John David Dignam

[7] Purification and properties of NADPH-Cytochrome P-450 reductase

Publisher Summary This chapter provides information on the purification and properties of NADPH-cytochrome P-450 reductase. NADPH—cytochrome P-450 reductase, a flavoprotein component of the endoplasmic reticulum of liver and other organs, catalyzes the transfer of electrons from NADPH to cytochrome P-450. Cytochrome P-450 is the terminal oxidase of the drug metabolism system that hydroxylates a variety of compounds, such as alkanes, fatty acids, drugs, and steroids. Several forms of this hemoprotein are purified to homogeneity, differing in minimum molecular weight and substrate specificity. The procedure reported in the chapter describes the purification to apparent homogeneity of NADPH cytochrome P-450 reductase by solubilization with Renex 690 and affinity chromatography. The affinity column used is an NADP ligand attached to Sepharose 4B through adipic acid dihydrazide. The reduction of cytochrome P-450 can be determined directly under anaerobic conditions in the presence of carbon monoxide by following the formation of the peak at 450 nm in the reduced carbon monoxide difference spectrum.

Preparation of extracts from higher eukaryotes

Publisher Summary Successful preparation of crude extracts from eukaryotic cells and tissues for enzyme purification requires close attention to conditions that may alter the activity or native structure of an enzyme because nonspecific inactivation can result in irreproducibility that may make interpretation of such studies difficult. This chapter describes some general problems that must be considered when developing an extraction procedure followed by some specific protocols regarding the same. In some cases, the exact source of an enzyme is not an important consideration. It is a good idea to examine several tissues inasmuch as one might find a tissue to have unexpectedly high levels of the activity. Solid tissues may pose more of a problem, although liver and other organs from small animals can be readily homogenized in hand-held or motor-driven homogenizers. This approach is not practical for large quantities of material or for organs from larger animals, where connective tissue and large blood vessels make homogenization difficult. Some tissues pose unique problems where some modification of the protocols might be required for application to specific problems.

A biochemical characterization of the adeno-associated virus Rep40 helicase

The human adeno-associated virus (AAV) has generated much enthusiasm as a transfer vector for human gene therapy. Although clinical gene therapy trials have been initiated using AAV vectors, much remains to be learned regarding the basic mechanisms of virus replication, gene expression, and virion assembly. AAV encodes four nonstructural, or replication (Rep), proteins. The Rep78 and Rep68 proteins regulate viral DNA replication, chromosomal integration, and gene expression. The Rep52 and Rep40 proteins mediate virus assembly. To better understand Rep protein function, we have expressed the Rep40 protein in Escherichia coli and purified it to near homogeneity. Like the other Rep proteins, Rep40 possesses helicase and ATPase activity. ATP is the best substrate, and Mg2+ is the most efficient divalent metal ion for helicase activity. A Lys to His mutation in the purine nucleotide-binding site results in a protein that inhibits helicase activity in a dominant negative manner. Rep40 unwinds double-stranded DNA containing a 3′ single-stranded end, or blunt end, unlike the Rep68 and Rep52 enzymes, which have a strict requirement for DNA duplexes containing a 3′ single-stranded end. Values for KATP in the ATPase assay are 1.1 ± 0.2 mm and 1.2 ± 0.2 mm in the absence and presence, respectively, of single-stranded DNA. Values for Vmax are 220 ± 10 and 1,500 ± 90 nmol/min/mg in the absence and presence, respectively, of single-stranded DNA. These studies provide the first enzymatic characterization of the AAV Rep40 protein and elucidate important functional differences between the AAV helicases.

Adeno-Associated Virus Rep Protein-Mediated Inhibition of Transcription of the Adenovirus Major Late Promoter In Vitro

