, and comparison of the quantity and composition of vDNA generated at discrete points during reverse transcription. Quantitative analysis of the reverse transcription intermediates for these species strongly suggests decreased stability of the DNA produced. Both Zn 2؉ finger mutants appear to be defective in DNA synthesis, with the minus-and plus-strand transfer processes being affected while interior portions of the vDNA remain more intact. Sequences obtained from PCR amplification and cloning of 2-LTR circle junction fragments revealed that the NC mutants had a phenotype similar to the IN mutant; removal of the terminal CA dinucleotides necessary for integration of the vDNA is disabled by the NC mutations. Thus, the loss of infectivity in these NC mutants in vivo appears to result from defective reverse transcription and integration processes stemming from decreased protection of the full-length vDNA. Finally, these results indicate that the chaperone activity of NC extends from the management of viral RNA through to the full-length vDNA.
In this report recombinant estrogen binding protein (EBP1), isolated originally from Candida albicans as a result of its high affinity for 17beta-estradiol, has been purified extensively using a modified affinity purification scheme originally developed for a homolog of EBP1, old yellow enzyme (OYE). It is shown that like OYE, the protein binds a variety of compounds with a phenolic structure, including 17beta-estradiol, and compounds with an alpha, beta-unsaturated keto or aldehyde structure. In addition, EBP1 exhibits an NADPH oxidoreductase activity, transferring electrons from NADPH to all alpha,beta-unsaturated ketones and aldehydes tested via the tightly bound FMN cofactor. Analysis of the steady-state kinetics of these reactions indicate a tetra uni ping-pong mechanism. Inhibition of the steady-state reaction by 17beta-estradiol gives a Ki = 10 +/- 2 nM, and indicates exclusive binding of this steroid to the enzyme in its oxidized state. In contrast, 19-nortestosterone binds to both oxidized and reduced forms of the enzyme with dissociation constants of 600 +/- 100 and 650 +/- 90 nM, respectively. EBP1 also catalyzes a disproportionation reaction with certain compounds, in which two molecules of a cylic alpha,beta-unsaturated ketone, including the steroid 19-nortestosterone, are individually aromatized and reduced to the corresponding saturated ketone. Despite the extensive similarity in sequence and enzymic activity, notable differences between EBP1 and the OYE family of proteins exist with regard to the binding behavior and reactivity with the two steroids tested here, estradiol and 19-nortestosterone.
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