sets of changes suggest a close link between the stability of the POR-PChlide 650 , membrane organization and NADPH binding.The low-pH driven spectral changes seen in maize PLB are shown to be accelerated by adenosine AMP, ADP and ATP. The significance of this is discussed in terms of current suggestions of the possible involvement of phosphorylation (or adenylation) in changes in the aggregational state of the POR complex.Keywords: protochlorophyllide oxidoreductase; prolamellar body; protochlorophyllide; oxidoreductase; chlorophyllide.Plant prolamellar bodies (PLB) found in the etioplasts of dark-grown (etiolated) seedlings, are the precursors of the chloroplast thylakoid membrane. The PLB membrane is dominated by the presence of a single protein species, protochlorophyllide oxidoreductase (EC 1.3.1.33) (POR) that catalyses the light-driven, NADPH-dependent reduction of protochlorophyllide (PChlide) to chlorophyllide (Chlide). Analyses of the absorption spectrum of PLB [1] and low-temperature fluorescence spectra of etioplast inner membrane preparations (EPIM) and PLB [2], indicate the presence of three major pools of PChlide; a nonphotoconvertible form PChlide 628)633 and two photoconvertible forms PChlide 640)645 and PChlide 650)657 . The suffix numbers relate to the wavelengths of the absorption and emission maxima, respectively. To emphasize the fact that the two photoconvertible forms are bound to POR, they will be referred to here to as POR-PChlide 640 and POR-PChlide 650 .Under in vivo conditions, exposure of etioplasts to a flash of bright white light leads to a conversion of the photoconvertible PChlide pigments to Chlide resulting in a rapid shift of the main absorption maximum from 650 nm, initially to about 678 nm and then to 684 nm. Over a period of about 20 min, this absorption maximum shifts back to 672 nm. This latter shift, r...
Transmission electron microscopy (TEM) indicates that maize prolamellar bodies (PLBs) are built up of tetrapodal units based on a highly convoluted but continuous lipid bilayer exhibiting diamond cubic (Fd3m) symmetry. Such lattices are often described in terms of infinite periodic minimal surfaces (IMPS) exhibiting zero net curvature and dividing the system into two identical subvolumes. If so, X-ray diffraction measurements would be expected to index on a double-diamond (Pn3m) lattice with a unit cell length half that of the TEM lattice. Our measurements index on a Fd3m lattice with a similar repeat distance to the TEM images. The PLB membrane is thus inherently asymmetric, probably as the result of the distribution of membrane protein.z 1998 Federation of European Biochemical Societies.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.