ABSTRACT Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four AAV replication proteins (Rep78, Rep68, Rep52, and Rep40) are pleiotropic effectors of virus integration, replication, transcription, and virion assembly. These proteins exert effects on Ad gene expression and replication. In transient plasmid transfection assays, Rep proteins inhibit gene expression from a variety of transcription promoters. We have examined Rep protein-mediated inhibition of transcription of the Ad major late transcription promoter (AdMLP) in vitro. Rep78/68 are the strongest transcription suppressors and the purine nucleotide binding site in the Rep proteins, and by implication, the ATPase activity or conformational change induced by nucleotide binding is required for full repression. Rep52 has modest effects, and Rep40 exerts no significant effect on transcription. Rep78/68 and their N-terminal 225-residue domain bind to a 55-bp AdMLP DNA fragment in gel shift assays, suggesting that protein-DNA interactions are required for inhibition. This interaction was confirmed in DNase I protection assays and maps to a region extending from the TATA box to the transcription initiation site. Gel shift, DNase I, and chemical cross-linking assays with TATA box-binding protein (TBP) and Rep68 indicate that both proteins interact with each other and with the promoter at adjacent sites. The demonstration of Rep interaction with TBP and the AdMLP suggests that Rep78/68 alter the preinitiation complex of RNA polymerase II transcription. These observations provide new insight into the mechanism of Rep-mediated inhibition of gene expression.

Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter

ABSTRACT Adeno-associated virus (AAV) and other parvoviruses inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we investigated AAVs interaction with adenovirus (Ad), AAVs most efficient helper virus. Coinfection with Ad and AAV results in an AAV-mediated inhibition of Ad5 gene expression and replication. The AAV replication proteins (Rep) activate and repress gene expression from AAV and heterologous transcription promoters. To investigate the role of Rep proteins in the suppression of Ad propagation, we performed chromatin immunoprecipitation analyses that demonstrated in vivo AAV Rep protein interaction with the Ad E2a gene promoter. In vitro binding of purified AAV Rep68 protein to the Ad E2a promoter was characterized by electrophoretic mobility shift assays (Kd = 200 ± 25 nM). A 38 bp, Rep68-protected region (5′-TAAGAGTCAGCGCGCAGTATTTACTGAAGAGAGCCT-3′) was identified by DNase I footprint analysis. The 38-bp protected region contains the weak E2a TATA box, sequence elements that resemble the Rep binding sites identified by random sequence oligonucleotide selection, and the transcription start site. These results suggest that Rep binding to the E2a promoter contributes to the inhibition of E2a gene expression from the Ad E2a promoter and may affect Ad replication.

Immunoelectron Microscopic Localization of Glutamyl-/ Prolyl-tRNA Synthetase within the Eukaryotic Multisynthetase Complex

A high molecular mass complex of aminoacyl-tRNA synthetases is readily isolated from a variety of eukaryotes. Although its composition is well characterized, knowledge of its structure and organization is still quite limited. This study uses antibodies directed against prolyl-tRNA synthetase for immunoelectron microscopic localization of the bifunctional glutamyl-/prolyl-tRNA synthetase. This is the first visualization of a specific site within the multisynthetase complex. Images of immunocomplexes are presented in the characteristic views of negatively stained multisynthetase complex from rabbit reticulocytes. As described in terms of a three domain working model of the structure, in “front” views of the particle and “intermediate” views, the primary antibody binding site is near the intersection between the “base” and one “arm.” In “side” views, where the particle is rotated about its long axis, the binding site is near the midpoint. “Top” and “bottom” views, which appear as square projections, are also consistent with the central location of the binding site. These data place the glutamyl-/prolyl-tRNA synthetase polypeptide in a defined area of the particle, which encompasses portions of two domains, yet is consistent with the previous structural model.

Control of the levels of alanyl-, glycyl-, and seryl-tRNA synthetases in the silkgland of Bombyx mori☆

We have examined the levels of glycyl-, alanyl-, and seryl-tRNA synthetases and the levels of their corresponding translatable mRNAs in the posterior and middle silkglands of the silkworm, Bombyx mori. Analysis of Western blots reveals that the change in the abundance of these enzymes during the fifth instar in crude extracts derived from posterior and middle silkgland correlates with changes in enzymatic activity; most of the change in activity for seryl- and alanyl-tRNA synthetases can be accounted for by the corresponding change in enzyme concentration, while the apparent specific activity of glycyl-tRNA synthetase appears to be elevated in the posterior silkgland. Accompanying the changes in enzyme activity and enzyme concentration are changes in the levels of the corresponding mRNAs as determined by immunoprecipitation of in vitro translation products. The levels of all three enzymes are 10 to 20 times higher in the posterior and middle silkglands than in ovarian tissue. A form of alanyl-tRNA synthetase with a slightly higher apparent molecular weight is detected in the posterior silkgland early in the fifth instar and in ovarian tissue.